Study On Anti Atherosclerotic Effect And Mechanism Of Mailuo Shutong Pill Based On NF-κB

Objective:1.Through the establishment of atherosclerotic（AS）rat model,the anti-AS mechanism of Mailuoshutong pill（MLSTP）was studied based on NF-κB pathway.2.To observe the changes of blood indexes and the effect of MLSTP on NF-κB pathway in AS rats,and to explore the mechanism of MLSTP inhibiting inflammation and the therapeutic mechanism of slowing down the occurrence and development of AS,so as to provide theoretical basis for clinical medication.Methods:1.In vivo experiment:6-week-old SD rats were fed adaptively for one week and randomly divided into seven groups according to body weight:blank control group（Control）,model control group（Model）,Atto vastatin control group(ATO,10mg·kg^-1),MLSTP high dose group（MH,6g·kg-1）,MLSTP medium dose group（MM,3g·kg-1）,MLSTP low dose group(ML,1.5g·kg^-1),NF-κB inhibitor group(MMP,MLSTP medium dose 3g·kg^-1+PDTC 100mg·kg^-1).The rat model of AS was induced by feeding with high cholesterol diet（HCD）for 12 weeks and intragastric administration of VD₃ every 4 weeks.12 weeks later,according to the changes of blood indexes and the degree of aortic disease in rats,the success of AS rat model was observed.The drug was then given by intragastric administration and continued to be fed with high-fat diet（D12492）,Control group and Model group were fed with ultra-pure water.After continuous intragastric administration for eight weeks,the changes of food intake and body weight of rats were observed every week,and the changes of plasma GLU,TC,TG,LDL-C and HDL-C were observed every 4 weeks.At the end of the eighth week,the rats were anesthetized with pentobarbital sodium,and the blood samples were taken from the abdominal aorta,and the changes of GLU,TC,TG,LDL-C and HDL-C were measured by blood index kit,the pathological changes of aorta and plaque and the pathological changes of liver and heart were observed by HE staining,and the levels of plasma glycosylated hemoglobin（GHb）and apolipoprotein B（apo B）were detected by ELISA kit.The distribution of NF-κB in rat aortic plaque was observed by immunohistochemistry,the mRNA level of inflammatory factors in NF-κB pathway was detected by RT-PCR,and the expression of NF-κB,IκBαand P-IκBαwas detected by Western Blot technique.2.Cell experiment:the model of HUVEC cell inflammation induced by LPS was selected.Firstly,the optimal concentration was screened by CCK-8 cell activity test,then when HUVEC grew and fused to about 80%in a 6-well plate,the culture medium was removed and PBS was washed once.The residual PBS,was absorbed and added to ATO(10μmol·L^-1)and 20%control serum（CS,Control serum）,20%MLSTP drug-containing serum,PDTC（100μmol·L-1）,PM（100μmol·L-1drug-containing serum）,LPS group only added culture medium,2 mL per well,6 holes in each group,and the Control group without medicine was further cultured in the incubator for 24 hours,then the medium containing drugs in the hole was discarded,washed by PBS,the Control group was removed,and the 2mL medium containing 10μg·mL-1 LPS was added.The cells were cultured in the incubator for 24 hours.The morphological changes of the cells were observed,and the expressions of NF-κB,IκBαand P-IκBαwere detected by Western Blot technique.Results:1.According to the results of blood indexes of rats,compared with Model group,there was no difference in fasting blood glucose between Control group and treatment group,but the blood indexes such as TG（P<0.01）,TC（P<0.001）,LDL-C（P<0.001）and HDL-C（P<0.01）in blank group were significantly lower than those in Model group.By measuring the blood indexes of rats in other administration groups,it was found that the high,middle and low dose of MLSTP and ATO group could significantly reduce the plasma levels of TG（P<0.01）,TC（P<0.001）,LDL-C（P<0.001）and Non HDL-C（P<0.001）in AS rats,and in a dose-dependent manner.According to the results of plasma ELISA detection in rats,compared with Model group,The level of GHb in the Control group was significantly lower than that in the model group（P<0.001）.It was found that the level of GHb in the administration group did not decrease the level of GHb in rats after administration.It was inferred that MLSTP could not reduce the fasting blood glucose of AS rats.After observing the level of plasma apoB in rats,it was found that the level of plasma apoB in rats treated with MLSTP was significantly lower than that in the model group（P<0.001）.2.According to the lipid deposition state of rat liver and the morphological changes of heart and aorta,compared with Control group,Model group showed obvious liver lipid deposition（P<0.001）,liver lipid metabolism disorder,inflammation,and a large amount of neutrophil infiltration and obvious inflammation in the enlarged and enlarged myocardial fiber space.The carotid intima-media thickness（CIMT）of rat aorta increased significantly（P<0.001）,and obvious atherosclerotic plaques appeared（P<0.001）.Compared with the Model group,the ectopic lipid deposition was improved in different degrees in the treatment group,and the aortic lesion was alleviated in the treatment group（P<0.001）,and the effect was the best in the MH and ATO groups.3.The expression of inflammatory cytokines in rat aorta was detected by RT-PCR.The results showed that NF-κB p50,IκBα,IL-1βand TNF-αin Model group were significantly higher than those in Model group（P<0.05）.Compared with Model group,the expression levels of NF-κB p50,IκBα,TNF-α,IKKβand IRAK4 in MH and ATO groups were significantly decreased,while the mRNA expression of NF-κB p65,IL-1β,IRAK1 and TAK1 genes had no statistical difference,but had a downward trend.The results of NF-κB p65（brown staining）in paraffin sections showed that compared with Control group,NF-κB p65 staining was stronger in Model group and drug treatment group（MH and ATO group）.Compared with the Model group,the NF-κB signal of aorta in the drug treatment group was significantly decreased.Consistent with the results of RT-PCR,the effect of MLSTP on the activation of NF-κB in AS rat aorta was investigated by detecting the expression of IκBαphosphorylation,IκBαand NF-κB.it was found that the phosphorylation level of IκBαand the expression of NF-κB in rat aorta were significantly decreased by MLSTP（P<0.001）.4.In vivo and in vitro verification experiment,it was found that after inhibition of NF-κB,the ability of MLSTP to reduce blood lipid,reduce aortic plaque area and CIMT in rats decreased,and could not play its normal anti-inflammatory effect.The cells in PM group were slender,loosely arranged,and had inflammatory reaction,and the number of parietal cells in PM group was higher than that in MLSTP group and PDTC group,and WB results showed that compared with PDTC group,The expressions of IKKβ,TAK1,NF-κB p65 and P-IκBαprotein in PM group were not further decreased.Conclusion:1.MLSTP can significantly reduce blood lipids and improve the state of ectopic lipid deposition in the liver.2.MLSTP can improve the morphological changes of heart and aorta in AS rats,and slow down the occurrence and development of AS.3.MLSTP can significantly reduce the mRNA and protein levels of inflammatory cytokines such as IκBα,TNF-αand IKKβin NF-κB pathway,and the ability of MLSTP to resist AS is decreased by inhibiting NF-κB.combined with in vivo and in vitro verification experiments,it is proved that NF-κB is the key target of MLSTP anti-AS.