Study On The Effect Of Improving Ovarian Function By Bushen-Huoxue Formula Enema On The Model Of Deficiency Of The Kidney And Blood Stasis Combined With Diminished-ovarian Reserve In Rats Based On Lipid Metabolomics

Objective:To study on the effect of improving ovarian function by Bushen-Huoxue Formula enema on the model of deficiency of the kidney and blood stasis combined with diminished-ovarian reserve in rats from metabolomics level based on Ultra High Performance Liquid Tandem Chromatography Quadrupole Time of Flight Mass Spectrometry（UHPLC-QTOFMS）.METHODS:Twenty-four SPF-grade non-mating adult female SD rats were selected,randomly divided into enema group（A group）,DHEA group（B group）,blank group（C group）and model group（D group）,Six rats in each group.For the model group and the enema group,the western medicine group were given tripterygium wilfordii polyglycoside suspension 50 mg/kg·d for 14 days,on the 4th day,adrenaline was injected intradermally for 0.3 mg/kg·d for 7 days,establishment of kidney deficiency,blood stasis and decline in ovarian reserve function（DOR）models.Blank group was given 0.9%sodium chloride injection for 5 ml/kg for 14days,on the 4th day,0.9%sodium chloride injection was injected subcutaneously for0.5 ml/kg for 7 days.Behavioral changes and weight（g）changes were recorded during mold molding.After successful modeling,each group was given corresponding drugs or saline for 10 days,and behavioral changes and body weight（g）were recorded during administration.After the intervention,observe the general condition of each group of rats,wet weight of ovaries,ovarian organ index.To observe morphological changes of ovarian tissue by hematoxylin-eosin staining（HE）.TO test anti-müllerian hormone（AMH）,estradiol（E₂）and follicle stimulating hormone（FSH）by enzyme-linked immunosorbent assay（ELISA）.UHPLC-QTOFMS technique performed quantitative testing of ovarian tissue in rats.By principal component analysis（PCA）and orthogonal projections to latent structures-discriminant analysis（OPLS-DA）to look for differential metabolites.Results:（1）Compared with the blank group,the model group had poor general condition,decreased ovarian wet weight and organ index（P<0.05）,decreased number of normal follicles at all levels,increased atresia follicles,decreased AMH、E2 value（P<0.05）and increased FSH value（P<0.05）,indicating the success of the kidney deficiency blood stasis-DOR model;（2）After drug intervention,the general conditions of rats in the guide group and western medicine group were obviously improved,the wet weight and organ index of ovaries were increased（P<0.05）,the number of normal follicles was increased,the atresia follicles were decreased,the AMH、E2 value was increased（P<0.05）,and the FSH value decreased（P<0.05）,indicating that Bushen-Huoxue Formula and dehydroepiandrosterone could improve ovarian function;（3）Analysis of rats in each group by principal component analysis（PCA）,most of the samples are distributed within the 95%confidence interval of the scatter plot,quality control（QC）there is no obvious separation of sample overlap.Analysis of inter-group relationship models by Orthogonal projections to latent structures-discriminant analysis（OPLS-DA）,R²X>0.50 and 0.50 R²Y>0.50,nor did the permutation test find overfitting.The results showed that the metabolomics model was reliable and reproducible;（4）A total of 10 types of lipid metabolites were selected by rat ovarian lipid metabolomics:Cholesterol Esters（CE）,Diacylglycerols（DAG）,Hexosylceramides（HCER）,Lysophosphatidylcholines（LPC）,Lysophosphatidylcholines（LPC）,Phosphatidylethanolamines（PE）,Sphingomyelins（SM）,Ceramides（CER）,Free Fatty Acids（FFA）and Triacylglycerols（TAG）as a DOR lipid biomarker;（5）After the intervention of tonifying the kidney and activating blood circulation,Relative increase in CE and DAG content（VIP>1,P<0.05）,Relative decline in TAG and FFA（VIP>1,P<0.05）,Conclusion Bushen Huoxue can improve CE、DAG level,Downregulation of TAG、FFA levels to improve ovarian function.Conclusion:（1）Tripterygium wilfordii polyglycoside tablets combined with epinephrine can successfully replicate the DOR model of kidney deficiency and blood stasis;（2）Bushen-Huoxue Formula enema on rats can improve the general condition,body weight,wet weight of ovarian and ovarian organ index of model rats.And it can improve reproductive endocrine level in rats;（3）A total of 78 lipid metabolites were screened.The decline in ovarian function can cause disorders of lipid synthesis and metabolism,CE、DAG、HCER、LPC、PC、PE、SM、CER and TAG can be used as a DOR lipid biomarker.Bushen-Huoxue Formula can correct abnormal lipid metabolism and improve ovarian function,by raising CE and DAG levels,lowering TAG and FFA levels.