Effects And Mechanisms Of Desulfovibrio Desulfuricans On Atherosclerosis In Apoe^-/- Mice

According to the data published in 2018,the number of cardiovascular diseases（CVD）patients in China is about 290 million and will continue to increase in the future.Atherosclerosis（AS）is considered to be the main pathological basis of CVD and one of the difficulties in the field of CVD.The pathogenesis of AS is complex,previous studies showed that there exists a close connection between AS and gut microbiota.At present,most studies focus on the correlation between gut microbiota and the development of AS,however,the exact role of specific gut microbe on AS has been far less studied.Therefore,a gut microbe Desulfovibrio desulfuricans（D.desulfuricans）which can affect the development of AS was screened by modified linear discriminant analysis（M-LDA）,and the mechanism of D.desulfuricans on the development of AS was explored.The main research contents and conclusions are as follows:1.Screening of AS-associated gut microbes:AS and non-AS rodent model was successfully constructed through dietary intervention.The gut microbiota structure of the two groups was compared by 16S high throughput sequencing.It was found that there was a significant difference in gut microbiota structure between AS and non-AS mice.Significant differences in theα-diversity index,β-diversity index,gram-positive/negative ratio,Bacteroidetes/Firmicutes ratio,and enterotype between the two groups were observed.Then M-LDA（Modified Linear Discriminant Analysis）was used to score the features of the gut microbiota,and according to the abundance and scores of the strains,D.desulfuricans was selected.2.The impact of D.desulfuricans on the development of AS:AS plaque in the aorta and aortic root of Apoe^-/-mice increased significantly with the administration of D.desulfuricans.We next verified the proportion of gut microbiota at the phylum level after administrating with D.desulfuricans,as drastic changes in the composition of gut microbiota could worsen the development of AS.It was found that D.desulfuricans did not make any significant change in the proportion of the six common phyla.Then,the factors that affect the development of AS,such as inflammation,lipid metabolism,glucose metabolism were explored.It was found that D.desulfuricans can increase the level of systemic and local inflammation in mice,but has no significant effect on lipid metabolism and glucose metabolism in mice.3.The mechanism of D.desulfuricans on the development of AS:It was found that the thickness of the intestinal mucus layer,the number of goblet cells,and the expression of intestinal permeability-related proteins Occludin,and ZO-1 decreased significantly administrated with D.desulfuricans.Assess of the administration of FITC-dextran and q PCR also showed that D.desulfuricans could increase intestinal permeability in mice.Then,Caco-2 cells were co-cultured with D.desulfuricans medium,immunofluorescence results showed that the fluorescence area and intensity of Occludin and ZO-1 in Caco-2 cells decreased significantly,and Western Blot results showed that the grayscale values of Occludin and ZO-1 in Caco-2 cells decreased significantly.We found that D.desulfuricans could reduce the abundance of intestinal permeability protective related bacteria.These results suggest that D.desulfuricans can increase intestinal permeability in mice.The level of LPS（Lipopolysaccharide）in mice were detected,and we found that D.desulfuricans could increase the level of LPS in blood and mesentery of mice,and the expression of TLR4（Toll-like receptor 4）,a receptor for LPS,and P-p65,a key molecule involves in the TLR4/NF-κB signaling pathway,were also significantly increased.According to the above results,we considered that the proatherogenic effect of D.desulfuricans was attributed to its ability to increase intestinal permeability and subsequent raise in the transit of LPS from the intestine to the bloodstream.Excessive LPS in the blood can elicit local and systemic inflammation and activate TLR4/NF-κB signaling of endothelial cells and finally exacerbated AS.TAK-242,a specific inhibitor of TLR4,was used to verify that the activation of the TLR4/NF-κB signal pathway induced by LPS is the main reason for the development of AS.The results showed that TAK-242 could inhibit the development of AS;TAK-242 did not affect the level of LPS,but the expression of P-p65 and TLR4decreased significantly;TAK-242 could significantly reduce systemic and local inflammation in mice;TAK-242 had no significant effect on lipid metabolism,glucose metabolism and intestinal permeability in mice.These results suggest that the inhibition of the LPS-mediated TLR4/NF-κB signal pathway by TAK-242 can ameliorate the development of D.desulfuricans-induced AS.In summary,this study identified a proatherogenic gut bacterial species D.desulfuricans with M-LDA.It was found that D.desulfuricans could increase intestinal permeability in mice and consequently excessive LPS transit into the blood circulation and activate TLR4/NF-κB signal pathway,which eventually leads to the development of AS.With the inhibition of TLR4/NF-κB signal pathway by TAK-242,AS was significantly improved.However,TAK-242 has no effect on LPS concentration,lipid metabolism,and intestinal permeability,which again demonstrated that D.desulfuricans can enhance the development of atherosclerosis by increasing intestinal permeability and activating the LPS-mediated TLR4/NF-κB signal pathway.