Study On The Effect Of Electroacupuncture On Atherosclerosis Based On Intestinal Flora Regulating Bile Acid Metabolis

Purpose:The atherosclerotic mouse model was prepared by high-fat diet induction method.Taking the intestinal flora and bile acid metabolism as the entry point,electroacupuncture was used to intervene the atherosclerotic mice,observe the effect of electroacupuncture on the distribution of intestinal flora and bile acid metabolism of atherosclerotic mice,and explore the effect and possible mechanism of electroacupuncture on improving atherosclerosis,In order to provide experimental basis for electroacupuncture treatment of atherosclerosis.Materials and methods:1.Modeling and intervention:25SPF grade 6-week-old male Apo E^-/-mice were fed with5g/high-fat diet for 14 weeks after adaptive feeding for 1 week,and 9 SPF grade 6-week-old male C57BL/6J mice were fed with 5g/ordinary diet for 14 weeks after adaptive feeding for 1week.After modeling,1 Apo E^-/-mouse and 1 C57BL/6J mouse were randomly selected and killed.The formation of aortic plaque in mice was observed by HE staining to judge the modeling situation.After successful modeling,the remaining 24 Apo E^-/-mice were randomly divided into model control group,positive drug control group（AS model+simvastatin suspension gavage）and acupuncture treatment group（AS model+electroacupuncture treatment）.8 mice in each group were fed with 5g/mouse daily.The remaining 8 6-week-old C57BL/6J male SPF mice were used as normal control group,and 5g/mouse daily was fed with ordinary diet at the same time.From the 16th week,the positive drug control group was treated with simvastatin suspension by gavage once a day,and fixed on the acupuncture treatment fixator for 10 minutes every other day for 11 weeks;The acupuncture treatment group was treated with Neiguan point,Xinshu point and Geshu point,and was treated with Zhongyan Taihe acupuncture needle（0.16mm×7mm）,after local disinfection,connect the electroacupuncture instrument,use the density wave of 2/15Hz,the current of 0.3m A,and the stimulation intensity should be the slight vibration at the acupuncture site consistent with the frequency of electroacupuncture.Keep the needle for 10 minutes,treat once every other day,and gavage the same volume of distilled water as the positive drug control group every day for 11 weeks;The normal control group and the model control group were not intervened.They were given distilled water of the same volume as the positive drug control group every day and fixed on the acupuncture treatment fixator for 10 minutes every other day.2.Index detection:He staining was used to observe the formation of aortic plaque in mice.The levels of TC,TG,HDL-C and LDL-C in mice were detected by enzyme-linked immunosorbent assay.The distribution of intestinal flora was detected by 16S r RNA amplicon sequencing.The expressions of ABCG5,ABCG8,FGFR4 and CYP7A1 in liver and the expressions of ABCG5,ABCG8,SREBP2,NPC1L1,FXR and FGF15 in intestinal tissue were detected by Western blot.The expressions of ABCG5,ABCG8,FGFR4 and CYP7A1m RNA in liver tissue and the expressions of ABCG5,ABCG8,SREBP2,NPC1L1,FXR and FGF15 m RNA in intestinal tissue were detected by fluorescence quantitative PCR.Results:1.He staining resultsModel evaluation:After modeling,the aorta was observed under electron microscope.The tissues of each layer of the aorta of C57BL/6J mice were normal,the intima of the aorta was smooth and complete,and no plaque and foam cells were found;The aortic intima of Apo E^-/-mice is significantly thickened,and a large number of foam cells can be seen under the arterial intima.Plaque formation under the intima can be observed,suggesting that the atherosclerosis model is successfully established.Treatment outcome:After the intervention,compared with the normal control group,the arterial intima of the model control group was significantly thickened,a large number of foam cells were observed under the arterial intima,and the formation of atherosclerotic plaque was observed under the arterial intima.After treatment,compared with the model control group,the aortic intima of the positive drug control group and the electroacupuncture treatment group became thinner,a small amount of foam cells accumulated under the intima,and the area of atherosclerotic plaque under the intima decreased significantly.2.Expression levels of blood lipid and cholesterol metabolism related proteinsBlood lipid expression level:After the intervention,compared with the normal control group,the levels of serum TC,TG and LDL-C in the model control group increased significantly（P<0.01）,and the level of serum HDL-C in the model control group decreased significantly（P<0.01）.After treatment,compared with the model control group,the levels of serum TC,TG and LDL-C in the electroacupuncture treatment group and the positive drug control group decreased significantly（P<0.01）,and the levels of serum HDL-C in the electroacupuncture treatment group and the positive drug control group increased significantly（P<0.01）.Expression level of cholesterol metabolism related proteins in liver:After the intervention,compared with the normal control group,the expression of ABCG5 protein in the liver tissue of the model control group decreased significantly（P<0.01）,and the expression of ABCG8 protein in the liver tissue of the model control group decreased significantly（P<0.01）.After treatment,compared with the model control group,the expression of ABCG5 protein in liver tissue of mice in electroacupuncture treatment group and positive drug control group increased significantly（P<0.01）,and the expression of ABCG8 protein in liver tissue of mice in electroacupuncture treatment group and positive drug control group increased significantly（P<0.01）.Expression level of cholesterol metabolism related protein m RNA in liver tissue:After the intervention,compared with the normal control group,the expression of ABCG5 m RNA in the liver tissue of model control group decreased significantly（P<0.01）,and the expression of ABCG8 m RNA in the liver tissue of model control group decreased significantly（P<0.01）.After treatment,compared with the model control group,the expression of ABCG5 m RNA in liver tissue of mice in electroacupuncture treatment group and positive drug control group increased significantly（P<0.01）,and the expression of ABCG8 m RNA in liver tissue of mice in electroacupuncture treatment group and positive drug control group increased significantly（P<0.01）.Expression level of cholesterol metabolism related proteins in intestinal tissue:After the intervention,compared with the normal control group,the expression of ABCG5 protein in the intestinal tissue of model control group decreased significantly（P<0.01）,the expression of ABCG8 protein in the intestinal tissue of model control group decreased significantly（P<0.01）,the expression of SREBP2 protein in the intestinal tissue of model control group increased significantly（P<0.01）,and the expression of NPC1L1 protein in the intestinal tissue of model control group increased significantly（P<0.01）.After treatment,compared with the model control group,the expression of ABCG5 protein in intestinal tissue of mice in electroacupuncture treatment group and positive drug control group increased significantly（P<0.01）,the expression of ABCG8 protein in intestinal tissue of mice in electroacupuncture treatment group and positive drug control group increased significantly（P<0.01）,the expression of SREBP2 protein in intestinal tissue of mice in electroacupuncture treatment group and positive drug control group decreased significantly（P<0.01）,and the expression of NPC1L1 protein in intestinal tissue of mice in electroacupuncture treatment group and positive drug control group decreased significantly（P<0.01）.Expression level of cholesterol metabolism related protein m RNA in intestinal tissue:After the intervention,compared with the normal control group,the expression of ABCG5m RNA in the intestinal tissue of the model control group decreased significantly（P<0.01）,the expression of ABCG8 m RNA in the intestinal tissue of the model control group decreased significantly（P<0.01）,the expression of SREBP2 m RNA in the intestinal tissue of the model control group increased significantly（P<0.01）,and the expression of NPC1L1 m RNA in the intestinal tissue of the model control group increased significantly（P<0.01）.After treatment,compared with the model control group,the expression of ABCG5 m RNA in intestinal tissue of mice in electroacupuncture treatment group and positive drug control group increased significantly（P<0.01）,the expression of ABCG8 m RNA in intestinal tissue of mice in electroacupuncture treatment group and positive drug control group increased significantly（P<0.01）,the expression of SREBP2 m RNA in intestinal tissue of mice in electroacupuncture treatment group and positive drug control group decreased significantly（P<0.01）,and the expression of NPC1L1 m RNA in intestinal tissue of mice in electroacupuncture treatment group and positive drug control group decreased significantly（P<0.01）.3.Expression of relative abundance at the level of intestinal floraAfter the intervention,compared with the normal control group,the relative abundance of Bacteroidetes in the model control group decreased significantly（P<0.01）,and the relative abundance of Firmicutes in the model control group increased significantly（P<0.01）.After treatment,compared with the model control group,the relative abundance of Bacteroidetes in the positive drug control group and electroacupuncture treatment group increased significantly（P<0.01）,and the relative abundance of Firmicutes in the electroacupuncture treatment group and positive drug control group decreased significantly（P<0.01）.4.Expression of target genes and proteins in FXR/FGF15/FGFR4 pathwayExpression of FXR/FGF15/FGFR4 pathway target protein:After the intervention,compared with the normal control group,the expression of FXR protein in the intestinal tissue of model control group increased significantly（P<0.01）,the expression of FGF15 protein in the intestinal tissue of model control group increased significantly（P<0.01）,the expression of FGFR4 protein in the liver tissue of model control group increased significantly（P<0.01）,and the expression of CYP7A1 protein in the liver tissue of model control group decreased significantly（P<0.01）.After treatment,compared with the model control group,the expression of FXR protein in intestinal tissue of mice in positive drug control group and electroacupuncture treatment group decreased significantly（P<0.01）,the expression of FGF15 protein in intestinal tissue of mice in positive drug control group and electroacupuncture treatment group decreased significantly（P<0.01）,the expression of FGFR4 protein in liver tissue of mice in positive drug control group and electroacupuncture treatment group decreased significantly（P<0.01）,and the expression of CYP7A1 protein in liver tissue of mice in positive drug control group and electroacupuncture treatment group increased significantly（P<0.01）.Expression of FXR/FGF15/FGFR4 pathway target genes:After the intervention,compared with the normal control group,the expression of FXR m RNA in the intestinal tissue of the model control group increased significantly（P<0.01）,the expression of FGF15m RNA in the intestinal tissue of the model control group increased significantly（P<0.01）,the expression of FGFR4 m RNA in the liver tissue of the model control group increased significantly（P<0.01）,and the expression of CYP7A1 m RNA in the liver tissue of the model control group decreased significantly（P<0.01）.After treatment,compared with the model control group,the expression of FXR m RNA in intestinal tissue of mice in positive drug control group and electroacupuncture treatment group decreased significantly（P<0.01）,the expression of FGF15 m RNA in intestinal tissue of mice in positive drug control group and electroacupuncture treatment group decreased significantly（P<0.01）,the expression of FGFR4 m RNA in liver tissue of mice in positive drug control group and electroacupuncture treatment group decreased significantly（P<0.01）,and the expression of CYP7A1 m RNA in liver tissue of mice in positive drug control group and electroacupuncture treatment group increased significantly（P<0.01）.Conclusions:1.Electroacupuncture treatment can effectively inhibit the damage of arterial intima,the formation of foam cells and atherosclerotic plaque in atherosclerotic mice.2.Electroacupuncture treatment can effectively improve the dyslipidemia of atherosclerotic mice.3.Electroacupuncture can effectively regulate the expression level of cholesterol metabolism related proteins in atherosclerotic mice.4.Electroacupuncture can effectively regulate the relative abundance of Bacteroidetes and Firmicutes in atherosclerotic mice and improve the intestinal flora.5.Electroacupuncture treatment can improve atherosclerosis by correcting the metabolic disorder of intestinal flora in atherosclerotic mice,down regulating the expression of bile acid receptor FXR,inhibiting FGF15 activity,affecting its binding with FGFR4,inhibiting FXR/FGF15/FGFR4 pathway,promoting the expression of CYP7A1 and increasing bile acid secretion.