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edited by  編輯

Ruiwen Zhang, University of Houston, United States
Ruiwen Zhang，美國休斯頓大學

REVIEWED BY  校訂者

Xueqi Liu,  劉雪琪，
First Affiliated Hospital of Anhui Medical University, China
中國安徽醫科大學第一附屬醫院
Jiahong Wang,  王佳紅，
Shenyang Pharmaceutical University, China
Shenyang Pharmaceutical University， 中國

*CORRESPONDENCE  *通信

Chih-Yang Huang,  Chih-Yang Huang 黃 ，
Kuan-Ho Lin,  林寬和，
区 lin839@mail.cmu.edu.tw
區 lin839@mail.cmu.edu.tw
† † ^(†){ }^{\dagger} These authors have contributed equally to this work
† † ^(†){ }^{\dagger} 這些作者對這項工作做出了同樣的貢獻
RECEIVED 04 February 2025
收稿日期 2025 年 2 月 4 日
accepted 28 April 2025
接受日期 2025 年 4 月 28 日
published 23 May 2025
發佈時間 2025 年 5 月 23 日

CITATION  引文

Lin S-Z, Kuo W-W, Tsai BC-K, Paul CR, Kuo C-H, Hsieh DJ-Y, Kao S-W, Pai P-Y, Chen S-J, Huang C-Y and Lin K-H (2025) Therapeutic potential of Cordyceps militaris cultivated with Ginkgo biloba seeds for alleviating western diet-induced type 2 diabetes and diabetic nephropathy.
Lin S-Z， Kuo W-W， Tsai BC-K， Paul CR， Kuo C-H， Hsieh DJ-Y， Kao S-W， Pai P-Y， Chen S-J， Huang C-Y and Lin K-H （2025） 用銀杏葉種子種植的蛹蟲夏草治療西方飲食誘導的 2 型糖尿病和糖尿病腎病的潛力。
Front. Pharmacol. 16:1562116.
前面。藥理學。16:1562116.
doi: 10.3389/fphar.2025.1562116
doi： 10.3389/fphar.2025.1562116
© 2025 Lin, Kuo, Tsai, Paul, Kuo, Hsieh, Kao, Pai, Chen, Huang and Lin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
© 2025 Lin， Kuo， Tsai， Paul， Kuo， Hsieh， Kao， Pai， Chen， Huang 和 Lin.這是一篇根據知識共用署名許可 （CC BY） 條款分發的開放獲取文章。允許在其他論壇中使用、分發或複製，前提是註明原作者和版權擁有者，並按照公認的學術慣例引用本期刊上的原始出版物。不允許使用、分發或複製不符合這些條款的內容。

Therapeutic potential of Cordyceps militaris cultivated with Ginkgo biloba seeds for alleviating western diet-induced type 2 diabetes and diabetic nephropathy
銀杏種子培養的蛹蟲夏草治療西餐誘導的 2 型糖尿病和糖尿病腎病

Shinn-Zong Lin 1 , 2 1 , 2 ^(1,2){ }^{1,2}, Wei-Wen Kuo 3 , 4 , 5 3 , 4 , 5 ^(3,4,5){ }^{3,4,5}, Bruce Chi-Kang Tsai 6 6 ^(6){ }^{6}, Catherine Reena Paul 6 6 ^(6){ }^{6}, Chia-Hua Kuo 7 , 8 7 , 8 ^(7,8){ }^{7,8}, Dennis Jine-Yuan Hsieh 9 , 10 9 , 10 ^(9,10){ }^{9,10}, Shih-Wen Kao 11 , 12 11 , 12 ^(11,12){ }^{11,12}, Pei-Ying Pai 13 , 14 13 , 14 ^(13,14){ }^{13,14}, Shan-Jun Chen 15 15 ^(15){ }^{15}, Chih-Yang Huang © 6 , 16 , 17 , 18 , 19 ∗ † 6 , 16 , 17 , 18 , 19 ∗ † ^(6,16,17,18,19**†){ }^{6,16,17,18,19 * \dagger} and Kuan-Ho Lin 13 , 20 ∗ † 13 , 20 ∗ † ^(13,20**†){ }^{13,20 * \dagger}
Shinn-Zong Lin 1 , 2 1 , 2 ^(1,2){ }^{1,2} ， Wei-溫 Kuo 3 , 4 , 5 3 , 4 , 5 ^(3,4,5){ }^{3,4,5} ， Bruce Chi-Kang Tsai 6 6 ^(6){ }^{6} ， Catherine Reena Paul 6 6 ^(6){ }^{6} ， Chia-華 Kuo 7 , 8 7 , 8 ^(7,8){ }^{7,8} ， Dennis Jine-Yuan Hsieh 9 , 10 9 , 10 ^(9,10){ }^{9,10} ， Shih-溫 Kao 11 , 12 11 , 12 ^(11,12){ }^{11,12} ， Pei-Ying Pai 13 , 14 13 , 14 ^(13,14){ }^{13,14} ， Shan-Jun Chen 15 15 ^(15){ }^{15} ， Chih-Yang Huang © 6 , 16 , 17 , 18 , 19 ∗ † 6 , 16 , 17 , 18 , 19 ∗ † ^(6,16,17,18,19**†){ }^{6,16,17,18,19 * \dagger} 和 Kuan-Ho Lin 13 , 20 ∗ † 13 , 20 ∗ † ^(13,20**†){ }^{13,20 * \dagger} 1 1 ^(1){ }^{1} Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan, 2 2 ^(2){ }^{2} Department of Neurosurgery, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan, 3 3 ^(3){ }^{3} Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan, 4 4 ^(4){ }^{4} Ph.D. Program for Biotechnology Industry, China Medical University, Taichung, Taiwan, 5 5 ^(5){ }^{5} School of Pharmacy, China Medical University, Taichung, Taiwan, 6 6 ^(6){ }^{6} Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan, 7 7 ^(7){ }^{7} Laboratory of Exercise Biochemistry, University of Taipei, Taipei, Taiwan, 8 8 ^(8){ }^{8} School of Physical Education and Sports Science, Soochow University, Suzhou, China, 9 9 ^(9){ }^{9} Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, 10 10 ^(10){ }^{10} Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan, 11 11 ^(11){ }^{11} Department of Orthopedic Surgery, Chung Shan Medical University Hospital, Taichung, Taiwan, 12 12 ^(12){ }^{12} School of Medicine, Chung Shan Medical University, Taichung, Taiwan, 13 13 ^(13){ }^{13} School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan, 14 14 ^(14){ }^{14} Division of Cardiovascular Medicine, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan, 15 15 ^(15){ }^{15} Home Run Biotechnology Co., Ltd., Tainan, Taiwan, 16 16 ^(16){ }^{16} Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan,
1 1 ^(1){ }^{1} 臺灣花蓮市佛教慈濟醫學基金會生物創新中心， 2 2 ^(2){ }^{2} 臺灣花蓮市佛教慈濟醫學基金會， 中國醫藥大學生命科學學院生物科學與技術 3 3 ^(3){ }^{3} 系， 臺灣台中市， 4 4 ^(4){ }^{4} 中國醫藥大學生物技術產業博士課程， 臺灣台中市， 5 5 ^(5){ }^{5} 中國醫藥大學藥學院， 臺灣台中， 6 6 ^(6){ }^{6} 花蓮慈濟醫院心血管及線粒體相關疾病研究中心， 臺灣花蓮， 臺灣， 臺北大學運動生化 7 7 ^(7){ }^{7} 實驗室， 8 8 ^(8){ }^{8} 蘇州大學， 中國蘇州 9 9 ^(9){ }^{9} ， 中山醫學大學醫學檢驗與生物技術系， 10 10 ^(10){ }^{10} 臺灣 台中， 中山醫學大學醫院臨床檢驗， 臺灣 11 11 ^(11){ }^{11} 中， 中山醫學大學醫院 12 12 ^(12){ }^{12} 骨外科， 臺灣 中， 中山醫學大學醫學院， 臺灣 台中 13 13 ^(13){ }^{13} ， 中國醫藥大學醫學院， 臺灣 台中， 14 14 ^(14){ }^{14} 臺灣， 中國醫藥大學醫院內科心血管內科， 臺灣台中， 15 15 ^(15){ }^{15} 家樂生物科技股份有限公司， 臺灣台南， 16 16 ^(16){ }^{16} 中國醫藥大學生物醫學研究所， 臺灣， 17 17 ^(17){ }^{17} Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan, 18 18 ^(18){ }^{18} Department of Medical Laboratory Science and Biotechnology, Asia University, Taichung, Taiwan, 19 19 ^(19){ }^{19} Center of General Education, Tzu Chi University, Hualien, Taiwan, 20 20 ^(20){ }^{20} Department of Emergency Medicine, China Medical University Hospital, Taichung, Taiwan
17 17 ^(17){ }^{17} 中國醫藥大學附屬醫院醫學研究部， 18 18 ^(18){ }^{18} 臺灣台中， 臺灣台中亞洲大學醫學檢驗科學與技術系， 19 19 ^(19){ }^{19} 臺灣花蓮慈濟大學通識教育中心， 20 20 ^(20){ }^{20} 臺灣台中市中國醫藥大學醫院急診醫學科

Background: Diabetic nephropathy (DN), a leading cause of chronic kidney disease and end-stage renal disease, is a serious complication of type 2 diabetes mellitus (T2DM). Current therapies primarily slow disease progression but are unable to reverse kidney damage, highlighting the need for novel therapy to treat DN.
背景： 糖尿病腎病 （DN） 是慢性腎病和終末期腎病的主要原因，是 2 型糖尿病 （T2DM） 的嚴重併發症。目前的療法主要減緩疾病進展，但無法逆轉腎損傷，這凸顯了治療 DN 的新療法的必要性。
Objective: This study evaluated the therapeutic potential of Cordyceps militaris (C. militaris) cultivated on Ginkgo biloba (G. biloba) seeds in ameliorating T2DM and its complications, especially DN. A T2DM mouse model was established using ApoE knockout mice fed a Western diet (WD).
目的： 本研究評價了在銀杏葉 （G. biloba） 種子上種植的蛹蟲夏草 （C. militaris） 在改善 T2DM 及其併發症，尤其是 DN 方面的治療潛力。使用飼餵西方飲食 （WD） 的 ApoE 敲除小鼠建立 T2DM 小鼠模型。
Results: Treatment with the specially cultivated C. militaris ameliorated hyperglycemia, dyslipidemia and hepatic dysfunction, while mitigating T2DMinduced renal damage. Key biochemical markers, including blood glucose, triglycerides, cholesterol, blood urea nitrogen (BUN), and creatinine, were significantly improved after treatment. Histopathologic analysis revealed restored renal morphology, reduced fibrosis and decreased amyloid
結果： 用特殊培養的 C. militaris 治療可改善高血糖、血脂異常和肝功能障礙，同時減輕 T2DM 誘導的腎損傷。治療后血糖、甘油三酯、膽固醇、血尿素氮 （BUN） 和肌酐等關鍵生化標誌物得到顯著改善。組織病理學分析顯示腎臟形態恢復，纖維化減少，澱粉樣蛋白減少

deposition. Mechanistic studies showed downregulation of fibrosis-related proteins such as α α alpha\alpha-SMA, COL1, TIMP-1, CTGF, TGF β 1 β 1 beta1\beta 1 and fibronectin, and upregulation of E-cadherin, Smad7 and Klotho, proteins with anti-fibrotic and renoprotective properties.
Conclusion: These results suggest that the specially cultivated C. militaris enhances metabolic regulation and renal repair mechanisms, effectively attenuating T2DM-induced renal damage. This unique cultivation approach enriches the bioactive properties of C. militaris and offers a promising natural therapeutic strategy for T2DM and DN. Further studies are needed to validate these results in clinical settings and to explore long-term efficacy and safety.
口供。機制研究表明，纖維化相關蛋白（如 α α alpha\alpha -SMA、COL1、TIMP-1、CTGF、TGF β 1 β 1 beta1\beta 1 和纖連蛋白）下調，以及 E-鈣粘蛋白、Smad7 和 Klotho 蛋白（具有抗纖維化和腎臟保護特性）的上調。 結論： 這些結果表明，特殊培養的 C. militaris 增強了代謝調節和腎臟修復機制，有效減輕了 T2DM 誘導的腎損傷。這種獨特的培養方法豐富了 C. militaris 的生物活性特性，併為 T2DM 和 DN 提供了一種有前途的自然治療策略。需要進一步的研究來在臨床環境中驗證這些結果，並探索長期療效和安全性。

KEYWORDS  關鍵字

diabetes mellitus, diabetic nephropathy, Cordyceps militaris, Ginkgo biloba seeds, kidney
糖尿病， 糖尿病腎病， 蛹蟲夏草， 銀杏仁， 腎

1 Introduction  1 引言

Diabetes mellitus (DM) is a common and serious metabolic disorder that poses a significant challenge to modern healthcare systems. The global prevalence of DM has increased significantly in recent decades. According to the classification system established by the American Diabetes Association, there are two popular types of DM. Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the destruction of insulin-producing β β beta\beta-cells in the islets of Langerhans, accounting for approximately 5 % − 10 % 5 % − 10 % 5%-10%5 \%-10 \% of cases. In contrast, type 2 diabetes mellitus (T2DM) is the more common DM, accounting for up to 90 % 90 % 90%90 \% of cases, and is attributed to a lack of insulin secretion, impaired insulin action, or both. The global rise in obesity is closely associated with a significant increase in the incidence of T2DM and is increasingly observed in younger obese individuals (Sagoo and Gnudi, 2020). One of the causes of T2DM is changes in the dietary habits of modern people, such as the Western Diet (WD) (Clemente-Suárez et al., 2023). Prolonged DM is associated with multiple organ failure and microvascular complications, including neuropathy, retinopathy, and nephropathy (Beckman and Creager, 2016). Diabetic nephropathy (DN), also known as diabetic kidney disease, is one of the major complications of DM and a leading cause of chronic kidney disease and end-stage renal disease. T2DM often progresses to DN, which is the leading cause of end-stage renal disease worldwide and is associated with significant morbidity and mortality (Maggiore et al., 2017). However, standard treatments for DN primarily slow its progression, but are unable to halt or reverse the disease. Therefore, the development of novel therapeutic strategies for the effective management of DN has become imperative (Samsu, 2021).
糖尿病 （DM） 是一種常見且嚴重的代謝紊亂，對現代醫療保健系統構成重大挑戰。近幾十年來，DM 的全球患病率顯著增加。根據美國糖尿病協會建立的分類系統，DM 有兩種流行的類型。1 型糖尿病 （T1DM） 是一種自身免疫性疾病，其特徵是朗格漢斯胰島中產生 β β beta\beta 胰島素的細胞被破壞，約佔 5 % − 10 % 5 % − 10 % 5%-10%5 \%-10 \% 病例數。相比之下，2 型糖尿病 （T2DM） 是更常見的 DM，佔多達 90 % 90 % 90%90 \% 一例，歸因於胰島素分泌不足、胰島素作用受損或兩者兼而有之。全球肥胖率的上升與 T2DM 發病率的顯著增加密切相關，並且在年輕的肥胖個體中越來越多地觀察到（Sagoo 和 Gnudi，2020 年）。T2DM 的原因之一是現代人飲食習慣的變化，例如西方飲食 （WD）（Clemente-Suárez 等人，2023 年）。長期 DM 與多器官衰竭和微血管併發症有關，包括神經病變、視網膜病變和腎病（Beckman 和 Creager，2016 年）。糖尿病腎病 （DN），也稱為糖尿病腎病，是 DM 的主要併發症之一，也是慢性腎病和終末期腎病的主要原因。T2DM 經常進展為 DN，這是全球終末期腎病的主要原因，與顯著的發病率和死亡率相關（Maggiore 等人，2017 年）。然而，DN 的標準治療主要減緩其進展，但無法阻止或逆轉疾病。 因此，開發有效管理 DN 的新型治療策略已成為當務之急 （Samsu， 2021）。
Traditional Chinese medicines and their bioactive metabolites possess protective properties against various stresses and diseases. These properties make them promising candidates for the development of innovative therapeutic approaches to effectively treat DN (Chang et al., 2013; Liu et al., 2020; Chang et al., 2021;
中藥及其生物活性代謝物具有對各種壓力和疾病的保護特性。這些特性使它們成為開發有效治療 DN 的創新治療方法的有希望的候選者（Chang et al.， 2013;Liu et al.， 2020;Chang et al.， 2021;
Ho et al., 2024). Cordyceps militaris (C. militaris), a fungus belonging to the Ascomycota, is highly regarded for its medicinal and nutritional benefits. Known in Asia for centuries, C. militaris has been used both as a dietary supplement and in traditional medicine. C. militaris is a rich source of various bioactive metabolites such as cordycepin, adenosine, carotenoids, pentostatin, polysaccharides, proteins, ergosterol, and myriocin, among others (Zhang et al., 2019; Zeng et al., 2024). Pharmacological research has shown that C. militaris offers remarkable therapeutic benefits for a wide range of health problems. These include diseases of the neurological, respiratory, cardiovascular, hepatic, and renal systems (Zhang et al., 2019). For the treatment of DM and DN, C. militaris has been shown to modulate the gut microbiota by enhancing beneficial bacteria and regulating metabolites and metabolic pathways, thereby ameliorating T2DM in mice (Liu et al., 2023). Previous research shows that C. militaris extract has potent anti-diabetic and renal protective effects, making it a promising candidate for the development of new treatments for diabetes (Dong et al., 2014). In addition, cordycepin from C. militaris has ability against DN by reducing oxidative stress, inflammation, and apoptosis in HK-2 cells through the miR-193b-5p/MCL-1 pathway. In a mouse model, cordycepin improved renal function and pathology (Zheng et al., 2023). C. militaris polysaccharides are observed to improve renal function and mitigate inflammation and podocyte injury while restoring autophagy in a mouse model of streptozotocin-induced DN (Chen et al., 2019). In addition, C. militaris mitigated DN in a mouse model by reducing blood glucose, markers of renal dysfunction, pathological renal changes, and fibrosis-related proteins while improving lipid metabolism (Yu et al., 2016). These studies indicate the potential of C C CC. militaris and its metabolites as a natural therapy for DM and diabetic kidney disease.
Ho et al.， 2024）。冬蟲夏草 （C. militaris） 是一種屬於子囊菌門的真菌，因其藥用和營養價值而備受推崇。幾個世紀以來，C. militaris 在亞洲廣為人知，既被用作膳食補充劑，也被用作傳統醫學。C. militaris 是各種生物活性代謝物的豐富來源，如蟲草素、腺苷、類胡蘿蔔素、噴司他丁、多糖、蛋白質、麥角甾醇和肉豆蔻素等（Zhang 等，2019 年;Zeng et al.， 2024）。藥理學研究表明，C. militaris 對各種健康問題具有顯著的治療益處。這些疾病包括神經、呼吸系統、心血管、肝臟和腎臟系統的疾病（Zhang et al.， 2019）。對於 DM 和 DN 的治療，C. militaris 已被證明可以通過增強有益細菌和調節代謝物和代謝途徑來調節腸道微生物群，從而改善小鼠的 T2DM （Liu et al.， 2023）。先前的研究表明，C. militaris 提取物具有強大的抗糖尿病和腎臟保護作用，使其成為開發糖尿病新療法的有前途的候選者（Dong et al.， 2014）。此外，來自 C. militaris 的蟲草素通過 miR-193b-5p/MCL-1 通路減少 HK-2 細胞中的氧化應激、炎症和細胞凋亡，從而具有對抗 DN 的能力。在小鼠模型中，蟲草素改善了腎功能和病理（Zheng 等人，2023 年）。在鏈脲佐菌素誘導的 DN 小鼠模型中，觀察到 C. militaris 多糖可改善腎功能並減輕炎症和足細胞損傷，同時恢復自噬（Chen 等人，2019 年）。此外，C. militaris 通過降低血糖、腎功能不全標誌物、病理性腎臟變化和纖維化相關蛋白來減輕小鼠模型中的 DN，同時改善脂質代謝（Yu 等人，2016 年）。這些研究表明了 C C CC 的潛力。militaris 及其代謝物作為 DM 和糖尿病腎病的自然療法。
Ginkgo biloba (G. biloba), often referred to as the “living fossil,” is a deciduous tree belonging to the genus Ginkgo in the family Ginkgoaceae. Native to East Asia, this species has a long history of use in traditional Chinese medicine. It has been used in the treatment of several conditions, including cognitive impairment, respiratory diseases, and gastrointestinal disorders (Liu et al., 2022; Biernacka et al., 2023). Previous studies have highlighted the diverse therapeutic properties of G. biloba, including its anti-inflammatory and anticancer activities. In addition, G. biloba has been shown to exert protective effects on various physiological systems, such as the nervous and cardiovascular systems. It also plays a role in preventing
銀杏 （G. biloba），通常被稱為“活化石”，是屬於銀杏科銀杏屬的落葉喬木。該物種原產於東亞，在傳統中藥中的使用歷史悠久。它已被用於治療多種疾病，包括認知障礙、呼吸系統疾病和胃腸道疾病（Liu et al.， 2022;Biernacka 等人，2023 年）。以前的研究強調了 G. biloba 的多種治療特性，包括其抗炎和抗癌活性。此外，G. biloba 已被證明對各種生理系統（如神經和心血管系統）具有保護作用。它還起著預防
damage associated with diabetes, particularly in mitigating the development of diabetes-induced cataracts (Belwal et al., 2019). G. biloba seeds, which are rich in starch, protein, and ginkgo oil, serve a dual purpose as both a food and a medicinal agent. They show significant potential in combating inflammation, oxidative stress, bacterial infections, tumor progression, and nerve damage (Liu et al., 2022). A study shows that G. biloba seeds alleviate hyperglycemia-induced oxidative stress, reduce insulin resistance, enhance antioxidant defenses, and reduce inflammation in pancreatic β β beta\beta-cells, liver, and kidneys in a T2DM mouse model (Jing et al., 2021). However, G. biloba seeds contain toxic metabolites that pose potential health risks and require the development and implementation of specific detoxification strategies, especially when used as a food or medicinal agent (Boateng and Yang, 2021; Biernacka et al., 2023).
與糖尿病相關的損害，特別是在減輕糖尿病誘發的白內障的發展方面（Belwal 等人，2019 年）。G. biloba 種子富含澱粉、蛋白質和銀杏油，具有食品和藥劑的雙重用途。它們在對抗炎症、氧化應激、細菌感染、腫瘤進展和神經損傷方面顯示出巨大的潛力（Liu et al.， 2022）。一項研究表明，在 T2DM 小鼠模型中，G. biloba 種子可緩解高血糖誘導的氧化應激，降低胰島素抵抗，增強抗氧化防禦，並減少胰腺 β β beta\beta 細胞、肝臟和腎臟的炎症（Jing 等人，2021 年）。然而，G. biloba 種子含有有毒代謝物，這些代謝物會帶來潛在的健康風險，需要制定和實施特定的解毒策略，尤其是用作食品或藥物時（Boateng 和 Yang，2021 年;Biernacka 等人，2023 年）。
Although G. biloba seeds have the potential to alleviate complications associated with DM, their toxicity limits their widespread applicability and safe use. C. militaris shows significant health benefits in the management of DM. The bioactive metabolites in C. militaris contribute to improved metabolic regulation, antioxidant protection, and antiinflammatory effects, making them valuable in alleviating complications associated with DM, particularly DN. However, improving the efficacy of C. militaris remains an area of research that warrants further investigation. Therefore, in this study, G G GG. biloba seeds were used as a culture medium for C. militaris to explore its potential therapeutic effects in the context of DM and DN . The research aimed to evaluate the functional properties of C C CC. militaris cultivated on G. biloba seeds and its efficacy in alleviating the complications associated with DM. Using a T2DM mouse model, the study demonstrated that this specially cultivated C C CC. militaris mitigated hyperglycemia, dyslipidemia, liver dysfunction and renal damage, especially DN. The results highlighted the bioactive properties of the specially cultivated C. militaris in improving metabolic health and reversing kidney injury, underscoring its potential as a novel treatment strategy for DM and DN .
儘管 G. biloba 種子有可能緩解與 DM 相關的併發症，但它們的毒性限制了它們的廣泛適用性和安全使用。C. militaris 在 DM 的管理中顯示出顯著的健康益處。C. militaris 中的生物活性代謝物有助於改善代謝調節、抗氧化保護和抗炎作用，使其在緩解與 DM 相關的併發症（尤其是 DN）方面具有價值。然而，提高 C. militaris 的功效仍然是一個值得進一步研究的研究領域。因此，在本研究中， G G GG .將 biloba 種子用作 C. militaris 的培養基，以探索其在 DM 和 DN 背景下的潛在治療效果。該研究旨在評估的功能 C C CC 特性。在 G. biloba 種子上培養的 militaris 及其在緩解與 DM 相關的併發症方面的功效。使用 T2DM 小鼠模型，研究表明這種專門培養的 C C CC .Militaris 減輕了高血糖、血脂異常、肝功能障礙和腎功能損害，尤其是 DN。結果突出了專門培養的 C. militaris 在改善代謝健康和逆轉腎損傷方面的生物活性特性，強調了其作為 DM 和 DN 新型治療策略的潛力。

2 Materials and methods
2 材料和方法

2.1 Materials  2.1 材料

All chemicals used in this study were analytical grade. They were purchased primarily from Sigma-Aldrich (St. Louis, MO, United States) and Merck (Darmstadt, Germany). Any exceptions were explicitly mentioned. The original C. militaris (Cordycipitaceae; C. militaris (L.) Link) was obtained from the Bioresource Collection and Research Center (BCRC34380; Food Industry Research and Development Institute, Hsinchu, Taiwan; Hsinchu, Taiwan). Prof. Tsung-Jung Ho of the Department of Chinese Medicine, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (Hualien, Taiwan) and the technical staff of HOME RUN Biotechnology Co., Ltd. (Tainan, Taiwan) performed the identification of the specially cultivated C. militaris. The specially cultivated C. militaris was examined morphologically and its identity was then confirmed using the 2018 (Version III, Chinese Edition) of the Taiwan Herbal Pharmacopoeia. The specially
本研究中使用的所有化學品均為分析級化學品。它們主要從 Sigma-Aldrich（美國密蘇里州聖路易斯）和 Merck（德國達姆施塔特）購買。明確提到了任何異常。最初的 C. militaris （Cordycipitaceae;C. militaris （L.） Link） 來自生物資源收集和研究中心 （BCRC34380;食品工業研究與發展研究所，臺灣新竹;Hsinchu， Taiwan）。臺灣花蓮慈濟醫院中醫科、佛教慈濟醫學基金會何宗貞教授和家潤生物科技股份有限公司（臺灣台南）的技術人員對特殊培育的軍化蚜蟲進行了鑒定。對專門培養的 C. militaris 進行形態學檢查，然後使用 2018 年臺灣草藥藥典（第三版，中文版）確認其身份。特別的
cultivated C. militaris was also analyzed using HPLC-MS (Protech Technology Enterprise Co., Ltd., Taipei, Taiwan) to identify its primary metabolites (Supplementary Material). The C C CC. militaris cultivated on G. biloba seeds (Patent No. I687170 in Taiwan and CN112913572B in China) was processed into pills and manufactured by HOME RUN Biotechnology Co., Ltd. (Tainan, Taiwan). The WD diet (D12079B, carbohydrate: 43 % kcal 43 % kcal 43%kcal43 \% \mathrm{kcal}, fat: 40 % 40 % 40%40 \% kcal, and protein: 17 % kcal 17 % kcal 17%kcal17 \% \mathrm{kcal} ) was purchased from Research Diets, Inc. (New Brunswick, NJ, Unites States). A variety of primary antibodies were used for analysis in the current study. These primary antibodies included mouse monoclonal anti-alpha smooth muscle actin ( α α alpha\alpha SMA; ab7817, Abcam, Cambridge, United Kingdom), rabbit polyclonal anti-collagen I (COL1; GTX20292, GeneTex, Irvine, CA, Unites States), mouse monoclonal anti-fibronectin (sc-8422, Santa Cruz Biotechnology, Santa Cruz, CA, Unites States), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc-32233, Santa Cruz Biotechnology), anti-connective tissue growth factor (CTGF; sc-365970, Santa Cruz Biotechnology), anti-tissue inhibitor of metalloproteinase-1 (TIMP-1; sc-365905, Santa Cruz Biotechnology), anti-E-cadherin (sc-8426, Santa Cruz Biotechnology), goat polyclonal antitransforming growth factor β 1 β 1 beta1\beta 1 (TGF β 1 β 1 beta1\beta 1; sc-31609, Santa Cruz Biotechnology), rabbit polyclonal anti-Smad7 (sc-11392, Santa Cruz Biotechnology), rabbit polyclonal anti-Klotho (A12028, Abclonal, Woburn, MA, Unites States), and mouse monoclonal anti-beta-actin ( β β beta\beta-actin, sc-47778, Santa Cruz Biotechnology). The corresponding secondary antibodies (sc-2357, sc-2354, and sc516102) were purchased from Santa Cruz Biotechnology.
還使用 HPLC-MS （Protech Technology Enterprise Co.， Ltd.， Taipei， Taiwan） 分析培養的 C. militaris 以鑒定其主要代謝物 （補充材料）。的 C C CC .在 G. biloba 種子（臺灣專利號 I687170 號和中國 CN112913572B 號）上培養的軍事力量被加工成丸劑，由家潤生物科技有限公司（臺灣台南）生產。WD 飲食（D12079B、碳水化合物： 43 % kcal 43 % kcal 43%kcal43 \% \mathrm{kcal} 、脂肪： 40 % 40 % 40%40 \% kcal 和蛋白質： 17 % kcal 17 % kcal 17%kcal17 \% \mathrm{kcal} ）購自 Research Diets， Inc.（美國新澤西州新不倫瑞克省）。在本研究中，使用了多種一抗進行分析。這些一抗包括小鼠單克隆抗 α 平滑肌肌動蛋白 （ α α alpha\alpha SMA;ab7817， Abcam， Cambridge， United Kingdom）、兔多克隆抗膠原 I （COL1;GTX20292，GeneTex，加利福尼亞州，美國加利福尼亞州），小鼠單克隆抗纖連蛋白（sc-8422，Santa Cruz Biotechnology，美國加利福尼亞州聖克魯斯），小鼠單克隆抗甘油醛-3-磷酸脫氫酶（GAPDH;sc-32233，Santa Cruz Biotechnology），抗 結締組織生長因數（CTGF;sc-365970，Santa Cruz Biotechnology），抗金屬蛋白酶-1 組織抑製劑（TIMP-1;sc-365905，Santa Cruz Biotechnology），抗 E-鈣粘蛋白（sc-8426，Santa Cruz Biotechnology），山羊多克隆抗體抗轉化生長因數 β 1 β 1 beta1\beta 1 （TGF β 1 β 1 beta1\beta 1 ;sc-31609，Santa Cruz Biotechnology）、兔多克隆抗 Smad7（sc-11392，Santa Cruz Biotechnology）、兔多克隆抗 Klotho（A12028，Abclonal，Woburn，MA，美國）和小鼠單克隆抗 β-肌動蛋白（ β β beta\beta -actin，sc-47778，Santa Cruz Biotechnology）。相應的二抗（sc-2357、sc-2354 和 sc516102）購自 Santa Cruz Biotechnology。

2.2 Animal experimental design
2.2 動物實驗設計

The Institutional Animal Care and Use Committee (IACUC) of Hualien Tzu Chi Hospital, Hualien, Taiwan, approved the animal experiment (approval number: 113-43). Four-week-old male C57BL/ 6JNarl mice ( n = 9 n = 9 n=9\mathrm{n}=9, RMRC11005) and male Apoeem1Narl/Narl mice (ApoE KO mice, n = 27 n = 27 n=27\mathrm{n}=27, RMRC13302) were obtained from the National Institutes of Applied Research National Center for Biomodels in Taipei, Taiwan (http://www.nlac.org.tw/RMRC/webc/ html/data/show.aspx?ix=1&page=1&kw=13302). The ApoE KO mice were generated using CRISPR/Cas9 technology. All animals were housed under controlled conditions, maintained at 22 ∘ C ± 2 ∘ C 22 ∘ C ± 2 ∘ C 22^(@)C+-2^(@)C22^{\circ} \mathrm{C} \pm 2^{\circ} \mathrm{C} with a 12-h light-dark cycle and 55 % ± 5 % 55 % ± 5 % 55%+-5%55 \% \pm 5 \% relative humidity, and provided with standard rodent chow and water ad libitum. Mice were allowed a 2-week acclimation period before the start of experimental procedures. The C57BL/6JNarl mice ( n = 9 n = 9 n=9\mathrm{n}=9 ) served as the control group, while the A p o E A p o E ApoEA p o E KO mice were randomly divided into three experimental groups ( n = 9 n = 9 n=9\mathrm{n}=9 per group): A p o E KO A p o E KO ApoEKOA p o E \mathrm{KO} mice fed a standard diet (ApoE KO mice), A p o E A p o E ApoEA p o E KO mice fed a WD (ApoE KO mice with WD), and A p o E A p o E ApoEA p o E KO mice fed a WD and this specially cultivated C C CC. militaris (ApoE KO mice with WD + C. militaris). The experimental period was 16 weeks. Starting at week 8, two C. militaris pills ( 30 mg each, containing 111.3 μ g 111.3 μ g 111.3 mug111.3 \mu \mathrm{~g} cordycepin and 9.4 μ g 9.4 μ g 9.4 mug9.4 \mu \mathrm{~g} adenosine) were administered orally every 2 days. Fasting serum glucose was monitored weekly using Accu-Chek ( 8 ) ( 8 ) ^((8)){ }^{(8)} guide test strips and AccuChek guide meter (Fritz Hoffmann-La Roche AG, Basel, Switzerland), and a serum glucose level higher than 200 mg / dL 200 mg / dL 200mg//dL200 \mathrm{mg} / \mathrm{dL} was considered indicative of T2DM (Chiang et al., 2021; Ohno
台灣花蓮慈濟醫院機構動物護理及使用委員會 （IACUC） 批准該動物實驗 （批准編號：113-43）。4 周齡雄性 C57BL/6JNarl 小鼠 （ n = 9 n = 9 n=9\mathrm{n}=9 ， RMRC11005） 和雄性 Apoeem1Narl/Narl 小鼠 （ApoE KO 小鼠， n = 27 n = 27 n=27\mathrm{n}=27 RMRC13302） 購自臺灣臺北國立應用研究院國家生物模型中心 （http://www.nlac.org.tw/RMRC/webc/ html/data/show.aspx？ix=1&page=1&kw=13302）。使用 CRISPR/Cas9 技術生成 ApoE KO 小鼠。所有動物均在受控條件下飼養，維持 22 ∘ C ± 2 ∘ C 22 ∘ C ± 2 ∘ C 22^(@)C+-2^(@)C22^{\circ} \mathrm{C} \pm 2^{\circ} \mathrm{C} 12 小時的明暗循環和 55 % ± 5 % 55 % ± 5 % 55%+-5%55 \% \pm 5 \% 相對濕度，並隨意提供標準的啮齒動物食物和水。小鼠在實驗程序開始前被允許 2 周的馴化期。C57BL/6JNarl 小鼠 （ n = 9 n = 9 n=9\mathrm{n}=9 ） 作為對照組，而 A p o E A p o E ApoEA p o E KO 小鼠被隨機分為三個實驗組（ n = 9 n = 9 n=9\mathrm{n}=9 每組）： A p o E KO A p o E KO ApoEKOA p o E \mathrm{KO} 餵食標準飲食的小鼠（ApoE KO 小鼠）、 A p o E A p o E ApoEA p o E 餵食 WD 的 KO 小鼠（帶 WD 的 ApoE KO 小鼠）和 A p o E A p o E ApoEA p o E 餵食 WD 的 KO 小鼠，這是專門培養的 C C CC 。militaris（具有 WD + C. militaris 的 ApoE KO 小鼠）。實驗期為 16 周。從第 8 周開始，每 2 天口服兩粒 C. militaris 藥丸 （每片 30 毫克，含有 111.3 μ g 111.3 μ g 111.3 mug111.3 \mu \mathrm{~g} 蟲草素和 9.4 μ g 9.4 μ g 9.4 mug9.4 \mu \mathrm{~g} 腺苷）。每周使用 Accu-Chek ( 8 ) ( 8 ) ^((8)){ }^{(8)} 導紙和 AccuChek 導引儀（Fritz Hoffmann-La Roche AG，瑞士巴塞爾）監測空腹血糖，並且血糖水平高於 200 mg / dL 200 mg / dL 200mg//dL200 \mathrm{mg} / \mathrm{dL} 被認為表明 T2DM（Chiang 等人，2021 年;大野
et al., 2022). At the end of 16 weeks, all mice were sacrificed and the organs were harvested for further analysis. The different biochemical indicators in serum were measured by Arkray Automated analyzer for clinical chemistry (SPOTCHEM EZ SP-4 430, Arkray Inc., Kyoto, Japan).
等人，2022 年）。在 16 周結束時，處死所有小鼠並收穫器官用於進一步分析。使用愛科來臨床化學自動分析儀（SPOTCHEM EZ SP-4 430，愛科來公司，日本京都）測量血清中不同的生化指標。

2.3 Preparation of paraffin-embedded tissue sections
2.3 石蠟包埋組織切片的製備

Kidney tissues from each experimental group were fixed in 10% formalin for 2 weeks. The samples were then dehydrated through a graded series of ethanol solutions and embedded in paraffin. The paraffin-embedded tissue blocks were then sectioned at 4 μ m 4 μ m 4mum4 \mu \mathrm{~m} thickness for further analysis. Kidney sections were deparaffinized with xylene and rehydrated through a graded series of ethanol solutions for further staining (Tsai et al., 2020a; Tsai et al., 2020b).
將各實驗組的腎組織用 10% 福馬林固定 2 周。然後通過一系列分級乙醇溶液對樣品進行脫水，並包埋在石蠟中。然後將石蠟包埋的組織塊切 4 μ m 4 μ m 4mum4 \mu \mathrm{~m} 成厚度以進行進一步分析。用二甲苯對腎切片進行脫蠟，並通過一系列分級乙醇溶液再水化以進行進一步染色（Tsai 等人，2020a;Tsai et al.， 2020b）。

2.4 Hematoxylin and eosin staining
2.4 蘇木精和伊紅染色

The rehydrated kidney sections were sequentially stained with hematoxylin and eosin followed by rinsing in water. The slides were then dehydrated through a graded ethanol series and immersed twice in xylene. Images of the stained sections were captured using an OLYMPUS BX53 microscope (Olympus ® ®  ^("® "){ }^{\text {® }} Corporation, Shinjukuku, Tokyo, Japan) (Lin et al., 2021).
依次用蘇木精和伊紅對再水合的腎切片進行染色，然後用水沖洗。然後通過分級乙醇系列對載玻片進行脫水，並在二甲苯中浸入兩次。使用奧林巴斯 BX53 顯微鏡（奧林巴斯 ® ®  ^("® "){ }^{\text {® }} 公司，日本東京新宿）捕獲染色切片的圖像（Lin 等人，2021 年）。

2.5 Masson's trichrome staining
2.5 Masson 三色染色

The rehydrated kidney sections were stained with Masson’s trichrome dye for 5 min and then rinsed with water. After staining, the slides were dehydrated through a graded ethanol series and immersed twice in xylene. Images of stained tissues were captured using an OLYMPUS BX53 microscope (Olympus ( 8 ) ( 8 ) ^((8)){ }^{(8)} Corporation) (Lai et al., 2023).
將再水化的腎臟切片用 Masson 三色染料染色 5 分鐘，然後用水沖洗。染色后，通過分級乙醇系列對載玻片進行脫水，並在二甲苯中浸入兩次。使用奧林巴斯 BX53 顯微鏡（奧林巴斯 ( 8 ) ( 8 ) ^((8)){ }^{(8)} 公司）（Lai 等人，2023 年）捕獲染色組織的圖像。

2.6 Picrosirius red staining
2.6 Picrosirius 紅染色

Rehydrated kidney tissue sections were stained for collagen type I and type III fibers using the Picrosirius Red Stain Kit (ab150681, Abcam) according to the manufacturer’s protocol. After staining, the sections were thoroughly rinsed to remove excess dye. High-resolution images of the stained tissues were captured using an OLYMPUS BX53 microscope (Olympus ( 8 ) ( 8 ) ^((8)){ }^{(8)} Corporation) (Lattouf et al., 2014).
根據製造商的方案，使用 Picrosirius Red 染色試劑盒（ab150681，Abcam）對再水化的腎組織切片進行 I 型和 III 型膠原纖維染色。染色后，徹底沖洗切片以去除多餘的染料。使用奧林巴斯 BX53 顯微鏡（奧林巴斯 ( 8 ) ( 8 ) ^((8)){ }^{(8)} 公司）（Lattouf 等人，2014 年）捕獲染色組織的高解析度圖像。

2.7 Immunohistochemical staining
2.7 免疫組化染色

Rehydrated kidney tissue sections were treated with a permeabilization solution and blocking buffer to reduce nonspecific binding, followed by thorough washing with PBS. The sections were incubated with the primary antibody, anti- α α alpha\alpha-SMA (diluted in 1 % 1 % 1%1 \% horse serum), for 1 h to target specific protein expression. After further washing with PBS, the slides were processed using a horseradish peroxidase-conjugated avidin-biotin complex (ABC) from the
用透化溶液和封閉緩衝液處理再水化的腎組織切片以減少非特異性結合，然後用 PBS 徹底洗滌。將切片與一抗抗 anti- α α alpha\alpha -SMA（在馬血清中 1 % 1 % 1%1 \% 稀釋）孵育 1 小時以靶向特異性蛋白表達。用 PBS 進一步洗滌后，使用辣根過氧化物酶偶聯的親和素-生物素複合物 （ABC） 處理載玻片。
Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). The chromogenic detection was performed using NovaRed substrate (Vector Laboratories, Burlingame, CA), and slides were counterstained with hematoxylin for nuclei visualization. Finally, photomicrographs of the stained sections were captured using an OLYMPUS BX53 microscope (Olympus ( 0 ) ( 0 ) ^((0)){ }^{(0)} Corporation) (Tsai et al., 2020a; Tsai et al., 2020b).
Vectastain Elite ABC 試劑盒（Vector Laboratories，Burlingame，CA）。使用 NovaRed 底物 （Vector Laboratories， Burlingame， CA） 進行顯色檢測，並用蘇木精複染載玻片以進行細胞核可視化。最後，使用奧林巴斯 BX53 顯微鏡（奧林巴斯 ( 0 ) ( 0 ) ^((0)){ }^{(0)} 公司）捕獲染色切片的顯微照片（Tsai 等人，2020a;Tsai et al.， 2020b）。

2.8 Toluidine blue staining
2.8 甲苯胺藍染色

Rehydrated kidney tissue sections were stained with toluidine blue solution (ScyTek Laboratories, West Logan, UT, Unites States) according to the manufacturer’s protocol. Upon completion of the staining process, the sections were thoroughly rinsed to remove excess dye. High-resolution images of the stained tissues were captured using an OLYMPUS BX53 microscope (Olympus ( 8 ) ( 8 ) ^((8)){ }^{(8)} Corporation) (Niculae et al., 2017).
根據製造商的方案，用甲苯胺藍溶液（ScyTek Laboratories，West Logan，UT，美國）對再水化的腎組織切片進行染色。染色過程完成後，徹底沖洗切片以去除多餘的染料。使用奧林巴斯 BX53 顯微鏡（奧林巴斯 ( 8 ) ( 8 ) ^((8)){ }^{(8)} 公司）（Niculae 等人，2017 年）捕獲染色組織的高解析度圖像。

2.9 Tissue protein extraction and western blotting
2.9 組織蛋白提取和蛋白質印跡

Kidney tissues from each experimental group were homogenized in lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1 % 1 % 1%1 \% nonylphenoxypolyethoxylethanol, 0.25% deoxycholic acid, and supplemented with phosphatase inhibitor cocktail 2 (Cat# P5726, Sigma-Aldrich) and protease inhibitor cocktail (Cat# S8830, SigmaAldrich) at a ratio of 100 mg tissue per 1 mL lysis buffer. Homogenized samples were stored at − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} overnight and then centrifuged at 10 , 000 g 10 , 000 g 10,000g10,000 \mathrm{~g} for 30 min at 4 ∘ C 4 ∘ C 4^(@)C4^{\circ} \mathrm{C}. The supernatant was collected and protein concentrations were determined using the Lowry protein assay. Calculations were performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, Unites States). Protein aliquots were prepared by mixing with 5 × 5 × 5xx5 \times loading dye and then heated at 95 ∘ C 95 ∘ C 95^(@)C95^{\circ} \mathrm{C} for 5 min . Equal amounts of protein from each sample were separated by 8 % , 10 % 8 % , 10 % 8%,10%8 \%, 10 \%, or 12 % 12 % 12%12 \% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.45 μ m 0.45 μ m 0.45 mum0.45 \mu \mathrm{~m} pore size polyvinylidene difluoride (PVDF) membranes (GE Healthcare UK, Ltd., Amersham, United Kingdom). Membranes were blocked in a buffer containing 5 % 5 % 5%5 \% skim milk in TBST buffer ( 20 mM Tris-HCl, pH 7.6, 150 mM sodium chloride, and 0.1 % 0.1 % 0.1%0.1 \% polysorbate 20 ) for 1 h at 25 ∘ C 25 ∘ C 25^(@)C25^{\circ} \mathrm{C} to prevent non-specific binding. After blocking, the membranes were incubated overnight at 4 ∘ C 4 ∘ C 4^(@)C4^{\circ} \mathrm{C} with primary antibodies diluted in blocking buffer. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and protein bands were visualized using an Image Bright 1,500 imaging system (Thermo Fisher Scientific, Waltham, MA, Unites States) with Immobilon Western chemiluminescent horseradish peroxidase substrate (WBKLS0500, Merck Millipore, Burlington, MA, Unites States) (Lin et al., 2023; Lin et al., 2024). GAPDH or β β beta\beta-actin were used as internal control proteins.
將每個實驗組的腎組織在含有 50 mM Tris-HCl pH 7.4、150 mM 氯化鈉、1 mM 乙二胺四乙酸、 1 % 1 % 1%1 \% 壬氧氧基聚乙氧基乙醇、0.25% 去氧膽酸的裂解緩衝 液中勻漿，並補充磷酸酶抑製劑混合物 2（Cat# P5726，Sigma-Aldrich）和蛋白酶抑製劑混合物（Cat# S8830，SigmaAldrich），比例為 100 mg 組織/1 mL 裂解緩衝液。將勻漿樣品儲存 − 80 ∘ C − 80 ∘ C -80^(@)C-80^{\circ} \mathrm{C} 過夜，然後在 下離心 10 , 000 g 10 , 000 g 10,000g10,000 \mathrm{~g} 30 分鐘 4 ∘ C 4 ∘ C 4^(@)C4^{\circ} \mathrm{C} 。收集上清液，並使用 Lowry 蛋白測定法測定蛋白質濃度。使用 Microsoft Excel（Microsoft Corporation，Redmond，WA，美國）進行計算。通過與 5 × 5 × 5xx5 \times 上樣染料混合製備蛋白質等分試樣，然後在 5 分鐘下 95 ∘ C 95 ∘ C 95^(@)C95^{\circ} \mathrm{C} 加熱。通過 8 % , 10 % 8 % , 10 % 8%,10%8 \%, 10 \% 十二烷基硫酸 12 % 12 % 12%12 \% 鈉-聚丙烯醯胺凝膠電泳分離每個樣品中等量的蛋白質，並轉移到 0.45 μ m 0.45 μ m 0.45 mum0.45 \mu \mathrm{~m} 孔徑的聚偏二氟乙烯 （PVDF） 膜（GE Healthcare UK， Ltd.，Amersham，United Kingdom）上。將膜封閉在含有 5 % 5 % 5%5 \% TBST 緩衝液（20 mM Tris-HCl、pH 7.6、150 mM 氯化鈉和 0.1 % 0.1 % 0.1%0.1 \% 聚山梨酯 20）中的脫脂牛奶的緩衝液中 1 小時 25 ∘ C 25 ∘ C 25^(@)C25^{\circ} \mathrm{C} ，以防止非特異性結合。封閉后，將膜 4 ∘ C 4 ∘ C 4^(@)C4^{\circ} \mathrm{C} 與在封閉緩衝液中稀釋的一抗一起孵育過夜。 第二天，將膜與辣根過氧化物酶偶聯的二抗一起孵育，並使用 Image Bright 1,500 成像系統（Thermo Fisher Scientific，Waltham，MA，美國）和 Immobilon Western 化學發光辣根過氧化物酶底物（WBKLS0500，Merck Millipore，Burlington，MA，美國）（Lin 等人，2023 年;Lin 等人，2024 年）。GAPDH 或 β β beta\beta -actin 用作內部對照蛋白。

2.10 Statistical analysis
2.10 統計分析

Statistical analyses were performed using GraphPad Prism software (version 6.0, CA, Unites States). Data are presented as the mean ± ± +-\pm
使用 GraphPad Prism 軟體 （版本 6.0，CA，美國） 進行統計分析。數據以平均值 ± ± +-\pm 表示
FIGURE 1  圖 1
Changes in body weight of ApoE KO mice under different experimental conditions. The group of ApoE KO mice fed a Western diet (WD) without treatment showed an initial increase in body weight followed by a decrease after 14 weeks. In contrast, ApoE KO mice fed a WD and treated with this specially cultivated Cordyceps militaris from week 8 maintained a stable body weight throughout the experimental period. Data are expressed as mean ± ± +-\pm SD. *** = p < 0.001 = p < 0.001 =p < 0.001=p<0.001 compared to ApoE KO mice fed a standard diet group; ## = p < 0.01 = p < 0.01 =p < 0.01=p<0.01 compared to ApoE KO mice fed with WD without any treatment.
不同實驗條件下 ApoE KO 小鼠體重的變化。飼餵西方飲食 （WD） 且未治療的 ApoE KO 小鼠組顯示體重最初增加，然後在 14 周後下降。相比之下，ApoE KO 小鼠餵食 WD 並從第 8 周開始用這種特殊培養的蛹蟲夏草處理，在整個實驗期間保持穩定的體重。數據表示為平均 ± ± +-\pm SD。*** = p < 0.001 = p < 0.001 =p < 0.001=p<0.001 與飼喂標準飲食組的 ApoE KO 小鼠相比;## = p < 0.01 = p < 0.01 =p < 0.01=p<0.01 與未經任何治療餵食 WD 的 ApoE KO 小鼠相比。
Serum Glucose level  血清葡萄糖水準
FIGURE 2  圖 2
Serum glucose levels in ApoE KO mice under different experimental conditions. The ApoE KO mice fed a Western diet (WD) alone showed a significant increase in serum glucose levels by week 8. After initiation of this specially cultivated Cordyceps militaris treatment at week 8, a progressive decrease in serum glucose levels was observed, which continued consistently until week 16. Data were expressed as mean ± ± +-\pm SD. *** = p < = p < =p <=p< 0.001 compared to ApoE KO mice fed a standard diet group; ### = p < 0.001, # = p < 0.05 compared to ApoE KO mice fed with WD without any treatment.
不同實驗條件下 ApoE KO 小鼠的血糖水準。單獨餵食西方飲食 （WD） 的 ApoE KO 小鼠在第 8 周時顯示血糖水平顯著升高。在第 8 周開始這種特殊培養的蛹蟲夏草治療后，觀察到血糖水平進行性下降，並持續到第 16 周。數據表示為平均 ± ± +-\pm SD。*** = p < = p < =p <=p< 與飼喂標準飲食組的 ApoE KO 小鼠相比為 0.001;### = p < 0.001，# = p < 0.05 與未經任何治療餵食 WD 的 ApoE KO 小鼠相比。
standard deviation (SD) of independent experiments. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to assess the statistical significance of differences between group means. A p p pp-value of less than 0.05 was considered statistically significant, whereas p p pp-values of less than 0.01 and 0.001 were considered highly significant ( p < 0.01 p < 0.01 p < 0.01p<0.01 and p < 0.001 p < 0.001 p < 0.001p<0.001, respectively).
獨立實驗的標準差 （SD）。單因素方差分析 （ANOVA） 後使用 Tukey 事後檢驗來評估組均值之間差異的統計顯著性。小於 0.05 的 p p pp -值被認為具有統計顯著性，而 p p pp 小於 0.01 和 0.001 的 -值被認為具有高度顯著性（分別為 p < 0.01 p < 0.01 p < 0.01p<0.01 和 p < 0.001 p < 0.001 p < 0.001p<0.001 ）。

3 Results  3 結果

3.1 Beneficial effects of the specially cultivated C. militaris on mice with T2DM
3.1 特殊培養的 C. militaris 對 T2DM 小鼠的有益影響

First, to evaluate the therapeutic potential of this specially cultivated C. militaris in the treatment of T2DM, experiments were conducted using an established T2DM mouse model to evaluate the effect of C C CC. militaris on body weight and serum glucose levels in A p o E A p o E ApoEA p o E KO mice fed a WD over a 16-week
首先，為了評估這種專門培養的 C. militaris 治療 T2DM 的治療潛力，使用已建立的 T2DM 小鼠模型進行了實驗，以評估 C C CC 的效果。在 16 周內飼喂 WD 的 KO 小鼠的 A p o E A p o E ApoEA p o E 體重和血糖水準
period. The results showed significant differences in body weight between the experimental groups. In the group of A p o E KO A p o E KO ApoEKOA p o E \mathrm{KO} mice receiving a WD without any treatment, a significant increase in body weight was observed during the early weeks of the study. However, this trend was reversed and body weight began to decrease after 14 weeks in the WD group (Figure 1). Conversely, the group of A p o E A p o E ApoEA p o E KO mice that received WD and C. militaris treatment starting at week 8 showed stable body weight throughout the experimental period (Figure 1). In addition to body weight, the study also monitored serum glucose levels, a critical biomarker in T2DM. Mice exposed to WD alone showed a significant increase in serum glucose levels by week 8 of the study (Figure 2). Treatment with this specially cultivated C. militaris began at this week, and a significant reduction in serum glucose levels was observed after 4 weeks of supplementation. This downward trend in serum glucose levels continued consistently through week 16 in the group treated with both WD and this specially cultivated C. militaris (Figure 2). These results indicate the potential of this specially cultivated C. militaris as
時期。結果顯示實驗組之間的體重存在顯著差異。在接受 WD 且未進行任何治療的 A p o E KO A p o E KO ApoEKOA p o E \mathrm{KO} 小鼠組中，在研究的最初幾周觀察到體重顯著增加。然而，這種趨勢發生了逆轉，WD 組的體重在 14 周後開始下降（圖 1）。相反，從第 8 周開始接受 WD 和 C. militaris 治療的 A p o E A p o E ApoEA p o E KO 小鼠組在整個實驗期間表現出穩定的體重（圖 1）。除了體重，該研究還監測了血糖水準，這是 T2DM 的關鍵生物標誌物。到研究第 8 周，單獨暴露於 WD 的小鼠的血糖水平顯著升高（圖 2）。從本周開始用這種特殊培養的 C. militaris 進行治療，補充 4 周後觀察到血糖水平顯著降低。在接受 WD 和這種特殊培養的 C. miitaris 治療的組中，血糖水準的這種下降趨勢一直持續到第 16 周（圖 2）。這些結果表明，這種專門培養的 C. militaris 的潛力為

(A)  （一）



(B)  （二）

FIGURE 3  圖 3
Biochemical analysis of blood lipid levels and liver function in ApoE KO mice under different experimental conditions. (A) The ApoE KO mice fed a Western diet (WD) showed significantly elevated levels of triglycerides and total cholesterol, which were reduced after this specially cultivated Cordyceps militaris treatment. (B) Elevated serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), markers of liver dysfunction induced by WD, were significantly reduced in the Cordyceps militaris-treated group. Data were expressed as mean ± ± +-\pm SD. *** = p < = p < =p <=p< 0.001 compared to ApoE KO mice fed a standard diet group; ### = p < 0.001 compared to ApoE KO mice fed with WD without any treatment.
不同實驗條件下 ApoE KO 小鼠血脂水準和肝功能的生化分析。（A） 餵食西方飲食 （WD） 的 ApoE KO 小鼠顯示甘油三酯和總膽固醇水平顯著升高，在這種專門培養的蛹蟲夏草治療后，這些水準降低。（B） WD 誘導的肝功能障礙標誌物天冬氨酸氨基轉移酶 （AST） 和丙氨酸氨基轉移酶 （ALT） 血清濃度升高在蛹蟲草治療組中顯著降低。數據表示為平均 ± ± +-\pm SD。*** = p < = p < =p <=p< 與飼喂標準飲食組的 ApoE KO 小鼠相比為 0.001;### = p < 0.001 與未經任何治療餵食 WD 的 ApoE KO 小鼠相比。
a therapeutic agent in the management of DM. Its ability to attenuate hyperglycemia in a T2DM mouse model highlighted its feasibility for further investigation in the treatment of DM.
DM 治療的治療劑。它在 T2DM 小鼠模型中減輕高血糖的能力突出了其進一步研究治療 DM 的可行性。

3.2 The specially cultivated C. militaris alleviated dyslipidemia and hepatic dysfunction in a T2DM mouse model
3.2 專門培養的 C. militaris 減輕了 T2DM 小鼠模型中的血脂異常和肝功能障礙

Next, to evaluate the potential therapeutic effects of this specially cultivated C. militaris on metabolic health, a series of biochemical analyses were performed in a T2DM mouse model. The present study used A p o E A p o E ApoEA p o E KO mice fed WD to mimic the metabolic disturbances of T2DM. The results showed that WD significantly elevated blood lipid levels, as evidenced by marked increases in triglyceride and total cholesterol levels (Figure 3A). In addition, WD resulted in significant liver dysfunction as evidenced by elevated serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Figure 3B), two well-established markers of liver injury. Interestingly, when the A p o E KO A p o E KO ApoEKOA p o E \mathrm{KO} mice with WD received this
接下來，為了評估這種特殊培養的 C. militaris 對代謝健康的潛在治療作用，在 T2DM 小鼠模型中進行了一系列生化分析。本研究使用 A p o E A p o E ApoEA p o E 餵食 WD 的 KO 小鼠來類比 T2DM 的代謝紊亂。結果表明，WD 顯著升高了血脂水準，甘油三酯和總膽固醇水準顯著增加證明瞭這一點（圖 3A）。此外，WD 導致嚴重的肝功能障礙，天冬氨酸氨基轉移酶 （AST） 和丙氨酸氨基轉移酶 （ALT） 的血清濃度升高證明瞭這一點（圖 3B），這是兩個公認的肝損傷標誌物。有趣的是，當具有 WD 的 A p o E KO A p o E KO ApoEKOA p o E \mathrm{KO} 小鼠接受此
specially cultivated C. militaris treatment, a marked improvement in these pathological markers was observed. Specifically, triglyceride and total cholesterol levels were significantly reduced in the C. militaris-treated group compared to the WD-only group (Figure 3A). Similarly, the elevated levels of AST and ALT, which are indicative of liver injury, were also significantly reduced after C. militaris administration (Figure 3B). These findings suggest that the metabolic abnormalities induced by T2DM, including dyslipidemia and liver dysfunction, were ameliorated. It suggested that this specially cultivated C. militaris appeared to exert a protective effect by ameliorating lipid abnormalities and reducing liver damage induced by T2DM.
專門培養的 C. militaris 處理，觀察到這些病理標誌物的顯著改善。具體來說，與僅 WD 組相比，C. militaris 治療組的甘油三酯和總膽固醇水平顯著降低（圖 3A）。同樣，表明肝損傷的 AST 和 ALT 水準升高在 C. militaris 給葯后也顯著降低（圖 3B）。這些發現表明 T2DM 誘導的代謝異常，包括血脂異常和肝功能障礙得到改善。它表明，這種專門培養的 C. militaris 似乎通過改善脂質異常和減少 T2DM 引起的肝損傷來發揮保護作用。

3.3 C. militaris reduced renal dysfunction in a mouse model of T2DM
3.3 C. militaris 在 T2DM 小鼠模型中減少腎功能不全

DN is recognized as one of the primary and most serious complications of T2DM, often leading to progressive renal dysfunction. To evaluate the beneficial effects of this specially
DN 被認為是 T2DM 的主要和最嚴重的併發症之一，通常會導致進行性腎功能障礙。為了特別評估其有益效果
FIGURE 4  圖 4
Evaluation of renal dysfunction and histopathologic changes in ApoE KO mice under different experimental conditions. (A) The ApoE KO mice fed a Western diet (WD) showed changes in renal morphology and a decrease in renal weight. (B) The ApoE KO mice showed significantly elevated blood urea nitrogen (BUN) and serum creatinine levels, indicating renal dysfunction. However, this specially cultivated Cordyceps militaris treatment improved the kidney and significantly decreased the BUN and serum creatinine levels. Data were expressed as mean ± ± +-\pm SD. *** = = == P < 0.001 < 0.001 < 0.001<0.001 compared to ApoE KO mice fed a standard diet group; ### = P < 0.001 compared to ApoE KO mice with WD without any treatment.
不同實驗條件下 ApoE KO 小鼠腎功能不全和組織病理學變化的評價。（A） 飼喂西方飲食 （WD） 的 ApoE KO 小鼠顯示腎臟形態的變化和腎臟重量的降低。（B） ApoE KO 小鼠的血尿素氮 （BUN） 和血清肌酐水平顯著升高，提示腎功能不全。然而，這種專門培養的蛹蟲夏草治療改善了腎臟，並顯著降低了 BUN 和血清肌酐水準。數據表示為平均 ± ± +-\pm SD。*** = = == P < 0.001 < 0.001 < 0.001<0.001 與飼喂標準飲食組的 ApoE KO 小鼠相比;### = P < 0.001 與未接受任何治療的 WD 的 ApoE KO 小鼠相比。
cultivated C. militaris on renal dysfunction in T2DM, various indicators of renal health were evaluated in this study. The values such as kidney weight, BUN and serum creatinine levels were measured to determine the extent of renal damage and the therapeutic potential of this specially cultivated C. militaris. The results showed that the fibrosis morphologies, including dark brown and shrinking morphology, were observed in ApoE KO mice fed with WD. Similarly, a significant reduction in kidney weight was observed in A p o E A p o E ApoEA p o E KO mice fed with WD, accompanied by markedly elevated levels of BUN and serum creatinine. (Figures 4A,B). Because glomerular abnormalities are the hallmark of DN and lead to structural destruction of the kidney (Kaya et al., 2021), histopathologic analysis of renal glomeruli was performed using Hematoxylin and eosin staining. The stained tissue sections revealed pronounced morphological abnormalities in renal glomeruli, in the WDfed A p o E A p o E ApoEA p o E KO mice (Figure 5). Remarkably, treatment with this specially cultivated C. militaris was found to alleviate these pathological changes. In T2DM mice receiving C. militaris, kidney weight was improved. Similarly, elevated BUN and serum creatinine levels were significantly reduced (Figure 4). Histological examination further confirmed these results, as the structural integrity of renal glomeruli was visibly restored in C C CC. militaris-treated mice, with morphology comparable to that of healthy controls (Figure 5). The data suggest that this specially cultivated C. militaris not only improved biochemical markers of renal health, but also facilitated the recovery of renal morphology associated with DN in a T2DM mouse model.
培養的 C. militaris 對 T2DM 腎功能不全的影響，本研究評價了腎臟健康的各項指標。測量腎臟重量、尿素氮和血清肌酐水準等值，以確定腎損傷的程度和這種特殊培養的 C. militaris 的治療潛力。結果表明，在飼喂 WD 的 ApoE KO 小鼠中觀察到纖維化形態，包括深棕色和縮小形態。同樣，在餵食 WD 的 KO 小鼠中 A p o E A p o E ApoEA p o E 觀察到腎重顯著降低，並伴有 BUN 和血清肌酐水平顯著升高。（圖 4A、B）。由於腎小球異常是 DN 的標誌並導致腎臟結構破壞 （Kaya et al.， 2021），因此使用蘇木精和伊紅染色對腎小球進行組織病理學分析。染色的組織切片顯示 WD 餵養的 A p o E A p o E ApoEA p o E KO 小鼠腎小球存在明顯的形態異常（圖 5）。值得注意的是，發現用這種特殊培養的 C. militaris 治療可以緩解這些病理變化。在接受 C. militaris 的 T2DM 小鼠中，腎臟重量得到改善。同樣，升高的 BUN 和血清肌酐水平顯著降低（圖 4）。組織學檢查進一步證實了這些結果，因為腎小球的結構完整性在 中 C C CC 明顯恢復。蟬聯治療的小鼠，其形態與健康對照相當（圖 5）。數據表明，這種專門培養的 C. militaris 不僅改善了腎臟健康的生化標誌物，還促進了 T2DM 小鼠模型中與 DN 相關的腎臟形態的恢復。

3.4 Therapeutic effects of C. militaris on diabetes-induced renal fibrosis
3.4 C. militaris 對糖尿病誘導的腎纖維化的治療作用

Renal fibrosis is recognized as a hallmark pathological feature of DN and contributes significantly to its progression and severity (Xue et al., 2023). In this study, the potential therapeutic effects of this specially cultivated C. militaris on DM-induced renal fibrosis were thoroughly investigated by applying various tissue staining techniques and Western blot analysis. Masson’s trichrome staining (Figure 6A, blue area) and picrosirius red staining (Figure 6B, red area) were used to evaluate collagen deposition within the renal tissue. The staining results showed a significant increase in collagen accumulation in the kidneys of WD-fed A p o E A p o E ApoEA p o E KO mice. Remarkably, C. militaris treatment attenuated this collagen deposition as shown in the staining images (Figure 6). Furthermore, the expression of α α alpha\alpha-smooth muscle actin ( α α alpha\alpha-SMA), a widely recognized marker of fibrosis, was significantly increased in the kidneys of A p o E A p o E ApoEA p o E KO mice fed with WD. However, after C. militaris administration, α α alpha\alpha-SMA levels were significantly reduced, highlighting the potential antifibrotic effects of this specially cultivated C. militaris in diabetic kidneys (Figure 7A). To further evaluate the extent of amyloid deposition, toluidine blue staining was performed. Toluidine blue staining revealed pronounced amyloid deposits in the renal glomeruli of WD-fed A p o E A p o E ApoEA p o E KO mice. Notably, C. militaris treatment attenuated these deposits, suggesting its protective role against amyloid accumulation in the kidneys (Figure 7B). The results of these histologic stains were further supported by Western blot analysis, which detects the expression of key fibrosis-related proteins. In the kidneys of WD-fed A p o E KO A p o E KO ApoEKOA p o E \mathrm{KO} mice, proteins such as collagen type I (COL1), fibronectin,
腎纖維化被認為是 DN 的標誌性病理特徵，並對其進展和嚴重程度有重大影響 （Xue et al.， 2023）。在這項研究中，通過應用各種組織染色技術和 Western blot 分析，徹底研究了這種特殊培養的 C. militaris 對 DM 誘導的腎纖維化的潛在治療作用。Masson 三色染色 （圖 6A，藍色區域） 和 picrosirius 紅染色 （圖 6B，紅色區域） 用於評估腎組織內的膠原蛋白沉積。染色結果顯示 WD 餵養的 A p o E A p o E ApoEA p o E KO 小鼠腎臟中膠原蛋白積累顯著增加。值得注意的是，C. militaris 處理減弱了這種膠原沉積，如染色圖像所示（圖 6）。此外，廣泛認可的纖維化標誌物 α α alpha\alpha -smooth muscle actin （ α α alpha\alpha -SMA） 的表達在餵食 WD 的 A p o E A p o E ApoEA p o E KO 小鼠的腎臟中顯著增加。然而，在 C. militaris 給葯后， α α alpha\alpha -SMA 水平顯著降低，突出了這種專門培養的 C. militaris 在糖尿病腎臟中的潛在抗纖維化作用（圖 7A）。為了進一步評估澱粉樣蛋白沉積的程度，進行了甲苯胺藍染色。甲苯胺藍染色顯示 WD 餵養的 A p o E A p o E ApoEA p o E KO 小鼠腎小球中有明顯的澱粉樣蛋白沉積。值得注意的是，C. militaris 治療減少了這些沉積物，表明它對腎臟中澱粉樣蛋白積累具有保護作用（圖 7B）。這些組織學染色的結果得到了 Western blot 分析的進一步支援，該分析可檢測關鍵纖維化相關蛋白的表達。在 WD 餵養小鼠 A p o E KO A p o E KO ApoEKOA p o E \mathrm{KO} 的腎臟中，I 型膠原 （COL1）、纖連蛋白、
FIGURE 5  圖 5
Restoration of renal glomerular morphology by Cordyceps militaris treatment in ApoE KO mice. Histopathological analysis using Hematoxylin and eosin staining showed severe morphological abnormalities in renal glomeruli, including shrinkage and atrophy, in WD-fed mice. These pathological changes were ameliorated in the Cordyceps militaris-treated group and the glomerular morphology resembled that of healthy controls.
ApoE KO 小鼠中蛹蟲夏草治療腎小球形態的恢復。使用蘇木精和伊紅染色的組織病理學分析顯示，WD 餵養的小鼠腎小球出現嚴重的形態學異常，包括縮小和萎縮。這些病理變化在 Cordyceps militaris 治療組中得到改善，腎小球形態類似於健康對照者。
connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinases (TIMPs), and transforming growth factor β 1 β 1 beta1\beta 1 (TGF β 1 β 1 beta1\beta 1 ) were upregulated. Conversely, E-cadherin (Chang et al., 2022) and Smad7 (Li et al., 2002), key proteins known for their anti-fibrotic properties, was remarkably downregulated. Importantly, C. militaris treatment reversed these pathological trends, demonstrating a reduction in the levels of pro-fibrotic proteins and an upregulation of Smad7 expression (Figure 8). In addition, the study observed that C C CC. militaris treatment increased the expression of Klotho, an anti-aging protein with renoprotective properties, which was decreased in the kidneys of WD-fed A p o E A p o E ApoEA p o E KO mice (Figure 8). Taken together, the reversal of fibrotic markers, the reduction of amyloid deposition, and the enhancement of protective protein expression highlighted the therapeutic potential of this specially cultivated C. militaris in mitigating DM-induced renal fibrosis and kidney damage in DN.
結締組織生長因數 （CTGF） 、金屬蛋白酶組織抑製劑 （TIMP） 和轉化生長因數 β 1 β 1 beta1\beta 1 （TGF β 1 β 1 beta1\beta 1 ） 上調。相反，E-鈣粘蛋白 （Chang et al.， 2022） 和 Smad7 （Li et al.， 2002） 是以其抗纖維化特性而聞名的關鍵蛋白，顯著下調。重要的是，C. militaris 治療逆轉了這些病理趨勢，表明促纖維化蛋白水準降低，Smad7 表達上調（圖 8）。此外，該研究還觀察到 C C CC .軍事化處理增加了 Klotho 的表達，Klotho 是一種具有腎臟保護特性的抗衰老蛋白，在 WD 餵養的 A p o E A p o E ApoEA p o E KO 小鼠的腎臟中表達降低（圖 8）。綜上所述，纖維化標誌物的逆轉、澱粉樣蛋白沉積的減少和保護性蛋白表達的增強突出了這種特殊培養的 C. militaris 在減輕 DM 誘導的腎纖維化和 DN 腎損傷方面的治療潛力。

4 Discussion  4 討論

A previous study mentions that C. militaris extracts significantly reduce blood glucose, cholesterol, and triglycerides, improve antioxidant capacity, and combat DN by reducing uric acid, creatinine, BUN, and urinary protein levels (Dong et al., 2014).
之前的一項研究提到，C. militaris 提取物可顯著降低血糖、膽固醇和甘油三酯，提高抗氧化能力，並通過降低尿酸、肌酐、尿素氮和尿蛋白水準來對抗 DN（Dong et al.， 2014）。
The present study also demonstrates that C. militaris cultivated on G. biloba seeds has remarkable therapeutic potential by effectively addressing critical complications associated with DM. These included reducing hyperglycemia, alleviating dyslipidemia, and improving liver dysfunction. In addition, it specifically alleviated DN, a serious complication of DM, by reducing renal fibrosis while supporting renal function and metabolic balance. T2DM is a chronic metabolic disorder characterized by insulin resistance and relative insulin deficiency, resulting in elevated blood glucose levels (GaliciaGarcia et al., 2020). The present study demonstrates that this specially cultivated C. militaris effectively reduces serum glucose levels in a T2DM mouse model. This finding is consistent with previous research showing the hypoglycemic effects of C. militaris (Cheng et al., 2012). The diabetic serum lipid abnormalities commonly observed in T2DM are characterized by an atherogenic lipid profile with high levels of triglycerides, elevated low-density lipoprotein cholesterol, and decreased high-density lipoprotein cholesterol. This lipid imbalance significantly increases the risk of cardiovascular disease in people with DM (Kalra and Raizada, 2024). Moreover, an increase in total cholesterol is associated with a higher risk of DM-related cardiovascular diseases in diabetic patients, whereas a decrease in total cholesterol is associated with a lower risk of cardiovascular
本研究還表明，在 G. biloba 種子上培養的 C. militaris 通過有效解決與 DM 相關的嚴重併發症具有顯著的治療潛力。這些方法包括降低高血糖、緩解血脂異常和改善肝功能障礙。此外，它通過減少腎纖維化，同時支持腎功能和代謝平衡，專門緩解了 DN，這是 DM 的一種嚴重併發症。T2DM 是一種慢性代謝紊亂，其特徵是胰島素抵抗和相對胰島素缺乏，導致血糖水準升高（GaliciaGarcia 等人，2020 年）。本研究表明，這種專門培養的 C. militaris 可有效降低 T2DM 小鼠模型中的血糖水準。這一發現與先前顯示 C. militaris 的降血糖作用的研究一致（Cheng et al.， 2012）。在 T2DM 中常見的糖尿病血脂異常的特徵是致動脈粥樣硬化血脂譜，甘油三酯水準高，低密度脂蛋白膽固醇升高，高密度脂蛋白膽固醇降低。這種脂質失衡顯著增加了 DM 患者患心血管疾病的風險（Kalra 和 Raizada，2024 年）。此外，總膽固醇的增加與糖尿病患者患 DM 相關心血管疾病的風險較高有關，而總膽固醇的降低與心血管疾病的風險較低有關
FIGURE 6  圖 6
Evaluation of collagen deposition in renal tissue using histological staining. (A) Masson’s trichrome staining and (B) Picrosirius red staining revealed significant collagen accumulation in the kidneys of ApoE KO mice fed a Western diet (WD). This specially cultivated Cordyceps militaris treatment remarkably reduced collagen deposition as evidenced by reduced staining intensity and improved tissue morphology.
使用組織學染色評估腎組織中膠原蛋白沉積。（A） Masson 三色染色和 （B） Picrosirius 紅染色顯示，飼餵西方飲食 （WD） 的 ApoE KO 小鼠的腎臟中膠原蛋白顯著積累。這種專門培養的 Cordyceps militaris 處理顯著減少了膠原蛋白沉積，染色強度降低和組織形態改善證明瞭這一點。
FIGURE 7  圖 7
Analysis of fibrosis and amyloid deposition in the kidneys of ApoE KO mice. (A) Immunohistochemical staining revealed significantly increased expression of α α alpha\alpha-smooth muscle actin ( α α alpha\alpha-SMA), a marker of fibrosis, in the kidneys of mice fed a Western diet (WD). This specially cultivated Cordyceps militaris treatment significantly reduced α α alpha\alpha-SMA expression, demonstrating its antifibrotic effects. (B) Toluidine blue staining showed extensive amyloid deposits in the renal glomeruli of WD-fed mice, which were attenuated after this specially cultivated Cordyceps militaris administration, indicating its protective role against amyloid accumulation.
分析 ApoE KO 小鼠腎臟中的纖維化和澱粉樣蛋白沉積。（A） 免疫組織化學染色顯示，在餵養西方飲食 （WD） 的小鼠腎臟中平滑肌肌動蛋白 （ α α alpha\alpha -SMA） 的表達 α α alpha\alpha 顯著增加，這是一種纖維化標誌物。這種專門培養的蛹蟲夏草治療顯著降低了 α α alpha\alpha -SMA 表達，證明瞭其抗纖維化作用。（B） 甲苯胺藍染色顯示 WD 餵養小鼠腎小球中大量澱粉樣蛋白沉積，在這種特殊培養的蛹蟲夏草給葯后，澱粉樣蛋白沉積減弱，表明其對澱粉樣蛋白積累具有保護作用。
diseases (Khil et al., 2023). Non-alcoholic fatty liver disease has a significant and well-documented association with T2DM. It is estimated that fatty liver affects approximately 70 % − 80 % 70 % − 80 % 70%-80%70 \%-80 \% of individuals diagnosed with T2DM, highlighting its high prevalence in this population. Diabetic dyslipidemia contributes significantly to the pathogenesis of fatty liver. The dysregulated
疾病（Khil et al.， 2023）。非酒精性脂肪性肝病與 T2DM 有顯著且有據可查的關聯。據估計，脂肪肝影響了大約 70 % − 80 % 70 % − 80 % 70%-80%70 \%-80 \% 被診斷患有 T2DM 的個體，這凸顯了脂肪肝在該人群中的高患病率。糖尿病血脂異常對脂肪肝的發病機制有顯著影響。失調
lipid metabolism associated with diabetic dyslipidemia leads to excessive lipid accumulation in the liver, thereby promoting the development of hepatic steatosis (Han et al., 2019). Furthermore, associated liver enzymes such as AST and ALT are often significantly elevated in patients with diabetes, reflecting liver dysfunction (AlJameil et al., 2014). The current study confirms that WD
與糖尿病血脂異常相關的脂質代謝導致肝臟脂質過度積累，從而促進肝脂肪變性的發展（Han et al.， 2019）。此外，糖尿病患者的相關肝酶如 AST 和 ALT 通常顯著升高，反映肝功能障礙（AlJameil 等人，2014 年）。目前的研究證實 WD
FIGURE 8  圖 8
Western blot analysis of fibrosis-related proteins and Klotho expression in the kidneys of ApoE KO mice. Western diet (WD)-fed mice showed upregulation of fibrosis-associated proteins, including collagen type I (COL1), fibronectin, connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP-1), and transforming growth factor β 1 β 1 beta1\beta 1 (TGF β 1 β 1 beta1\beta 1 ), and downregulation of anti-fibrotic proteins E-cadherin and Smad7. This specially cultivated Cordyceps militaris treatment reversed these trends by reducing pro-fibrotic proteins, restoring E-cadherin and Smad7 expression, and increasing Klotho level.
ApoE KO 小鼠腎臟中纖維化相關蛋白和 Klotho 表達的蛋白質印跡分析。西方飲食 （WD） 餵養的小鼠顯示纖維化相關蛋白上調，包括 I 型膠原蛋白 （COL1） 、纖連蛋白、結締組織生長因數 （CTGF）、金屬蛋白酶組織抑製劑 1 （TIMP-1） 和轉化生長因數 β 1 β 1 beta1\beta 1 （TGF β 1 β 1 beta1\beta 1 ），以及抗纖維化蛋白 E-cadherin 和 Smad7 的下調。這種專門培養的蛹蟲夏草治療通過減少促纖維化蛋白、恢復 E-鈣粘蛋白和 Smad7 表達以及增加 Klotho 水平來逆轉這些趨勢。
significantly increases blood lipid levels (triglycerides and cholesterol) and causes liver dysfunction as indicated by elevated serum AST and ALT levels. However, treatment with this specially cultivated C. militaris improves these markers by reducing lipid abnormalities and mitigating liver damage. These results highlight the protective effects of C. militaris against T2DM-induced metabolic disorders, including dyslipidemia and liver injury. One study shows that patients with diabetic nephropathy have significantly higher levels of total cholesterol and triglycerides than those without nephropathy (Palazhy and Viswanathan, 2017). These findings underscore the link between lipid abnormalities and progression of diabetes-related kidney damage.
顯著增加血脂水準（甘油三酯和膽固醇）並導致肝功能障礙，如血清 AST 和 ALT 水準升高所示。然而，用這種專門培養的 C. militaris 治療通過減少脂質異常和減輕肝損傷來改善這些標誌物。這些結果突出了 C. militaris 對 T2DM 誘導的代謝紊亂（包括血脂異常和肝損傷）的保護作用。一項研究表明，糖尿病腎病患者的總膽固醇和甘油三酯水平明顯高於沒有腎病的患者（Palazhy 和 Viswanathan，2017 年）。這些發現強調了脂質異常與糖尿病相關腎損傷進展之間的聯繫。
DN, the most common complication of DM, is the leading cause of end-stage renal disease in DM patients. Renal fibrosis mediated by TGF- β 1 β 1 beta1\beta 1 upregulation plays a key role in the onset and progression of diabetic nephropathy by promoting type I collagen synthesis and suppressing its degradation (Cheng et al., 2013; Zhang et al., 2021). Smad proteins are essential components of the downstream signaling pathway involved in TGF- β 1 β 1 beta1\beta 1-mediated renal fibrosis. While Smad2-4 are activated by TGF- β 1 β 1 beta1\beta 1 to drive renal fibrosis, Smad7, an inhibitory Smad, counteracts the process of renal fibrosis by suppressing the expression of fibrotic cytokines (Zhang et al., 2021). Additionally, the TGF- β β beta\beta signaling pathway promotes the downregulation of E-cadherin and the expression of mesenchymal markers such as α α alpha\alpha-SMA and fibronectin, leading to the transformation of injured renal epithelial cells into a mesenchymal-like state. This transition may contribute to increased extracellular matrix production and accumulation (Nagae et al., 2008; Gewin and Zent, 2012). CTGF is a known
糖尿病神經病變是糖尿病最常見的併發症，是糖尿病患者終末期腎病的主要原因。TGF 上 β 1 β 1 beta1\beta 1 調介導的腎纖維化通過促進 I 型膠原蛋白合成並抑制其降解，在糖尿病腎病的發生和發展中起關鍵作用（Cheng et al.， 2013;Zhang et al.， 2021）。Smad 蛋白是參與 TGF β 1 β 1 beta1\beta 1 介導的腎纖維化的下游信號通路的重要組成部分。雖然 Smad2-4 被 TGF- β 1 β 1 beta1\beta 1 啟動以驅動腎纖維化，但 Smad7 是一種抑制性 Smad，通過抑制纖維化細胞因數的表達來抵消腎纖維化的過程（Zhang 等人，2021 年）。此外，TGF- β β beta\beta 信號通路促進 E-鈣粘蛋白的下調和間充質標誌物如 α α alpha\alpha -SMA 和纖連蛋白的表達，導致受傷的腎上皮細胞轉化為間充質樣狀態。這種轉變可能有助於細胞外基質產生和積累的增加（Nagae 等人，2008 年;Gewin 和 Zent，2012 年）。CTGF 是已知的
profibrotic mediator. It is also upregulated in human kidney proximal tubular cells (HKC-8) after TGF- β 1 β 1 beta1\beta 1 stimulation (Phanish et al., 2006). Additionally, TIMP-1, which inhibits interstitial collagenases and promotes fibrosis progression, is also induced by TGF- β 1 β 1 beta1\beta 1 stimulation (Zhou et al., 2007; Kim et al., 2018). Together, these studies demonstrate that TGF- β 1 β 1 beta1\beta 1 plays a central role in the initiation and progression of renal fibrosis. Moreover, Klotho, a protein known for its anti-aging properties, has been shown to be downregulated in T2DM and its associated complication, DN (Tang et al., 2023). In the current study, this specially cultivated C. militaris attenuates TGF- β 1 β 1 beta1\beta 1 expression to reduce fibrotic markers such as α α alpha\alpha SMA, COL1, CTGF, TIMP-1 and fibronectin, and increases the expression of anti-fibrotic proteins such as Smad7 and E-cadherin to limit DM-induced renal fibrosis in a T2DM mouse model. Treatment with this specially cultivated C. militaris also increases Klotho expression. And then, this specially cultivated C. militaris presents ability to restore renal morphology and weight as well as normalize BUN and serum creatinine levels through changes in molecular mechanisms. Furthermore, amyloid plaques are observed in over 70 % 70 % 70%70 \% of patients with T2DM, and these deposits readily develop into mature fibrils (Smith et al., 2022). The kidney is one of the most common organs affected by amyloid deposition (Dember, 2006; Feitosa et al., 2022). The present study shows amyloid deposition in the renal glomeruli of WD-fed A p o E A p o E ApoEA p o E KO mice, which was reduced by the specially cultivated C C CC. militaris treatment, indicating its potential protective effect in the kidney of T2DM. These findings highlight the therapeutic potential of this specially cultivated C. militaris in ameliorating renal injury and fibrosis in DN.
促纖維化介質。在 TGF 刺激 β 1 β 1 beta1\beta 1 后，它在人腎近端腎小管細胞 （HKC-8） 中也上調 （Phanish et al.， 2006）。此外，抑制間質膠原酶並促進纖維化進展的 TIMP-1 也由 TGF- β 1 β 1 beta1\beta 1 刺激誘導（周 et al.， 2007;Kim et al.， 2018）。總之，這些研究表明 TGF- β 1 β 1 beta1\beta 1 在腎纖維化的發生和發展中起著核心作用。此外，Klotho 是一種以其抗衰老特性而聞名的蛋白質，已被證明在 T2DM 及其相關併發症 DN 中下調（Tang et al.， 2023）。在目前的研究中，這種專門培養的 C. militaris 減弱 TGF- β 1 β 1 beta1\beta 1 表達以減少纖維化標誌物，如 α α alpha\alpha SMA、COL1、CTGF、TIMP-1 和纖連蛋白，並增加抗纖維化蛋白如 Smad7 和 E-cadherin 的表達，以限制 DM 誘導的 T2DM 小鼠模型中的腎纖維化。用這種特殊培養的 C. militaris 治療也會增加 Klotho 的表達。然後，這種專門培養的 C. militaris 具有通過分子機制的變化恢復腎臟形態和重量以及使 BUN 和血清肌酐水準正常化的能力。此外，在超過 70 % 70 % 70%70 \% 2DM 患者中觀察到澱粉樣斑塊，這些沉積物很容易發育成成熟的原纖維（Smith 等人，2022 年）。腎臟是受澱粉樣蛋白沉積影響的最常見器官之一（Dember，2006 年;Feitosa 等人，2022 年）。本研究顯示澱粉樣蛋白沉積在 WD 餵養的 A p o E A p o E ApoEA p o E KO 小鼠的腎小球中，其通過特殊培養的 C C CC .軍事治療，表明其在 T2DM 腎臟中的潛在保護作用。 這些發現突出了這種專門培養的 C. militaris 在改善 DN 腎損傷和纖維化方面的治療潛力。
Although this study highlights the therapeutic potential of C C CC. militaris cultivated on G. biloba seeds in mitigating T2DM and its related complications, it still has some research limitations. In terms of detection indicators, the focus is primarily on a few metabolic indicators, renal function indicators, and fibrosis-related indicators, with a lack of data on other physiological processes such as inflammatory factors and oxidative stress indicators. This limits a comprehensive understanding of the mechanism of action of C C CC. militaris in the body. Protein detection is also limited to a few fibrosis-related and anti-fibrosis proteins and does not cover all key proteins involved in renal fibrosis. Regarding the experimental design, the lack of different dosage groups for C. militaris hinders the identification of an optimal therapeutic dose, reducing the clinical applicability of the results. Furthermore, the lack of comparative trials with clinical medications makes it difficult to evaluate the relative advantages of this specially cultivated C C CC. militaris in clinical application. The study also relies on an animal model that may not fully capture the complexity of DN in humans, thus caution should be exercised in translating these results to clinical practice. In addition, the short duration of the study does not address the long-term safety and efficacy of C. militaris. Future research should include clinical trials to validate these findings in humans, explore potential side effects, and optimize dosing regimens. Nevertheless, the results underscore the promising potential of this specially cultivated C. militaris as a natural and effective therapeutic strategy for the management of T2DM and its associated complications.
儘管這項研究強調了 C C CC 的治療潛力。在 G. biloba 種子上培養的 militaris 減輕 T2DM 及其相關併發症，它仍然存在一些研究局限性。在檢測指標方面，重點主要集中在少數代謝指標、腎功能指標和纖維化相關指標上，缺乏炎症因數和氧化應激指標等其他生理過程的數據。這限制了對 的作用 C C CC 機制的全面理解。軍人在體內。蛋白質檢測也僅限於少數纖維化相關和抗纖維化蛋白，並不涵蓋與腎纖維化有關的所有關鍵蛋白質。關於實驗設計，缺乏 C. militaris 的不同劑量組阻礙了最佳治療劑量的確定，降低了結果的臨床適用性。此外，缺乏與臨床藥物的比較試驗使得很難評估這種專門培養的相對優勢 C C CC 。軍事在臨床中的應用。該研究還依賴於一種動物模型，該模型可能無法完全捕捉人類 DN 的複雜性，因此在將這些結果轉化為臨床實踐時應謹慎。此外，該研究的持續時間短並不能解決 C. militaris 的長期安全性和有效性。未來的研究應包括臨床試驗，以在人體中驗證這些發現，探索潛在的副作用，並優化給葯方案。儘管如此，結果強調了這種特殊培養的 C. militaris 作為管理 T2DM 及其相關併發症的天然有效治療策略的潛力。

5 Conclusion  5 總結

In conclusion, this study highlighted the therapeutic potential of C. militaris cultivated on G. biloba seeds in the treatment of T2DM and its complications, especially DN. The results showed that this unique cultivation approach enhanced the bioactive properties of C C CC. militaris, enabling it to effectively alleviate hyperglycemia, dyslipidemia, liver dysfunction, and renal damage. In addition, the treatment reduced renal fibrosis while promoting renal repair and metabolic balance. These results highlight the promise of this specially cultivated C. militaris as a natural and effective therapeutic option for T2DM and its associated complications, which warrants further investigation and clinical validation.
總之，本研究強調了在 G. biloba 種子上培養的 C. militaris 在治療 T2DM 及其併發症（尤其是 DN）方面的治療潛力。結果表明，這種獨特的培養方法增強了 C C CC 的生物活性特性。軍事化，使其能夠有效緩解高血糖、血脂異常、肝功能障礙和腎功能損害。此外，該治療減少了腎纖維化，同時促進了腎臟修復和代謝平衡。這些結果突出了這種特殊培養的 C. militaris 作為 T2DM 及其相關併發症的天然有效治療選擇的前景，這需要進一步的研究和臨床驗證。

Data availability statement
數據可用性聲明

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
研究中介紹的原始貢獻包含在文章/補充材料中，進一步查詢可直接聯繫通訊作者。

Ethics statement  道德聲明

The animal study was approved by the Institutional Animal Care and Use Committee of Hualien Tzu Chi Hospital (approval number: 113-43). The study was conducted in accordance with the local legislation and institutional requirements.
該動物研究獲花蓮慈濟醫院機構動物照護與使用委員會核准（批准文號：113-43）。該研究是根據當地立法和機構要求進行的。

Author contributions  作者貢獻

S-ZL: Conceptualization, Methodology, Project administration, Writing - review and editing. W-WK: Conceptualization, Methodology, Project administration, Writing - review and editing. BC-KT: Investigation, Methodology, Validation, Visualization, Writing - original draft. CP: Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing - review and editing. C-HK: Formal Analysis, Investigation, Validation, Writing - review and editing. DJ-YH: Formal Analysis, Validation, Writing - review and editing. S-WK: Validation, Data curation, Writing - review and editing. P-YP: Data curation, Methodology, Writing - review and editing. S-JC: Funding acquisition, Resources, Visualization, Writing - review and editing. C-YH: Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review and editing. K-HL: Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review and editing.
S-ZL：概念化、方法論、專案管理、寫作 - 審查和編輯。W-WK：概念化、方法論、專案管理、寫作 - 審查和編輯。BC-KT：調查、方法、驗證、可視化、寫作 - 原始草案。CP： 形式分析、調查、方法論、驗證、可視化、寫作 - 審查和編輯。C-HK： 形式分析、調查、驗證、寫作 - 審查和編輯。DJ-YH： 形式分析、驗證、寫作 - 審查和編輯。S-WK：驗證、數據管理、寫作 - 審查和編輯。P-YP：數據管理、方法、寫作 - 審查和編輯。S-JC：資金獲取、資源、可視化、寫作 - 審查和編輯。C-YH： 概念化、資金獲取、專案管理、監督、寫作 - 審查和編輯。K-HL：概念化、資金獲取、專案管理、監督、寫作 - 審查和編輯。

Funding  資金

The author(s) declare that financial support was received for the research and/or publication of this article. The authors acknowledge the Home Run Biotechnology Co., Ltd. (TCJ112002), China Medical University (CMU103-TC-03), China Medical University Hospital (DMR-108-057), Ministry of Science and Technology (Taiwan; MOST 110-2320-B-303-001MY2 and MOST 111-2314-B-303-008-MY3), and National Science and Technology Council (Taiwan; NSTC 113-2811-B-303-002; NSTC 113-2314-B-303-024 and NSTC-113-2314-B-303-024) to support this study.
作者聲明已獲得本文研究和/或出版的財政支援。作者感謝家潤生物科技股份有限公司 （TCJ112002）、中國醫藥大學 （CMU103-TC-03）、中國醫藥大學附屬醫院 （DMR-108-057）、科學技術部（臺灣;MOST 110-2320-B-303-001MY2 和 MOST 111-2314-B-303-008-MY3）和美國國家科學技術委員會（臺灣;NSTC 113-2811-B-303-002;NSTC 113-2314-B-303-024 和 NSTC-113-2314-B-303-024） 來支援這項研究。

Acknowledgments  確認

The authors gratefully acknowledge Prof. Tsung-Jung Ho of the Department of Traditional Chinese Medicine, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (Hualien, Taiwan), for his expertise in the morphological and related identification of this specially cultivated Cordyceps militaris. The authors also thank the Laboratory Animal Center at Tzu Chi University, Hualien, Taiwan, for their support in animal husbandry and care and the core facilities provided by Advanced Instrumentation Center of Department of Medicine Research, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan.
作者衷心感謝佛教慈濟醫學基金會（台灣花蓮）花蓮慈濟醫院中醫科的 Tsung-Jung Ho 教授，感謝他在這種特殊培養的蛹蟲夏草的形態學和相關鑒定方面的專業知識。作者還感謝臺灣花蓮慈濟大學實驗動物中心在畜牧和護理方面的支援，以及臺灣花蓮慈濟醫學基金會花蓮慈濟醫院醫學研究部先進儀器中心提供的核心設施。

Conflict of interest  利益衝突

Author S-JC was employed by Home Run Biotechnology Co., Ltd.
作者 S-JC 曾受雇於 Home Run Biotechnology Co.， Ltd.。
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
其餘作者聲明，該研究是在沒有任何可能被解釋為潛在利益衝突的商業或財務關係的情況下進行的。

Generative AI statement  生成式 AI 聲明

The author(s) declare that Generative AI was used in the creation of this manuscript. DeepL Writing was only used to check the English grammar error.
作者聲明，本手稿的創作使用了生成式 AI。DeepL Writing 僅用於檢查英語語法錯誤。

Publisher's note  出版商注

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated
本文中表達的所有聲明僅代表作者的觀點，並不一定代表其附屬公司的觀點

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organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
組織，或者出版商、編輯和審稿人的組織。本文中可能評估的任何商品或製造商可能提出的聲明均不受出版商的保證或認可。

Supplementary material  補充材料

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2025.1562116/ full#supplementary-material
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  1. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BNU, blood urea nitrogen; C. militaris, Cordyceps militaris; DM, Diabetes mellitus; DN, Diabetic nephropathy; G. biloba, Ginkgo biloba; T2DM, type 2 diabetes mellitus; WD, western diet.
    縮寫：ALT，丙氨酸氨基轉移酶;AST，天冬氨酸氨基轉移酶;BNU，血尿素氮;C. militaris， 蛹蟲夏草;DM，糖尿病;DN，糖尿病腎病;G. biloba， 銀杏;T2DM，2 型糖尿病;WD，西方飲食。