| Objective:Atherosclerosis(AS)is the main pathological basis of acute coronary syndrome,hypertension,cerebral infarction and other cardiovascular and cerebrovascular diseases.Through the study of the pathogenesis of AS,it is particularly important to seek an effective mechanism inhibition the formation of AS and reduce the occurrence of cardiovascular and cerebrovascular events.AS is closely related to oxidative stress.Reducing the stress damage could block the further development of the AS,and NADPH oxidase(NOX)/reactive oxygen species(ROS)/nuclear factor kappa B(NF-κB)signal pathway is the important pathway in the process of oxidative stress,activate the signal pathway can stimulate the body oxidation reaction,accelerate the formation of foam cells,promote the proliferation and migration of vascular smooth muscle cells(VSMCs),aggravate the stress injury and expanding the lesion area can help reduce oxidative stress injury by inhibiting this signal pathway.At the same time,this signal pathway exists in different cells and plays an important role in AS,which has become a hot spot for treatment and intervention.In addition,gut microbiota,an important immune organ of the body,could initiate the growth and rupture of AS plaques by activating the immune system,changing cholesterol metabolism and producing bacterial metabolites.Therefore adjusting the structure of the gut microbiota would be a new therapeutic strategy for the prevention of AS cardiovascular disease.Jianpi huazhuo tiaozhi granules(JHTG),is an in-hospital preparation of the affiliated hospital of Jiangxi university of traditional Chinese medicine,which takes strengthening the spleen and activating turbidity as the core,and mainly target on the heart,spleen and gut,has been widely used in the prevention and treatment of AS.However,there is no systematic and detailed theoretical study about JHTG on anti-AS.In previous studies,it was found that JHTG can reduce oxidative stress injury response and play a role in preventing and treating AS,but the specific mechanism was still unclear.In addition,JHTG has been found to significantly improve the stool of AS patients in clinical practice,and a large number of studies have shown that Chinese medicine monomer or compound preparation has the ability to regulate intestinal flora.Therefore,this study systematically elaborated and analyzed the traditional Chinese medicine theory of JHTG in the treatment of AS,and proposed that the lesions of AS were closely related to the viscera of "heart-spleen-gut".Based the theory,the main treatment methods of invigorating spleen,invigorating qi and activating blood to turn turbidity can effectively delay the pathological changes of AS.Moreover,the theory of "heart-spleen-gut" was verified by cell experiment and animal experiment,and the role of JHTG in NOX/ROS/NF-κB signal pathway and gut microbiota structural was discussed respectively.Methods:Experiment 1: RAW264.7 macrophages were stimulated by oxidized low density lipoprotein(ox-LDL)with a final concentration of 100μg/mL to form an oxidative injury model.The cells were divided into 7 groups: blank group(conventional medium culture),ox-LDL group(ox-LDL stimulation),2.5% JHTG group(2.5% JHTG drug serum added to ox-LDL group),5% JHTG group(5% JHTG drug serum added to ox-LDL group),10% JHTG group(10% JHTG drug serum added to ox-LDL group),NF-κB inhibitor group(adding 200μmol/L NF-κB inhibitor to ox-LDL group)and ox-LDL+statin group(adding 1μmol/L of atorvastatin to ox-LDL group);cell apoptosis and survival were detected by flow cytometry and MTT assay,malondialdehyde(MDA)and superoxide dismutase(SOD)were determined by enzyme-linked immunosorbent assay,and NOX/ROS/NF-κB signal pathway was detected by fluorescence probe,western blot and real-time fluorescence assay for ROS,NOX4,p22 phox and inhibitor of NF-κB kinase(IKK)-α、IKK-β.Experiment 2: the ox-LDL stimulating VSMCs form the oxidative damage model,grouping like experiment 1.Using the determined by MTT method to test the cell proliferation,also using enzyme-linked immunosorbent method of oxidative damage of MDA and SOD parameter detection,and using fluorescence detection,protein immunoblot method and real-time fluorescence quantitative method for testing NOX/ROS/NF-κB signal pathways related parameters.Experiment 3: The AS rabbit model was established with high-fat feed,and 60 rabbits were divided into: blank group(ordinary feed),high-fat group(high-fat feed),JHTG low-dose group(high-fat feed with 1 g/kg/d JHTG),JHTG medium-dose group(high-fat feed with 2 g/kg/d JHTG),the JHTG high-dose group(high-fat feed with 4 g/kg/d JHTG)and high-fat+statin group(high-fat feed with 2 mg/kg/d atorvastatin),the use of ultrasonic detection,HE and oil red staining for morphological evaluation,kits were used to detect serum lipid levels,highthroughput sequencing was used to detect the structure of gut microbiota.Results:Experiment 1: Compared with blank group,ox-LDL group apoptosis rate increased,survival rate decreased,MDA content increased significantly,SOD activity decreased,and NOX/ROS/NF-κB signal pathway expression higher(P﹤0.05);compared with ox-LDL group,5% JHTG group,10% JHTG group of cell survival rate increased,MDA and SOD levels were significantly decreased/increased,NOX/ROS/NF-κB signal pathway has significantly down-regulated(P﹤0.05).Experiment 2: Compared with blank group,the abnormal proliferation of VSMCs in ox-LDL group,MDA and ROS content increased significantly,SOD activity decreased,NOX/ROS/NF-κB signal pathway expression higher(P﹤0.05).Compared with ox-LDL group,the cell proliferation in 5% JHTG group and 10% JHTG group was inhibited,significantly change in MDA and SOD,the expression of NOX/ROS/NF-κB signal pathway was significantly down-regulated(P﹤0.05),and 10% JHTG group showed the most significant change(P﹤0.01).Experiment 3: Compared with blank group,high-fat group is clearly plaques visible at cervical aortic,has plenty of foam cells in microscopically,serum lipid levels were increased,Bacteroidetes,Bacteroides and Akkermansia decreased,Firmicutes and Clostridium increased(P﹤0.05).Compared with high-fat group,JHTG group and highfat+statin group of plaque reduce,foam cells and TC,TG,LDL-C levels decreased significantly,HDL-C levels continue to rise(P﹤0.05),TC and LDL-C in high-dose JHTG group decreased significantly(P﹤0.01).JHTG groups appear Bacteroidetes,Bacteroides,Akkermansia,Lactobacillus and Bifidobacterium increased gradually,Firmicutes and Clostridium decreased gradually(P﹤0.05).Conclusion:1.JHTG down-regulates the NOX/ROS/NF-κB signal pathway and reduces the damage to macrophages caused by oxidative stress,reduces the excessive proliferation of VSMCs.It is suggested that JHTG could improve the antioxidant capacity of cells to prevent and treat of AS lesions through this signal pathway.2.JHTG can change the structure of gut microbiota,appear Bacteroidetes,Bacteroides,Akkermansia,Lactobacillus and Bifidobacterium increased gradually,Firmicutes and Clostridium decreased gradually,reduce the formation of AS plaque and reduce the level of blood lipid,which provides experimental basis for the application and promotion of JHTG in the clinical treatment of AS. |
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