| As is essentially a chronic inflammatory disease driven by lipid metabolism disorders,and is the main pathological basis for cardiovascular adverse events such as coronary heart disease,stroke and myocardial infarction.In recent years,TMAO produced by the metabolism of choline-rich diet has been confirmed to be a new independent risk factor for the progression of AS,which can mediate the activation of inflammatory factors by activating the PI3K/Akt signaling pathway and the NF-κB signaling pathway,initiating vascular inflammatory response and leading to endothelial dysfunction,ultimately accelerating AS progression.Preliminary research of the research group has confirmed that the phlegm and blood stasis Tongzhi Recipe has antiinflammatory,anti-oxidative and lipid-lowering effects,and has good curative effect on various cardiovascular diseases such as hyperlipidemia and myocardial ischemiareperfusion injury.Therefore,in this study,ApoE-/-mice induced by high-fat and highcholine diet were used as the research objects for AS.In vivo experiments,blood lipid levels,plaque area,aortic inflammatory factor expression and TMA/TMAO levels were detected after treatment with Tanyu Tongzhi Decoction.On this basis,the PI3K/Akt/NF-κB signaling pathway was used as an entry point to conduct in vitro experiments to study the molecular mechanism of Tanyu Tongzhi Decoction’s effects on TMAO-stimulated endothelial cell microenvironment and inflammatory response changes.Finally,the effect and mechanism of Tanyu Tongzhi Fang in delaying chronic vascular inflammation in AS were described from the two aspects of the overall system level and the cellular and molecular level.Part 1 Study on the intervention effect of Tanyu Tongzhi Decoction on AS vascular inflammation in ApoE-/-miceObjective:To establish the AS model in ApoE-/-mice induced by high-fat and high-choline diet,and to observe the correlation between TMAO level and the development of chronic inflammation in AS and the intervention effect of Tanyu Tongzhi Decoction.Methods:After 75 ApoE-/-mice were fed with high-fat-choline diet for 7 weeks,they were randomly divided into model,atorvastatin,and low-dose,medium-dose and high-dose groups of Tanyu Tongzhi Decoction.Each group was given drug intervention for 3 weeks,and 15 C57BL/6J mice were fed with normal diet for 10 weeks as control group.Blood lipids,local aortic root pathology,percentage of aortic plaque lesions,expression of IL-1β,TNF-α,MCP-1 cytokines in aortic root,serum TMA/TMAO levels and the expression of aortic PI3K/Akt/NF-κB signaling pathway proteins were detected in mice.Results:(1)High-fat and high-choline diet can induce the abnormal increase of serum TMA and TMAO levels in ApoE-/-mice,and accelerate the progression of AS,which is mainly manifested in the significant increase of serum TC,TG and LDL-C levels in mice(p<0.01),the significant decrease of serum HDL-C(p<0.01),the plaques formed in the whole aorta and at the valve(p<0.01),the arterial intima was thickened with the appearance of cholesterol crystals,the significantly increased expression of inflammatory factors around the arterial(p<0.01),and significantly up-regulated of the protein expression of aortic PI3K/Akt/NF-κB signaling pathway(p<0.01).Spearman correlation analysis results showed that serum TMAO and TMA levels were significantly forwardly correlated with blood lipid levels(TC,TG,LDL-C),aortic root plaque area,inflammatory cytokine expression and PI3K/Akt/NF-κB signaling pathway(IL-1β,MCP-1)(p<0.01),slightly positively correlated with the overall aortic plaque area and TNF-α expression(p>0.05),and negatively correlated with HDL-C(p>0.05).(2)All dose groups which are utilized Tanyu Tongzhi Decoction can significantly reduce the serum TG and TC levels of ApoE-/-mice in the model group,and the Tanyu Tongzhi Decoction high-dose group can significantly reduce the LDL-C serum content(p<0.05),however,all Tanyu Tongzhi Decoction dose groups have no significant effect on HDL-C.(3)Both the middle and high dose groups of Tanyu Tongzhi Decoction significantly improved the increase in the lipid core of aortic plaques in the model group(p<0.05),accompanied by a reduction in the degree of inflammatory infiltration in the lesion area and an increase in the fibrous cap.Among them,the effect of the high-dose group is particularly prominent.(4)All Tanyu Tongzhi Decoction dose groups could significantly down-regulate the antigen expression levels of IL-1β and MCP-1 in the aortic root(p<0.01),and the TNF-α expression level showed a downward trend after Tanyu Tongzhi Decoction treatment.(5)All dose groups of Tanyu Tongzhi Decoction can significantly reduce the serum TMA and TMAO levels in the model group(p<0.05),and the decreasing trend is dose-dependent.(6)The expression of p-PI3K/PI3K and p-NF-κB/NF-κB in all Tanyu Tongzhi Decoction dose groups were significantly decreased(p<0.01),and the expression of p-Akt/Akt in Tanyu Tongzhi Decoction high dose group was significantly decreased(p<0.05).Conclusion:Long-term high-fat and high-choline diet can cause abnormal increase of circulating TMA and TMAO levels in AS mice,and at the same time leading to accumulation of blood lipids,plaque formation and vascular inflammation,and activation of PI3K/Akt/NF-κB signaling pathway.Tanyu Tongzhi Decoction treatment can reduce the inflammatory damage of arterial vessels and delay the process of AS by down-regulating serum TMA and TMAO levels,reducing blood lipids and the release of vascular inflammatory factors,reducing plaque area,and inhibiting the activation of PI3K/Akt/NF-κB signaling pathway.Part 2:Tanyu Tongzhi Decoction regulates TMAO-induced endothelial cell inflammation through PI3K/Akt/NF-κB pathwayObjective:To observe the activation effect of TMAO on the PI3K/Akt/NF-κB signaling pathway of endothelial cells and the mechanism and specific targets of Tanyu Tongzhi Decoction.Methods:HUVEC were intervened with 100μM TMAO for 24 h to establish endothelial cell injury model.All HUVEC were divided into 6 groups:control group,TMAO intervention group(100μM TMAO),PI3K inhibitor group(20μM LY294002),low(5%),medium(10%)and high(10%)dose of medicated serum of Tanyu Tongzhi Decoction.Each group was given corresponding drug intervention for 24h.Cell viability,cytokine content in supernatant and expression of PI3K/Akt/NF-κB signaling pathway protein in cells were detected.Results:(1)HUVEC treated with different concentrations of TMAO and medicated serum for 24 h had no obvious inhibitory effect on the cell viability(p>0.05).(2)Similar to the serum concentration of TMAO in the in vivo experiment,100μM TMAO treatment in HUVEC cells for 24h could significantly up-regulate the expression levels of NF-κB total protein and its phosphorylated proteins(p<0.01),and significantly increased the secretion levels of IL-1β,IL-6,IL-8,MCP-1 and TNF-α(p<0.01).(3)All dose groups of medicated serum and PI3K inhibitor can significantly inhibit the secretion of cytokines induced by TMAO,such as IL-1β,IL-6,IL-8,MCP1 and TNF-α(p<0.05).(4)The expression levels of the ratio of PI3K,Akt,NF-κB phosphorylated protein to total protein in HUVEC cells in each medicated serum dose group and PI3K inhibitor group were significantly down-regulated(p<0.05).Conclusion:TMAO can activate the PI3K/Akt/NF-κB signaling pathway in HUVEC,and promote the release of inflammatory factors,such as IL-1β,IL-6,IL-8,MCP-1 and TNF-α.Medicated serum of Tanyu Tongzhi Dection can significantly inhibit the activation of PI3K/Akt/NF-κB signaling pathway and release inflammatory cytokines in HUVEC induced by TMAO,playing an indispensable role of alleviating AS vascular inflammation and sheltering endothelial cells. |
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