Study On The Effect And Mechanism Of Sanjie Tongmai Recipe On Atherosclerosis By Regulating Autophagy Factors

Objective:Under the theoretical guidance of"Blood stasis can be turned into phlegm and retained fluid"in traditional Chinese medicine,the effect of Sanjie Tongmai Recipe on atherosclerosis（AS）and its relationship with lipid metabolism and macrophage autophagy were explored through experimental studies.To clarify the mechanism of Sanjie Tongmai Recipe in the treatment of atherosclerosis,so as provides a modern theoretical basis for traditional Chinese medicine to treat atherosclerosis and the theory of"Blood stasis can be turned into phlegm and retained fluid".Methods:Ten male C57BL/6Cnc mice of 7 weeks old were selected as the blank group（Control Group）,and 50 male ApoE^-/-mice of 7 weeks old with C57BL/6cnc mice as background.After being fed with ordinary feed to 10 weeks of age,ApoE^-/-mice were divided into 5 groups of 10 mice each by random number table method,divided into model group,positive drug atorvastatin group（LPT Group）,Sanjie Tongmai Recipe with low,medium,and High-dose group（SJTM-L group,SJTM-M group,SJTM-H group）.Except that the blank group was given a normal diet,the other groups were fed with high-fat diet for 9 weeks to establish an AS model.Then the positive drug group was given atorvastatin calcium tablets（0.0026mg/g/d）,the Sanjie Tongmai low,medium and high dose groups were given the crude drug concentrations of 0.0085g/g/d,0.017g/g/d and 0.034g/g/d,the blank group and the model group were given 0.025ml/g/d saline.During the experimental intervention period,mice were not controlled for feed and water.After 4 weeks of intragastric administration,mice in each group were killed to isolate the heart and thoracoabdominal aorta.The following experiments were carried out,Experiment 1:To observe the effects of Sanjie Tongmai Recipe on aortic plaque tissue in mice and blood lipid.The pathological changes of mouse aorta were detected by HE staining,and Serum blood lipids（TC,TG,LDL-C,HDL-C）were measured by automatic biochemical analyzer.Experiment 2:To observe the effect of Sanjie Tongmai Recipe on lipid metabolism related proteins in aortic plaque tissue.Immunohistochemical method was used to detect the expression of lipid reverse transport related proteins ABCA1,ABCG1 and lipid uptake SR-A1,CD36 protein;and RT-PCR was used to detect the expression of ABC1,ABCG1,SR-A1,CD36 mRNA.Experiment 3:To observe the effect of Sanjie Tongmai Recipe on autophagy-related factors in macrophages of aortic plaque tissue.Immunohistochemical method was used to detect the expression level of autophagy protein LC3II/I.Western Blot method and RT-PCR method were used to detect the expression of autophagy factors Beclin1,p62 protein and mRNA.Results:Experiment 1:（1）After the intervention of drugs,the blood lipid level of ApoE^-/-mice AS model was improved in varying degrees,among which the blood lipid TC and LDL-C levels of the SJTM-H group,SJTM-M group and LPT group were significantly decreased（P<0.001,P<0.01,P<0.05）,the TG levels of SJTM-H group and LPT group were significantly decreased（P<0.05）,the TG levels of SJTM-M group were not significantly decreased（P>0.05）,the TC,TG and LDL-C levels of SJTM-L group were no significant difference（P>0.05）,but there was a downward trend;In terms of HDL-C,the levels of SJTM-H group,SJTM-M group and LPT group were significantly higher（P<0.001,P<0.01,P<0.05）,but there was no significant difference in SJTM-L group（P>0.05）,with an upward trend.（2）HE staining results:The aortic structure of the Model group was incomplete,the intima was not smooth,and there was significant plaques formation.After the intervention of drugs,SJTM groups’plaque showed a concentration dependent decrease,and no macrophages,foam cells and other cytokines were infiltrated,SJTM-H group and LPT group could significantly reduce plaque lesion.Experiment 2:（1）The results of immunohistochemistry showed that after drug intervention,the expressions of ABCA1 and ABCG1 in SJTM-H group and LPT group increased significantly（P<0.001,P<0.05）,the expression of ABCG1 in SJTM-M group increased significantly（P<0.05）,and there was no significant difference in ABCA1expression（P>0.05）;there was no statistical difference in ABCA1 and ABCG1 expression in SJTM-L group（P>0.05）.After drug intervention,the expression of lipid uptake protein decreased.The expression of SR-A1 and CD36 in SJTM-H group,SJTM-M group and LPT group decreased significantly（P<0.001,P<0.01,P<0.05）.There was no significant difference in the expression of SR-A1 and CD36 in SJTM-L group（P>0.05）,there is a downward trend.（2）Detection by RT-PCR method:After drug intervention,the expression of ABCA1and ABCG1mRNA in SJTM-H group,SJTM-M group and LPT group was significantly increased（P<0.01,P<0.05）,there was no statistically significant increase in ABCA1 and ABCG1mRNA in the SJTM-L group（P>0.05）;SR-A1 mRNA expression was significantly down-regulated in SJTM-H group,SJTM-M group,SJTM-L group and LPT group（P<0.001,P<0.01,P<0.05）,the expression of CD36mRNA in SJTM-H group and LPT group was significantly down-regulated（P<0.01）.Although there was no statistically significant difference in CD36mRNA expression in SJTM-M group and SJTM-L group（P>0.05）,there was still a downward trend.Experiment 3:（1）The results of immunohistochemistry showed that after drug intervention,the LC3II/I ratio in the SJTM-H group,SJTM-M group and LPT group was significantly increased（P<0.001,P<0.05）.There was no significant difference in the ratio of LC3II/I in SJTM-L group（P>0.05）.（2）Western Blot test results showed that the expression of Beclin 1 protein in SJTM-H group,SJTM-M group,SJTM-L group and LPT group was significantly up-regulated（P<0.001,P<0.05）;the expression of p62 protein in SJTM-H group,SJTM-M group and LPT group decreased significantly（P<0.001）,the expression of p62 protein in SJTM-L group decreased slightly,but there was no statistical difference（P>0.05）.（3）RT-PCR method found that the expression of Beclin-1 mRNA in SJTM-H group,SJTM-M group and LPT group was significantly up-regulated（P<0.01,P<0.05）,and there was no significant difference in the expression of Beclin-1 mRNA in SJTM-L group（P>0.05）;the expression of p62 mRNA in SJTM-H group and LPT group decreased significantly（P<0.01,P<0.05）,there was no significant difference in p62 mRNA expression between SJTM-M group and SJTM-L group（P>0.05）,but there was a downward trend.Conclusion:1.Sanjie Tongmai recipe can effectively improve the degree of aortic plaque lesions in ApoE^-/-mice AS model,and play an anti-arteriosclerotic effect.2.Sanjie Tongmai Recipe can significantly reduce the level of blood lipids（TG,TC,LDL-C）and increase the level of HDL-C in ApoE^-/-mice AS model,it can also up regulate the expression of cholesterol anti-transporter ABCA1,ABCG1,and down regulate the expression of cholesterol intake protein SR-A1 and CD36,thereby inhibiting the accumulation of lipid in aortic tissue.3.Sanjie Tongmai Recipe can regulate the autophagy related protein level of macrophages in aortic plaque tissue.By up-regulating the expression of LC3II/I and Beclin1,and down-regulating p62 expression,it can be seen that Sanjie Tongmai Recipe can regulate Macrophages autophagy plays a role in the prevention and treatment of AS.To sum up,Sanjie Tongmai recipe can prevent and treat AS by promotingautophagy of macrophages and regulating lipid metabolism in aortic plaque tissue.