Clinical Characteristics Of Gut Microbiota In Phlegm And Blood Stasis Syndrome And The Intervention Effect Of Compound SSXL On Fecal Microbiota Transplantation Mice

Background:With the acceleration of the pace of life in modern society,the living habits of residents such as diet,work and rest are becoming more and more irregular,and dyslipidemia occur frequently,which has become an important incentive for cardiovascular and cerebrovascular diseases.The existing lipid-lowering drugs are mainly western drugs,which have single curative effect and strong side effects,so it is difficult to achieve comprehensive treatment of the disease.There are common syndrome characteristics of Phlegm and Blood Stasis Syndrome in the process of dyslipidemia evolving into atherosclerosis combined with dyslipidemia.At present,the research on Phlegm and Blood Stasis Syndrome is lack of comprehensive representation and systematic cognition based on system biology.Shuangshen Xionglian granule（SSXL）,as a traditional chinese medicine compound that has obtained the clinical approval of new drugs,has definite pharmacological effects,such as regulating blood lipids,improving hemorheology,anti-platelet aggregation,anti-thrombosis,anti-inflammatory,and improving the characterization of Phlegm and Blood Stasis Syndrome.Further study on the effective mechanism of SSXL in the intervention of dyslipidemia through intestinal pathway can provide new ideas and methods for TCM in the prevention and treatment of dyslipidemia syndrome of Phlegm and Blood Stasis through intestinal microbiological pathway.Objective:To study the characteristics of intestinal microecology,clinical serological indicators and Phlegm and Blood Stasis Syndrome in healthy subjects（NOR group）,patients with dyslipidemia（DYS group）and patients with atherosclerosis complicated with dyslipidemia（AS group）.The differences in the characteristics of the disease evolution process were analyzed from the overall level of animal experiments to explore the mechanism of SSXL intervening in dyslipidemia from the intestinal pathway,and to explore new targets for traditional Chinese medicine to treat dyslipidemia from the intestinal flora.Methods:Using 16S rDNA high-throughput sequencing and metabolomics liquid chromatography-mass spectrometry（LC-MS）technology to analyze and study the differential characteristics of intestinal flora in patients with dyslipidemia,atherosclerosis complicated with dyslipidemia and healthy people in clinical practice,using flow cytometry to detect changes in monocyte subsets,to clarify the relationship between intestinal flora-fecal metabolites-inflammatory markers-monocyte subsets-dyslipidemia-AS.The fecal samples of patients with clinical typical atherosclerosis complicated with dyslipidemia were selected and transplanted into germ-free C57BL/6J mice by gavage,and fed with high-fat diet for 3 months to establish a model of dyslipidemia with phlegm and blood stasis.To observe the improvement effect of SSXL on dyslipidemia related indicators of Phlegm and Blood Stasis Syndrome and intestinal microecology after intervention,and further study the biological mechanism of SSXL’s effective effect on dyslipidemia through intestinal pathway.Results:In the first part of the experiment,we observed the following results.①The clinical manifestations of dyslipidemia and atherosclerosis combined with dyslipidemia were:systolic blood pressure,Phlegm and Blood Stasis Syndrome scores were significantly higher than those of the normal population;The elasticity of the arterial wall is weakened,the distensibility is reduced,the speed and acceleration of the active expansion of the arterial wall are reduced,and the elasticity of the carotid artery gradually decreases;serum biochemical indicators show that the levels of total cholesterol,LDL cholesterol,and apolipoprotein B are increased and increased.Density lipoprotein cholesterol decreased,accompanied by coagulation dysfunction,and the levels of MCP-1,M-CSF and other inflammatory factors increased;with the aggravation of dyslipidemia,the proportion of intermediate monocyte subsets gradually increased,and non-classical monocytes gradually increased.The proportion of monocyte subsets gradually decreased.For every 1 mmol/L increase of TG,the possibility of aggravation of Phlegm and Blood Stasis Syndrome is 4.402 times higher than the original;The possibility of aggravation of LDL-C was 3-7 times higher than that of the original;For every 1 mm2/kPa increase in AC,the possibility of aggravation is 0.074 times higher than the original;For every 1 percentage point increase in FMD,the possibility of aggravation is 0.825 times higher than the original.②Atherosclerosis combined with dyslipidemia leads to a gradual increase in the F（Firmicutes）/B（Bacteroidetes）value.Further analysis found that Firmicutes and very low density lipoprotein cholesterol（VLDL-C）,triglycerides（TG）,total cholesterol（TC）,apolipoprotein-b（ApoB）and carotid artery thickness（IMT）were positively correlated,indicating that the increase in Firmicutes in the gut is the cause of atherosclerosis and dyslipidemia-related biomarkers increase s reason.The clinical scores of Phlegm and Blood Stasis Syndrome were significantly positively correlated with Blautia,Bifidobacterium,Parabacteroides,Lachnoclostridium,Prevotella₉ and Subdoligranulum.Among them,Bifidobacterium was positively correlated with vascular elasticity（EP）and lipoprotein a,Subdoligranulum was positively correlated with VLDL-C,and Prevotella₉ was positively correlated with TC,indicating that phlegm stasis the clinical score and related clinical indicators of mutual association were affected by the above-mentioned gut microbiota.③ Fecal metabolomic analysis showed that bile acid metabolism pathway was closely related to atherosclerosis complicated with dyslipidemia.In the second part of the experiment,we found that SSXL can significantly reduce serum TC,TG,LDL-C,body weight,inflammatory cytokines MCP-1,M-CSF,TNF-α in germ-free mice model of dyslipidemia with Phlegm and Blood Stasis Syndrome,monocyte subset Ly6C level,increase serum HDL-C level,improve liver pathological morphology,and play a role in significantly improving the disease degree of the germ-free mouse model of dyslipidemia with Phlegm and Blood Stasis Syndrome.In the third part of the experiment,we obtained the following results:① Dyslipidemia was associated with phlegm and blood stasis and caused disturbance of the intestinal microenvironment in germ-free mice.AS-FMT led to the intestinal flora Firmicutes(Firmicutes/Bacteroidetes ratio imbalance,high-fat diet led to high levels of Desulfobacterota and Desulfovibrionaceae relative abundance in the gut microbiota of mice.②The content of fecal metabolites in AS-FMT mice and SSXL-treated mice was determined.Significant differences were found among 135 compounds.11%of the metabolites in the AH group were significantly enriched,20%of the metabolites in the AHS group were significantly enriched,and 37%of the metabolites in the AH group were depleted compared to the NH group.③SSXL can reshape the structure of intestinal flora,which may be related to ovarian steroidogenesis and arachidonic acid metabolism.Conclusions:①The clinical manifestations of dyslipidemia and atherosclerosis combined with dyslipidemia are:systolic blood pressure and the score of Phlegm and Blood Stasis Syndrome are significantly higher than those of the normal population;blood lipid levels of TC,LDL-C,and ApoB are increased and HDL-C levels are decreased.Accompanied by coagulation dysfunction,the levels of MCP-1,M-CSF and other inflammatory factors increase;the F（Firmicutes）/B（Bacteroidetes）value of intestinal flora gradually increases,and the increase of Firmicutes in the intestinal tract is the cause of atherosclerosis.Causes of elevated sclerosis-like sclerosis complicated with dyslipidemia-related biomarkers.②TG、TC、LDL-C、ApoA1、Ep、AI、PWV β、and β were a low positive correlation between IMT and the score of Phlegm and Blood Stasis Syndrome.There was a moderate positive correlation between ApoB and the score.There was a low negative correlation between AC and the score.The clinical score of Phlegm and Blood Stasis Syndrome was significantly positively correlated with Blautia,Bifidobacterium,Parabacteroides,Lachnoclostridium,Prevotella₉ and Subdoligranulum.Among them,Bifidobacterium was positively correlated with vascular elasticity（EP）and lipoprotein a,Subdoligranulum was positively correlated with very low density lipoprotein cholesterol VLDL-C,and Prevotella₉ was positively correlated with TC,indicating that phlegm stasis mutual association is influenced by the above-mentioned intestinal flora.③SSXL can play an improving effect on dyslipidemia by promoting the balance of intestinal flora and regulating bile acid metabolism.