Effect Of Zuogui Pill On Premature Ovarian Insufficiency Induced By Cisplatin

OBJECTIVE:To explore the treating mechanism of Zuogui Pill for cisplatin-induced premature ovarian insufficiency from the perspective of apoptosis and autophagy.METHODS:1.As model group,human ovarian granulosa cell line COV434 was cultured in vitro and injured with 10 mg/L for 24 h.Normal control group,blank serum group,low-and high-dose Zuogui Pill serum containing group were set.Levels of estradiol（E₂）,follicle stimulating hormone receptor（FSHR）and luteinizing hormone receptor（LHR）of cell culturing supernatant were detected by enzyme-linked immuno sorbent assay（ELISA）.The mRNA expression of Bcl-2,Bax,Beclin-1 gene were detected by quantitative polymerase chain reaction（QPCR）.2.Female SD rats were intraperitoneally injected with 6 mg/kg cisplatin on the 1^stt and 8^thh day.4 rats were executed on both 5 and 10 day after the last administration of cisplatin,then the ovarian tissue was taken for pathology.Afterwards,the survival rats were randomly divided into model group and Zuogui Pill group.Normal young female SD rats were served as control.Pathological changes of rat ovarian tissue were observed by hematoxylin-eosin（HE）staining.Levels of peripheral blood E₂ and FSH detected by ELISA.The expression of Bcl-2,Bax,Beclin-1 and LC3II proteins in the ovary were detected by immunohistochemistry.RESULTS:1.Compared with the blank control group,the levels of FSHR and LHR in the model group decreased（P<0.01）,while the level of E₂ had no statistical significance（P>0.05）and varied in the group.Compared with the model group,levels of FSHR,and LHR increased（P<0.05,P<0.01）after the intervention of Zuogui Pill containing serum,E₂ level was also increased but had no statistical significance（P>0.05）.2.Compared with the normal control group,the expression of Bcl-2 mRNA was decreased in the model group（P<0.01）,and the expression of Bax and Beclin-1 mRNA were increased（P<0.05,P<0.01）.Compared with the model group,the expression of Bcl-2 mRNA was increased in the low-dose Zuogui Pill serum containing group（P<0.01）,and the expression of Beclin-1 mRNA decreased in both low-and high-dose group（P<0.01）.3.HE staining results showed large amounts of atretic follicles in the model group that at different stages;meanwhile,primordial,preantral and antral follicles were all seen in the Zuogui Pill group.4.In the animal experiment,the FSH levels of both model group and Zuogui Pill group were increased（P<0.05）,compared with the normal group.After the Zuogui Pill treatment,the FSH level was decreased and the E₂ level was increased but had no statistical significance（P>0.05）,compared with the model group.5.Immunohistochemistry results showed that compared with the normal group,the expression of Bcl-2 protein in the model group and Zuogui Pill group was significantly reduced;after the treatment of Zuogui Pill,the expression of Bcl-2 protein was increased compared with the model group.Compared with the normal group,the expression of Caspase-3,Beclin-1,and LC3II protein in the model group increased significantly;after the Zuogui Pill treatment,the protein expression was decreased compared with the model group and was similar to the normal group.CONCLUSIONS:1.Both apoptosis and autophagy are involved in the pathogenesis of POI.2.In vitro and in vivo studies show that Zuogui Pill can repair cisplatin-induced premature ovarian insufficiency to some extent by improving autophagy and apoptosis,whose interaction mechanism remains to be further studied.