Protection Of Kangshuanyihao Formula On Inflammatory Reaction Through Sirt1/TLR4/NF-κB Signaling Pathway In Endothelial Injury

Objective:To observe the improvement of "kangshuanyihao" formula(safflower,leeches and hematoxylin)on the endothelial injury induced by high fat.The endothelial injury rat model is induced by intraperitoneal injection of vitamins(VitaminD3 VD3)combined with high-fat diet.To observe the effect of "kangshuanyihao" formula on improving the blood lipid level of the rat model.To observe the regulation ability on various inflammatory factors of "kangshuanyihao"formula.Methods:To make the analysis of the active ingredients of "kangshuanyihao" formula by high performance liquid chromatography(HPLC);(2)animal experiments:Modeling the endothelial injury model refer to intraperitoneal injection of VD3 combined with high fat diet.Except the blank group,all rats would be dealed with modeling treatment after one week adaptive feeding with normal diet.One-t ime intraperitoneal injection of VD3 of 600,000U.kg-1,and add VD3 100,000 U · kg-1 in the next 3,6,9 week,then feed the rats with high-fat diet for 9 weeks instead of nor mal diet.60 SD rats were randomly divided into 7 groups:high dose group,middle dos e group,low dose group,positive drug group(rosuvastatin),combination group(positive medicine group and middle dose group),And another 10 SD rats as blank control grou p.To observe HE staining results of aortic endothelium.Serum levels of total cholesterol(T-CHO),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lip oprotein cholesterol(HDL-C)were measured.Interleukin-1 β(IL-1 β),interleukin-6(IL-6),int erleukin-12(IL-12)and tumor necrosis factor-α(TNF-α)were measured by ELISA.The le vels of IL-1β,interleukin-6(IL-6),interleukin-12(IL-12)and tumor necrosis factor-α(TNF-α)were detected by immunohistochemistry.Using Western Blot and realtime-PCR metho d to study the mechanism of anti-inflammatory effect of "kangshuanyihao" formula.(3)Cell experiments:The HUVEC model was induced by ox-LDL in reference to the related literature.Using HUVEC cells transferred to 5-7 generation.After pre-culture,differ ent concentrations of ox-LDL(10ug/ml～160ug/ml)were given to observed HUVEC pr oliferation inhibition with ox-LDL by CCK8 test,in order to determine the induced conce ntration of ox-LDL;Cells of 5-7 generation were first grown in starvation culture,then add different concentrations of "kangshuanyihao" formula(Final concentration of 5%,10%,20%,40%).CCK8 method was also used to observe the effect of "kangshuanyihao" form ula on proliferation and cytotoxicity of HUVEC,to determine intervention concentration of the "kangshuanyihao" formula drug-containing serum.The cells were divided into blank g roup,control group,low serum concentration group,middle serum concentration group,a nd high serum concentration group.The levels of interleukin-1 β(IL-1 β),interleukin-6(IL-6),interleukin-12(IL-12)and tumor necrosis factor-α(TNF-α)were measured by cell sup ernatant in the cell ELISA experiment.The Sirtl/TLR4/NF-κB signal pathway was further validated by Western Blot assay with extracted protein of each group.Result:(1)High-performance liquid chromatography:According to the experimental steps of high performance liquid chromatography,isolated from the standard we found:1.Urine nucleoside;2.Hypoxanthine;3.Carnosine;Safflower yellow A;5.Brazilian hematoxylin.(2)Animal experiment:①After 9 weeks of modeling,the emergence of high-fat-induced endothelial injury was performed in SD rats.Through the HE staining of the thoracic aorta,the rats in the model group were found to have irregulr neointimal manifestation.And endothelial cells arranged disorder with poor continuity.At the same time,there were observed accumulation of a large number of foam cells,proliferation of vascular smooth muscle cells;In addition,the body weight of the model group was significantly reduced compared with the blank group.②HE staining results:After the rats were treated with the drug,HE staining results show that,Compared with the model group,staining results of the endometrium of’kangshuanyihao"formula,positive drug group and combination group have been greatly improved,endometrium had more relative arrangement and was more smooth.Only a small amount of foam cells accumulated and smooth muscle hyperplasia;③Blood lipid levels:Compared with the blank group,The levels of TC,TG and LDL in the model group were increased,HDL decreased,and was statistically significant(p<0.01);Compared with the model group,in addition to the low dose group,the level of T C were significantly decreased,with statistical significance(p<0.01)in high and middle d ose group,positive drug group,combination group.Compared with the model group,the le vel of TC in low dose group downward trend is smaller,but still statistically significant(p<0.05).The degree of TG level is just similar to TC level,with statistical significance(p<0.01,p<0.05).LDL-c decreased and was statistically significant(p<0.01,p<0.05)in the in tervention group compared with the model group(p<0.01,p<0.05).Compared with the mod el group,the improvement of HDL-c in the high dose group and the middle dose group was statistically significant(p<0.01 and p<0.05).There was no significant difference betwee n the low dose group and the model group(p>0.05).④ Levels of Inflammatory factor:Immunohistochemical results suggest that,compare d with the blank group,IL-1β,IL-6,IL-12 and TNF-α level were increased in model g roup,Compared with the model group,The expression of inflammatory factors decreased;E LISA method was used to determine the level of inflammatory factors in serum,The level s of IL-1β,IL-6,IL-12 and TNF-α in the model group were significantly higher than those in the blank group and the model group(p<0.01).Compared with the model group,i nflammatory factors were decreased,with statistical significance(p<0.01);⑤ Sirtl/TLR4/NF-κB signaling pathway experimental results:By WB and rt-PCR ex perimental methods,the results showed that the level of Sirtl protein in model group was significantly lower than that in blank group(p<0.01),TLR4 and NF-κB were significa ntly up-regulated(p<0.01).After intervened by the intervention group of "kangshuanyihao"formula,the expression level of Sirtl protein increased,TLR4 and NF-κB decreased(p<0.01 or p<0.05),and the level of adjustment was correlated with the dose of’kangshuanyiha o" formula.PCR results suggest that,the expression of Sirtl in the model group was signi ficantly lower than that in the blank group(p<0.01),and the expression of TLR4 and NF-κB was significantly increased(p<0.01),the results of "kangshuanyihao" formula intervent ion were consistent with WB results.(3)Cell experiments:①ox-LDL and "kangshuanyihao" formula drug-containing serum intervention concentr ation:1.In the same phase of drug treatment,With the increase of ox-LDL concentration,Th e relative viability of HUVEC cells decreased gradually,The inhibitory rate of ox-LDL on HUVEC cell proliferation was gradually increased,and the inhibitory effect was dose-de pendent.After 24 hours of intervention,it was found that 80 μg/mL ox-LDL could inhibit the proliferation of HUVEC cells,the relative viability of HUVEC cells was significantly decreased(p<0.05).Finally,HUVEC cells were treated with ox-LDL at a concentration o f 80 μg/mL,to carry out the relevant role and mechanism of the study.2.Under the same drug treatment time,the experimental results show that,with the in crease of serum concentration,HUVEC cells grew better,and in a dose-dependent manner.T he survival rate of HUVEC cells treated with 40%concentration of serum was significan tly different from that of 20%(p<0.05).Finally,we selected 5%,10%,20%of the drug-c ontaining serum concentration as the three intervention dose.The effects of ox-LDL-induce d HUVEC cells on the damage and the mechanism of action were investigated by interv ention with the three concentrations of serum.② inflammatory factor levels:ELISA method was used to test the level of inflammat ory factors in cell supernatant,the results showed that the levels of IL-1β,IL-6,IL-12 a nd TNF-α in ox-LDL group were significantly higher than those in blank group(p<0.01).Compared with ox-LDL intervention group,the inflammatory cytokines decreased with the statistical significance(p<0.01),and was correlated with the concentration.③ Sirtl/TLR4/NF-κB signal pathway experimental results:Compared with the blank control group,the level of Sirtl protein in the ox-LDL inte rvention group was significantly down-regulated,and the levels of TLR4 and NF-κB pr otein were significantly up-regulated(p<0.01).After "kangshuanyihao" formula drug-contai ning serum intervention,Sirtl protein levels were significantly increased,the corresponding,TLR4,NF-κB protein levels were significantly reduced,and the level of protein adjustm ent and the concentration of drug-containing serum are related.The protein concentration of 10%and 20%serum was statistically significant(p<0.01)compared with the model gr oup,while compared with the model group,the adjustment trend is lower in 5%concentrat ion of drug-containing serum to adjust the protein(p<0.05);After adding the Sirtl protein inhibitor,in the "kangshuanyihao" formula drug-containi ng serum intervention in ox-LDL-induced HUVEC,Sirtl up-regulation had a significant di fference compared with the absence of protein inhibitors(p<0.01).Meanwhile,there were si gnificant differences in TLR4 and NF-κB protein expression between the two groups(p<0.05).After adding TLR4 protein inhibitor,in the "kangshuanyihao" formula drug-containing serum in ox-LDL-induced HUVEC,there was a significant difference in TLR4 downregul ation(p<0.01)compared with that without protein inhibitor.In addition,NF-κB protein e xpression between the two groups were also significantly different(p<0.05).Sirtl protein expression between the two groups was without significant differences.Conclusion:(1)high performance liquid chromatography can be used to carry out the analysis of active ingredients of traditional Chinese medicine compound;(2)To give SD rats intraperitoneal injection of VD3 combined with high fat diet can be used as an animal model of endothelial injury model;(3)"kangshuanyihao,formula can improve the blood lipid level in serum of rats wit h endothelium injury induced by high fat,and can reduce TC,TG,LDL-c,increase HDL-c to relieve endothelial injury;(4)"kangshuanyihao" formula can reduce the levels of IL-1β,IL-6,IL-12 and TNF-a in supernatant of HUVEC cells induced by ox-LDL and serum induced by high fat,and the levels of IL-1β,IL-6,IL-12 and TNF-α in the thoracic aorta can also be decr eased "kangshuanyihao" formula,which means "kangshuanyihao" formula plays a role in i nhibiting inflammation to improve endothelial injury;(5)Sirtl/TLR4/NF-κB signaling pathway may be a new pathway for inflammatory intervention in endothelial injury;"kangshuanyihao" formula with Yiqi Huoxue function can improve the inflammatory response through Sirtl/TLR4/NF-κB signaling pathway to inhibit high-fat induced endothelial injury,thereby alleviating the process of atherosclerosis.