Study On The Mechanism Of The Anti-atherogenic Effect Of Enterobacter Aerogenes ZDY01 Based On The FXR-FGF15 Axis

Objective:Atherosclerosis is the leading cause of cardiovascular diseases worldwide.Trimethylamine N-oxide（TMAO）,a metabolite of intestinal flora,has been shown to be closely related to the development of atherosclerosis.Previous studies have shown that Enterobacter aerogenes ZDY01 significantly reduces the serum concentration of TMAO and cecal trimethylamine（TMA）in Balb/c mice;however,its role in the inhibition of TMAO-induced atherosclerosis in ApoE^-/-mice remains unclear.Methods:1.Animals and groups:female（six to eight-weeks old）ApoE^-/-mice were randomly divided into the following four groups:Chow group,Choline+PBS group,Choline+ZDY01 group and Choline+ZDY08 group,with 10 mice in each group.2.Model and administration:mice received a chow diet containing 1.3%choline chloride to established atherosclerosis model and treated with sterile PBS or strain containing 15%glycerol for 16 weeks.3.Detection:Atherosclerotic lesion formation in whole aorta was detected by Oil red O staining.Hematoxylin-eosin,Oil red O,and Masson staining were used to detect the atherosclerotic area,lipid deposition and the collagen content in the aortic roots.Immunofluorescence staining was used to detect macrophage（CD68 positive）and smooth muscle cell（α-SMA positive）in cross-sections of the aortic root.Serum lipids were measured using the analysis kits.The serum TMAO,feces TMA and 7 bile acids contents were analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry（UHPLC/MS/MS）.The changes of mice intestinal microbial diversity in each group was detected by 16S rDNA high-throughput sequencing technology.The genes related to reverse cholesterol transport,related to bile acid metabolism in small intestine and Fmo3 transcription and translation levels in liver were detected by RT-qPCR and Western blot.The contents of total bile acid in serum and feces was measured using an analysis kit.Results:Our results show that:1.Compared with Choline+PBS group,the atherosclerotic lesions（P<0.001）in the entire aorta was significantly decreased in Choline+ZDY01 group.The plaque area（P<0.05）,lipid deposition（P<0.05）and macrophage content（P<0.01）were markedly decreased after treatment with E.aerogenes ZDY01 compared with Choline+PBS group,but not lesion smooth muscle cell or collagen content.2.Compared with Choline+PBS group,the contents of cecum TMA（P<0.01）and serum TMAO（P<0.05）were significantly decreased in Choline+ZDY01 group,but not FMO3 enzyme and intestinal flora.3.Compared with Choline+PBS group,we found that the levels of serum TC（P<0.05）were significantly decreased while the levels of serum HDL-C（P<0.001）and the genes related to cholesterol transport and decomposition,Abcg5/g8（P<0.01）and Cyp7a1（P<0.05）were significantly increased in Choline+ZDY01 group.4.Compared with Choline+PBS group,the content of bile acid in the cecum of mice was significantly decreased after treatment with E.aerogenes ZDY01,while the content of bile acid in serum was significantly increased（P<0.01）and there was no significant difference in fecal bile acid excretion.Furthermore,the study on the intestinal FXR-FGF15 axis regulated by bile acid showed that E.aerogenes ZDY01down-regulating the FXR-FGF15 axis and promoting the m RNA expression of bile acid transporter Asbt and Ost-α（P<0.01）,thereby promoting the decomposition of cholesterol in the liver.Conclusion:E.aerogenes ZDY01 used cecum TMA and reduced TMAO derived from TMA while promoting the expression of bile acid transporter ASBT and accelerating the resorption of bile acids into portal blood,reducing the levels of intestinal bile acids,lowering the ileal FXR-FGF axis,promoting the expression of CYP7A1,accelerating the decomposition of cholesterol into bile acids,further lowering the levels of cholesterol and inhibiting atherosclerosis in mice.