| Atherosclerosis(AS)is a chronic inflammatory disease occurring in the arterial wall.Pyroptosis is a newly discovered and confirmed proinflammatory programmed cell death form.Pyroptosis is accompanied by the maturation and release of inflammatory factors such as interleukin-1 beta(IL-1?)and interleukin-18(IL-18),and induces inflammatory cascade reaction.Studies have shown that macrophage pyroptosis plays an important role in the formation,rupture and immune inflammation of vulnerable plaques in AS.NLRP3 inflammosome is an important regulator mediating pyroptosis.The regulation of NLRP3 inflammasome mediated macrophage pyroptosis is expected to become the new strategy of stabilizing vulnerable plaque AS.Qingxin Jieyu Formula(QXJYF)is simplified from Yugeng Tongyu Decoction,an experienced prescription of Academician Chen Keji in treating myocardial infarction.QXJYF has the effect of supplementing Qi and activating blood circulation,dissolving turbidity and detoxicating,and is a representative prescription for the "blood-stasis and toxin" etiology and pathogenesis of coronary heart disease.Previous clinical studies have shown that QXJYF can reduce cardiovascular endpoint events and serum levels of inflammatory factors in patients with stable coronary heart disease;network pharmacological research suggests that anti-inflammation is one of the main mechanisms of action;proteomics research has shown that QXJYF can regulate the programmed death of AS-related cells.However,can QXJYF play an anti-inflammatory role through NLRP3 inflammosome pathway at the molecular level?Does it have a regulatory effect on the inflammatory programmed cell death form of pyroptosis at the cellular level?What is the mechanism of anti-inflammatory and stabilizing AS vulnerable plaques at animal level?All the above scientific questions need to be further explored and answered.Based on this,we propose the following scientific hypothesis:QXJYF may play an anti-inflammatory and stabilizing role in AS vulnerable plaque by inhibiting the activation of NLRP3 inflammosome,regulating the occurrence of pyroptosis in macrophages and reducing the release of inflammatory factors IL-1? and IL-18.Based on the above hypothesis,in vivo experiments,in vitro experiments and network pharmacology research were carried out to explore the intervention effect of QXJYF on vulnerable plaques of AS and its relationship with macrophage pyroptosis and activation of NLRP3 inflammosome.The aim of this study was to elucidate the effects and mechanism of QXJYF in regulating macrophage pyroptosis and stabilizing AS vulnerable plaque at three levels:animal,cellular molecular and biological networksPart 1 Effects and mechanisms of QXJYF on AS vulnerable plaque in ApoE-/-miceObjective:To observe the effects of QXJYF on vulnerable plaque in ApoE-/-mice,and to explore its mechanism from the perspective of NLRP3 inflammosome mediated macrophage pyroptosis.Methods:Fifty ApoE-/-mice fed with high fat diet were randomly divided into model control group,positive control group(atorvastatin,5mg/Kg/d),QXJYF low-dose group(4.7 g/Kg/d),QXJYF medium-dose group(9.4 g/Kg/d)and QXJYF high-dose group(18.8 g/Kg/d),and 10 C57BL/6J mice as normal control group;the corresponding drugs were given orally for 8 weeks;the levels of serum total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),triglyceride(TG)and high density lipoprotein cholesterol(HDL-C)were measured by automatic biochemical analyzer;AS lesion and plaque stability were evaluated by Sudan IV staining,hematoxylin-eosin(HE)staining and Movat staining;the levels of serum C-reactive protein(CRP),IL-1? and IL-18 in ApoE-/-mice were detected by ELISA;the protein expression of NLRP3,Caspase-1 and GSDMD were detected by immunohistochemistry and Western blot(WB);the mRNA expression of NLRP3,Caspase-1 and GSDMD were detected by quantitative real-time PCR(qPCR).Results:Compared with the model group,the levels of total cholesterol(TC)in atorvastatin group,QXJYF low-dose,medium-dose and high-dose groups were significantly decreased(P<0.05);compared with the model group,the levels of low density lipoprotein cholesterol(LDL-C)in atorvastatin group,QXJYF medium-dose and high-dose groups were significantly decreased(P<0.05);Sudan IV staining showed that aortic plaque was most obvious in the model group,and aortic plaque of atorvastatin group,QXJYF low-dose,medium-dose,high-dose were alleviated in varying degrees;HE staining showed that compared with the model group,the proportion of plaque area in the aortic roots in the atorvastatin group,QXJYF medium-dose and high-dose groups were significantly decreased(P<0.05),and the proportion of lipid in plaque in the atorvastatin group,QXJYF low-dose,middle-dose and high-dose groups were significantly decreased(P<0.05);Movat staining showed that compared with model group,the proportion of foam cells in atorvastatin group and QXJYF high-dose group were significantly decreased(P<0.05)and the proportion of collagen fiber in atorvastatin group and QXJYF high-dose group were significantly increased(P<0.05);ELISA detection showed that compared with model group,the serum levels of CRP,IL-1? and IL-18 in atorvastatin group,QXJYF middle-dose group and high-dose group were significantly decreased(P<0.05);Immunohistochemical and WB detections showed that compared with the model group,the expression of NLRP3 protein in the atorvastatin group,the QXJYF medium-dose and high-dose groups was significantly decreased(P<0.05),the expression of Caspase-1 protein in the atorvastatin group and the QXJYF high-dose group were significantly decreased(P<0.05),the expression of GSDMD-N protein in atorvastatin group and QXJYF high-dose group were significantly decreased(P<0.05);qPCR detection showed that compared with the model group,the expression of NLRP3 mRNA in atorvastatin group and QXJYF high-dose group were significantly decreased(P<0.05),the expression of Caspase-1 mRNA in atorvastatin group and QXJYF medium-dose and high-dose groups was significantly decreased(P<0.05),the expression of GSDMD in the atorvastatin group and the QXJYF high-dose group were significantly decreased(P<0.05).Conclusion:QXJYF can decrease the levels of blood lipid and serum inflammatory factors in ApoE-/-mice,improve lipid metabolism and inflammatory reaction,reduce macrophage infiltration and lipid deposition in aortic plaque,improve local inflammatory microenvironment of plaque,and stabilize vulnerable plaque of AS.The mechanism may be related to regulating the NLRP3 inflammosome mediated macrophages pyroptosis and inflammatory cascade reaction.Part 2 Effects and mechanisms of QXJYF on pyroptosis in J774A.1 macrophages.Objective:To observe the effects of QXJYF drug-containing serum on J774A.1 macrophages pyroptosis,and to explore its mechanism through NLRP3 inflammosome pathway.Methods:J774A.1 macrophages stimulated by lipopolysaccharide(LPS)and adenosine triphosphate(ATP)were divided into model group,positive control group(MCC 950,50?M)and QXJYF group(8%QXJYF drug-containing serum),and J774A.1 macrophages cultured routinely were used as blank control group;the corresponding drug intervention was given for 24 hours;the pyroptosis of J774A.1 macrophages were detected by Hoechst 33342/propidium iodide(PI)double staining,acridine orange(AO)/ethidium bromide(EB),flow cytometry labelled with PI and 7-Aminoactinomycin D(7-AAD)respectively;the levels of IL-1? and IL-18 were detected by ELISA;the protein expression of NLRP3,Caspase-1 and GSDMD were detected by immunofluorescence and WB methods;the mRNA expression of NLRP3,Caspase-1 and GSDMD were detected by qPCR.Results:Hoechst 33342/PI double staining showed that compared with model group,PI positive rate of MCC 950 group and QXJYF group was significantly decreased(P<0.05);AO/EB double staining showed that compared with model group,EB positive rate of MCC 950 group and QXJYF group was significantly decreased(P<0.05);PI flow cytometry showed that compared with model group,PI positive rate of MCC 950 group and QXJYF group was significantly decreased(P<0.05);7-AAD flow cytometry showed that compared with model group,7-AAD positive rate in MCC 950 group and QXJYF group was significantly decreased(P<0.05);ELISA detection showed that compared with model group,the level of IL-1? in MCC 950 group and QXJYF group was significantly decreased(P<0.05);immunofluorescence and WB detections showed that compared with model group,the protein expression of NLRP3,Caspase-1 and GSDMD-N in MCC 950 group and QXJYF group were significantly decreased(P<0.05);qPCR detection showed that compared with model group,the mRNA expression of NLRP3,Caspase-1 and GSDMD in MCC 950 group and QXJYF group were significantly decreased(P<0.05).Conclusion:The QXJYF drug-containing serum can regulate the J774A.1 macrophages pyroptosis induced by LPS and ATP,and its mechanism may be related to the inhibition of NLRP3 inflammosome activation.Part 3 Network pharmacology research of QXJYF on inhibiting the activation of NLRP3 inflammosome.Objective:To explore the characteristics and potential biological mechanisms of QXJYF on inhibiting the activation of NLRP3 inflammosome by means of network pharmacology,and to provide basis for further research in the futureMethods:Gene chip information published in GEO database was used to screen genes related to NLRP3 inflammosome activation;BATMAN-TCM database was used to screen active ingredients and target genes of QXJYF;Cytoscape 3.2.1 software and STING database were used to construct and analyze the "herb-active ingredient-target" network of QXJYF inhibiting the activation of NLRP3 inflammosome;FunRich 3.1.3 software was used for gene function enrichment analysis.Results:The 115 active ingredients in QXJYF can inhibit the activation of NLRP3 inflammosome by acting on 64 target genes.Among them,neotanshinone C,tanshiquinone B,gamma-sitosterol,miltionone I,neocryptotanshinone ?,miltirone,dehydromiltirone and estriol from Danshen and Chuanxiong were the main active ingredients.Gene function enrichment analysis showed that QXJYF can regulate energy pathways,metabolism,immune response,fatty acid metabolism and other 10 biological processes;IL-1 signaling pathway,IL-12 signaling pathway,TNF signaling pathway,IFN-gamma signaling pathway,AP-1 signaling pathway,mTOR signaling pathway,ARF6 signaling pathway,PI3K/Akt signaling pathway and other 20 biological signaling pathways to inhibit the activation of NLRP3 inflammosome.Conclusion:QXJYF can inhibit NLRP3 inflammosome activation with the characteristics of multi-component,multi-target and multi-pathway integration and regulation.In the future,the mechanisms of QXJYF regulating macrophage pyroptosis can be further explored from the perspective of interaction between different biological signaling pathways and NLRP3 inflammosome. |
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