| Background:With the changes of diet and lifestyle,hyperlipidemia has become an important public health matter.The long-term application of drugs for the treatment of hyperlipidemia will be along with varying probability and degrees of adverse reactions.Chinese medicine has been treating hyperlipidemia for more than 2000 years.In recent years,the lipid-lowering effect of Chinese herbal medicine has also received more and more attention.In the previous study,our group found that the lipid-lowering effect of the main effective components of Coptis chinensis and Fructus evodiae at 1:1 could significantly reduce the levels of plasma TG and LDL-C,reduce the steatosis of liver tissue in hyperlipidemic rats,and regulate lipid metabolism by acting on Leptin-AMPK pathway and Leptin-Jak2-Stat3 pathway.And it will affect the expression of SREBP2,HMGCR,PPAR ?,LXR ?,CYP7A1,NPC1L1,SR-BI,ABCG5,ABCG8 genes in order to inhibit cholesterol synthesis in liver,promote cholesterol decomposition,and inhibit cholesterol absorption in small intestine.Also,it can increase the diversity of intestinal flora,reduce the expression of harmful bacteria and increase the expression of beneficial bacteria and intestinal barrier repair bacteria in hyperlipidemic rats.Bile acid is the final product of cholesterol catabolism,and the conversion of cholesterol to bile acid is one of the main ways of human cholesterol excretion.It is essential to maintain cholesterol homeostasis and prevent the accumulation of cholesterol,triglycerides and toxic metabolites as well as damage to the liver and other organs.Therefore,in addition to the effect on the synthesis,absorption and decomposition of cholesterol,the effect of compatibility of Coptis chinensis and Evodia rutaecarpa on bile acid metabolism should also be shown.In order to further clarify the lipid-lowering mechanism of the compatibility of Coptis chinensis and Evodia rutaecarpa,we propose whether the compatibility of Fructus Evodia will affect this important process.Objective:To observe the effect of compatibility of Coptis chinensis and Evodia rutaecarpa on the expression of FXR/FGF15/FGFR4 related genes and proteins in bile acid metabolism pathway by real-time fluorescence quantitative PCR and Western blotting,and to observe the effect of compatibility of Coptis chinensis and Evodia rutaecarpa on bile acid metabolism by chromatography-mass spectrometry(GC-MS),using hyperlipidemia model rats as the research carrier and bile acid metabolism as the starting point,real-time fluorescence quantitative PCR and Western blotting were used to observe the effect of Coptis chinensis and Evodia rutaecarpa compatibility on bile acid metabolism pathway.To explore the mechanism of lipid-lowering effect of Coptis chinensis and Evodia rutaecarpaa on regulating bile acid metabolism.Methods: 1.Animal grouping and drug administration.Forty-eight male C57BL/6 rats were fed normally for 3 days.They were weighed and recorded.After fasting for 12 hours,the blood was collected from the tail vein of each rat for about 0.5ml,3500 rpm,and centrifuged 15 min to separate serum,and the content of serum total cholesterol in each group was detected.According to the value of serum total cholesterol and the weight,the rats were randomly divided into 6 groups: blank group(n = 8),model group(n = 8),atorvastatin group(n = 8),low dose group(n = 8),middle dose group(n = 8)and high dose group(n = 8).There was no difference in initial total cholesterol and weight among each group.The rats in the blank group were fed with normal diet,and the rats in other groups were fed with high-fat diet.All animals were fed and drank freely during the experiment.The decoction of traditional Chinese medicine was prepared and the corresponding drugs were given to the stomach according to the body weight of the animals in each group every day.The volume of the medicine was 1ml / 100 g.The rats in the blank group and model group were given normal saline once a day for 8 weeks.2.Detection of serum lipids and inflammatory factors.The contents of total cholesterol,triglyceride,low density lipoprotein cholesterol,high density lipoprotein cholesterol,interleukin 6,interleukin 10 and tumor necrosis factor ? in serum were determined by enzyme chemical method.The effects of Coptis chinensis and Evodia rutaecarpa on serum lipid and inflammatory factors in hyperlipidemic rats were observed.3.Pathological observation of liver.HE staining was used to observe the effect of Coptis chinensis and Evodia rutaecarpa compatibility on liver pathomorphology of hyperlipidemic rats.4.Detection of gene expression in FXR/FGF15/FGFR4 pathway.The effect of compatibility of Coptis chinensis and Evodia rutaecarpa on the expression of genes related to FXR/FGF15/FGFR4 pathway was detected by real-time fluorescence quantitative PCR.5.Detection of protein expression of FXR/FGF15/FGFR4 pathway.Western blotting was used to detect the effect of compatibility of Coptis chinensis and Evodia rutaecarpa on the expression of proteins related to FXR/FGF15/FGFR4 pathway.6.Detection of fecal bile acid metabolism profileChromatography-mass spectrometry was used to observe the effect of the compatiblity of Coptis chinensis and Evodia rutaecarpa on fecal bile acid metabolism in rats with hyperlipidemia.Results: 1.Effect of compatibility of Coptis chinensis and Evodia rutaecarpa on Blood Lipid levelin Hyperlipidemia Model rats.Compared with the blank group,the levels of serum low density lipoprotein cholesterol,triglyceride and total cholesterol in the model group increased significantly(P < 0.01),while the level of high density lipoprotein cholesterol decreased significantly(P < 0.01).Compared with the model group,the levels of low density lipoprotein cholesterol,triglyceride and total cholesterol in the low,middle and high dose groups and Atto vastatin group were lower than those in the model group,while the level of high density lipoprotein cholesterol in the model group was higher than that in the model group.The differences were statistically significant(P < 0.05).2.Effect of compatibility of Coptis chinensis and Evodia rutaecarpa on liver Index and Pathomorphology of Hyperlipidemia ratsFrom the liver index,the liver index of the model group was significantly higher than that of the blank group(P < 0.01),and the liver index of each treatment group was lower than that of the model group(P < 0.01).There was no difference among low,middle and high dose group(P > 0.05).From the pathological morphology of the liver,steatosis,fat vacuoles and inflammatory cell infiltration were found in the liver of the model group,and all the intervention groups could improve the above performances.The low,middle and high dose groups were better than the atorvastatin group.3.Effect of compatibility of Coptis chinensis and Evodia rutaecarpa on the level of inflammatory factors in hyperlipidemic rats.The levels of IL-6 and TNF-? in the model group were significantly higher than those in the blank group(P < 0.01),while the levels of IL-10 in the model group were significantly lower than those in the blank group(P < 0.01).Compared with the model group,the level of IL-6 in the low,middle and high dose groups was significantly lower than that in the model group(P < 0.05),and the level of IL-6 in the atorvastatin group was higher than that in the model group(P > 0.05).The level of tumor necrosis factor-? in each intervention group was significantly lower than that in the model group(P < 0.01).The level of IL-10 in the low and middle dose groups was significantly higher than that in the model group,the level of IL-10 in the high-dose group was higher than that in the model group,and the level of IL-10 in the Atto vastatin group was lower than that in the model group.4.Effect of compatibility of Coptis chinensis and Evodia rutaecarpa on target genes of FXR/FGF15/FGFR4 pathway in hyperlipidemic rats.Compared with the blank group,the gene expression levels of CYP7A1 and FXR in the liver of the model group were lower than those of the blank group,while the gene expression levels of FXR,FGF15 and FGFR4 in the ileum of the model group were significantly higher than those of the blank group.Atto vastatin group and low dose group of Cornus officinalis could down-regulate the expression of FXR gene in ileum.Atto vastatin group,low-dose group and high-dose group of Cornus officinalis could downregulate the expression of FGF15 gene in ileum.Low-dose group,middle-dose group and Atto vastatin group could all down-regulate the expression of FGFR4 gene in liver.Lowdose group and high-dose group of Cornus officinalis could up-regulate the expression level of CYP7A1 gene in liver(P < 0.05).Atto vastatin group,low-dose group and highdose group could up-regulate the expression level of FXR gene in liver(P < 0.05,P < 0.05,P < 0.01).5.Effect of compatibility of Coptis chinensis and Evodia rutaecarpa on target protein of FXR/FGF15/FGFR4 pathway in hyperlipidemic rats.Compared with the blank group,the expression levels of liver CYP7A1 and liver FXR in the model group were lower than those in the blank group,and the levels of ileal FXR,ileal FGF15 and liver FGFR4 in the model group were higher than those in the blank group(P < 0.01,P < 0.01,P < 0.05).Compared with the model group,the expression of FXR and FGF15 protein in ileum was down-regulated in all intervention groups(P < 0.01).The expression of FGFR4 protein in liver was down-regulated in the low-dose group,middledose group and the high-dose group(P < 0.05,P < 0.05).The expression of CYP7A1 and FXR in liver was up-regulated in the low-dose group and the high-dose group(P < 0.05).The low dose group,middle dose group and the high dose group of Cornus officinalis could up-regulate the expression of FXR protein in liver.6.Effect of compatibility of Coptis chinensis and Evodia rutaecarpa on fecal bile acid metabolic spectrum in hyperlipidemic rats.In terms of the classification of bile acids:(1)primary bile acid and secondary bile acid: the primary bile acid in the blank group accounted for 42.39% of the total bile acid spectrum in the feces,and the secondary bile acid accounted for 57.61% of the total bile acid spectrum in the feces.The primary bile acid in the model group accounted for 32.90% of the total bile acid spectrum in the feces,and the secondary bile acid accounted for 67.10% of the total bile acid spectrum in the feces.In the middle dose group,the primary bile acid accounted for 46.92% of the total bile acid spectrum of feces,and the secondary bile acid accounted for 53.08% of the total bile acid spectrum of feces.(2)Free bile acid and conjugated bile acid: the free bile acid in the blank group accounted for 97.39% of the total bile acid spectrum in the feces,and the conjugated bile acid accounted for 2.61% of the total bile acid spectrum in the feces.The free bile acid in the model group accounted for 98.34% of the total bile acid spectrum in the feces,and the conjugated bile acid accounted for 1.66% of the total bile acid spectrum in the feces.In the middle dose group,the primary bile acid accounted for 94.15% of the total bile acid spectrum of feces,and the conjugated bile acid accounted for 5.85% of the total bile acid spectrum of feces.In terms of differential bile acids,compared with the blank group,the expression of 12-KLCA,GDCA,HDCA,Apo CA,CDCA,DCA,Iso LCA and LCA in the feces of the model group was up-regulated(p < 0.05).The expression of TMCA,TUDCA,TCDCA and ?-MCA was down-regulated.Compared with the model group,the fecal bile acids 7-KDCA,UDCA,CA and GCA in the middle dose group were up-regulated,while 12-KLCA,Apo CA and Iso LCA were down-regulated.Conclusions: 1.The pathogenesis of hyperlipidemia in Traditional Chinese medicine is relatively complex.The main pathogenesis of hyperlipidemia is spleen dysfunction and abnormal ascending and descending.When treating it is necessary to lower the bitter and clear the turbid,and should take the principle of recovering the Qi mechanism of spleen and stomach.As the representative of acid opening and bitter downbearing,the compatibility of Coptis chinensis and Evodia retaecarpa has obvious lipid regulating and anti-inflammatory effects.2.To inhibit the pathway of FXR/FGF15/FGFR4,up-regulate CYP7A1 expression to promote the synthesis of bile acids,the compatibility of Coptis chinensis and Evodia retaecarpa can promote the metabolism of bile acid and play the role of lipid regulation and anti-inflammatory. |
PDF Full Text Request