Mechanism Research Of Huoxue JieduⅠ Prescription On Treating Atherosclerosis In ApoE-/- Mouse Model Based On The Inflammasomes/Caspases Pathway

Vascular stenosis,plaque rupture and thrombosis caused by atherosclerotic plaque are the fundamental causes of acute myocardial ischemia and acute myocardial infarction.Pyroptosis is a new type of pro-inflammatory cell death mediated by inflammasomes.During pyroptosis,a large number of inflammatory factors are released,which affects the occurrence,development and stability of atherosclerosis.Apoptosis is classic programmed cell death pathway,which can participate in the occurrence and progress of atherosclerosis by affecting thrombosis and plaque rupture.Huoxue Jiedu I Prescription was flavored by Academician Chen Keji and his team on the basis of Xiong Shao capsule.The previous study has confirmed that the effect of Huoxue Jiedu I Prescription on stabilizing plaque is superior to that of pure huoxue medicine.Its mechanism was related to inhibiting inflammatory response,regulating blood lipid,improving collagen deposition and metabolism,especially inhibiting inflammatory response.Huoxue Jiedu I Prescription is an effective recipe for the prevention and treatment of atherosclerosis,which has experimental evidence based on the intervention of immune inflammatory response in the development of atherosclerosis and the stabilization of plaque.How does it play anti-inflammatory and immunomodulatory effect through the inflammasomes pathway?Inflammasomes-mediated immune inflammation plays an important role in pyroptosis and apoptosis of endothelial cells and macrophages.The downstream caspases family is the key protein in the cross talk and transformation between pyroptosis and apoptosis.What is the effect of Huoxue Jiedu I Prescription on it?The above questions remained to be answered.Based on this,we proposed the following hypotheses:Huoxue Jiedu I Prescription can reduce the release of inflammatory factors,inhibit pyroptosis and apoptosis,play an anti-inflammatory and immunomodulatory role,and play a role in inhibiting the formation of atherosclerosis and stabilizing plaque by regulating the inflammasomes/caspases pathway.Based on the above hypothesis,network pharmacology research and in vivo experiment were carried out respectively.Network pharmacology used network database.ApoE-/-mice fed with high fat diet were used as atherosclerosis model in vivo.The effects of blood lipid,atherosclerotic lesion degree,plaque stability,pyroptosis level,apoptosis and the expression of related inflammatory factors in ApoE-/-mice were observed in order to explain its mechanisms on regulating pyroptosis and apoptosis,inhibiting atherosclerosis formation,stablizing plaque,anti-inflammatory and immune regulation.Part1 Mechanism exploration of Huoxue Jiedu Ⅰ Prescription on atherosclerosis based on network pharmacologyObjective To evaluate the molecular mechanism of Huoxue Jiedu Ⅰ Prescription on atherosclerosis based on network pharmacology.Methods Active components and their target proteins of Huoxue Jiedu Ⅰ Prescription were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),and target proteins were standardized by UniProt protein database.Atherosclerosis related targets were obtained from OMIM,GeneCards and DRUGBANK database.After intersecting,the targets of Huoxue Jiedu Ⅰ Prescription against atherosclerosis were collected.The intersection targets were screened using Cytoscape 3.8.2 software to construct a "component-target"network to screen key active ingredients.Protein-protein interaction(PPI)network and core targets analysis were conducted through STRING network platform and Cytoscape 3.8.2 software.Matascape database was used to perform Gene Ontology(GO)and Kyoto Encyclopaedia of Genes and Genome(KEGG)pathway enrichment analysis of intersection targets.The " component-target-pathway" was constructed by Cytoscape 3.8.2 software.Results The results showed that there were 25 active components and 214 potential targets were related to Huoxue Jiedu Ⅰ Prescription,and 1265 targets were associated with atherosclerosis.The Venn analysis revealed that 118 intersection targets were correlated with the anti-atherosclerosis effect of Huoxue Jiedu I Prescription.The key active components of Huoxue Jiedu I Prescription against atherosclerosis were quercetin,baicalein,bata-sitosterol,ellagic acid,berlambine,palmatine and berberine.PPI network and core targets analysis suggested that TNF,AKT1,IL6,VEGFA,TP53,IL1B,Jun,CASP3,PTGS2,PPARG were key targets against atherosclerosis.Enrichment analysis indicated that Huoxue Jiedu I Prescription participated in various biological processes,such as apoptosis,cell migration,cell mobility,vascular progression and reactive oxygen species metabolism,via TNF pathway,Toll like receptor pathway,NF-κ B pathway,HIF-1 pathway,PI3K-Akt pathway,apoptosis,T cell receptor pathway,MAPKs pathway and FoxO pathway.Conclusion This study preliminarily revealed the mechanism of multi-component,multi-target and multi-channel of Huoxue Jiedu I Prescription against atherosclerosis,providing theoretical basis and research ideas for further study.Part2 Mechanism research of Huoxue Jiedu I Prescription on treating atherosclerosis in ApoE-/-mouse model based on inflammasomes/caspases pathwayObjective To observe the effect of Huoxue Jiedu I Prescription on blood lipid,atherosclerotic lesion degree and plaque stability,pyroptosis,apoptosis and expression of related inflammatory factors in mice,and explain its partial mechanism based on inflammasomes/caspases pathway.Methods 84 ApoE-/-mice fed with high fat diet were randomly divided into model group,statin group(atorvastatin,10mg/Kg/d),Huoxue Jiedu I Prescription group(2.171g/Kg/d),Huoxue Jiedu I Prescription+statin group(Huoxue Jiedu I Prescription 2.171g/Kg/d+atorvastatin 10mg/Kg/d),AC inhibitor group(2 mg/Kg/time)and AC inhibitor+Huoxue Jiedu I Prescription group.10 ApoE-/-mice were used as common diet group,and 14 C57BL/6J mice were used as normal control group.AC inhibitor was intraperitoneally injected at the first day of week13,17 and 21.The other groups were gavaged for 12 weeks.The levels of serum total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)were detected by automatic biochemical analyzer.The pathological degree and plaque stability of atherosclerosis were assessed by HE staining and Movat staining.The morphology of cells in atherosclerosis plaque was observed by transmission electron microscopy and the expression of GasderminD in aortic tissue was detected by immunohistochemistry.Serum IL-1β and IL-18 level were detected by ELISA,and gene expression of NLRP1,NLRP3,AIM2,ASC,Caspase-1,IL-1β and IL-18 were detected by qPCR.The protein expression of NLRP1,NLRP3,ASC,Caspase-1,IL-1β and IL-18 were detected by Western blot.Western blot was used to detect the protein expression level of Caspase-3,Caspase-8 and Caspase-9,which were related to apoptosis.The expression of inflammatory factors IL-6,TNF-α,hs-CRP and NF-κBp65 were detected by Western blot.Results TC,TG and LDL-C in model group were significantly increased than those in normal control group and common diet group(P<0.05),while HDL-C was significantly decreased(P<0.05).Compared with model group,TC,TG and LDL-C levels of statin group,Huoxue Jiedu Ⅰ Prescription group,Huoxue Jiedu ⅠPrescription+statin group,AC inhibitor group and AC inhibitor+Huoxue Jiedu ⅠPrescription group were significantly decreased(P<0.05),while HDL-C level was significantly increased(P<0.05).Compared with statin group,TC and TG in Huoxue Jiedu Ⅰ Prescription+ statin group were further decreased(P<0.05).The results of HE staining showed that the aortic wall of the normal control group was smooth and no plaque deposition was observed.The aortic lumen of other groups showed different degrees of plaque deposition,and model group was the most obvious.The plaque deposition degree in each intervention group was reduced to varying degrees compared with model group.Compared with common diet group,model group significantly increased the proportion of plaque to vascular area(P<0.05).Compared with model group,the proportion of plaque to vascular area in statin group,Huoxue Jiedu Ⅰ Prescription group,Huoxue Jiedu Ⅰ Prescription+statin group,AC inhibitor group and AC inhibitor+Huoxue Jiedu Ⅰ Prescription group was significantly decreased(P<0.05).The results of Movat staining showed that compared with common diet group,model group significantly increased plaque vulnerability index(P<0.05).Compared with model group,plaque vulnerability index of statin group,Huoxue Jiedu Ⅰ Prescription group,Huoxue Jiedu Ⅰ Prescription+statin group,AC inhibitor group and AC inhibitor+Huoxue Jiedu Ⅰ Prescription group was significantly decreased(P<0.05).Electron microscopy showed endothelial cell apoptosis and macrophage pyroptosis in atherosclerotic plaque.Expression of GasderminD protein in model group was significantly higher than that in normal control group and common diet group(P<0.05).Compared with model group,expression of GasderminD protein in statin group,Huoxue Jiedu Ⅰ Prescription group,Huoxue Jiedu Ⅰ Prescription+statin group,AC inhibitor group and AC inhibitor+Huoxue Jiedu Ⅰ Prescription group was significantly decreased(P<0.05).ELISA showed that the serum IL-1β and IL-18 in model group were significantly increased than those in normal control group and common diet group(P<0.05).Compared with model group,serum IL-1β and IL-18 in statin group,Huoxue Jiedu Ⅰ Prescription group,Huoxue Jiedu Ⅰ Prescription+statin group,AC inhibitor group and AC inhibitor+Huoxue Jiedu Ⅰ Prescription group were significantly decreased(P<0.05).Compared with statin group,IL-1β in Huoxue Jiedu Ⅰ Prescription+atorvastatin group was further decreased(P<0.05).QPCR showed that expression of NLRP1,NLRP3,AIM2,ASC,Caspase-1,IL-1β and IL-18 mRNA in aorta tissues of model group were significantly increased than those of normal control group and common diet group(P<0.05).Compared with model group,expression of NLRP3,AIM2,ASC,IL-1β and IL-18 mRNA in statin group were significantly decreased(P<0.05),expression of NLRP1,NLRP3,AIM2,ASC,Caspase-1,IL-1β and IL-18 mRNA in Huoxue Jiedu Ⅰ Prescription group and Huoxue Jiedu Ⅰ Prescription+statin group were significantly decreased(P<0.05),expression of NLRP3,AIM2,Caspase-1,IL-1β and IL-18 mRNA in AC inhibitor group were significantly decreased(P<0.05),expression of NLRP3,AIM2,ASC,Caspase-1,IL-1β and IL-18 mRNA in AC inhibitor+Huoxue Jiedu Ⅰ Prescription group were significantly decreased(P<0.05).Compared with statin group,expression of NLRP1,NLRP3,AIM2 and Caspase-1 mRNA in Huoxue Jiedu Ⅰ Prescription+statin group were further decreased(P<0.05),expression of Caspase-1 mRNA in AC inhibitor group and AC inhibitor+Huoxue Jiedu Ⅰ Prescription group were further decreased(P<0.05).WB showed that expression of NLRP1,NLRP3,ASC,Caspase-1,IL-1β and IL-18 protein in aorta tissues of model group were significantly increased than those of normal control group and common diet group(P<0.05).Compared with model group,expression of ASC,Caspase-1,IL-1β and IL-18 protein in aorta tissues of statin group were significantly decreased(P<0.05),expression of NLRP1,NLRP3,ASC,Caspase-1 and IL-1β protein in Huoxue Jiedu Ⅰ Prescription group and Huoxue Jiedu Ⅰ Prescription+statin group were significantly decreased(P<0.05),expression of NLRP3,ASC,Caspase-1 and IL-1β protein in AC inhibitor group and AC inhibitor+Huoxue Jiedu I Prescription group were significantly decreased(P<0.05).Compared with statin group,expression of NLRP1,NLRP3 and Caspase-1 protein were further decreased in Huoxue Jiedu Ⅰ Prescription+atorvastatin group(P<0.05).Expression of Caspase-3,Caspase-8 and Caspase-9 protein in aorta tissues in model group were significantly increased than those in normal control group and common diet group(P<0.05).Compared with model group,expression of Caspase-3,Caspase-8 and Caspase-9 protein in statin group,Huoxue Jiedu Ⅰ Prescription group,Huoxue Jiedu ⅠPrescription+statin group,AC inhibitor group and AC inhibitor+Huoxue Jiedu ⅠPrescription group were significantly decreased(P<0.05).Compared with statin group,expression of Caspase-9 protein in Huoxue Jiedu Ⅰ Prescription+statin group was further decreased(P<0.05).Expression of IL-6,TNF-α,hs-CRP and NF-κBp65 protein in aorta tissue of model group were significantly higher than those of normal control group and common diet group(P<0.05).Compared with model group,expression of IL-6,TNF-α,hs-CRP and NF-κBp65 protein in statin group,Huoxue Jiedu Ⅰ Prescription group,Huoxue Jiedu Ⅰ Prescription+statin group were significantly decreased(P<0.05),expression of NF-κBp65 protein in AC inhibitor group were significantly decreased(P<0.05),expression of TNF-α,hs-CRP and NF-κBp65 protein in AC inhibitor+Huoxue Jiedu Ⅰ Prescription group were significantly decreased(P<0.05).Compared with statin group,expression of IL-6 and TNF-α in Huoxue Jiedu Ⅰ Prescription+statin group were further decreased(P<0.05).Conclusion Huoxue Jiedu Ⅰ Prescription reduced the level of blood lipid and inflammatory factors,improved lipid metabolism and inflammatory response,inhibited the formation of aortic plaque and increased the stability of plaque,so as to play the role of anti-atherosclerosis.The mechanisms of its anti-atherosclerosis may be related to the regulation of the NLRP1-NLRP3/ASC/Caspase-1/IL-1β pathway and inhibition of pyroptosis.In addition,Huoxue Jiedu Ⅰ Prescription inhibited Caspase-3,Caspase-8,Caspase-9 protein expression and regulated endogenous and exogenous apoptosis pathways to inhibit apoptosis,which may be one of its anti-atherosclerosis mechanisms.