| Ob jective:The purpose of the study was through animal experiment to observe the effects of sappanwood ethyl acetate extract on abdominal aorta pathological morphology,blood fat,interleukin-1?,abdominal aorta TLR4mRNA,N F-?B p65mRNA,and TLR4,NF-?B p65 proteins of atherosclerosis rats model.And through cell experiment observe the effects of containing serum of sappanwood ethyl acetate extract on apoptosis,tumor necrosis factor-a,cell supernatant TLR4mRNA,N F-?B p65mRNA,and TLR4,NF-?Bp65 proteins of lipopolysaccharide induced endothelial cell injury model.Methods:Experiment One:We selecteed 60 SD rats(male,200±20g)and randomly divided into 6 groups according to random number table,namely blank control group,model control group,low dose group,middle dose group,high dose group and simvastatin group.Each group had 10 rats.Rats of the blank control group were fed with normal diet.Rats of other groups were given intraperitoneal injection of vitamin D3 combined with high fat diet for 12 weeks.After 12 weeks,rats of eblank control group and model control group were given equal volume of sodium carboxymethyl cellulose suspension,other groups were givein the corresponding dose of sappanwood ethyl acetate extract and simvastatin suspension,once time a day for 4 weeks.After 4 weeks,pathological changes of abdominal aorta endothelial morphology were observed by HE staining.Serum blood fat,interleukin-1? levels were detected by EL AS A method.Expression of abdominal aorta TLR4mRNA?NF-?B p65mRNA was detected by Real-time fluorescent quantitative PCR.Experiment Two:Divided human umbilical vein endotlhelial cells into 7 groups,namely blank control group,model control group,low dose group,middle dose group,high dose group,simvastatin group and prevention group.Control group:human umbilical vein endothelial cells were incubated in endothelial cell culture medium 24h.Model control group:LPS solutio-n(1?g/mL)was used to treat human umbilical vein endothelial cells 12h,and then incubated with 12h.Low dose group:LPS solution(1?g/mL)was used to treat human umbilical vein endothelial cells 12h,and then treated with low dose serum containing ethyl acetate extract 12h.Middle dose group:LPS solution(1?g/mL)was used to treat human umbilical vein endothelial cells 12h,and then treated with middle dose serum containing ethyl acetate extract 12h.High dose group:LPS solution(1?g/mL)was used to treat human umbilical vein endothelial cells 12h,and then treated with high dose serum containing ethyl acetate extract 12h.Simvastatin group:LPS solution(1?g/mL)treatment of endothelial cells 12h,after the introduction of a newe generation of drugs containing simvastatin treatment of 12h.The middle dose prevention group:pretreatment with 12h in the middle dose of ethyl acetate extract then the human umbilical vein endothelial cells were incubated in the complete medium by 12h.Cell apoptosis was detected by flow cytometry.Serum TNF-? levels were detected by ELISA method.Expression of abdominal aorta TLR4mRNA and NF-?B p6 5mRNA was detected by Real-time fluorescent quantitative PCR.Expressions of channel proteins TLR4 and NF-?B p65 were determined by Western-blot method.Results:Experiment One:1.Pathological changes of abdominal aorta endothelial morphology were observed by HE staining results showed:the endothelial cells of abdominal aorta of low dose group,middle dose group,high dose group,simvastatin group and prevention group were swelling,smoot muscle cells arranged in mild disorder,the number of nuclei increased,compared with the model control group,the pathological injury was significantly reduced.2.Blood fat results:showed that compared with the model control group,the serum triglyceride and total cholesterol level of the middle dose group,high dose group,simvastatin grorup were decreased(P<0.05).Compared with the model control group,the low density lipoprotein-cholesterol level of the low dose group,middle dose group,high dose group,simvastatin group were decreased(P<0.05)..3.ELISA results showed compared with the model control group,the serum interleukin-1? level of the middle dose group,high dose group,simvastatin group were decreased(P<0.05).4.Real-time PCR results showed that compared with the model control group,the expression of TLR4mRNA,N F-?B p65mRNA of the middle dose group,high dose group and simvastatin group were significantly decreased(P<0.05).5.The cell apoptosis results showed:compared with the human umbilical vein endothelial cells injury induced by LPS in the model control group,the apoptosis ratio of middle dose group,high dose group,simvastatin group and prevention group was decreased,the difference was statistically significant(P<0.05).6.ELISA results showed that the tumor necrosis factor-? level of the umodel control group was signaificantly increased compared with the blank control group(P<0.05).Compared with the model control group,the serum tumor necrosis factor-? level of the middle dose group,high dose group,simvastatin group and the middle dose prevention group were decreased(P<0.05).7.Real-time PCR results showed that compared with the model control group,the expression of TLR4mRNA,NF-?B p65mRNA of the middle dose group,high dose group,simvastatin group and prevention group were significantly decreased(P<0.05).8.Western Blot results showed that compared with the model control group,the expression of TLR4,NF-?B p65 protein of the middle dose group,high dose group,simvastatin group and prevention group was significantly decreased(P<0.05).Conclusion:1.The sapaanwood ethyl acetate extract could improve the p athological changes of abdominal aorta in atherosclerosis rats m odel,reduce the triglyceride,total cholesterol,low density lipo protein-cholesterol and interleukin-1 level of atherosclerosis rast model.2.The sapaanwood ethyl acetate extract could improve the morphology of human umbilical vein endothelial cells induced by LPS,reduce the apoptosis rate of endothelial cells,reduce the TNF-?level of human umbilical vein endothelial cells induced by LPS3.The capanwood ethyl acetate extract could decrease the expression of TLR4mRNA and NF-?B p65mRNA of the atherosclerosis rast model and human umbilical vein endothelial cells induced by LPS,inhibit activation of TLR4/NF-?B pathway,which may be one of the mechanisms of prevention and treatment of atherosclerosis. |
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