| Objective:(1)To detect the changes of serum metabonomics of compound stress erectile dysfunction(ED)rat models before and after the intervention of Yimusake tablets,and to find the serum metabonomic biomarkers and the possible targets of medicine;(2)Based on the validation of TMAO in serum,respectively detecte the expression of FXR1/2and FMO 3 in liver and penis tissues to further study the role of FXR/FMO 3/TMAO lipid metabolism pathway changes in ED rat model and the mechanism of medicine regulation.Methods:(1)150 normal mature male rats were selected in this study,30 of them were randomly chosed as the normal group(N group),the remaining 120 rats were used as model group,the compound stress ED rat model(60 rats)was established by environmental estrogen-like diet combined with cold stress intervention for 20 weeks,then screened into compound stress ED model group(ED group,30)and other rats of them as Yimusake tablet intervention group(Yimusake group,30).After 2 weeks of intervention,the changes of serum metabolites in each group were detected by~1H-NMR.(2)Based on the results of the first part of metabonomics screening,the changes of TMAO concentration in serum of rats in N group,ED group and Yimusake group were re-validated by ELISA method;The Changes of expression levels of FXR1/2 and FMO3in liver and penis tissues of rats were detected by Western-blot and immunohistochemistry.Results:(1)PLS-DA results showed that the samples of ED group,Y group and N group showed a trend of separation;R~2X,R~2Y and Q~2 of the model were 0.627,0.482 and 0.41,respectively.The results of OPLS-DA analysis in ED group and N group showed that there were significant differences between the two groups,which could effectively distinguish the differences between the two groups,and the R~2X,R~2Y and Q~2 of the two groups were 0.45,0.652 and 0.596,respectively.OPLS-DA analysis of Yimusake group and ED group showed that there were also significant differences between the two groups,R~2X,R~2Y and Q~2 were 0.524,0.609 and 0.485,respectively.(2)The serum levels of VLDL,leucine,choline,glutamine,acetone,valine,gamma-aminobutyric acid,pyruvate,glutamic acid,citric acid,creatine,taurine,methionine and aspartic acid in ED group were lower than those in N group,while the levels of lactic acid,alanine,acetoacetic acid,trimethylamine oxide,beta-glucose,guanylacetic acid and beta-furose in ED group were higher than those in N group(P<0.05).(3)The expression of lactic acid,alanine,trimethylammonium oxide,beta-glucose,guanidine acetic acid and beta-furose in serum of rats in group Yimusake was lower than that in ED group,(P<0.05).(4)Changes of TMAO concentration in serum of rats in each group:ELISA detection results showed that compared with group N,the concentration of TMAO in serum of rats in group ED increased by 33.83%with significant difference(P<0.05);compared with group ED,the level of TMAO in Yimusake group decreased by 15.03%,the difference was statistically significant(P<0.05).(5)Changes of FXR1,FXR2 and FMO3 in rat liver tissues:Western-blot results showed that FXR1 expression in the liver of rats in ED group increased by 65.38%(P<0.05)compared with that in N group,and decreased by 17.05%in Yimusake group(P<0.05).The expression of FXR2 in the liver of rats in ED group increased by 352.38%compared with that in N group(P<0.05),while that in Yimusake group decreased by 45.26%(P<0.05).Compared with group N,the expression of FMO3increased by 103.49%in the liver of rats in ED group(P<0.05),and decreased by 40.00%in Yimusake group(P<0.05).(6)Changes of FXR1,FXR2 and FMO3 in rat penis tissues:IHC results showed that the expression of FXR1 in penis tissues of rats in ED group increased by 172.46%compared with that in N group(P<0.05);decreased by 39.27%in Yimusake group compared with that in ED group(P<0.05).Compared with N group,the expression of FXR2 in penis tissues of rats in ED group increased by 64.76%(P<0.05),while that in Yimusake group decreased by 23.42%(P<0.05).Compared with N group,the expression of FMO3 in penis tissues of rats in ED group increased by 162.30%(P<0.05),and decreased by 52.78%in Yimusake group(P<0.05).Conclusion:(1)There were significant differences in serum metabolites between compound stress ED rat model and group N,21 metabolites such as VLDL,leucine,choline and trimethylammonium oxide could be the candidate serum metabolic markers for ED rat model,and 8metabolites such as lactic acid,alanine and trimethylammonium oxide could be the candidate serum metabolic markers for Yimusake tablets in the treatment of compound stress ED.(2)TMAO can be initially identified as a serum metabolic marker of ED rat model and a target for Yimusake treatment of ED.(3)Activation of FXR/FMO3/TMAO lipid metabolic pathway may play an important role in the development of compound stress ED rat model,Yimusake tablets may play a therapeutic role in ED by regulating FXR/FMO 3/TMAO lipid metabolic pathway. |
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