Mechanism of Wenjing Decoction regulating activating transcription factor 6/C/EBP homologous protein pathway and inhibiting endoplasmic reticulum stress to improve model of decreased ovarian reserve
Objective To explore the mechanism of Wenjing Decoction (温经汤) regulating the endoplasmic reticulum stress (ERS) mediated activating transcription factor 6/C/EBP homologous protein (ATF6/CHOP) pathway in the treatment of decreased ovarian reserve (DOR). Methods The DOR rat model was established by multi-glycosides of Tripterygium glycosides,then the rats were divided into control group,model group,femoston (FMT) group (Sequential therapy involving the administration of estradiol valerate suspension at a dosage of 0.2 mg/kg on days 1 to 2,followed by estradiol and dydrogesterone tablets at a dosage of 1.2 mg/kg on days 3 to 5),low-,medium-and high-dose groups (4.85,9.70,19.40 g/kg) of Wenjing Decoction. The ovarian and uterine indexes were calculated after four weeks of treatment,the levels of serum anti mullerian hormone (AMH),follicle stimulating hormone (FSH),estradiol (E2),and luteinizing hormone (LH) were detected by ELISA method,hematoxylin eosin (HE) staining was used to observe the pathological structure of ovary,and the follicles at all levels were counted,the mRNA expressions of ERS markers glucose regulatory protein 78 (GRP78),ATF6,CHOP in ovarian tissues of rats in each group were detected by qRT-PCR method,the protein expressions of GRP78,ATF6,CHOP and cysteine aspartic acid specific protease-12 (Caspase-12) were detected by Western blotting. The modeling concentration of GTW was screened using the CCK-8 method,and Human ovarian granulosa cells KGN cells were divided into control group,GTW group,GTW+5％ blank serum group (GTW+KBXQ),GTW+5％ Wenjing Decoction containing serum group (GTW+WJTXQ),and ERS agonists thapsigargin (TG) group. The effect of Wenjing Decoction on the viability of KGN cells treated with GTW was detected by CCK8 assay,then Ca2+concentration in each group was detected by Fluo-4 AM calcium fluorescent probe,the protein expression levels of GRP78,ATF6,CHOP and Caspase-12 in cells of each group were analyzed by western blotting. Results Compared with control group,the ovarian and uterine indexes in the model group were significantly reduced (P<0.05,0.01),serum AMH and E2 levels were decreased (P<0.01),FSH and LH levels were increased (P<0.01),the number and layers of granulosa cells in the ovarian tissue of the model group were less,the arrangement was sparse,and the ovarian parenchyma was empty,and the number of developing follicles in the ovarian tissue of the model group decreased significantly (P<0.01),and the number of atretic follicles were increased (P<0.01),and the mRNA and protein expressions of GRP78,ATF6,and CHOP,and the protein expressions of Caspase-12 in ovarian tissue were significantly increased (P<0.05,0.01). Compared with the model group,the ovarian and uterine indexes in FMT group,WJTZ group,and WJTG group were increased obviously (P<0.05,0.01),the serum AMH level of rats in each administration group were significantly increased (P<0.01),and the FSH level were significantly decreased (P<0.01),and the serum LH level of rats in the FMT group,WJTZ group and WJTG group were significantly decreased (P<0.01),and the E2 level of rats in the FMT group,WJTZ group and WJTG group were significantly increased (P<0.05,0.01).The number of developing follicles at all levels in the ovarian tissue of each administration group were increased,and the number of atretic follicles were decreased (P<0.05,0.01). The mRNA and protein expressions of GRP78,ATF6,and CHOP,and the protein expression of Caspase-12 in the FMT group,WJTZ group and WJTG group were significantly reduced (P<0.05,0.01). In vitro experiments showed that GTW of 40,80,120,200 and 500 μg/mL could significantly inhibit KGN cells proliferation (P<0.05). Compared with the GTW group,there was no significant change in cell viability,Ca2+concentration,GRP78,ATF6,CHOP,and Caspase-12 protein expression levels in the GTW+KBXQ group (P>0.05),while the cell viability was significantly increased (P<0.01),and the Ca2+concentration and protein expression levels of GRP78,ATF6,CHOP,and Caspase-12 were significantly decreased in the GTW+WJTXQ group (P<0.05,0.01). Conclusion Wenjing Decoction could significantly improve the ovarian reserve function of rats and has a good therapeutic effect on DOR,the mechanism might be related to regulating the ATF6/CHOP pathway and inhibiting ERS.
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