| Objective:To establish a model of rats with chronic stress premature ovarian failure(POF)and to intervene with a traditional Chinese medicine Munziq,to detect the changes of metabolites in the serum of rats for search for serum metabolic markers with chronic stress POF and study of the action target of Munziq.Methods:(1)90 female SD rats with normal sexual cycle were selected,10 of them were randomly selected as normal group(N Group)and 80 as chronic stress model group,The rats in the normal group were fed under normal environment(temperature T is 18-22?,humidity H is 55-65%),and the rats in the model group were modeled in a wet and cold environment(temperature T is 7?,humidity H is 85%).Observe and record the biological characteristics of all rats during modeling.After this model is established,the serum FSH,E2 and LH contents of all rats were detected.Then,according to the biological characteristics of the rats,the disorder of the estrous cycle and the changes of serum estrogen levels,the rats that have become premature ovarian failure are selected from this model group,were divided into stress POF model group(S-POF group)and stress POF drug intervention group(S-PTD group);Rats that had not developed POF fully were divided into stress model group(S group)and stress drug intervention group(STD group).After two weeks of drug gavage in the stress POF group and the stress drug intervention group,the levels of FSH,E2 and LH in peripheral blood of all the experimental rats were detected by ELISA.(2)Changes of metabolites in blood of rats in each group were detected using by ~1H-NMR technique.(3)The content of lactic acid in the serum of rats in each group was tested and verified by ELISA,immunohistochemistry was used to detect the expression of Smad2,TGF-TGF 1in the ovaries of rats in each group.Results:(1)Compared with N group,the content of E2 in the peripheral serum of rats in S-POF group and S group had decreased significantly(P<0.05),and the content of FSH and LH had increased significantly(P<0.05);After drug intervention,the hormone content of rats in each group could be regulated;(2)The content of?-ketoisovaleric acid,lactic acid,acetylaspartic acid,?-aminobutyric acid,lysine,tyrosine,choline,ascorbic acid and?-glucose in S-POF group were significantly lower than those in N group.The content of?-hydroxybutyric acid,pyruvate,glutamic acid,aspartic acid,guanidylacetic acid,creatine and phenylalanine had significantly increased,and the differences were statistically significant(P<0.05).(3)Compared with S-POF group,the content of lactate,acetylaspartic acid,lysine,choline,ascorbic acid and?-glucose in serum of S-PTD group increased significantly,while the content of?-hydroxybutyrate,pyruvate,glutamate and creatine decreased significantly(P<0.05).(4)The results of ELISA showed that the serum lactate content of S-POF group was significantly lower than that of N group(P<0.05),and the lactate content of S-PTD group was significantly higher than that of S-POF group(P<0.05).(5)The results of immunohistochemistry showed that TGF-?1 protein was mainly expressed in primary follicle oocytes,membrane cells and granulosa cells.The expression of TGF-?1 protein in ovarian tissue cells of S-POF group and S group was significantly lower than that of N group(P<0.05).After drug treatment,the expression of TGF-?1 protein in ovarian tissue cells of S-PTD group and STH group had increased significantly;Smad2 was highly expressed in luteal and atretic follicles,and low expressed in primordial and growing follicles,The expression of Smad2 protein in ovarian tissue cells of S-POF group and S group was significantly lower than that of N group(P<0.05).Conclusion:(1)There was a significant difference in serum metabolites content between the stress POF rat model and group N,among which 16 metabolites such as?-ketoisovaleric acid,lactic acid and acetylaspartic acid could be used as candidate serum metabolic markers in the stress POF rat model.Ten metabolites such as lactic acid,?-hydroxybutyric acid,pyruvate and glutamic acid can be used as serum candidate metabolic marker targets for the treatment of chronic stress premature ovarian failure with munizi.(2)Lactic acid can be preliminarily identified as a serum glucose metabolic marker in the stress POF rat model and as a serum metabolic marker in the treatment of the stress POF model with Munziq.(3)Abnormal changes in the lactate content of the metabolite may be through affecting the normal expression of TGF-?1 and Smad2 proteins,thereby playing an important role in the occurrence of chronic stress premature ovarian failure rat model,the Chinese medicine Munziq may be through repair of rat endocrine The disorder of the system regulates abnormal changes in the content of metabolites and affects the normal expression of ovarian function-related proteins,improving POF. |
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