Exploration of the Mechanism of Jianpi Huazhuo Tiaozhi Recipe in Treating Atherosclerosis Based on the Gut Microbiota Driving TMA/FMO3/TMAO Pathway

HUANG Langlang; LIU Zhongyong; LIU Yanwei

Abstract：Objective To explore the possible mechanism of Jianpi Huazhuo Tiaozhi recipe in anti-atherosclerosis(AS).Methods Animal model of AS was established by high choline diet.Sixty ApoE-/-mice were randomly divided into ApoE-/-control group,model group,antibiotic group and low,middle and high dose groups of Jianpi Huazhuo Tiaozhi recipe,with 10 mice in each group.Another 20 C57BL/6J mice were randomly divided into blank control group and choline control group.The blank control group and ApoE-/-control group were given the same volume of 0.9%NaCl solution,and the other groups were given 1%choline chloride.After 16 weeks of modeling,the pathological changes of mouse aorta were observed by HE staining,the blood lipid level was detected by automatic biochemical analyzer,the expression of CRP and IL-6 in the aorta was detected by immunohistochemistry,and the gut microbiota of cecum contents was detected by 16S rRNA sequencing.LC/MS was used to measure the levels of TMA and TMAO in the serum.Western blot was used to detect the expression of FMO3 protein in the liver.RT-qPCR was used to detect changes in hepatic FMO3 mRNA.Results The endothelium was smooth and intact in the blank control group,and thickened in the choline control and ApoE-/-control groups.Compared with the model group,the antibiotic group and the Jianpi Huazhu Tiaozhi recipe groups of various dosages showed a reduction in the degree of plaque lipid deposition,significant improvement in blood lipid levels,and the levels of aortic CRP,IL-6,and serum TMA,TMAO,as well as liver FMO3 mRNA were obviously decreased(P<0.05).Compared with the model group,the antibiotic group and the medium and high dose groups of the Jianpi Huazhu Tiaozhi recipe showed a significant reduction in the liver FMO3 protein levels(P<0.01).The results of gut microbiota sequencing showed that the abundance and diversity of gut microbiota(Shannon,ACE,Chao1 index)were significantly lower in the antibiotic group compared with the model group,and the abundance and diversity of gut microbiota were significantly higher after the intervention of various dosages of Jianpi Huazhu Tiaozhi recipe(P<0.05).At the phylum level of the microbiota,compared with the model group,after intervention with various doses of the Jianpi Huazhu Tiaozhi recipe,the proportion of Firmicutes and Proteobacteria significantly decreased,while the proportion of Bacteroidetes significantly increased.At the genus level of the microbiota,after the intervention with various doses of the Jianpi Huazhu Tiaozhi recipe,the proportion of Akkermansia and Lactobacillus significantly increased,while the proportion of Staphylococcus significantly decreased.Conclusion Jianpi Huazhu Tiaozhi recipe may delay the onset and progression of AS by regulating the structure of the gut microbiota,inhibiting the TMA/FMO3/TMAO pathway,improving serum lipid levels,and reducing inflammatory responses.