The Effect Mechanism Of Moxibustion On Improving Chronic Vascular Inflammation Based On PI3K/AKT/NF-κB Signaling Pathway

Objective:This study takes the chronic vascular inflammation induced by high-fat diet in rats as an entry point to study the therapeutic effect and related parameters of moxibustion Zusanli on chronic vascular inflammation,and explores the chronic vascular inflammation and vascular damage induced by high-fat diet through bioinformatics analysis.characteristics to study the related mechanisms of moxibustion in regulating chronic vascular inflammation.Focused on examining the efficacy of moxibustion on chronic vascular inflammation,including the differences in the effects of different moxibustion courses and acting on different vascular parts,and revealing the regulatory mechanism of moxibustion on chronic vascular inflammation through the PI3K/AKT/NF-κB signaling pathway.Methods:Experiment 1:36 male Sprague-Dawley rats were divided into a blank group,a model group,and a moxibustion group.To explore the differences in treatment duration,they were further subdivided into six subgroups for 2-week and 4-week interventions,systematically assessing the impact of moxibustion.The moxibustion group received treatment at the "Zusanli" acupoint for 10 minutes per session,once a day,for either 2 or 4 weeks.HE staining observed the morphology of the thoracic aorta in each group;Oil Red O staining assessed lipid deposition in the thoracic aorta;ELISA measured the contents of TC,TG,HDL,LDL,ox-LDL,ICAM-1,VCAM-1,IL-6,TNF-α,ET-1 in serum,and NO,ox-LDL,ET-1 in the thoracic aorta;qPCR evaluated the relative expression of IL-6,TNF-α in the thoracic aorta.Experiment 2:Similar to Experiment 1,focusing on the abdominal aorta.HE staining observed the abdominal aorta morphology;ELISA detected NO,ox-LDL,ET-1 contents;qPCR and Western Blot assessed IL-6,TNF-α’s protein and mRNA expression;immunofluorescence identified IL-6 positive expression.Experiment 3:Involved 18 rats divided into three groups with moxibustion applied for 4 weeks.HE and Oil Red O staining observed the morphology and lipid deposition of thoracic and abdominal aortas.Western Blot and qPCR measured the protein and mRNA expression levels of IL 1β,IL2,IL-6,IL12,TNF-α in thoracic and abdominal aortas.Experiment 4:1.Using the GSE6735,GSE45927 data sets and the cardiovascular disease-related gene list,inflammation negative regulatory gene list,and atherosclerosis susceptibility gene list in Malacard as data sources,the limma package identifies differentially expressed genes,and the ggplot2 package(version 3.3.6)and Complex Heatmap package(version 2.13.1)for result visualization.2.Analyze the unique and common parts between each group of data,and visualize the results with ggplot2[3.3.6]and VennDiagram[1.7.3].The differentially expressed gene data of each data set were integrated and analyzed with Malacards’ related gene list to identify key molecular pathways related to cardiovascular disease and inflammation regulation.4.Use GO and KEGG databases to annotate genes and analyze gene functions and metabolic pathways.The R package clusterProfiler[4.0]performs ID conversion,and the clusterProfiler package performs enrichment analysis.The GO plot package calculates the z score value of enriched items,and combines GO/KEGG with FC for analysis.Use the ggplot2 package,igraph package,and ggraph package for visualization.5.clusterProfiler[4.0]performs GSEA analysis,and the ggplot2 package(version 3.3.6)visualizes the enrichment analysis results.Experiment 5:Fifty male Sprague-Dawley rats were used as subjects and divided into blank group,model group,moxibustion group,moxibustion+PI3K inhibitor group,and moxibustion+NF-κB agonist group.The moxibustion group received moxibustion "Zusanli" intervention,10 minutes/time,once/d,for 4 consecutive weeks.Moxibustion+PI3K inhibitor group:Rats were intraperitoneally injected with LY294002(PI3K inhibitor)(1.2 mg/kg/d)before each moxibustion intervention.The method,time and frequency of moxibustion were consistent with those in the moxibustion group.Moxibustion+NF-κB agonist group:Rats were given intraperitoneal injection of Prostratin(NF-κB agonist)(50 mg/kg/d)before each moxibustion intervention.The method,time and frequency of moxibustion were consistent with those in the moxibustion group.Western Blot method to detect IL-1β,IL-2,IL-6,IL-8,IL-12,TNF-α,TRAF-1,p-PI3K,PI3K,p-Akt,Akt,IKKa in rat abdominal aorta/IKKβ,IAP1,IAP2,NF-κBp65,NF-KBp50 protein relative expression;real-time fluorescence quantitative PCR method was used to detect IL-1β,IL-2,IL-6,IL-8,IL-in the abdominal aorta of rats in each group 12.Relative expression of TNF-α,TRAF-1,PI3K,Akt,IKKα/1KKβ,IAP1,IAP2,NF-κBp65,NF-κBp50;immunofluorescence staining method to detect the positive expression of NF-κB in rat abdominal aorta.Results:Experiment 1:Compared with the model group(2 weeks),the serum LDL,ICAM-1,and ET-1 of rats in the moxibustion group(2 weeks)decreased(P<0.05),and IL-6 and TNF-α increased(P<0.01,P<0.05);thoracic aorta ox-LDL and ET-1 decreased(p<0.01,p<0.05),IL-6 and TNF-α mRNA decreased(p<0.01,p<0.001),HE staining color Deeper,the blood vessel wall is smooth,the cell nucleus and surrounding tissue structure are relatively clear,and there is no significant change in the Oil Red O staining area;Compared with the model group(4 weeks),the serum LDL,TC,TG,ox-LDL,VCAM-1,ICAM-1,IL-6,TNF-α,and ET-1 of rats in the moxibustion group(4 weeks)decreased,increased HDL(P<0.05,P<0.01),increased thoracic aortic NO(p<0.001),decreased ox-LDL(p<0.01)and ET-1(p<0.001),IL-6 and TNF The mRNA of-a decreased(p<0.01,p<0.001),the HE staining color was darker,the blood vessel wall was smooth,the cell nucleus and surrounding tissue structure were relatively clear,and the oil red O staining area was significantly reduced(p<0.01).Compared with the moxibustion group(2 weeks),the serum HDL and VCAM-1 of rats in the moxibustion group(4 weeks)increased(P<0.05),and TNF-α decreased(P<0.001).IL-6 mRNA expression decreased,TNF-α expression increased(p<0.01),blood vessel walls were smooth,cell nuclei and surrounding tissues were clearer,and lipid deposition was reduced(p<0.001).Experiment 2:Compared with the model group(2 weeks),HE staining in the moxibustion group(2 weeks)showed that the abdominal aorta endothelium was neat,the blood vessel wall hierarchical structure was clear,NO levels were increased(p<0.05),and ox-LDL and ET-1 levels were decreased(p<0.01),and the protein and mRNA expressions of IL-6 and TNF-α increased slightly,but the difference was not significant.Compared with the model group(4 weeks),HE staining in the moxibustion group(4 weeks)showed that the abdominal aorta endothelium was smooth and the vessel wall structure was clear.Compared with the moxibustion group(2 weeks),it was improved and the NO level increased.(p<0.01),ox-LDL and ET-1 levels decreased(p<0.05,p<0.01),IL-6 and TNF-α protein and mRNA expression decreased(p<0.01,p<0.05),IL-6 The positive expression of immunofluorescence decreased(P<0.05)..Compared with the moxibustion group(2 weeks),the expression of IL-6 in the abdominal aorta of the moxibustion group(4 weeks)was reduced(p<0.001).Experiment 3:HE staining and Oil Red O staining showed that compared with the model group,the HE staining color of the thoracic and abdominal aorta of rats in the moxibustion group became darker,the thoracic and abdominal aorta blood vessel walls were smooth,and the cell nuclei and surrounding tissue structures were relatively clear.Fat coloring was significantly reduced,with only a small amount of lipid distributed around the vascular endothelium.Serum IL6 and TNF-αlevels were reduced(p<0.0001,p<0.01).Thoracic aorta IL-1β,IL-2,IL-6,IL-12.The protein and mRNA expression of TNF-α decreased(p<0.01,p<0.05,p<0.0001,p<0.0001,p<0.05),and the protein expression of IL-2,IL-6,and TNF-α in abdominal aorta expression and mRNA expression of IL-1β,IL-2,IL-6,IL-12,and TNF-α were reduced(p<0.05,p<0.001,p<0.01).Compared with the thoracic aorta,the abdominal aorta in the model group was darker in color,and the texture of the adventitia,media,intima layer,and endothelial layer of the blood vessel were clearer.Obvious oily red coloring and lipid droplet distribution were seen deep in the wall;while after Moxibustion intervention showed that the vascular endothelium of the thoracic aorta was smoother than that of the abdominal aorta,the red coloring of blood vessels and the distribution of lipid droplets were improved,and the protein expression of TNF-α in the abdominal aorta was reduced(p<0.01).Compared with the abdominal aorta,high-fat diet led to a significant increase in IL-1β,IL-6,and TNF-α protein and mRNA in the thoracic aorta(p<0.05),and an increase in the protein expression of IL-2(p<0.05),while the mRNA levels of IL-2 and IL-12 were reduced(p<0.05).Moxibustion reduced the protein expression of IL-1β,IL-6,and IL-12 in the thoracic aorta and the expression of IL-2,IL-6,The mRNA levels of IL-12 and TNF-α(p<0.01,p<0.05).Experiment 4:①A high-fat diet up-regulated 294 genes and down-regulated 193 genes in the GSE6735 data set;it up-regulated 48 genes and down-regulated 235 genes in the GSE45927 data set.②Significantly changed genes are mainly involved in key biological processes such as inflammatory response,lipid localization,and response to external stimuli.③GSEA enrichment analysis confirmed that under high-fat diet conditions,oxidative phosphorylation,lipogenesis and metabolism,and myogenesis pathways were significantly up-regulated;TNFα through NF-κB signaling and IL6 through JAK-STAT3 signaling pathways were also significantly enriched.set.Experiment 5:Moxibustion treatment can significantly reduce IL-1β(p<0.01,p<0.001),IL-2(p<0.01),IL-6(p<0.01,p<0.001),IL-8(p<0.05,p<0.001),TNF-α(p<0.05,p<0.01),IKK-α/IKK-(β(p<0.001,p<0.0001),IAP-2(p<0.001,p<0.01)and NF-The protein and mRNA expression levels of KBp50/NF-κBp65(p<0.0001)decreased the mRNA level of IAP-1(p<0.0001).It can significantly increase the phosphorylated protein and mRNA expression levels of PI3K(p<0.05,p<0.0001)and AKT(p<0.05,p<0.01),and reduce the expression of NF-κB and its accumulation in the nucleus.Compared with the moxibustion group,the moxibustion+PI3K inhibitor group had IL-1β(p<0.01,p<0.05),IL-2(p<0.01,p<0.05),and TNF-α(p<0.05,p<0.001),IKK-α/IKK-β(p<0.05,p<0.01),IAP-2(p<0.05,p<0.05)protein and mRNA expression significantly increased,IL-8 and NF-KBp50/NF-The mRNA level of κBp65 increased significantly(p<0.0001,p<0.01),the phosphorylated protein and mRNA expression levels of PI3K(p<0.0001)and AKT(p<0.01,p<0.001)significantly decreased,and the expression levels of IAP-1(p<0.01,p<0.001)protein and mRNA levels decreased significantly,and the expression of NF-κB and its accumulation in the nucleus increased.Compared with the moxibustion group,moxibustion+NF-κB agonist IL-2(p<0.001,p<0.01),IL-6(p<0.05,p<0.01),IL-8(p<0.05,p<0.0001),TNF-α(p<0.05,p<0.0001),IAP-2(p<0.05,p<0.05),NF-κBp50/NF-κBp65(p<0.01,p<0.0001)protein and mRNA The levels of IL-1β and TRAF-1 were significantly increased(p<0.05).The phosphorylated protein and mRNA expression levels of PI3K decreased significantly(p<0.05,p<0.001),and the expression of NF-κB and its accumulation in the nucleus increased.Compared with the moxibustion+PI3K inhibitor group,the protein expressions of IL-1β(p<0.01)and IL-6(p<0.05)in the moxibustion+NF-κB agonist group were significantly decreased(p<0.01).-8.The mRNA expression of IAP-1 increased significantly(p<0.05,p<0.01).The phosphorylated protein and mRNA expression levels of PI3K(p<0.01)and AKT(p<0.05)were significantly increased,and the expression levels of IAP-2(p<0.01,p<0.05),NF-κBp50/NF-κBp65(p<0.05,The protein and mRNA levels of IKK-α/IKK-β were significantly increased(p<0.01),the protein and mRNA levels of IKK-α/IKK-β were significantly decreased(p<0.05),and the expression of NF-κB and its accumulation in the nucleus were reduced.Conclusion:1.Moxibustion intervention can significantly reduce the expression of ox-LDL,VCAM-1,ICAM-1,ET-1,IL-6,and TNF-α by improving the roughness and lipid deposition of vascular endothelium,regulating blood lipids,increasing NO levels,and Improved vascular endothelial damage and chronic inflammatory response.2.In the effect of moxibustion on improving chronic vascular inflammation in different courses of moxibustion,there is the selectivity of the thoracic aorta and the abdominal aorta and the joint effects of "pro-inflammatory" and "anti-inflammatory".The 2-week moxibustion protects vascular endothelial function,improves damage and reduces ox-LDL through"pro-inflammatory" effects;the 4-week intervention exerts its effects through"anti-inflammatory" effects.3.The damage and inflammatory response of high-fat diet to blood vessels are common and site-selective.The thoracic aorta is more directly attacked by inflammation,while the lipid deposition in the abdominal aorta is more serious.Moxibustion can significantly improve the inflammatory damage of the thoracic and abdominal aorta.The changes in the abdominal aorta are mainly manifested by the significant decrease of TNF-α,while the thoracic aorta is mainly manifested by the decrease of IL-1β and IL-6,suggesting that The anti-inflammatory effects of moxibustion are common and selective in blood vessels at different locations.4.High-fat diet significantly changes the expression of genes related to key biological functions such as cardiovascular function,lipid metabolism,and inflammatory response in aortic tissue,suggesting that the PI3K/AKT/NF-κB signaling pathway plays a role in high-fat diet-induced Blood vessels play a central role in the process of chronic inflammation.5.Moxibustion can inhibit the activation of downstream IKK-α/IKK-β,IAP-1,IAP-2,NF-κBp50/NF-κBp65,and reduce the protein and mRNA expression of pro-inflammatory factors by activating the PI3K/AKT signaling pathway.,reducing the nuclear transfer of NF-κB and exerting its anti-inflammatory effect through the PI3K/AKT/NF-κB signaling pathway.