The Role And Mechanism Of Intestinal Flora In The Progression Of Atheroscletic Vulnerable Plaque And Tongxinluo Intervention

Background Atherosclerosis(AS)is a chronic inflammatory disease of the arterial wall.Ischemic heart disease,ischemic stroke,and peripheral arterial disease caused by atherosclerotic plaques are the leading causes of death and loss of quality of life worldwide,placing an enormous burden on human health and socioeconomics.Vulnerable plaques formed by atherosclerosis,with large lipid cores,thin fibrous caps,and marked macrophage infiltration,are the underlying cause of most acute coronary events.Therefore,the improvement of vulnerable plaques has always been a major problem to be solved.At present,it is known that the pathogenesis of AS is complex,involving multiple factors such as chronic inflammation,oxidative stress,genetic susceptibility,immune dysregulation,and epigenetics.In recent years,the relationship between gut microbiota and atherosclerosis has also begun to receive more and more attention.There are 1013-1014 bacteria in the human gut that can regulate a variety of metabolic homeostasis in the host.Changes in intestinal metabolites accompanied by dysbacteriosis further affect the occurrence and development of diseases.However,the relationship between atherosclerosis and gut microbiota is very complex,and the relationship between vulnerable plaque and gut microbiota has not been studied yet,and further exploration is needed.Improving the vulnerable of plaque is the top priority for improving the prognosis of patients with atherosclerosis.Although a variety of ways have been proposed to induce plaque regression or slow down the atherosclerotic process,such as:inhibition of HMG-CoA reductase(statins),inhibition of NPC1L1(ezetimibe),inhibition of PCSK9(Repatha,Praluent),etc.,however,these treatments mainly stay at the lipid-lowering.Studies have shown that lipid-lowering drugs can greatly improve the prognosis of patients with atherosclerosis,but this method can only reduce the risk of cardiovascular and cerebrovascular events by about 25%to 30%,and there is still about 70%residual risk.It is well known that inflammation plays an important role in the development of vulnerable plaques,the activation of NLRP3 inflammasome is one of the important pathways.Early discovery of drugs and mechanisms that intervene in vulnerable plaques through inflammation is of great significance for the prevention of cardiovascular events.According to previous studies,the traditional Chinese medicine compound preparation Tongxinluo(TXL)has the functions of regulating lipids,dilating blood vessels and protecting vascular endothelium.TXL has been approved by the State Food and Drug Administration for the treatment of cardiovascular diseases such as angina pectoris and ischemic stroke,and its clinical effectiveness and safety have been well verified.It has been found that TXL intervention can reduce the arterial plaque load and plaque vulnerable of ApoE-/-mice.However,the mechanism of TXL stabilization of vulnerable plaques is still unclear.Whether Tongxinluo plays a role in stabilizing vulnerable plaques by inhibiting inflammatory pathways remains to be further studied.And whether Tongxinluo can reduce the inflammatory response by inhibiting the activation of the NLRP3 inflammasome and its downstream pathways also needs further study.More and more studies have confirmed that Chinese herbal medicine has a significant impact on the intestinal flora,and its role and metabolism are inseparable from the role of the intestinal flora.Does Tongxinluo affect the intestinal flora of vulnerable plaque model?Whether TXL can improve vulnerable plaques through intestinal flora?It’s worth exploring further.Objectives1.To explore changes in intestinal flora and metabolites in a high-cholesterol diet and balloon injury(HCB)-induced atherosclerotic vulnerable plaque model;2.To explore the effect of TXL intervention on vulnerable plaque in vulnerable plaque model,and the effect of Tongxinluo on intestinal flora and intestinal metabolites;3.To explore whether Tongxinluo inhibits inflammatory response and plays a role in stabilizing atherosclerotic vulnerable plaques by changing intestinal flora and metabolites;Methods Sixty-two New Zealand rabbits were randomly divided into CONTROL group(n=12)and experimental group(n=50).A high-cholesterol diet and balloon injury surgery were performed on the rabbits in the experimental group to establish a vulnerable plaque model of atherosclerosis.After operation,the rabbits in the experimental group were randomly divided into HCB group,Tongxinluo intervention group for 4 weeks(HCB+TXL4w),8 weeks(HCB+TXL8w)and 12 weeks(HCB+TXL12w).The concentrations of IL-1β,TNF-α in serum were determined by ELISA.The concentrations of triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)in serum were detected by enzyme method.Eosin-hematoxylin(HE)staining was used to observe the vascular morphology of different groups.Masson’s trichrome and Sirius red staining were used to detect the fibrosis of rabbit blood vessels.The localization and expression of NLRP3,caspasel,IL-1β and IL-18 were detected by IHC.Oil red O staining was performed on the gross and frozen sections of blood vessels to observe the difference of plaque and lipid content between different groups.The mRNA and protein expressions of NLRP3,caspasel,IL-1β and IL-18 were analyzed by RT-PCR and Western blot.16S rRNA gene sequencing and untargeted metabolomics sequencing were used to analyze the composition of intestinal flora and the differences of intestinal metabolites between groups.Statistical analysis data are expressed as mean ± standard error or median(minimum to maximum).P<0.05 or adjusted P<0.05 was considered statistically significant.All experiments were repeated at least three times.Results 1.HCB significantly promotes the formation of vulnerable arterial plaques,activates the NLRP3 inflammatory pathway,and enhances the inflammatory responseCompared with the control group fed with the normal diet,the contents of TG(2.6435±0.60,0.6450±0.16),TC(28.3522±3.62.0.9322±0.18)and LDL-C(10.5729±1.60,0.1599±0.04)in the serum of rabbits in the HCB group were significantly increased.In the HCB group,the intima of the thoracoabdominal aorta showed obvious plaque rupture and the existence of thrombus,and the gross oil red O staining also showed the existence of a large area of lipids and the formation of a large number of plaques.Also,staining of abdominal aorta sections revealed the presence of distinct thin fibrous cap plaques.At the same time,compared with the control group,the serum levels of IL-1β(43.35±1.82,33.0294±1.55)and TNF-α(246.4461±8.14.193.2904±10.33)in the HCB group were significantly increased,and the mRNA and protein expression levels of NLRP3 and its downstream caspase-1,IL-1β and IL-18 in the blood vessels were significantly increased.Localization analysis showed that these inflammatory factors were mainly expressed in vascular plaques,especially in vulnerable plaques,where the expression of these factors was extremely high.2.In the HCB-induced rabbit vulnerable plaque model,the composition of intestinal flora and intestinal metabolic profile were significantly changedThe study found that the composition of the intestinal flora of the rabbits in the HCB group was significantly different from that in the control group.The relative abundance of Bacteroidetes in the HCB group was significantly decreased,the ratio of Firmicutes/Bacteroidetes(F/B)was significantly increased,and the proportion of bacterial flora was seriously imbalanced.HCB significantly increased the negative correlation between many bacteria,especially Bacteroidetes and Firmicutes,and the symbiotic relationship between the flora was obviously disturbed.The results of non-targeted metabolomic sequencing analysis showed that compared with the control group,the contents of many amino acid derivatives,bile acids,carbohydrates,lipids,fatty acids,phenols and other intestinal metabolites changed significantly in the HCB group.Spearman correlation analysis showed that there were obvious correlations between various differential metabolites and differential bacteria,inflammatory factors and blood lipids.For example,the metabolite 5,10-methylenetetrahydrofolate was significantly negatively correlated with IL-1β,while cis-(Homo)3-aconitate was significantly positively correlated with IL-1β.Some differential bacteria,such as Akkermansia,islandicum,Alistipes,indisintinctus,intestinihominis and Succinispira mobilis,also had significant correlation with many metabolites.3.TXL stabilizes vulnerable arterial plaques through a lipid-independent pathway,and inhibits the NLRP3 inflammatory pathway,reducing the inflammatory responseCalculate the plaque vulnerable index:(macrophages%+lipids%)/(SMCs%+collagen fibers%).The results showed that compared with the HCB group,the abdominal aortic plaque vulnerable index of rabbits treated with TXL for 12 weeks was significantly reduced.Interestingly,the TXL intervention group did not reduce the levels of serum TG,TC,LDL-C,and had no significant effect on HDL-C.In contrast,TXL intervention significantly reduced serum TNF-α concentrations.In order to further clarify the mechanism of TXL inhibiting inflammatory response,we performed Western Blot and immunohistochemistry to detect the expression of NLRP3-related inflammatory factors in vascular tissue.The results showed that NLRP3,caspase1,IL-1β and IL-18 were mainly present in plaques,and their expressions in plaques were significantly reduced after TXL intervention.4.TXL stabilizes vulnerable plaques by altering gut microbiota and gut metabolic profilesThe study found that compared with the HCB group,the composition and structure of the intestinal flora of the rabbits in the HCB+TXL12w group were significantly changed.Both redundancy analysis and canonical correspondence analysis showed that the intestinal flora of the two groups of rabbits were significantly separated.The relative abundance of Bacteroidetes in HCB+TXL12w group was significantly increased,and the F/B value was significantly decreased.In addition,the relative abundance of Alistipes,Campylobacter,Rikenella,indistinctus,viscericola,Nordii,subantarcticus and other bacterial species in HCB+TXL12w group increased,while the relative abundance of Rulinococcus and albus decreased.Correlation analysis showed that negative correlations among many gut bacteria with significantly increased HCB weakened or even disappeared after TXL intervention,especially the correlations between Bacteroidetes and Firmicutes.Non-targeted metabolomic analysis showed that compared with the HCB group,the content of many metabolites,including various amino acid derivatives,bile acids,carbohydrates,lipids,fatty acids,phenols,etc.,were significantly different in the TXL intervention group for 12 weeks.Many of these metabolites are directly produced or regulated by gut bacteria.For example,Erucic acid,N-Acetylneuraminate,Chenodeoxycholate,6-Tuliposide B,etc.were significantly reduced in HCB+TXL12w group,while DL-3-Phenyllactic acid,1-Palmitoyl-2-hydroxy-sn-glycero-3phosphoethanolamine,Nitrilotriacetic acid,Hydroxyphenyllactic acid,trans-Ferulic acid,etc.increased significantly.Among them,DL-3-Phenyllactic acid,Nitrilotriacetic acid,Hydroxyphenyllactic acid,trans-Ferulic acid,etc.had obvious negative correlations with the vulnerable index,indicating that the changes of metabolites have a non-negligible relationship with plaque stability.5.TXL inhibits the NLRP3 inflammatory pathway by increasing the gut bacteria Alistipes indistinctus and the metabolite TFAAmong the TXL-altered microbiota and metabolites,the changes in the species indistinctus and metabolite TFA were particularly evident,which may be the potential mechanism of TXL to stabilize vulnerable plaques.Therefore,we further studied the effect of indistinctus in Alistipes genus in ApoE-/-mice,and found that indistinctus had no significant effect on the expression of NLRP3 inflammatory pathway protein in the blood vessels of mice fed a normal diet,but could significantly inhibit NLRP3 inflammatory pathway in blood vessels of high-fat-fed mice.At the same time,the study found that heat-inactivated indistinctus did not play a role in inhibiting the NLRP3 inflammatory pathway.This indicates that the active form of indistinctus is essential for suppressing the inflammatory response.TXL can stabilize vulnerable plaques by increasing the active form of indistinctus bacteria in the gut,thereby inhibiting the NLRP3 inflammatory pathway in blood vessels.Further studies on the differential metabolite TFA also found that TFA could inhibit the expression of NLRP3 and downstream inflammatory factors caspase-1,IL-1β and IL-18 in macrophages.We further studied TFA,which was significantly negatively correlated with vulnerable index,and found that TFA could inhibit the expression of NLRP3 and downstream caspase1,IL-1β and IL-18.Conclusions 1.HCB significantly promoted the formation of atherosclerotic vulnerable plaque and enhanced inflammatory response in rabbits;At the same time,HCB leads to obvious changes in intestinal flora and metabolites,and the F/B value of the flora increases significantly.The properties of Succinispira,Alistipes,Campylobacte,and Desulfovibrio have been significantly reduced,while Akkermansia,islandicul,Desulfovibrio have been significantly increased;2.In vulnerable plaque model,Tongxinluo inhibited NLRP3 inflammation pathway;It significantly improved the imbalance of intestinal flora,increased the content of bacteria such as Alistipes,and changed the composition of intestinal flora;Tongxinluo significantly increased the intestinal trans-ferulic acid and increased the content of some other beneficial metabolites;3.TXL can stabilize vulnerable plaques by increasing the active form of indistinctus bacteria in the intestine and increasing the content of intestinal metabolites TFA to inhibit NLRP3 inflammatory pathway in blood vessels.