Study On The Mechanism Of XBATF To Regulate Mir-214-3p/TLR4/MyD88 In The Treatment Of BCL With Phlegm,Blood Stasis And Heat Syndrome

Borderline Coronary Lesions(BCL)is a special stage of coronary artery disease,which refers to lesions with 30%to 70%stenosis as determined by coronary angiography diameter method,also called moderate coronary artery stenosis.Although this stage has not yet reached the indication of Percutaneous coronary intervention(PCI)surgery,patients have shown obvious symptoms of discomfort,and their plaques are mostly soft plaques and eccentric plaques,which are vulnerable and unstable plaques.If this period is neglected without active intervention,the stenosis will be further aggravated,leading to the deterioration of myocardial blood supply and cardiac function.Modern medicine uses lipid-lowering drugs,anti-platelet drugs and other secondary prevention treatments,but the incidence of adverse cardiovascular events caused by coronary artery lesions has not been reduced.Traditional Chinese medicine(TCM)has a very positive effect on the treatment of BCL,and making full use of the characteristics of TCM is expected to delay the course of the disease or even reverse it.In his clinical practice,Prof.Wang Jie found that the important pathological factors of BCL are phlegm,blood stasis and heat accumulation,and the method of clearing heat,resolving phlegm and activating blood can significantly improve patients’ symptoms,stabilize plaque and improve prognosis,thus improving patients’ survival quality.Xuanbi Antong Formula(XBATF)is a Chinese herbal formula formulated by Prof.Wang Jie based on the method of clearing heat,resolving phlegm and invigorating blood,which contains eight herbs,including Panax ginseng,Salvia miltiorrhiza,Coptidis Rhizoma,Pinellia ternata,Cistanche deserticola,Puerariae Lobatae Radix,Ginseng and Paeoniae rubra.We found that the formula can dilate coronary arteries and peripheral blood vessels,enhance myocardial contractility,reduce anterior and posterior cardiac load,improve cardiac function,and inhibit ventricular remodeling,and its mechanism may be related to the downregulation of inflammatory factors(TNF-α and IL-6)and the upregulation of IL-10 expression.Moreover,through the network pharmacology analysis,we know that the potential targets of Xuan bi an tong Formula for the treatment of BCL include TNF,IL-1β,NFκB1,LDL-R,IL-6,PTGS2,etc.,which may act through signaling pathways such as inflammatory response,NO biosynthesis process,lipopolysaccharide response,lipid synthesis and metabolism,and coagulation function.However,the key target genes and their molecular mechanisms of XBATF for the treatment of critical lesions in BCL are not yet clear.Therefore,the aim of this study is to explore the the mechanism of XBATF in the treatment of BCL with Phlegm,Blood stasis and Heat syndrome through clinical studies and cellular experiments.ObjectiveTo observe the efficacy and safety of XBATF in patients with phlegm-stasis-heat syndrome of BCL through clinical studies.To initially validate the targets of XBATF using RT-qPCR analysis.We further explored the mechanism of miR-2143p/TLR4/MyD88 pathway regulation by XBATF to inhibit the inflammation of macrophages through cellular analysis.The aim of the study was to reveal the mechanism of XBATF to improve the critical lesions of coronary arteries,and to establish a research model of "experience of famous Chinese medicine practitionerscombined with literature-clinical validation of efficacy-experiments to reveal the scientific connotation".MethodsThe first part was a clinical trial of XBATF to intervene in patients with phlegm-stasis-heat syndrome of BCL.29 subj ects with phlegm-stasis-heat syndrome of BCL were included in this study for 24 weeks.The morphological changes of plaque(percentage of plaque area stenosis,percentage of plaque volume stenosis,plaque volume),TCM symptom scores,lipid changes(CHO,TG,HDL,LDL,VLDL),HsCRP,safety indicators(BUN,CR,ALT,AST,WBC,RBC,HGB)were statistically analyzed to systematically observe the effectiveness and safety of XBATF in treating BCL.The second part was the effect of XBATF on miR-214-3p/TLR4/MyD88 in patients with BCL.To further investigate the mechanism,we included 20 patients with phlegm-stasis-heat evidence of BCL.RT-qPCR was selected to detect the changes of miRNA-214-3p/TLR4/MyD88 regulatory relationship before and after treatment in XBATF subjects,and to perform preliminary validation of the mechanism.The third part is the study of miR-214-3p/TLR4/MyD88 regulation by XBATF to inhibit M1 type macrophage polarization.We used the Human Monocyte Leukemia Cells(THP-1).Its culture conditions were:RPMI-1640 medium+10%fetal bovine serum+1%P/S+0.05 mM β-mercaptoethanol.The modeling was performed by first inducing THP-1 with 100 ng/mL of PMA for 48h and then differentiating into THP-derived macrophages.The differentiated THP-derived macrophages were implanted into 6-well plates at a density of 1×105 cells/well.After 24h of culture,they were induced into M1 macrophages by treatment with 100ng/ml LPS+20ng/ml IFN-γfor 24h.The cells were divided into 5 groups:control group(normal cultured THP-1derived macrophages),model group(100 ng/ml LPS+20 ng/ml IFN-y),XBATF group(model+XBATF),model+miR-214-3p inhibitor group,and model+miR-214-3p inhibitor+XBATF group.We first screened the non-toxic concentrations of XBATF by CCK8 method to determine the optimal non-toxic concentration of XBATF used in the experiment.Then,we measured the levels of IL-1 β,IL-6,TNF-α and the expression levels of miR-214-3p,TLR4 mRNA and MyD88 mRNA in five groups of cells to further verify the regulatory relationship of miRNA-214-3p/TLR4/MyD88 pathway.ResultsPart IXBATF could significantly improve the morphological status of plaques in subjects.Among them,the percentage of plaque area stenosis and the percentage of plaque diameter stenosis were significantly improved before and after treatment(P<0.01),and the plaque volume also tended to be smaller,but the difference was not statistically significant(P>0.05).XBATF could significantly improve the subjects’ TCM total symptom scores(P<0.01),the symptom scores of chest pain,chest tightness,phlegm,and so on(P<0.01).XBATF could stabilize the blood lipid level of the subjects.There was a trend of downregulation of CHO,TG,HDL,LDL and VLDL in the subjects before and after treatment with XBATF,but the difference was not statistically significant(P>0.05).No significant changes in safety indicators such as BUN,CR,ALT,AST,WBC,RBC and HGB were observed in the subjects before and after treatment with XBATF.Part IIPeripheral blood miR-214-3p was quantified in patients with phlegm-stasis syndrome of BCL.The results showed that the level of miR-214-3p was downregulated after treatment with XBATF,and the difference was statistically significant(P<0.01).This indicates that there is indeed a regulatory effect of XBATF on miR-2143p.In this study,RT-qPCR was performed to detect the genes of TLR4/MyD88 pathway.The results showed that the expression levels of TLR4 mRNA and MyD88 mRNA had a tendency to be down-regulated after XBATF treatment,but the difference was not statistically significant(P>0.05).Based on this,we suggest that XBATF may play a protective role in coronary adventitia by regulating miR-214-3p and thus TLR4/MyD88 inflammatory signaling pathway.Part IIIThe results of CCK8 method showed that the highest concentration of drug intervention for cell survival was 200 μg/mL.The levels of M1 macrophage markers IL-1β,IL-6 and TNF-α were measured in five groups of cells.The results of the study showed that LPS and IFN-γ-induced M1type macrophages showed increased levels of IL-6(P<0.01)and TNF-α(P<0.01)relative to the control group,and the level of IL-1β had a tendency to increase but the difference was not statistically significant(P>0.05).Relative to the model group,the intervention of XBATF given at the same time of modeling could effectively reduce the level of IL-6(P<0.01),could effectively reduce the level of TNF-α(P<0.01)and could effectively reduce the level of IL-1β(P<0.05).Relative to the model plus miR-214-3p inhibitor group,the model+miR-214-3p inhibitor+XBATF group showed a decrease in IL-6 content(P<0.01),a decrease in TNF-α content(P<0.05),and a significant decrease in IL-1β content(P<0.01).The expressions of miR-214-3p,TLR4 mRNA,and MyD88 mRNA were measured in five groups of cells.The results showed that LPS-and IFN-γ-induced M1type macrophages showed decreased expression of miR-214-3p(P<0.01),increased expression of TLR4 mRNA(P<0.05),and increased expression of MyD88 mRNA(P<0.01)relative to the control group.Relative to the model group,the intervention of XBATF given along with modeling effectively increased the expression of miR-214-3p(P<0.01)and effectively decreased the expression of TLR4 mRNA(P<0.01)and MyD88 mRNA(P<0.01),with statistically significant differences(P<0.01).Relative to the model plus miR-214-3p inhibitor group,the expression of miR-214-3p was decreased in the model+miR-214-3p inhibitor+Chinese medicine group(P<0.05),and the expression of TLR4 mRNA and MyD88 mRNA was significantly lower(P<0.01).The expression of miR-214-3p in the model+miR-214-3p inhibitor+XBATF group was lower than that in the model+Chinese medicine group,and the difference was statistically significant(P<0.01).In conclusion,XBATF can down-regulate the expression of miR-214-3p,upregulate the expression of TLR4 mRNA and MyD88 mRNA,and promote the secretion of inflammatory factors IL-6,TNF-α,and IL-1β.Conclusion1 XBATF has good clinical efficacy and safety in the treatment of phlegm-stasisheat evidence of critical coronary lesions.2 XBATF can regulate miRNA-214-3p/TLR4/MyD88 pathway,inhibiting LPS and IFN-y-induced macrophage inflammatory response.3 XBATF can improve the prognosis of patients with critical coronary lesions and improve their survival quality by inhibiting the inflammatory response.4 MiRNA-214-3p/TLR4/MyD88 pathway may be a potential target of XBATF in the treatment of phlegm-stasis-heat syndrome of BCL.