Studies On The Anti-atherosclerosis Mechanism Of Ethanol Extract Of Usnea Based On Intestinal Flora

Objective To investigate the potential mechanisms of anti-atherosclerosis（AS）effect of the ethanol extract of Usnea based on intestinal flora.Methods A rat model of AS was established by long-term high-fat diet feeding combined with intraperitoneal injection of vitamin D₃.After successful establishment of the model,the low（0.7 g/kg/d）,medium（1.4 g/kg/d）and high（2.8 g/kg/d）doses of the ethanol extract of Usnea and the positive drug simvastatin（0.004 g/kg/d）were administered by gavage for 4 consecutive weeks.At the end of drug administration,blood was taken from the femoral artery,and aortic,liver,ileal and ileum contents were also collected for the detection of relevant indexes.Hematoxylin-Eosin（HE）staining was used to observe the pathological morphology of the aorta,liver and ileum of rats in each group;The content of plasma of serum total cholesterol（TC）and triglycerides（TG）,high-density lipoprotein cholesterol（HDL-C）,low-density lipoprotein cholesterol（LDL-C）,bile acids（BAs）,lipopolysaccharide（LPS）,tumor necrosis factor（TNF-α）,interleukin-6（IL-6）and trimethylamine-N-oxide（TMAO）of rats was detected by enzyme-linked immunosorbent assay（ELISA）;The protein expression of cholesterol 7αhydroxylase（CYP7A1）and flavin monooxygenase-3（FMO3）in the liver and ZO-1 and Occludin in the ileum were measured by Western Blot;16S r DNA amplicon high-throughput sequencing analysis was used to detect the ileal intestinal flora in each group of rats and investigate the changes of ileal intestinal flora in AS model rats and the regulation and amelioration of the intestinal flora structure by the ethanol extract of Usnea combined community composition analysis at different taxonomic levels with Alpha and Beta diversity analysis.Results 1.After the completion of modeling,four rats were randomly selected for pathological examination.The results of HE staining showed that the aortic mesothelium of the modeled rats was damaged,combined with a large number of foamy cells in the intima and obvious plaque deposition,which suggested the successful replication of the AS rat model.2.After 4 consecutive weeks of intervention treatment,the ethanol extract of Usnea can significantly shrink the plaque deposition in the aorta of model rats,reduce the content of TC,TG,and LDL in AS model rats（P<0.01 or P<0.05）and increase the level of HDL（P<0.01）.3.The ethanol extract of Usnea reduced the protein expression of FMO3（P<0.01or P<0.05）and decreased the content of TMAO（P<0.01 or P<0.05）,while upregulating the protein expression of CYP7A1（P<0.01 or P<0.05）and increased the content of BAs（P<0.01 or P<0.05）.4.The ethanol extract of Usnea can enhance the intestinal mucosal barrier function by up-regulating the protein expression of ZO-1 and Occludin,thus reducing the content of serum LPS,TNF-αand IL-6（P<0.01 or P<0.05）.5.Compared with the normal group,the diversity and uniformity of the ileum intestinal flora of AS model rats are significantly varied（P<0.01）,and the structure of the intestinal flora is disordered.The ethanol extract of Usnea could correct the intestinal flora of AS model rats to the normal level by reducing conditional pathogenic bacteria,increasing the relative abundance of beneficial bacteria（P<0.01 or P<0.05）and ameliorating the state of excessive abundance but the disorders of diversity and balance in AS model rats at the same time（P<0.01 or P<0.05）.Conclusion The ethanol extract of Usnea can significantly improve the plaque deposition in the aorta of AS model rats,reduce blood lipid levels,promote lipid metabolism,and significantly reduce the concentration of inflammatory factors,thus exerting therapeutic effect on the pathological changes of AS.The underlying mechanisms may be related to improving the disorder of intestinal flora,reducing conditional pathogenic bacteria and increasing the relative abundance of beneficial bacteria,thereby regulating the content of TMAO and BAs to promote lipid metabolism by the metabolic pathway and reducing the formation of foam cells,and enhancing the intestinal mucosal barrier function to reduce the absorption of intestinal-derived LPS and extenuate the inflammatory response by the non-metabolism pathway.This can provide more theoretical basis for Usnea to improve the mechanism of AS.