| Objective: Based on the CD40/CD40 L pathway,the effects of Sanjie Tongmai Recipe on the pathological changes of the common carotid artery,blood lipid levels and signal molecules related to CD40/CD40 L pathway in the common carotid artery of atherosclerosis rats were observed by means of animal experiments and biological research methods.We hope to find out the anti-Atherosclerosis mechanism of Sanjie Tongmai Recipe,and provide reliable experimental evidence for theory of "blood stasis can change into phlegm and retained fluid".Methods:Experiment one: 60 male Sprague-Dawley rats were fed for 1 week,and 10 rats were randomly selected as sham operation group.The rats in this group were only ligated with carotid artery and no balloon injury.The other 50 rats underwent carotid artery balloon injury.One week after surgery,except for the sham operation group,the rats in the other groups were fed with high-fat diet for 6 weeks.According to the preliminary pre-experiment results,the entire modeling period was determined to be 7 weeks.At the end of 7th weak of modeling,the AS model can be successfully replicated.Measure the blood lipids of each group rats and pick up the ungrouped rats.Based on blood lipid results,these ungrouped rats were randomly divided into AS model group,simvastatin group,the low-dose of traditional Chinese medicine group,the medium-dose of traditional Chinese medicine group and the high-dose of traditional Chinese medicine.After the group was completed,the rats in each drug group were given the corresponding drug intervention.Simvastatin group was given simvastatin(10 mg/Kg/d),The low-dose,medium-dose and high-dose of Chinese medicine groups were given separately the concentration of Sanjie Tongmai Recipe 16g/Kg/d,32g/Kg/d,64g/Kg/d.In order to ensure the balance of the experiment,the sham operation group and the AS model group was given an equal volume of saline(100g/1ml/d)for 4 weeks.The rats in each group were sacrificed at the end of the 12 th week.The pathological changes of the common carotid artery were observed by HE staining.The contents of TC,TG,LDL-C and HDL-C in the serum were detected by spectrophotometer.The Serum ox-LDL levels of the rats were detected by ELASA.Experiment two: The model establishment and drug intervention of the rats were the same as in Experiment one.After the rats were sacrificed,the serum levels of TNF-?,sCD40 L and IL-1? were detected by ELISA.The protein expression level of CD40,CD40 L,NF-?Bp65,MCP-1 and LOX-1 in the common carotid artery were detected by Western blotting;The protein expression of ICAM-1,VCAM-1 and MMP-9 in rat common carotid artery were detected by immunohistochemistry;The gene expression of CD40,CD40 L,MCP-1,LOX-1 and NF-?Bp65 in rat common carotid artery were detected by RT-PCR.Results:Experiment one:1.The results of HE staining of the common carotid artery showed that the common carotid artery structure was intact,the intima was continuous,the smooth muscle cells and the elastic fiber plate were arranged neatly;However,the structure of the common carotid artery in the AS model group was obviously destroyed,and a large number of foam cells and macrophages were observed and we can also find the phenomenon of lymphocyte infiltration;The low-dose group of Sanjie Tongmai Recipe was not smooth in the intima of the common carotid artery,and the accumulation of subendothelial foam cells and inflammatory cells was significantly reduced compared with the AS model group;the simvastatin group,Sanjie Tongmai prescription medium dose group,The common carotid artery lesions in the high-dose group of Sanjie Tongmai Recipe were significantly relieved,and no obvious foam cells and inflammatory cell infiltration were observed.2.The results of TC,TG,LDL-C and HDL-C in the serum of each group showed that,after drug intervention,the levels of TC,TG and LDL-C in each drug group were significantly lower than those in the AS model group,(P<0.01),the HDL-C in the low-dose group of Chinese medicine was significantly increased(P<0.05),and more significantly increased in the the other drug grops(P<0.01).Compared with the simvastatin group,the high-dose group of traditional Chinese medicine had a weaker effect on reducing TC(P<0.05),but it was effective in reducing LDL-C(P<0.05),and significantly reduced TG and elevated HDL-C(P<0.01),the effect of reducing TC in the middle dose group was weaker than that in the simvastatin group(P<0.05),but it was better than the simvastatin group in reducing TG(P<0.05),and there was no significant difference between the two groups in the improvement of LDL-C and HDL-C(P>0.05).The low-dose group of Chinese medicine was weaker than the simvastatin group in improving TC(P<0.01),but the results of TG,LDL-C,HDL-C were no significant difference(P>0.05).3.The results of ox-LDL in the serum of each group showed that,the ox-LDL in the low-dose group of Chinese medicine was significantly lower after drug intervention compared with the AS model group(P<0.05),while the ox-LDL in the simvastatin group,the high-dose group and the low-dose group of Chinese medicine was decline more significant(P<0.01),and the Chinese medicine high-dose group was superior to the simvastatin group(P<0.05).Experiment two:1.The serum levels of TNF-?,sCD40 L and IL-1? in rats were significantly higher than those in the sham operation group(P<0.01).After drug intervention,the serum levels of TNF-?,sCD40 L and IL-1? in AS rats were significantly decreased(P<0.01).The simvastatin group was equivalent to the Chinese medicine middle dose group in the reduction of TNF-?,sCD40 L,IL-1?(P>0.05),and its efficacy was significantly stronger than the traditional Chinese medicine low dose group,but weaker than the traditional Chinese medicine high dose group(P <0.05).2.The detection of CD40/CD40L-related protein in the common carotid artery of rats showed that,the expression of CD40,CD40 L,LOX-1,NF-?B p65(nucleus)and MCP-1 in the common carotid artery of the AS model group was extremely significant compared with the sham operation group(P<0.01).Compared with the AS model group,the expressions of CD40,CD40 L,LOX-1,and NF-?B p65(nucleus)in the simvastatin group,the low-dose group of Chinese medicine,the middle dose group,and the high-dose group of Chinese medicine were significantly lower(P<0.01).However,in the LOX-1 and MCP-1 groups,the low-dose group of traditional Chinese medicine showed a significant decrease trend(P<0.05),but the reduction rate was more obvious in the simvastatin group,the middle dose group and the high-dose group(P<0.01).3.The results of CD40/CD40 L related gene detection in rat common carotid artery showed that the expression of CD40 mRNA,CD40 L mRNA,LOX-1 mRNA,MCP-1 mRNA and NF-?Bp65 mRNA in AS model group was significantly increased(P<0.01).After the intervention of the corresponding drugs,the expression of LOX-1 mRNA in the common carotid artery of simvastatin group was significantly decreased(P<0.05),and the expression of CD40 mRNA,CD40 L mRNA,MCP-1 mRNA and NF-?Bp65 mRNA was decreased more significant(P<0.01);The low dose group of Chinese medicine can significantly inhibit the expression of CD40 L mRNA and LOX-1 mRNA(P<0.05),and have more powerful effect on inhibiting the expression of CD40 mRNA,MCP-1 mRNA and NF-?Bp65 mRNA(P<0.01).The expression of LOX-1 mRNA in the middle dose group of Chinese medicine was significantly decreased(P<0.05),and the expression of CD40 mRNA,CD40 L mRNA,MCP-1 mRNA and NF-?Bp65 mRNA was decreased more significant(P<0.01).The expressions of CD40 mRNA,CD40 L mRNA,LOX-1 mRNA,MCP-1 mRNA and NF-?Bp65 mRNA in the common carotid arteries of high dose group of traditional Chinese medicine were significantly lower than those in the AS model group(P<0.01).4.The immunohistochemical results of ICAM-1,VCAM-1 and MMP-9 in the common carotid artery of rats showed that,there was almost no positive staining in the sham operation group,indicating that no expression of ICAM-1,VCAM-1 and MMP-9 was observed.A large number of brown-yellow granules were observed in the AS model group,suggesting that there have a large number of ICAM-1,VCAM-1,and MMP-9 expression;Compared with the atherosclerosis model group,the brown-yellow granules decreased in simvastatin group,low-dose group of traditional Chinese medicine,medium-dose group of traditional Chinese medicine and high-dose group of traditional Chinese medicine,suggesting that the expression of ICAM-1,VCAM-1 and MMP-9 in the common carotid artery were decreased after drug intervention.According to the results of semi-quantitative analysis,the percentage of ICAM-1 positive area in the AS model group was significantly larger than that in the sham operation group(P<0.05),and the VCAM-1 and MMP-9 have a more pronounced increase(P<0.01).Compared with the AS model group,percentage of ICAM-1 positive area of the simvastatin group was decreased significant(P<0.05),and the percentage of VCAM-1 and MMP-9 were decreased more significant(P<0.01).The expression of ICAM-1 and VCAM-1 in the low-dose group of traditional Chinese medicine group were significantly decreased(P<0.05),the reduction of MMP-9 was more significantly(P<0.01).The percentage of positive area of ICAM-1,VCAM-1 and MMP-9 in the middle dose group and the high dose group of traditional Chinese medicine was significantly lower than that in AS model group(P<0.01).Conclusion:1.Rat AS lesion model can be established by balloon injury combined with high-fat diet feeding.The intervention of Sanjie Tongmai Recipe can effectively improve the lesions of common carotid artery AS in rats.2.Sanjie Tongmai Recipe can significantly reduce the levels of TC,TG,LDL-C and ox-LDL in serum of AS rats,and increase the level of HDL-C,and the low dose group of Chinese medicine is equivalent to the simvastatin group.The middle dose group and the high dose group of Chinese medicine were significantly better than the simvastatin group,and had good lipid-lowering effect.3.Sanjie Tongmai Recipe can reduce the expression of TNF-?,sCD40 L and IL-1? in serum of AS rats,and reduce the expression of proteins and genes of CD40,CD40 L,NF-?B p65,MCP-1 and LOX-1 in the common carotid arteries of AS rats.It can also reduce the expression of VCAM-1,ICAM-1 and MMP-9 in the common carotid artery of AS rats.All these results suggest that Sanjie Tongmai Recipe may exert anti-atherosclerosis effect by interfering with CD40/CD40 L signal transduction. |
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