Resveratrol Attenuates Atherosclerosis Via Gut Microbiota Remodeling

Atherosclerosis(AS) induced cardiovascular diseases(CVD) have become the leading cause of death threating to human health. Looking for an effective anti-AS strategy for reducing CVD morbidity and mortality has important scientific significance. Resveratrol(RSV) is a naturally occurring dietary polyphenolic phytochemicals, mainly containing in grapes and other berry fruits, peanuts and red wine, with a variety of biological activity. Numerous studies show that RSV has obvious anti-AS effects, however, the underlying mechanism is not yet clear. Meanwhile, accummulating evidence has indicated that the low plasma concentration and fast metabolic rate of RSV, forming a distinct "paradox" between its low bioavailability and its significant biological activity. How do low levels of RSV play a significant role in AS prevention in vivo, whether there are other mechanisms responsible for its anti-AS effect, how to explain this "paradox" reasonablely, are important problems remain to be solved.Recent studies found that the changes and dysfunction of intestinal flora play an important role in the development of AS, which have become a new target for prevention and treatment of AS. Intestinal flora contain trillions of symbiotic microbial composition and use human nutrition to survive and metabolism in the body, and respond to external environmental factors with human body, and have metabolic and immune activity to maintain human health. Studies confirmed that on the one hand, the intestinal flora can metabolize dietary lecithin to produce trimethylamine(TMA), the TMA then is metabolized by the liver flavin monooxygenase(FMO) to generate trimethylamine-N-oxide(TMAO), a new independent risk factor for AS, thereby inducing AS. On the other hand the gut microbiota can inhibit bile acid hydrolysis in the gut, thereby inhibiting the synthesis of bile acids in the liver, causing cholesterol metabolism dysfunction and thus promote the process of AS. In addition, more and more evidence indicates that a variety of phytochemicals including RSV might perform their health benefits via influencing and reshaping the gut microbiota. Accordingly, we proposed the following hypothesis that RSV may remodel the gut microbiota and then reduced the TMAO generation and regulated bile acid metabolism, thereby reducing the risk of AS. To verify this hypothesis, the current study focused on the gut flora, and investigated RSV’s effect on the intestinal flora-dependent TMAO generation and bile acid metabolism in C57BL/6J and ApoE-/- mice, using the main technical method for gut flora research(16SrRNA sequencing, q PCR, bacterial culture) and molecular nutrition research techniques(immunoblot, qPCR, LC/MS), etc., to reveal the role of gut microbiota in RSV’s anti-AS effect as well as the underlying molecular mechanisms.The main results and conclusion of our present study:1.RSV attenuated AS in Apo E-/- mice, in which gut microbiota played an important role. Compared to chow-fed mice, the atherosclerotic lesion area and the cholesterol content in the whole aorta were much higher in mice fed with choline, which were markedly reversed by RSV or Abs administration(p<0.01). Moreover, when the gut microbiota were suppressed by Abs, RSV-induced inhibition of choline-caused AS was significantly abolished(p<0.05). Additionally, RSV-single treatment could also attenuate the pathogenesis of AS in ApoE-/- mice(p<0.01).2. RSV inhibited the synthesis of TMAO via gut microbiota remodeling.(1) RSV notably reduced high dietary choline induced TMAO generation. RSV could significantly inhibite single or long-term high dietary choline induced plasma TMA and TMAO generation(p<0.01); meanwhile, compared to the normal diet group, RSV-single treatment also remarkably reduced the content of mouse plasma TMA and with chow diet for 1 month, they failed to reduce TMA and TMAO contents after choline administration(p<0.01). These results indicated that the gut microbiome played a key role in RSV-induced decrease of TMAO synthesis in mice.3. RSV regulated bile acid metabolism through gut microbiota remodeling.(1) RSV could significantly affect bile acid metabolism and promote hepatic bile acid synthesis, thereby maintaining cholesterol homeostasis. RSV significantly decreased liver cholesterol level and increased BA pool size, gallbladder and SI luminal BA content(p<0.01). No differences were observed with respect to serum and hepatic BA content(p>0.05). In addition, RSV increased TCA/TβMCA ratio and induced CYP7A1 m RNA and protein expression(p<0.01).(2) RSV induced hepatic BA synthesis via gut microbiota remodeling. RSV treatment resulted in an increase in the proportion of Lactobacillus and Bifidobacterium that were present in the gut, contributing to its effects on BSH activity, subsequently promoted the generation of unconjugated bile acids from conjugated bile acids, thereby enhancing fecal bile acid loss, ultimately inducing hepatic bile acid synthesis. In addition, when intestinal microbiota were suppressed by Abs, the previously observed changes in hepatic bile acid synthesis, mediated by RSV, were abolished(p<0.05). These results suggested that the intestinal microbiota played a key role in RSV-induced regulation of bile acid metabolism.4. The enterohepatic FXR-FGF15 axis played a key role in RSV-induced BA synthesis. RSV treatment resulted in a significant decrease in ileal FGF15 mRNA and protein levels(p<0.01); however, ileal FXR mRNA and protein levels were unchanged(p>0.05). In the liver, RSV treatment appeared to have no significant effect on mRNA level of SHP and the mRNA and protein levels of FXR(p>0.05). Furthermore, RSV failed to further reduce the expression of FGF15 and induce CYP7A1 expression in mice treated with a natural antagonist of FXR. The synthetic FXR agonist was able to reverse RSV-induced alterations in the expression of FGF15 and CYP7A1 in mice. Taken together, these observations indicated that the gut-liver FXR-FGF15 axis played a key role in RSV-induced BA synthesis.In conclusion, RSV attenuated AS by decreased TMAO levels and increasing hepatic BA neosynthesis via gut microbiota remodeling, and the BA neosynthesis was partially mediated through the enterohepatic FXR-FGF15 axis. These results on the one hand provides new scientific experimental basis for clinical application of RSV, on the other hand will also provide new strategies and guidance for dietary prevention and treatment of CVD.