| Objective:1 Clinical researchBased on data mining technology,this paper analyzes the prescription law of Professor Huang Yongsheng in the treatment of coronary heart disease angina pectoris（phlegm and blood stasis syndrome）,to provide a reference for clinical practice.2 Experimental researchTo explore the effect of Sanjie Tongmai Fang on autophagy of macrophages,and take the PI3K/AKT/mTOR signaling pathway as an entry point for mechanism research to explore whether the prescription can mediate macrophage autophagy through the PI3K/AKT/mTOR signaling pathway to improve atherosclerosis,provide the experimental basis for the treatment of atherosclerosis with Sanjie Tongmai Fang,and provide a reference for traditional Chinese medicine treatment of coronary heart disease.Methods:1 Clinical researchThe traditional Chinese medicine prescriptions of patients with coronary heart disease angina pectoris（phlegm and blood stasis syndrome）diagnosed in the outpatient department of Professor Huang Yongsheng of the Affiliated Hospital of Changchun University of traditional Chinese medicine from 2010 to 2020 were collected,and data mining was carried out by using the methods of frequency analysis integrated by traditional Chinese Medicine Inheritance auxiliary system（v2.5）and SPSS cluster analysis.2 Experimental researchExperiment 1 was divided into six groups,namely control group（Control group）,model group（Model group）,Sanjie Tongmai Fang group（SJTMF group）,positive drug control group（Ato group）,and autophagy inhibitor group（3-MA group）,autophagy inhibitor+Sanjie Tongmai Fang group（3-MA+SJTMF group）;experiment 2 was divided into five groups,namely control group（Control group）,model group（Model group）,Sanjie Tongmai Fang group（SJTMF group）,PI3K inhibitor group（LY294002 group）,PI3K inhibitor+Sanjie Tongmai Fang group（LY294002+SJTMF group）.Apo E-/-mice were fed with high-fat diet in both experiments 1 and 2 to establish atherosclerosis model.2.1 HE staining and transmission electron microscope to observe the pathological tissue of the aorta.2.2 An automatic biochemical analyzer detects four blood lipids（TC,TG,LDL-C,HDL-C）.2.3 Enzyme linked immunosorbent assay（ELISA）to detect inflammatory factors（IL-1,IL-6,IL-10,TNF-α,MCP-1,CRP）in serum.2.4 RT-PCR was used to detect the expression of M1 and M2 macrophage-related markers in aortic tissue（M1:i NOS;M2:Arg-1）.2.5 Western Blot was used to detect the expression levels of autophagy-related proteins LC3-II/I,Beclin-1,and p62 in aortic tissue;Western Blot was used to detect the expression levels of PI3K,AKT,mTOR and phosphorylated proteins in aortic tissue.Results:1 Clinical researchA total of 325 prescriptions were selected,including 135 kinds of drugs,and 31high-frequency（frequency>50）drugs were excavated.Among the 31 drugs,the main ones were qi regulating drugs,water and dampness promoting drugs,blood activating and blood stasis removing drugs,dampness removing drugs and phlegm resolving drugs;In terms of drug properties,the use frequency of warm and cold drugs is the highest;In terms of medicine taste,pungent and sweet drugs are used the most frequently;Drugs belong to meridian,mainly liver meridian;After cluster analysis of high-frequency drugs,five groups were obtained.2 Experimental research2.1 HE staining results:After drug intervention,compared with the Model group,the aortic plaques in the SJTMF group and Ato group were significantly reduced;compared with the 3-MA group,the 3-MA+SJTMF group had significantly reduced plaques and enlarged lumen.It was shown that SJTMF could effectively reduce the formation of aortic plaques.2.2 Transmission electron microscopy results:After drug intervention,compared with the Model group,more autophagosomes and lipid droplets appeared in macrophages in the SJTMF group and Ato group;compared with the 3-MA group,autophagosomes were found in the 3-MA+SJTMF group.It was shown that SJTMF could promote autophagy in macrophages.2.3 Blood lipid results:After drug intervention,compared with the Model group,the levels of TC,TG and LDL-C in the SJTMF group and Ato group were significantly decreased（P<0.01）,and the level of HDL-C was significantly increased（P<0.01）;compared with the 3-MA group,the levels of TC,TG and LDL-C in the 3-MA+SJTMF group were significantly decreased（P<0.01）,and the level of HDL-C was significantly increased（P<0.01）.It was shown that SJTMF could effectively improve blood lipid levels.2.4 Results of inflammatory factors:After drug intervention,compared with the Model group,the level of IL-10 in the SJTMF group and Ato group were significantly increased（P<0.01）,IL-6,TNF-ɑ,MCP-1,CRP,IL-1 levels were significantly decreased（P<0.01）;compared with the 3-MA group,the level of IL-10 in the 3-MA+SJTMF group was significantly increased（P<0.01）,IL-6,TNF-ɑ,MCP-1,CRP,IL-1 levels were significantly decreased（P<0.01）.It was shown that SJTMF could effectively improve the level of inflammatory factors.2.5 RT-PCR results:Experiment 1:After drug intervention,compared with the Model group,the expression of i NOS m RNA in the SJTMF group and Ato group was significantly decreased（P<0.01）,and the expression of Arg-1 m RNA was significantly increased（P<0.01）;compared with the3-MA group,the expression of i NOS m RNA in the 3-MA+SJTMF group was significantly decreased（P<0.01）,and the expression of Arg-1 m RNA was increased（P<0.05）.Experiment 2:After drug intervention,compared with the Model group,the expression of i NOS m RNA in the SJTMF group and LY294002 group was significantly decreased（P<0.01）,and the expression of Arg-1 m RNA was significantly increased（P<0.01）;compared with the LY294002 group,the expression of i NOS m RNA in the LY294002+SJTMF group was significantly decreased（P<0.01）,and the expression of Arg-1 m RNA was significantly increased（P<0.01）.The results of Experiment 1 and Experiment 2 showed that Sanjie Tongmai Fang inhibited the transformation of macrophages to M1 type and promoted the transformation of macrophages to M2 type.2.6 Western Blot results:Experiment 1:After drug intervention,compared with the Model group,the protein expression levels of LC3-II/I and Beclin-1 in the SJTMF group and Ato group were significantly increased（P<0.01）,and the protein expression level of p62 was significantly decreased（P<0.01）;compared with the 3-MA group,the protein expression levels of LC3-II/I and Beclin-1 in the 3-MA+SJTMF group were significantly increased（P<0.01）,and the protein expression level of p62 was significantly decreased（P<0.01）.Experiment 2:After drug intervention,compared with the Model group,the expression level of LC3-II/I protein in the SJTMF group and LY294002 group was significantly increased（P<0.01）,and the protein expression level of Beclin-1 in the SJTMF group was increased（P<0.05）,Beclin-1 protein expression level in the LY294002 group was significantly increased（P<0.01）,p62 protein expression level in the SJTMF group was decreased（P<0.05）,and p62 protein expression level in the LY294002 group was significantly decreased（P<0.01）;compared with the LY294002 group,LY294002+SJTMF group LC3-II/I protein expression level increased,but no statistical difference（P>0.05）,Beclin-1 protein expression level was significantly increased（P<0.01）,p62 protein expression level was significantly decreased（P<0.01）.Compared with the Model group,the phosphorylation levels of PI3K and mTOR proteins in the SJTMF group and LY294002group were significantly decreased（P<0.01）,and the phosphorylation level of AKT protein in the SJTMF group decreased,but there was no statistical difference（P>0.05）,and the phosphorylation level of AKT protein in the LY294002 group was significantly decreased（P<0.01）;compared with the LY294002 group,the phosphorylation levels of PI3K and mTOR proteins in the LY294002+SJTMF group were significantly decreased（P<0.01）,and the phosphorylation level of AKT protein was decreased,but there was no statistical difference（P>0.05）.The results of Experiment 1 and Experiment 2 showed that Sanjie Tongmai Fang could promote autophagy of macrophages and inhibit the PI3K/AKT/mTOR pathway.Conclusion:1 Clinical researchProfessor Huang Yongsheng believes that the main pathogenesis of coronary heart disease angina pectoris is the mutual accumulation of phlegm and blood stasis.In the treatment,he advocates the simultaneous treatment of phlegm and blood stasis,pays attention to the role of qi during the treatment process,and has achieved significant clinical therapeutic effects.2 Experimental researchSanjie Tongmai Fang can promote macrophage autophagy and inhibit the inflammatory response,thereby reducing atherosclerosis.The mechanism may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway by Sanjie Tongmai Fang. |
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