Study On The Effect And Mechanism Of Taoren Honghua Decoction On Coronary Artery Disease

Objective:Through in vivo and in vitro experiment and clinical trial,to observe the effect of Tao Ren Honghua Decoction（THD）treating coronary artery disease（CAD）and chronic inflammation of atherosclerosis（AS）,especially the possible mechanism of anti-inflammation by regulating cardiac lymphatic vessels（CLVs）.Methods:（1）Mechanism research:explore the relationship between CLVs and chronic inflammation of Apo E^-/-AS model mice.Apo E^-/-mice were fed with high-fat diet for 8weeks to establish AS model.Body weight,blood lipid,plaque area and morphology of aortic sinus,number and morphology of CLVs,T cell number of mediastinal lymph node and expression of inflammatory factors were detected.（2）In vitro experiment of cells:explore the anti-inflammatory effect and potential signaling pathways of THD.Mouse RAW264.7 macrophages with lipopolysaccharide were used to simulate the local chronic inflammation of AS.After incubated with THD for 24h,inflammatory factors and inflammation-related pathways were detected.（3）In vivo experiment of mice:Apo E^-/-AS model mice were treated with THD for 12weeks.Body weight,blood lipids,plaque of aortic sinus,number and morphology of CLVs,T cell number in mediastinal lymph node,inflammatory factors and inflammation-related pathways were detected.（4）Clinical trial:1:1 randomized,double-blind,placebo-controlled study method was used.THD or THD placebo was given to included 80 CAD patients for 28 days.The Seattle angina questionnaire,interleukin,C-reactive protein,complement,other immunoinflammatory indexes and serum lipids of the subjects were compared before and after intervention.Specific test methods:The plaques of aortic sinus were detected by oil red O staining.The number and morphology of CLVs,the number of T cells in mediastinal lymph nodes and the NF-B p65 signaling pathway were detected by immunofluorescence staining.The gene expressions of inflammation factors（IL-6,IL-10,TNF-a）in cells and mouse aorta were detected by real-time quantitative PCR.Protein of inflammation factors（IL-6,IL-10,TNF-a）in cell supernatant and mice serum was detected by ELISA.Western blot was used to detect the inflammation-related pathways MAPKs（P38,JNK,ERK1/2）and ERK5/STAT3/AKT of cells and mouse aorta.Results:（1）The relationship between CLVs and chronic inflammation of Apo E^-/-AS model mice:the number of CLVs in model group increased and disordered with uncompleted lumen structure.Number of T cells in mediastinal lymph node increased,and the pro-inflammatory factors（IL-6 and TNF-a）gene and protein expressions were increased（P<0.05,P<0.01）.（2）In vitro experiment of cells:compared with cells in control group,gene and protein expression of IL-6 and TNF-a increased（P<0.01）,IL-10 gene expression increased while protein decreased（P<0.01）in model group.After intervened with THD,gene and protein expression of IL-6 and TNF-a decreased（P<0.01）,but IL-10 increased（P<0.01）.MAPKs,ERK5/STAT3/AKT and NF-B p65 signaling pathways were upregulated in model group compared with control group,however THD inhibited the activation of these pathways.（3）In vivo experiment of mice:compared with the control group,mice body weights,blood lipids,numbers of disordered CLVs and T cells in mediastinal lymph node increased（P<0.01）in model group.Gene and protein expression of IL-6 and TNF-a increased（P<0.01）and IL-10 also increased（P>0.05）.MAPKs and ERK5/STAT3/AKT signaling pathway upregulated.THD reduced the body weights（P<0.01）,blood lipids（P<0.05,P<0.01）,plaque areas（P<0.01）,numbers of CLVs and T cells in mediastinal lymph node（P<0.01）.Gene and protein expression of IL-6 and TNF-a also decreased,but IL-10 gene expression（P<0.01）and protein expression increased（P>0.05）.Activation of MAPKs and ERK5/STAT3/AKT signaling pathways was inhibited.（4）Clinical trial:Compared with THD placebo group,THD reduced the expressions of IL-1b（P<0.05）,IL-6（P<0.01）,complement C3（P<0.05）,CD3（P<0.05）,CD4（P<0.01）and improved Seattle angina questionnaire scores（P<0.01）of CAD patients.Conclusion:（1）CLVs hyperplasia can aggravate the chronic inflammation in AS the pathological process,which may be related to the numbers of activated T lymphocytes in mediastinal lymph nodes and the upregulated inflammatory signaling pathways.（2）The traditional Chinese medicine THD can inhibit CLVs hypertension,reduce the number of T lymphocytes in cardiac lymph nodes,and down-regulate inflammation-related signaling pathways.Finally,inhibit the chronic inflammatory response of AS,limit the AS pathological process and treat AS-related cardiovascular diseases.