Puerarin Inhibits Atherosclerosis In ApoE^-/-Mice Via Gut Microbiota Remodeling And The Underlying Mechanisms

Background:Atherosclerosis(AS)is an important pathological basis for arteriosclerotic cardiocerebrovascular disease.In recent years,the incidence of AS has been increasing,which seriously harms human health.However,there is still lack of effective prevention and treatment.Puerarin(PU)is the main active component of pueraria lobata(Gegen in China).Previous studies showed that PU has been widely used in the treatment of AS.However,the underlying mechanism has not yet been fully identified.Recent studies found that the gut microbiota and its metabolite trimethylamine oxide(TMAO)play important roles in promoting the occurrence and development of AS.Thus,the gut microbiota might be a new potential therapeutic target for the prevention and treatment of AS.Our previous research has shown that PU has low bioavailability and most of which is excreted by the intestine as a prototype.Therefore,we propose the hypothesis:PU attenuates AS via gut microbiota remodeling.Experimental Approach:To investigate whether PU inhibits AS via gut microbiota remodeling and the underlying mechanism.Method and key results:1.High-fat and high-cholesterol(HFHC)diet promoted AS plaque formation in ApoE-/-mice.After treatment with PU,the protective effect of PU on AS was evaluated by oil red O staining,hematoxylin and eosin(HE)staining,ELISA,Western Blot and Quantitative Real-Time Polymerase Chain Reaction(q-PCR).The results suggested that PU could improve blood lipid metabolism,reduce the inflammatory response,and significantly inhibit AS plaque formation in ApoE-/mice;2.Compared to chow-fed mice,AS plaques were much more obvious in mice fed with HFHC diets,which could be markedly reversed by Abs administration.Moreover,when the fecal samples were analyzed for 16S rRNA sequencing of the gut microbiota,it was confirmed that PU significantly changed the gut microbiota of AS mice,especially reduced amount of Prevotella.Furthermore,we performed a Fecal Microbiota Transplantation(FMT)experiment.Mice that received PU group feces developed less AS plaques than AS group feces recipients.The results showed that gut microbiota played an important role in the process of PU attenuated AS in ApoE-/-mice.3.We isolated Prevotella copri strains from human stool,cultured them in vitro,and then transplanted to ApoE-/-mice to monitor whether it was responsible for the formation of AS.Mice in transplantation group were enriched in Prevotella copri which increased potential to exacerbate the formation of AS plaques.At the same time,our findings indicated that Prevotella copri played an important role in the development of AS,potentially by damaging the intestinal barrier,thus promoting inflammation levels,and increasing TMAO plasma levels.4.Prevotella copri could exacerbate the formation of AS,which were markedly reversed by PU administration.Importantly,co-culture of PU and Prevotella copri in vitro indicated that PU could inhibit the growth of Prevotella copri.Conclusion:1.PU inhibits AS in ApoE-/-mice by reconstructing gut microbiota and reducing TMAO production;2.PU exerts an anti-AS effect by inhibiting the growth of Prevotella copri.