Study On Anti-atherosclerotic Effects Of Di’ao Xinxuekang In Rats Based On TLR4/MyD88/NF-κB Signaling Pathway

ObjectiveObserve the effect of Di’ao Xinxuekang（DXXK）on TLR4/My D88/NF-κB signaling pathway in atherosclerotic（AS）rats,and to explore its anti-atherosclerotic mechanism.MethodsMale SD rats were selected and reared adaptively for one week.Then they were randomly divided into normal control group,model group,atorvastatin group(2.0mg.kg^-1),DXXK high-dose group(100 mg.kg^-1),medium-dose group(30 mg.Kg^-1),low-dose group(10 mg.kg^-1),with 10 rats in each group.The normal group was fed with ordinary feed,and the other groups were fed with high-fat feed.Vitamin D₂（VD₂）6.0×10⁵IU·kg^-1was injected intraperitoneally at the beginning of the experiment,and VD₂1.0×10⁵IU·kg^-1was injected in the 3rd,6th,and 9th weeks.From the 10th week,it was administered by gavage for 8 weeks.Observe the general state of rats.The levels of triglyceride（TG）,total cholesterol（TC）,low density lipoprotein cholesterol（LDL-C）and high density lipoprotein cholesterol（HDL-C）in blood lipids were measured.Calculate rats cardiac index and atherosclerosis index（AI）.The levels of serum tumor necrosis factor（TNF-α）,interleukin（IL-6）and IL-1βwere detected by enzyme linked immunosorbent assay（ELISA.）The extent and distribution of AS plaque lesions in the aorta with Sudan IV stain.Hematoxylin-eosin staining（HE）was used to observe the pathological changes of the aorta.Masson staining was used to observe the expression of collagen fibers in aortic sinuses.The mRNA and protein expressions of TLR4,My D88 and NF-κB p65 in aortic tissues were detected by Real time quantitative polymerase chain reaction（RT-q PCR）and Western blot respectively.Results1.General state of rat:The normal control rats had shiny fur and good spirits,and their diet,urine output,and weight gain were stable.In the model group,the fur of the rats is dull,the color is yellow,the daily activities are significantly reduced,the spirit is depleted,the diet and water consumption are reduced,and the weight is significantly reduced.Compared with the model group,the rats in the drug-treated group gradually recovered their luster,and their weight increased to varying degrees,and the situation improved.2.Effects of DXXK on blood lipid levels in rats:Compared with the normal control group,the serum levels of TC,TG,and LDL-C in the model group increased significantly（P<0.01）,andthelevelsofHDL-Cdecreased significantly（P<0.05）.Compared with the model group,the serum TC,TG,LDL-C contents of the high and medium dose DXXK and atorvastatin groups were significantly reduced（P<0.05 or P<0.01）,and the HDL-C contents were significantly increased（P<0.01）.3.Effects of DXXK on cardiac index and AI in rats:Compared with the normal group,the cardiac index and atherosclerosis index of the model group were significantly higher than those of the normal group（P<0.01）.Compared with the model group,each dose of DXXK groups and atorvastatin group varying degrees of reduced the the cardiac index and AI（P<0.05 or P<0.01）.4.Effect of DXXK on aortic lesions in rats:After Sudan IV staining,the aortic intima of normal control rats was smooth and clean;The aortic intima of the model group contained a variety of red plaques（lipid droplets）,and the ratio of the plaque area to the total area of the aortic intima significantly increased（P<0.01）.Compared with the model group,the size and number of plaques in the aortic intima of the various dose groups of DXXK and the atorvastatin group were relatively reduced,and the ratio of plaque area to the total area of aortic intima significantly decreased（P<0.05 or P<0.01）.After aortic HE staining,the normal control group had normal aortic morphology,continuous and smooth vascular endothelium,and regular arrangement of vascular smooth muscle cells.In the model group,the aorta intimal thickened,the endothelium was discontinuous and incomplete,smooth muscle cells proliferated and arranged disorderly,and a large number of foam cells were gathered.The pathological changes of the aorta in each DXXK dose group and atorvastatin group were significantly reduced compared with the model group’s lesion severity and thickness.After Masson staining of the aortic sinus,the content of collagen fibers in the aortic sinus of the model group increased significantly,and the ratio of the area of collagen fibers to the total area of the arterial lumen significantly increased（P<0.01）.Compared with the model group,the content of collagen fibers in the various dose groups of DXXK and the atorvastatin group decreased to varying degrees,and the ratio of the area of collagen fibers to the total area of arterial lumen decreased（P<0.05 or P<0.01）.5.Effect of DXXK on serum IL-1β,IL-6 and TNF-αlevels in AS rats:Compared with the normal control group,the serum levels of TNF-α,IL-1β,and IL-6 in the model group were significantly increased（P<0.01）;Compared with the model group,the levels of TNF-α,IL-1β,and IL-6 in the middle and high dose groups of DXXK and the atorvastatin group were significantly reduced（P<0.01）,and the content of TNF-αin the low dose group of DXXK was reduced（P<0.01）.6.Effect of DXXK on expression of TLR4,My D88 and NF-κB p65 mRNA and protein in aorta of AS rats:RT-q PCR results show that compared with the normal control group,the expression levels of TLR4,My D88,NF-κB p65 mRNA in the aortic tissue of the model group were significantly increased（P<0.01）.Compared with the model group,the expression levels of TLR4,My D88,and NF-κB p65 mRNA in the aortic tissue of rats in each dose group of DXXK and atorvastatin group were significantly reduced（P<0.05 or P<0.01）.WB results show that compared with the normal control group,the expression levels of TLR4,My D88,NF-κB p65 protein in the aortic tissue of the model group were significantly increased（P<0.01）.Compared with the model group,the expression levels of TLR4,My D88,and NF-κB p65 protein in the aortic tissue of rats in each dose group of DXXK and atorvastatin group were significantly reduced（P<0.05 or P<0.01）.ConclusionDXXK has preventive and therapeutic effects on atherosclerosis in rats.One of its mechanisms of action may be by regulating TLR4/My D88/NF-κB signal transduction,reducing inflammatory responses and inhibiting atherosclerosis.