Exploring The Biological Basis Of Polycystic Ovary Syndrome Phlegm Syndrome From Mitochondria-mediated Apoptosis Pathway

Objective:In this study,dehydroepiandrosterone was used to replicate a PCOS rat model,and Erchen Decoction was used to observe the serum sex hormone levels,ovarian morphological changes,ovarian granulosa cell apoptosis rate,and mitochondrial-mediated Expression of apoptotic pathway-related genes.Analyzing the relationship between sputum syndrome and PCOS ovarian granulosa cell apoptosis and the biological basis of PCOS sputum syndrome from the mitochondrial-mediated apoptosis pathway,enriching the biological connotation of PCOS sputum syndrome,and preliminary clearing the role of Erchen Decoction in treating PCOS.Methods:Thirty-six 21-day-old SPF SD female rats were randomly divided into a normal group?10 rats?and a model?26 rats?.The model was injected with DHEA subcutaneously on the back of the neck every day,and the same amount of soybean oil was injected into the same part of the normal group for 20 days.According to the vaginal exfoliated cells,ovarian tissue morphology and serum sex hormone levels,observe whether the rats are modeled.The model rats were randomly divided into model group,Erchen Decoction group,and metformin group,and the normal group was used as a control.The Erchen Decoction group(4.35g·kg^-1·d^-1)and the metformin group(0.3g·kg^-1·d^-1)were given the corresponding dose of drugs for 4 weeks,and the model group and normal group were given the same volume of extinction Fungus drinking water.Enzyme-linked immunosorbent assay?ELISA?to detect testosterone?T?,luteinizing hormone?LH?,follicle stimulating hormone?FSH?,and estradiol?E₂?;hematoxylin-eosin staining?HE?to observe ovarian tissue Morphology;terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay?TUNEL?to detect ovarian granulosa cell apoptosis;real-time fluorescence quantitative PCR?q-PCR?,immunohistochemical ABC method,Western Blot detection Expression of genes involved in the apoptosis pathway.Results:1.On the 21st day of modeling,it was found that compared with the normal group,the body weight and serum T,LH,and E₂ levels of rats in the model group were significantly increased?P<0.01?.There was no significant difference between the two groups in FSH?P>0.05?.The rats in the model group lost regular estrous cycles.Under the light microscope,multiple corpus luteum and follicles of various levels were found in the ovary tissue of normal rats.The granular cells were arranged in multiple layers,most of which were 8-9layers.The arrangement was close and orderly.The structure of nearly mature follicles was complete,including the cumulus,oocytes and the radiation crown.There were fewer follicles and corpus luteum at different developmental stages of the model,the granular layers were reduced to 1 to 3 layers,the arrangement was sparse,the follicles were cystic expansion,and there were no oocytes.2.Comparison of various indicators after phlegm elimination:?1?Body mass and ovarian index:Compared with the normal group,the body mass and ovarian index of the model group were higher,and the difference was not statistically significant?P>0.05?;Compared with the model group,the rats in the Erchen Decotion group and the metformin group,the body mass and ovarian index decreased,and the difference was statistically significant?P<0.05?.?2?The levels of serum T,LH,FSH,and E₂ levels:Compared with the normal group,the levels of serum T and LH in the model group were significantly increased?P<0.01?,and serum E₂ and FSH levels were significantly decreased?P<0.01?.Compared with the model group,the serum T and LH levels of the Erchen Decoction group were significantly reduced?P<0.01?,the serum E₂ and FSH levels were significantly increased?P<0.01?;The FSH levels were significantly increased?P<0.01?,and the LH levels were decreased.?P<0.01?.?3?Ovarian morphological changes:In the normal group,multiple corpus luteum and follicles of different developmental stages were seen in the ovarian tissue of the rat.In the model group,luteal corpus luteum and follicles at all levels were rare,and the number of granular cell layers was reduced,most of which were 1³.The arrangement was loose.A large number of cystic dilated follicles were seen,and oocytes or radiation crowns in cystic follicles disappeared.There was improvement in Erchen Decoction group and metformin group.?4?Apoptosis index of ovarian granulosa cells:Compared with the normal group,the apoptosis index of ovarian granulosa cells in the model group has increased,and the difference was statistically significant?P<0.05?.Compared with the model group,the apoptosis index of ovarian granulosa cells in Erchen Decoction group and metformin group had a decreasing trend,and the difference was not statistically significant.?5?Relative expression levels of Cyt-C,Bax,Bcl-2,and Caspase-9 mRNA:There was no significant difference in the relative expression of Cyt-C mRNA in each group.Compared with the normal group,the expression levels of Bcl-2/Bax and Caspase-9 mRNA in the model group decreased?P<0.05?.Compared with the model group,The expression levels of Bcl-2/Bax and Caspase-9 mRNA in Erchen Decoction group and metformin group increased?P<0.05?.?6?Relative expression levels of Bax and Bcl-2 proteins:Compared with the normal group,the expression level of Bcl-2 protein in the model group decreased and the expression level of Bax protein increased?P<0.01?.Compared with the model group,the expression level of Bcl-2 in the Erchen Decoction group and metformin group increased,and the expression level of Bax protein decreased.?P<0.05?.?7?Relative expression of Cyt-C,Procaspase-9 and Clearedcaspase-9 protein in ovarian tissue:Compared with the normal group,the expression level of mitochondrial Cyt-C in the model group decreased,and the expression level of cytoplasmic Cyt-C and Procaspase-9protein increased?P<0.05?,Cleavedcaspase-9 protein expression level showed an upward trend,the difference was not statistically significant?P>0.05?;Compared with the model group,the cytoplasmic Cyt-C and Procaspase-9 protein expression in the Erchen Decotion group and the metformin group the level decreased?P<0.05?,the expression level of mitochondrial Cyt-C showed an upward trend,and the difference was not statistically significant?P>0.05?.Conclusions:1.The phlegm-reducing effect of Erchen Decoction is mainly manifested in improving the ovarian morphology and regulating sex hormone levels of PCOS rats.2.The improvement of PCOS by Erchen Decoction may be achieved by promoting the expression of Bcl-2 in the ovarian tissue and the low expression of Bax,Caspase-9 and Cyt-C in the cytoplasm.3.The excessive apoptosis of the ovarian granulosa cells caused by abnormal mitochondrial-mediated apoptotic pathways may be one of the biological foundations of sputum syndrome in PCOS.