Study On Establishment Of Chronic Stress Induced Premature Ovarian Failure Rat Model And Regulation Of Drug Treatment

Objective: To explore the morphological changes of gonadal axis and biological basis of chronic stress(CS)induced premature ovarian failure(POF)disease and pharmacological effects of Balgham Munziq(BMq)treatment.Methods: 90 mature Wistar female rats were selected,took 10 of them as normal randomly,rest of them were used to make chronic stress model in humid and cold environment for 23 weeks,quantitative and qualitative biological changes were observed during the modeling period.After chronic stress model was entablished,rats which were diagnosed with POF were subdivided into POF disease group(P),high(PTH),middle(PTM)and low doses of BMq treatment group(PTL);The rest of the rats which were not diagnosed with POF were subdivide into chronic stress group(S),high(STH),middle(STM)and low doses of BMq treatment group(STL).(1)Serum concentration levels of AMH,C3 and VEGF were measured by ELISA;plasma concentration levels of neurotransmitters such as DA,5-HT and EP were detected by UPLC-MS/MS.(2)Histological changes: pathologic changes of gonad axis(Hypothalamus-pituitary-ovary)were observed through HE staining;ultrastructures of ovarian sections were observed through TEM;Area of fibrosis were observed and calculated through Masson staining.(3)Expressions of C3 and CYP51 in ovary tissue were detected by immunohistochemical technique.Results:(1)Biological features: the activity of chronic stress model group rats were significantly reduced and showed tireness during the modeling period.The increment in body weight and water intake of chronic stress model group rats were significantly lower than normal,meanwhile,feed intake,urine volume and stool quantity were significantly higher than normal(P<0.05),the quantitive and qualitative biological features were improved in the different degree during the BMq treatment period.(2)The serum levels of AMH and VEGF in P and S groups were significantly lower than those in normal group(P<0.05),meanwhile,serum levels of C3 was significantly higher than normal group(P<0.05).Hormone levels of each group rats were adjusted by BMq treatment in different degrees.The plasma levels of DA in P group and S group were significantly higher than that in normal group(P<0.05),meanwhile,the plasma levels of DA in corresponding model groups were significantly reduced in PTH group and STH group(P<0.05);plasma levels of 5-HT and EP have showed increasement trend,but no significant difference compared with normal group(P>0.05).(3)Histopathological changes: P group and S group rats were showed interstitial edema in different degrees,besides,neural cells were necrosed and emerged karyopyknosis in hypothalamus through HE staining,which can be improved by different doses of BMq treatment in each group in the different degrees;Pituitary: P group and S group showed interstitial vacuolar changes and severe edema,basophils were increased,pathological changes mentioned above were improved by different doses of BMq treatment in each group in the different degrees;Ovary: P group and S group rats showed no obvious antral follicles,vacuolar changes,severe interstitial edema and associated with fibrosis,pathological changes mentioned above were improved by different doses of BMq treatment in each group in the different degrees.The results of ovarian electron microscopy showed that the P and S groups had obvious cell membrane lysis,matrix lysis,nucleus lysis,loss of basic morphological structure and multi-nucleus in one cell.Pathological changes mentioned above were improved by different doses of BMq treatment in each group in the different degrees.Results of Masson staining showed that the degree of ovarian fibrosis in P and S groups was significantly higher than that in normal group(P<0.05);The degree of fibrosis in model groups were significantly reduced in PTH,PTM and STH group(P<0.05).(4)The results of immunohistochemistry showed that C3 was mainly expressed in granular layer cells and follicular fluid in each group,and the expressions of C3 in P and S group were significantly higher than that in normal group(P<0.05),high expressions of C3 can be improved by different doses of BMq treatment.The expressions of CYP51 in the interstitial and corpus luteum in each group were significantly lower than that in the normal group(P<0.05),and the levels of CYP51 in the P and S groups were significantly lower than normal group,low expressions of CYP51 can be improved by different doses of BMq treatment.Conclusions: Long-term chronic stress can lead to the occurrence of POF.The histopathological changes of gonadal axis,the decline of ovarian storage function and the occurrence of endocrine disorders may be one of the important mechanisms of its pathogenesis.Balgham Munziq can relieve POF to a certain extent,the mechanism of its drug action may be related to the adjustment of endocrine disorders and pathological changes of gonadal axis.