Study on the relationship between AMPK/UCP2 pathway and mitochondrial dysfunction in granulosa cells of polycystic ovary syndrome

Abstract: Objective: To investigate the changes of the AMP-activated protein kinase (AMPK)/uncoupling protein 2 (UCP2) (AMPK/UCP2) pathway in ovarian granulosa cells with PCOS and its relationship with mitochondrial dysfunction. Methods: PCOS mouse models and normally fed mice, ovarian granulosa cells from the two mice were extracted, and the protein expression levels of AMPKα, p-AMPKα and UCP2 were detected by western blotting. The ROS and ATP content of granulosa cells were determined by colorimetric and chemiluminescence immunoassays to assess mitochondrial function. Pearson correlation analysis was used to determine the correlation between AMPK/UCP2 pathway-related proteins, ROS and ATP. Results: P-AMPKα/GAPDH (0.12±0.09), AMPKα/GAPDH (0.35±0.40), P-AMPKα/AMPKα (0.56±0.33) and ATP (0.36±0.04) pmol/mg in PCOS model mice were lower than those in non-POCS groups, while UCP2/GAPDH (1.18±0.28) and ROS (48810.92± were lower than those in non-POCS groups4498.08) The fluorescence intensity of DCF was higher than that of the non-POCS group, (P<0.05). AMPK was positively correlated with ATP and negatively correlated with ROS. UCP2 was positively correlated with ROS and negatively correlated with ATP. Conclusion: There are abnormal changes such as decreased AMPK expression and increased UCP2 expression in ovarian granulosa cells of PCOS, and AMPK is positively and negatively correlated with mitochondrial function indexes ATP and ROS, while UCP2 is the opposite, suggesting that the imbalance in the expression and activity of AMPK/UCP2 pathway in PCOS may be one of the molecular mechanisms leading to mitochondrial dysfunction. Regulation of AMPK/UCP2 pathway activity may be a potential therapeutic target to ameliorate PCOS-related mitochondrial dysfunction.

Keywords: Polycystic ovary syndrome; adenylate-activated protein kinases; Uncoupling protein 2; mitochondrial dysfunction; Granule cells

引言

Polycystic ovary syndrome (PCOS) is a common endocrine system disease characterized by long-term anovulation and hyperandrogenism, and its pathogenesis is still unknown. Existing studies have shown that there is mitochondrial dysfunction in PCOS, which is manifested by decreased oxidative phosphorylation function and increased ROS production ^[1-2] . Mitochondrial dysfunction can affect the maturation and ovulation process of ovarian granulosa cells, and plays an important role in the pathogenesis of PCOS ^[3] . In recent years, adenylate-activated protein kinase (AMPK) and uncoupling protein 2 (UCP2) have attracted clinical attention as two important regulators of cellular metabolism. AMPK is an intracellular energy sensor that is activated in the body under conditions of hypoxia and glucose, and is involved in the process of insulin resistance in PCOS rats ^[4] . Insulin resistance increases the risk of inhibition of cytoplasmic synthesis of cellular mitochondrial proteins ^[5] . UCP2 is a mitochondrial carrier protein that stimulates hyperandrogenic effects during the pathogenesis of PCOS ^[6] . The regulation of glucose and lipid metabolism by androgens and their receptors is closely related to the effects of mitochondrial content and function ^[7] . Therefore, the AMPK/UCP2 pathway may be involved in the mitochondrial dysfunction process of PCOS. However, changes in AMPK and UCP2 in animal models of PCOS and their relationship to mitochondrial dysfunction are unclear. Therefore, this study intends to use ovarian granulosa cells from PCOS mouse model as the research object to detect the expression levels of AMPK and UCP2, and evaluate mitochondrial function, so as to elucidate the changes of AMPK/UCP2 pathway in PCOS disease and its relationship with mitochondrial dysfunction, and provide a theoretical basis for elucidating the pathogenesis of PCOS and exploring therapeutic targets.

1 Materials and Methods

Cell source

Human ovarian granulosa cells KGN, purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. There were 50 SPF healthy female mice, routinely reared, with a temperature of 23 °C, a relative humidity of 60%, a free diet of water, and 12 hours of periodic light.

Instruments & Reagents

The main instruments include: Thermo company produces microplate readers; chemiluminescence immunoassay analyzer manufactured by Beckman; High-speed refrigerated centrifuges are produced for the Thermo Sinentific enterprise; The developer was Tanon 5200, and the main reagents included: HyClone's DMEM/F12 medium, BioSource penictomycin, 10% fetal bovine serum (manufactured by Thermo Fisher), trypsin trypsin, and TransGen's BCA protein assay kit; ATP assay kit of Nanjing Institute of Bioengineering; AMPKα, p-AMPKα, UCP2, GAPDH, Abcam secondary antibodies, Bausch Tak ELISA kits.

Animal modeling

The mice were divided into POCS model group and normal group with 25 mice in each of the vaginal smear to be determined by vaginal smear, and the blank group was given normal saline gavage, 10 ml/(kg·d) combined with conventional diet, and the POCS model group was used to dissolve trozole in 1% sodium carboxymethylcellulose, gavaged 1mg/(kg·d), combined with high-fat diet to feed for 21 days, and the preparation process of POCS model was completed. When the estrous cycle of the model mice is disordered and the ovarian tissue forms polycystic changes, the modeling is considered successful.

Cell isolation and culture

After successful modeling, the mice were sacrificed and dissected, bilateral fallopian tubes were cut, placed in M2 culture medium, the oviduct bulge was picked open with a disposable injection needle, and the outflow oocyte complex droplets were transferred into the egg cell washing medium dish for 5 minutes, and then the granule cells of each oocyte were separated by a syringe needle. The collected granulosa cells and purchased KGN cells were used for later use, and the fetal bovine serum, penicillin, and DMEM/F12 medium with a volume fraction of 0.10 were used for routine culture of granulosa cells in a 0.05 CO ₂ incubator at 37°C.

AMPK/UCP2 protein assay

Two groups of ovarian granulosa cells were taken and washed in PBS and an appropriate amount of RIPA lysate was added to lyse for 30min. Centrifuge at 4°C and 12000rpm for 5min to collect total protein. The protein concentration was determined by BCA, and the concentration of the samples in each group was adjusted. Equal amounts of protein Sample Buffer were used to prepare an equal concentration protein solution. Each histone was separated by SDS-PAGE electrophoresis, and the primary antibodies AMPK, p-AMPK, UCP2 and internal control GAPDH antibodies were added after transfer and blocking, and incubated at 4°C overnight. After washing the membrane, the secondary antibody was incubated for 1 h, and the ECL was luminescent. Image J software analyzed the gray scale of the bands and calculated the relative expression levels of AMPK, p-AMPK, and UCP2 (gray value of target protein/gray value of GAPDH). The mean value of the independent experiment was included in the statistics for 3 times, and the differences in the expression of histones were statistically analyzed. The ratios of P-AMPKα/GAPDH, AMPKα/GAPDH, P-AMPKα/AMPKα and UCP2/GAPDH were included in the statistics.

ATP content determination

The ATP content of granulosa cells in the two groups of mice was determined by colorimetric method, and the ovarian granulosa cells of the two groups were seeded in 96-well plates at a density of 5×10 ⁷ /mL, and each sample was set up with blank wells, standard wells, reference wells and assay wells. Only the detection reagent is added, and no sample is added to eliminate the absorbance of the reagent itself. A concentration of standard ATP solution (known concentration: 1×10 ³ μmol/L) was added to plot the standard curve. KGN cell samples were added to the control wells and were not subjected to experimental treatment. The particle cells of the samples to be tested were added to the wells of the PCOS group and the normal group. The protein concentration of granulosa cells in each sample was measured by BCA, and 30 μL of the sample to be tested was placed in the control tube and the assay tube respectively, and mixed in a water bath at 37°C for 30 min. Add 50 μL of precipitant to each tube, centrifuge at 4000 r/min for 5 min, take the supernatant, add stop solution and chromogenic solution to each well, and mix well. The OD value of each well was measured with a microplate reader at a wavelength of 636 nm. The concentration of ATP in the sample was calculated according to Formula 1: each sample repeated the experiment 3 times, and the mean value was included in the statistics.

公式1

Note: The OD value in the formula _测定 is the OD value of the measured product; In the formula, the OD value _对照 is the OD value of the reference wells. In the formula, the OD value _标准 is the OD value of the standard pores; In the formula, the OD value _空白 is the OD value of the blank hole; 1×10 ³ μmol/L was the standard concentration.

ROS content determination

Ovarian granulosa cells from two groups were seeded at a density of 2×10 ⁴ /well in 96-well plates, with 3 compound wells in each group. Incubate for 24 h. Discard the medium, add 100 μL of DCFH-DA working solution to each well, and incubate for 30 min in the dark. DCFH-DA can be taken up by cells and oxidized by ROS to the fluorescent DCF. Discard the working solution and rinse 1 time with serum-free medium per well to remove DCFH-DA that has not entered the cells. The fluorophotometer was set to 488 nm excitation wavelength and 525 nm emission wavelength, and the fluorescence intensity value of each well was detected as a relative quantitative indicator of ROS content, that is, the relative absorbance value of ROS content. Repeat the independent experiment 3 times, and the mean value will be included in the statistics.

Statistical methods

The experimental data are all continuous data, expressed by 'x ± s, analyzed by SPSS 16.0 software, the sameness analysis of variance was performed first, and the t-test was used for comparison between groups, and P < 0. 05 Statistically significant for the difference. Correlation was performed using Pearson correlation analysis. The origin2021 software was used to draw a cluster scatter plot for visual analysis.

2 Results

2.1 Comparison of AMPK/UCP2 pathway-related proteins

P-AMPKα/GAPDH (0.12±0.09), AMPKα/GAPDH (0.35±0.40) and P-AMPKα/AMPKα (0.56±0.33) in the POCS model group were lower than those in the non-POCS group, while UCP2/GAPDH (1.18±0.28) was higher than that in the normal group (P<0.05) (Table 1).

Table 1 Comparison of the relative expression levels of AMPK/UCP2 pathway-related proteins between the two groups ()

2.2 Comparison of mitochondrial function-related indicators

The ATP (0.36±0.04) pmol/mg in the POCS model group was lower than that in the non-POCS group, while the DCF fluorescence intensity in ROS (48810.92±4498.08) was higher than that in the normal group (t=16.147, -19.362, P<0.001) (see Table 2 for details).

Table 2 Comparison of ATP and ROS contents of granulosa cells in the two groups ()

2.3 Correlation Analysis

Pearson correlation analysis showed that there was a negative correlation between the group and P-AMPKα/GAPDH, AMPKα/GAPDH, P-AMPKα/AMPKα and ATP, and a positive correlation with UCP2/GAPDH and ROS. P-AMPKα/GAPDH, AMPKα/GAPDH, and P-ampk α/AMPK α were positively correlated with ATP (r=0.775, 0.432, 0.351, P=0.000, 0.002, 0.012), and UCP2/GAPDH was negatively correlated with ATP (r=-0.584, P=0.000). There were negative correlations between P-AMPKα/GAPDH, AMPKα/GAPDH, P-AMPKα/AMPKα and ROS (r=-0.837, -0.412, -0.359, P=0.000, 0.003, 0.010). There was a positive correlation between UCP2/GAPDH and ROS (r=0.759, P=0.000) (Fig. 1).

Fig.1 Scatter plot of clusters between AMPK/UCP2 pathway-related proteins and mitochondrial ATP and ROS

3讨论

PCOS is a common cause of infertility in women of childbearing age and is affected by abnormal epigenetic modifications in mitochondrial DNA (mtDNA ^[9] ). Some scholars have found that PCOS has mitochondrial structure and function disorders, such as decreased expression of cytochrome P450 oxidase, a key enzyme in sex hormone synthesis, and decreased oxidative phosphorylation function. This is thought to be related to insulin resistance and metabolic abnormalities in PCOS. However, the molecular mechanism of mitochondrial dysfunction and its relationship with the pathogenesis of PCOS need to be further elucidated. Deficiency of α1 AMPK in human granulosa cells affects the cell cycle, adhesion, and lipid metabolism, and induces a hyperandrogenic response ^[10] . AMPK is an important cellular energy sensor, and UCP2 is an inner mitochondrial membrane transporter. Both regulate mitochondrial function and oxidative phosphorylation. In this study, it was found that there was an imbalance in the expression of AMPK/UCP2 in ovarian granulosa cells in the animal model of PCOS, which was associated with the disorders of ATP and ROS, which were related to mitochondrial dysfunction. This provides a new perspective on the mechanism of mitochondrial damage and suggests that AMPK/UCP2 may be a new target for PCOS intervention. In this study, immunoblotting and colorimetric methods were used to comprehensively evaluate the changes of AMPK/UCP2 pathway and mitochondrial function parameters in ovarian granulosa cells of PCOS mice, and the results supported the above hypothesis, suggesting that the AMPK/UCP2 pathway may play an important role in the pathogenesis of PCOS. At the same time, it provides a theoretical basis and experimental data support for elucidating the pathogenesis of PCOS and developing related new drugs and new treatments.

The results of this study showed that the expression of AMPK was down-regulated and the expression of UCP2 was up-regulated in ovarian granulosa cells of PCOS model mice, which was negatively correlated. In skeletal muscle and liver, excess ATP leads to insulin resistance by inhibiting AMPK and activating mTOR. Persistent ATP excess leads to mitochondrial dysfunction as a result of mitotic inhibition, and this result validates the underlying mechanism of mitochondrial dysfunction in PCOS related to the underlying mechanism of ATP excess leading to insulin resistance through inhibition of AMPK and activation of mTOR ^[11] . An animal study on myocardial ischemia found that myocardial ischemia injury was reduced by activating the AMPK/PCG-1α signaling pathway to improve myocardial mitochondrial function ^[12] . Mitochondrial β oxidation, leading to oxidative stress and inflammation, stimulates AMP-activated AMPK activity and its downstream targets involved in mitochondrial biogenesis and antioxidant protection ^[13] . It can be seen that AMPK is closely related to mitochondrial dysfunction. PCOS is accompanied by mitochondrial mutations, which cause decreased ATP production and increased ROS production ^[14] . This study is consistent with previous results in terms of reduced AMPK expression, but we jointly detected changes in AMPK and UCP2 and performed correlation analysis with mitochondrial functional parameters, providing granulose cytological evidence that an imbalance in the AMPK/UCP2 pathway may be involved in the pathogenesis of PCOS. In the future, the sample size needs to be expanded, and animal models should be used to further verify the role of this pathway in the pathogenesis of PCOS. This study preliminarily elucidates the AMPK/UCP2 expression imbalance in PCOS disease and its relationship with mitochondrial dysfunction, and provides primary evidence. However, this study mainly proposed the possible relationship between the AMPK/UCP2 pathway and mitochondrial dysfunction through correlation analysis, and further animal experiments or cell function verification experiments were needed to further confirm the pathogenic role of this pathway. Follow-up studies will expand the sample size, use animal models to intervene in the AMPK/UCP2 pathway, observe the effects on ovarian function and metabolism, and conduct molecular mechanism studies to determine the specific role of AMPK/UCP2 in the pathogenesis of PCOS. It will also screen potential drugs targeting this pathway to provide a basis for targeted therapy for PCOS. This study lays a foundation for the development of new PCOS drugs targeting the AMPK/UCP2 pathway.

4 Conclusion

In this study, it was found that the expression of AMPK in ovarian granulosa cells of PCOS model mice was down-regulated and UCP2 expression was up-regulated. At the same time, mitochondrial dysfunction in ovarian granulosa cells in PCOS mice was manifested by a decrease in ATP production and an increase in ROS. It was found that there was an imbalance in the expression and activity regulation of the AMPK/UCP2 pathway in PCOS, which may be a key factor leading to mitochondrial dysfunction. This discovery reveals the possible mechanism of mitochondrial damage in PCOS at the molecular level. At the same time, this discovery also suggests that the AMPK/UCP2 pathway may become a new target for PCOS intervention, which provides a theoretical basis and experimental data support for the drug development of PCOS, and has important clinical translational significance. Overall, the research process is rigorous and the results are reliable, which can make up for the lack of research on the AMPK/UCP2 pathway and mitochondrial dysfunction in PCOS, fill the academic gap in this field, and have important academic innovation significance, providing new perspectives and possible new therapeutic targets for the basic research and clinical treatment of PCOS, and has important basic research and clinical translational value.