The Effect Of Qing-Xin-Jie-Yu Granule On Atherosclerosis And Plaque Stability Based On The Gut Microbiota And Bile Acid Metabolism

Cardiometabolic diseases mainly refers to a clinical syndrome with significant correlation between chronic metabolic disorders and targeted cardiovascular damage.With atherosclerosis（AS）,coronary heart disease,heart failure as the main outcome,intervention of related metabolic disorders can effectively improve the prognosis of cardiometabolic diseases.AS is an important pathological marker of cardiometabolic diseases,and the disturbance of cholesterol metabolism and its interaction with immunometabolic signals are important causes of the occurrence and development of AS.Bile acid,a cholesterol metabolite,is a signaling molecule involved in various cardiometabolic disease processes.Previous clinical studies have shown that Qing-xin Jie-yu Granule reduces the absolute risk of complex "hard" endpoints（including cardiovascular death,nonfatal myocardial infarction,and ischemic stroke）in patients with stable coronary artery disease.In this study,through the combination of animal and cell experiments,the anti-AS mechanism of Qing-xin-Jie-yu Granule through bile acid metabolism signals and their receptor TGR5/cAMP/NFκB signaling pathway was investigated.Part 1 Exploring the active ingredients of Qing-Xin-Jie-Yu Granule for anti-AS with regulation of bile acid based on the network pharmacology and molecular docking technologyObjective:To clarify the potential molecular biological mechanism of Qing-Xin-JieYu Granule regulating bile acid metabolism in the treatment of AS by using network pharmacology and molecular docking techniques,and to provide the basis for further animal or cell experiments.Methods:The pharmacologic technology platform of TCM system was used to search the active ingredients of Qing-Xin-Jie-Yu Granule.Genecards,TTD and DrugBank databases were searched to obtain the target information of AS and bile acid.Cytoscape3.9.1 software and String platform were used to construct and analyze the network of "drug,active ingredients,and pharmacological target".The Metascape online platform was used for functional enrichment analysis of target genes.AutoDockTools-1.5.6 software was used for molecular docking.Results:The 99 active components of Qing-Xin-Jie-Yu Granule regulated bile acid metabolism and anti-AS by acting on 94 target genes,among which the key target genes of PPI network analysis were AKT1,TP53,TNF,IL6 and VEGFA.Pathway function enrichment analysis showed that Qing-Xin-Jie-Yu Granule play an anti-AS role in regulating bile acid metabolism mainly via the pathways related to inflammatory mediations such as cAMP signaling pathway,VEGF signaling pathway,NFκB signaling pathway,AMPK signaling pathway,cell fate processes such as Wnt signaling pathway and programmed necrosis,organic system such as thyroid hormone signaling pathway,relaxin signaling pathway and estrogen signaling pathway.Molecular docking results showed that berberine and quercetin had good docking ability with key target genes of TGR5/cAMP/NFκB signaling pathway.Conclusion:The pharmacological effects of Qing-Xin-Jie-Yu Granule and its main active ingredients have multi-component,multi-target and multi-pathway characteristics.The anti-AS effect by regulating the signal targets of bile acid metabolism may be an important mechanism of the pharmacological effects of QingXin-Jie-Yu Granule.Part 2 To study the effect and mechanism of Qing-Xin-Jie-Yu Granule on gut microbiota and bile acid metabolism of ApoE-/-miceObjective:To observe the effects of Qing-Xin-Jie-Yu Granule on plaque stability and metabolic homeostasis in AS mice,and to explore the mechanism of Qing-Xin-Jie-Yu Granule from the perspective of gut microbiota-bile acid metabolism.Methods:ApoE-/-mice were fed high fat diet（HFD）for 12 weeks to establish AS model.They were divided into Chow group,model control group（HFD group）,QingXin-Jie-Yu Granule low-dose group（HFD+QXJYG（L）group）and Qing-Xin-Jie-Yu Granule high-dose group（HFD+QXJYG（H）group）,and were continuously intervened for 12 weeks.The weekly weight changes of each group were observed.At the end of the 12th week,the levels of serum lipid,serum inflammation and arterial inflammation were measured.The pathological damage of aortic root plaques was detected by oil red O and HE staining.The inflammatory cell infiltration in aortic plaques and the expression of TGR5 and NFκB protein were observed by immunohistochemical staining.Targeted metabolomics was used to detect the bile acid level of liver and ileum,16s rRNA was used to detect the structure and function of gut microbiota in the ileocecal contents,and RT-qPCR was used to detect the related genes expression of bile acid hepatoenteric circulation.Results:（1）AS related phenotypic indexes:compared with the Chow group,the body weight of mice in HFD group increased with time,and the weight difference was significant after 8 weeks of intervention（P<0.05）.Compared with HFD group,both low-dose and high-dose QXJYG could reduce weight gain induced by high-fat diet in a dose-dependent fashion.After 8 weeks of intervention,there was a significant difference in body weight between high-dose group and HFD group（P<0.05）.Blood lipid test showed that after 12 weeks of treatment,compared with Chow group,serum TC,TG and LDL-C levels in HFD group were significantly increased（P<0.05）,while serum HDL-C level was significantly decreased（P<0.05）.Compared with HFD group,QXJYG in both low-dose and high-dose groups could reduce serum TC,TG and LDLC levels in a dose-dependent manner（P<0.05）.QXJYG increased serum HDL-C level in high-dose group（P<0.05）.Oil red O staining showed that compared with Chow group,the AS plaque area of aortic root in HFD group was significantly increased（P<0.05）.Compared with HFD group,QXJYG could reduce plaque area in a dose-dependent way,and the effect was significant in HFD+QXJYG（H）group（P<0.05）.HE staining showed that compared with Chow group,the necrotic area in HFD group was significantly increased（P<0.05）.Compared with HFD group,QXJYG intervention in low-dose and high-dose groups could significantly reduce the necrotic core area in plaque,and the effect was dosedependent（P<0.05）.Immunohistochemical staining showed that,compared with the HFD group,QXJYG increased the content of aSMA-positive smooth muscle cells on the surface of AS plaques（P<0.05）.Compared with the HFD group,QXJYG also reduced the infiltration degree of CD68-positive macrophages and CD3-positive T cells in AS plaques（P<0.05）.Elisa results showed that compared with Chow group,the levels of serum inflammatory cytokines TNF-α,IL-1β and IL-6 in HFD group were significantly increased（P<0.05）.Compared with HFD group,QXJYG in both low-dose and highdose groups showed better intervention effect on serum inflammation level,and the IL1β level in low-dose and high-dose groups was significantly decreased（P<0.05）,while the IL-6 level in high-dose group was significantly decreased（P<0.05）.There was no significant difference in serum TNF-α level after administration.RT-qPCR analysis showed that mRNA levels of TNF-α,IL-1β and IL-6 in aorta of HFD group were significantly increased compared with Chow group（P<0.05）.Compared with HFD group,the mRNA level of IL-1β in aorta of QXJYG in low-dose and high-dose groups was significantly decreased（P<0.05）,and the mRNA level of IL-6 in high-dose group was significantly decreased（P<0.05）.There was no significant difference in mRNA level of TNF-α in aorta after administration.（2）Regulation of gut microbiota and bile acid metabolism:16s rRNA was used to detect gut microbiota in ileocecal contents.Comparing the diversity of microbial alpha in each group after intervention,ACE,Fisher and Observed index showed an increasing trend from low-dose to high-dose of QXJYG prescription（P<0.05）.Principal coordinate analysis（PCoA）showed that the gut microbiota structure of QXJYG showed a dose-dependent separation trend.Roseburia,Aerococcus,Enterobacter,Defluviitaleaceae_UCG₀11,Turicibacter,Papillibacter,Jeotgailcoccus and Ruminococcus showed a high abundance in high-dose QXJYG group,while the abundance of Eubacterium_fissicatena_group,Erysipelatoclostridium,Alistipes,Rikenella,Candidatus_Stoquefichus and Blautia in the HFD group was significant Rising.LEfSe analysis showed that QXJYG can cause specific bacterial enrichment.The bacteria in AS mice enriched in the high-dose QXJYG group were mainly Roseburia and Monoglobus,while Blautia,Alistipes,Rikenella and Dorea were the most active in the HFD group.The dose-dependent trend analysis of Beta diversity showed that the abundance of Turicibacter and Roseburia in QXJYG group was higher than that in HFD group,while the abundance of Alistipes,Rikenella and Blautia in QXJYG group was lower than that in HFD group,and the trend was dose-dependent（P<0.05）.Analysis of metabolic levels in serum samples by ultra performance liquid chromatography-mass spectrometry with non-targeted metabolomics.Principal component analysis showed that the metabolites of QXJYG group and HFD group showed obvious cluster separation.Cluster heat map showed 70 kinds of serum metabolites in HFD group,QXJYG（L）group and QXJYG（H）group,indicating that QXJYG has a significant effect on serum metabolic profile.The MetaboAnalyst enrichment analysis showed that the most significant metabolic pathways were histidine,bile acid,betaine,phenylalanine and tyrosine.The effects of QXJYG on the five bile acid differential metabolites were dose-dependent,and the serum TCA content was significantly increased（P<0.05）.The effects of QXJYG on TbMCA,TCA,TDCA,UDCA,and CA were dose-dependently increased.Spearman correlation analysis showed that the lesion progression of AS was negatively correlated with the abundance of Roseburia,Turicibacter and Candidatus_Saccharimonas,while UDCA,CA and TCA were positively correlated with the abundance of Roseburia.（3）Regulation of bile acid metabolism and signaling pathways:targeting metabonomics for qualitative and quantitative analysis of bile acid profiles in liver and ileal tissues.The results showed that,compared with the HFD group,the liver specific bile acids in QXJYG groups increased,and TCA,TDCA and CA increased in a dosedependent manner.The results of ileal bile acid spectrum showed that the specific secondary bile acids,including TαMCA,TβMCA,TUDCA and TCA,were decreased after QXJYG treatment.Gene expression levels of key enzymes of liver bile acid synthesis and intestinal bile acid response signal protein in hepatoenteric circulation were measured by RT-qPCR.The results showed that compared with HFD group,the mRNA expression levels of bile acid synthesis initiation enzymes CYP7A1 and CYP27A1 in QXJYG group were significantly increased in a dose-dependent way（P<0.05）,while the mRNA expression levels of CYP8B1,CYP7B1 and FXR had no significant changes.The mRNA expression levels of FXR and SHP in ileal tissues of QXJYG group were lower than those of HFD group,but the difference was not significant.The mRNA expression level of FGF15 in ileum of mice in QXJYG（H）group was significantly decreased（P<0.05）.The expression level of β-Klotho mRNA in liver tissues of QXJYG group was also decreased,while the expression of FGFR4 was not significantly changed.Immunohistochemical staining was used to observe the content of bile acid receptor TGR5 and downstream signal p-NFκB in plaques.Compared with HFD group,QXJYG could increase the expression of TGR5 protein in plaques of AS mice,but decreased the expression of p-NF κB protein,and the effect was dose-dependent（P<0.05）.Conclusion:（1）QXJYG can improve the levels of serum lipids,serum inflammation and arterial inflammation,and relieve AS,and the anti-AS effect of QXJYG is related to the remodeling of gut microbiota and the regulation of bile acid metabolism.（2）QXJYG promotes cholesterol catabolism by increasing liver bile acid production and serum bile acid level,thus producing metabolic improvement effects on ApoE-/-mice.（3）QXJYG regulates liver cholesterol-bile acid metabolism by mediating intestinal FXR feedback signals,and regulates plaque inflammation through bile acid receptor TGR5 inflammatory signals,thus playing a role in lowering blood lipids and antiinflammation.Part 3 To study the effect and mechanism of Qing-Xin-Jie-Yu Granule on the bile acid receptor signaling pathway of macrophagesObjective:To observe the inhibitory effect of Qing-Xin-Jie-Yu Granule on macrophage inflammatory activation,and to explore the metabolic regulation mechanism of QingXin-Jie-Yu Granule based on bile acid receptor TGR5/cAMP/NFκB signaling pathway.Methods:Lipopolysaccharide（LPS）was used to interfere with RAW264.7 macrophage to induce inflammatory activation,crystal purple staining was used to observe the morphological changes of macrophages,CCK8 method was used to detect the activity of macrophages,and the optimal intervention concentration of Qing-XinJie-Yu Granule was screened.Macrophages were divided into blank control group,model group,QXJYG group and positive control group（INT-777 group）.The secretion of inflammatory cytokines and TGR expression of macrophages were detected by RTqPCR.The inflammatory migration behavior of macrophages was observed by Transwell assay,and the expression of TGR5 receptor on the surface of macrophages was observed by immunofluorescence staining.Lentivirus transfection technique was used to target the knockdown receptor TGR5 expression,cAMP expression was detected by Elisa,and the expressions of TGR5,NFκB and p-NFκB were detected by Western Blot.Results:（1）Inhibition of inflammatory activation of macrophages:LPS significantly decreased the activity of macrophages compared with blank control group;Compared with model group,QXJYG could reduce the inhibition of LPS on macrophage activity,and 6%concentration had the best effect（P<0.05）.Crystal violet staining showed that,compared with the blank control group,macrophages in the model group were activated and elongated by LPS under microscope.Compared with the model group,the macrophage antennae decreased with QXJYG administration.RT-qPCR showed that compared with blank control group,LPS stimulation significantly increased the mRNA expression levels of macrophage inflammatory cytokines IL-1b,IL-6 and TNF-α（P<0.05）.Compared with model group,QXJYG and INT-777 could significantly inhibit the secretion of LPS induced macrophage inflammatory cytokines IL-1b,IL-6 and TNF-α（P<0.05）.The results of Transwell experiment showed that LPS significantly promoted the inflammatory migration behavior of macrophages compared with blank control group.Compared with model group,both QXJYG and the INT-777 could significantly inhibit the inflammatory migration behavior of macrophages induced by LPS（P<0.05）.（2）Inducing the expression of TGR5 receptor in macrophages:The results of immunofluorescence staining showed that the fluorescence expression of TGR5 on the surface of macrophages in blank control group was obvious and clear,and the level of TGR5 receptor was high.In model group,the fluorescence effect of TGR5 on macrophage surface was significantly weakened,and the content of TGR5 receptor was decreased.Compared with the model group,the fluorescence effect of TGR5 in QXJYG and INT-777 group was significantly enhanced,and the content of TGR5 receptor was increased.RT-qPCR showed that compared with the blank control group,LPS-induced TGR5 receptor mRNA expression of macrophages in the model group was decreased.Compared with the modelgroup,both QXJYG and INT-777 can significantly promote the mRNA expression of macrophage bile acid receptor TGR5（P<0.05）.（3）Regulation of the TGR5/cAMP/NFκB signaling pathway:RT-qPCR results showed that the expression levels of inflammatory cytokines IL-1b,IL-6 and TNF-a were increased in the TGR5 receptor knockout group compared with the unrelated sequence control group（P<0.05）.Compared with the unrelated sequence control group,the expression levels of macrophage inflammatory cytokines IL-1b,IL-6 and TNF-a in the unrelated sequence+QXJYG group were decreased.Compared with the unrelated sequence+QXJYG group,TGR5 receptor knockdown can weaken the inhibitory effect of QXJYG on the secretion of macrophage inflammatory cytokines IL-1b,IL-6 and TNF-a（P<0.05）.The results of Transwell assay suggested that the inflammatory migration behavior of macrophages increased in the TGR5 receptor knockout group compared with the control group.Compared with the unrelated sequence control group,the migration behavior of macrophages in the unrelated sequence+QXJYG group showed a decreasing trend.Compared with the unrelated sequence+QXJYG group,TGR5 receptor knockdown can weaken the inhibitory effect of QXJYG on inflammatory migration behavior of macrophages（P<0.05）.Compared with the control group with independent sequence,the intracellular cAMP level in the TGR5 receptor knockout group was decreased,but not significantly.The expression level of p-NFκB protein was increased（P<0.05）;Compared with the unrelated sequence control group,the intracellular cAMP level of macrophages in the unrelated sequence+QXJYG group was increased,and the p-NFκB protein level was decreased,but there was no significant difference.Compared with the unrelated sequence+QXJYG group,the intracellular cAMP level in the TGR5 receptor knockout+QXJYG group was decreased,but there was no significant difference,and the p-NFκB protein level was increased（P<0.05）.Conclusion:（1）QXJYG can inhibit the inflammatory activation of macrophages induced by LPS.（2）LPS-induced inhibition of TGR5 receptor activity can lead to nflammatory activation of macrophages,and QXJYG can induce TGR5 receptor expression in macrophages.（3）QXJYG inhibits the activation of downstream cAMP/NFκB inflammatory signal by activating TGR5 receptor of macrophages,thus reducing inflammatory migration behavior of macrophages and secretion of inflammatory factors.