| Background and Objective:Cardiovascular diseases caused by atherosclerosis（AS）,which is characterized by lipid deposition and inflammatory cell infiltration,have long been the leading cause of death in humans.Finding an effective way to interfere with AS is of practical significance for reducing the incidence and mortality of cardiovascular diseases.Due to the formation of AS is closely related to people’s unhealthy diet patterns,dietary strategies are particularly important for the prevention and treatment of AS.Gut microbiota,as the body’s"metabolic filter",regulates the absorption and metabolism of host nutrients,and has become a hotspot in the cardiovascular field in recent years.Meanwhile,trimethylamine-N-oxide（TMAO）is a metabolite of gut microbiota that has been frequently studied,whose precursors include choline and mainly mechanism on AS is to damage the process of reverse cholesterol transport and its catabolism.A number of meta-analyses have confirmed that TMAO is an independent risk factor for AS,and the application of gut microbiota regulating agents exert an anti-AS effect by reducing the level of TMAO.In addition,the antibacterial activity of bile acids（BAs）can regulate the composition of gut microbiota,which has an important influence on the production of TMAO.Farnesoid X receptor（FXR）/fibroblast growth factor 15（FGF15）is a pivotal signal pathway for BA synthesis,and activation of this pathway will inhibit liver cholesterol 7α-hydroxylase（Cyp7a1）,causing lipid metabolism disorders and aggravating plaque load.Inhibiting the enterohepatic FXR/FGF15 axis is beneficial to promote BA synthesis,reduce TMAO production and delay the course of AS.Therefore,inhibiting the enterohepatic FXR/FGF15 axis to reduce the production of TMAO and improve lipid metabolism may be the central link in the prevention and treatment of AS induced by gut microbiota dysbiosis.Jianpi Huazhuo Tiaozhi Granule（JHTG）was developed under the guidance of"vital qi deficiency with insidious turbidity".Preliminary studies have proved JHTG plays an anti-AS effect by adjusting the structure of gut microbiota in the AS rabbit model.However,the relevance of the treatment of turbidity has not been systematically explained,and the molecular mechanism of modern medicine against AS is unknown.Therefore,this study was committed to a systematic theoretical elaboration on JHTG’s treatment of AS from turbidity,and believed that"vital qi deficiency with insidious turbidity"was an important pathogenesis of AS.The treatment methods of turbidity include"give turbidity a way out"and"nourishing one’s vitality to drive away turbidity".Even more noteworthy was"transporting spleen and dispersing liver"as a common method for turbid diseases.Based on these,the concrete relationship between the spleen in Chinese medicine and gut microbiota was explored,further proposed that TMAO might be a material target of endogenous turbidity.JTHG’s turbidity treatment method,mainly centered on transporting spleen and dispersing live,was conducive to regulating the relationship between liver and spleen,which would provide a new perspective for treating AS from turbidity theory.In addition,the high-choline chloride diet was used to establish gut microbiota dysbiosis animal models,and the hypothesis that JHTG might play an anti-AS role by inhibiting the FXR/FGF15 axis to promote BA synthesis and reduce TMAO production was studied.Methods:20 C57BL/6J mice and 70 Apo E-/-mice were randomly allotted into 9 groups,each with 10 mice:（1）Blank control group（C57BL/6J mice+basal feed）;（2）Choline chloride control group（C57BL/6J mice+basal feed+1%choline chloride）;（3）Apo E-/-control group(Apo E-/-mice+basal feed);（4）AS model group(Apo E-/-Mice+basal feed+1%choline chloride);（5）Antibiotic group(Apo E-/-mouse+basal feed+1%choline chloride+0.5 g·L-1vancomycin,1.0 g·L-1ampicillin,1.0 g·L-1metronidazole,1.0g·L-1neomycin sulfate added to the drinking bottle);（6）JHTG low-dose group(Apo E-/-mice+basal feed+1%chlorinated Choline+9.49 g·kg-1·d-1JHTG);（7）JHTG medium-dose group(Apo E-/-mice+basal feed+1%choline chloride+18.98 g·kg-1·d-1JHTG);（8）JHTG high-dose group(Apo E-/-mice+basal feed+1%choline chloride+37.96 g·kg-1·d-1JHTG);（9）FXR agonist group(Apo E-/-mice+basal feed+1%choline chloride+18.98 g·kg-1·d-1JHTG+intraperitoneal injection of the FXR agonist GW4064 at the 15th week).Except for FXR agonists,the intervention time of other drugs was 16 weeks.Whole aorta oil red O staining and aortic arch HE staining to observe histomorphological changes,enzymatic method to detect aortic total cholesterol（TC）concentration,biochemical detection of blood lipid profile,and ELISA to detect serum interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）levels.Collect intestinal contents for 16Sr RNA Mi Seq sequencing,and perform alpha diversity,beta diversity,species composition analysis,LEf Se analysis and correlation analysis on the composition and phylogenetic relationship of gut microbiota in the intervention samples with high choline,antibiotics,different doses of JHTG and FXR agist GW4064.The serum TMAO level of mice was detected by LC/MS,the total BA concentration of the small intestine concentration（SI Iumenal BA）,liver（Liver BA）and gallbladder（Billary BA）were determined biochemically,and the protein expression of FXR,FGF15 and Cyp7a1 were determined by Western blot.Correlate the genera screened by LEf Se analysis with TMAO and BAs,comprehensively analyze the effect of BAs on TMAO and significant genera,observe the changes in BA metabolism caused by the FXR agonist GW4064 and then detect its relationship with TMAO and significant genera.Results:1.Aorta whole oil red O staining results showed that high choline can promote C57BL/6J mice aorta lipid deposition and Apo E-/-mice aorta AS plaque formation,antibiotics intervention in gut microbiota can obviously reduce the AS plaque formation caused by high choline,JHTG in each dose group can obviously reduce the aorta AS plaque area percentage,and FXR agonists GW4064 will significantly weaken JHTG inhibition of the AS plaque formation.2.Aortic arch HE staining results showed that high choline can promote the early AS lesions in C57BL/6J mice and the formation of a large number of typical AS plaques in Apo E-/-mice.Antibiotic intervention in the gut microbiota can significantly inhibit AS caused by high choline.For plaque formation,each dose group of JHTG can effectively reduce the area of AS plaque.The addition of FXR agonist GW4064 will significantly weaken the JHTG-induced inhibition of high choline-caused AS.3.Aortic TC concentration and blood lipid profile results:After intervention with high choline,the aortic TC concentration,serum TC and low density lipoprotein cholesterol（LDL-C）levels were significantly increased in the choline chloride control group,while the high density lipoprotein cholesterol（HDL-C）levels were significantly decreased in the choline chloride control group.Compared with Apo E-/-control group,aortic TC concentration,serum TC,triglyceride（TG）and LDL-C level in AS model group were significantly increased,and HDL-C level was significantly decreased.Compared with choline chloride control group,aortic TC concentration and serum TC,TG and LDL-C levels in AS model group were further increased,while HDL-C level was further decreased.The aortic TC concentration and serum TC,TG and LDL-C levels in antibiotic group were lower than those in AS model group,and the HDL-C level was higher than that in AS model group.Compared with the AS model group,the aortic TC concentration and serum LDL-C level of the JHTG low-dose group decreased,while the HDL-C level increased;the JHTG medium and high-dose groups further down-regulated the aortic TC concentration and serum TC,TG,LDL-C level,up-regulate the serum HDL-C level.Compared with JHTG medium dose group,aortic TC concentration and serum TC level in FXR agonist group were significantly increased.4.Serum IL-6 and TNF-αlevels:high choline can significantly up-regulated the levels of TNF-α,and the levels of IL-6 and TNF-αin AS model group were higher than those in choline chloride control group.The levels of Serum IL-6 and TNF-αin the antibiotic group and JHTG groups were significantly decreased,and the levels of IL-6 and TNF-αin the low-dose JHTG group were higher than those in the medium-dose and high-dose JHTG groups.After the intervention of FXR agonist,the inhibition effects of JHTG on IL-6 and TNF-αwere significantly weakened.5.High-throughput sequencing results of gut microbiota:the richness and diversity of gut microbiota of AS model group were significantly decreased,while the medium-dose and high-dose JHTG groups could significantly improve the richness and diversity of gut microbiota.High choline caused significant changes in gut microbiota.At phylum level,the abundance of Firmicutes increased in the choline chloride control group and the AS model group,while the abundance of Bacteroidetes and Bacteroidetes/Firmicutes（B/F）ratio decreased.After the antibiotic and JHTG intervention,the B/F ratio was significantly increased,while FXR agonist GW4064could inhibit the regulatory effect of JHTG on B/F ratio.At genus level,the abundance of Ruminococcus,an obesity-promoting harmful bacteria,increased in the choline chloride control group and AS model group,while the abundance of Lactobacillus,an anti-obesity probiotic bacteria,was decreased in the choline chloride control group and AS model,and the abundance of Ruminococcus in the AS model group was significantly higher than that of the choline chloride control group.In addition,the abundance of Turicibacter,Desulfovibrio,Alistipes,Streptococcus,Oscillibacter and Prevotella,six pro-obesity and/or pro-inflammatory bacteria,were significantly increased in the AS model group,while probiotics include Akkermansia and Bacteroides were significantly reduced in the AS model group,and the abnormal changes of the above two probiotics and the five harmful bacteria except Alitipes in the AS model group were more obvious than those in the choline chloride control group.The abundance of Akkermansia in the antibiotic group was higher than that in the AS model group,and the abundance of 6 harmful bacteria such as Alistipes was lower than those in the AS model group.JHTG increased the abundance of anti-obesity and/or anti-inflammatory bacteria such as Bacteroides,Akkermansia,Lactobacillus and Bifidobacterium,while reduced the seven types of obesity-promoting and/or pro-inflammatory bacteria,including Ruminococcus,Turicibacter,Desulfovibrio and Alipipes.After the intervention of FXR agonist GW4064,the abundance of pro-obesity and pro-inflammatory harmful bacteria such as Turicibacter,Desulfovibrio,Alipipes were significantly increased compared with the JHTG medium-dose group.6.The correlation study between biochemical indicators and the genera screened by LEf Se analysis showed that the dominant genera altered by JHTG were closely related to the improvement of serum pro-inflammatory factors and lipid metabolism disorder indicators:Oscillibacter and other 7 harmful bacteria（of which 6 harmful bacteria were significantly reduced after JHTG intervention）were positively correlated with aortic TC and serum TC,TG,LDL-C,IL-6,TNF-α,while were negatively correlated with serum HDL-C.Alistipes（JHTG could effectively reduce the abundance of this bacteria）was positively correlated with aortic TC and serum TC,TG,IL-6,TNF-α.In addition,5 probiotics such as Akkermansia（Among them,Akkermansia increased significantly after JHTG intervention）were negatively correlated with aortic TC and serum TC,TG,LDL-C,IL-6,TNF-α,while were positively correlated with serum HDL-C.Bacteroides,Bifidobacterium,Lactobacillus（JHTG could effectively increase the abundance of these three probiotics）were negatively correlated with aortic TC and serum TC,TG,LDL-C,and positively correlated with serum HDL-C,Bacteroides was also negatively correlated with serum TNF-α.7.Serum TMAO concentration results:After high choline intervention,TMAO concentration of choline chloride control group was higher than that of blank control group,TMAO concentration of AS model group was higher than that of Apo E-/-control group,and TMAO concentration of AS model group was higher than that of choline chloride control group.Compared with the AS model group,the antibiotic group and JHTG dose groups could effectively reduce the concentration of TMAO,and the antibiotic group,JHTG medium and high-dose groups were better than JHTG low dose group in reducing the concentration of TMAO in serum.After the intervention with FXR agonist GW4064,the TMAO concentration in the FXR agonist group was significantly higher than that in the JHTG medium-dose group.8.BA results:After the intervention of high choline,the total BA concentration of the gallbladder（Billary BA）and small intestine contents（SI lumenal BA）of the choline chloride control group and the AS model group were significantly reduced,and SI lumenal BA of the AS model group was lower than that of chlorine Choline control group.SI lumenal BA of the JHTG low-dose group were significantly increased compared with the AS model group.The antibiotic group,JHTG medium-dose group,JHTG high-dose group’s Billary BA and SI lumenal BA were significantly increased compared with the AS model.Billary BA and SI lumenal BA in the FXR agonist group were significantly lower than those in the JHTG medium-dose group.9.Interfering effects of JHTG and FXR agonists on the enterohepatic FXR/FGF15Axis:the expressions of FXR and FGF15 proteins in the AS model group were significantly higher than those in the Apo E-/-control group and the choline chloride control group,and the expression of Cyp7a1 protein in the liver was significantly lower than that in the Apo E-/-control group and the choline chloride control group.Compared with the AS model group,the expressions of intestinal FXR and FGF15protein were significantly decreased in the antibiotic group,JHTG medium-dose group and JHTG high-dose group,while the expression of liver Cyp7a1 protein was significantly increased.In addition,the expressions of intestinal FXR and FGF15protein in FXR agonist group were significantly higher than those in JHTG medium-dose group,and the expression of liver Cyp7a1 protein was significantly lower than that in JHTG medium-dose group.10.The correlation study of TMAO,BAs and the genus selected by LEf Se analysis suggested that JHTG’s inhibition of TMAO production may be related to changes in the resistance of the gut microbiota to BAs:In the correlation analysis between TMAO and BAs,serum TMAO concentration was negatively correlated with Billary BA and SI lumenal BA.In the correlation analysis among the changed genus,BAs and TMAO,Akkermansia,an anti-obesity probiotic that can survive in a high BA environment,was negatively correlated with TMAO.Alipipes,a harmful bacteria with BA resistance,were positively correlated with TMAO.Desulfovibrio and Turicibacter,which were harmful bacteria intolerant to BAs,were positively correlated with TMAO.Additionally,the correlation results among Turicibacter,Desulfovibrio,Alitipes,BAs and TMAO were consistent with the direction of the FXR agonist GW4064 on JHTG.Conclusion:JHTG can inhibit the enterohepatic FXR/FGF15 axis to promote BA synthesis and reduce TMAO production,decreasing the abundance of Turicibacter,Desulfovibrio,and Alipipes,improving blood lipid profile,reducing IL-6 and TNF-αlevels,consequently fight against AS induced by gut microbiota dysbiosis. |
PDF Full Text Request