Effect And Mechanism Of YiqiWenyang Huoxue Lishui Method In Regulating MiR-351/MLK3 Pathway In Preventing Myocardial Remodeling In CHF

Objective:To investigate the effect of mi R-351/MLK3-mediated Pyroptosis and Ferroptosis in different stages of chronic heart failure and the intervention effect of Yiqi Wenyang Huoxue Lishui method.Methods:1.Animal experimental studies（1）Animal model establishmentC57BL/6J mice were adopted Transverse Aortic Constriction（TAC）surgery to build pressure overload of chronic heart failure（CHF）model.（2）The first part of experimental study was divided the animals into eight groups:（1）Sham,（2）TAC,（3）TAC+U-099,（4）Sham+AAV^NC,（5）TAC+AAV^NC,（6）TAC+AAV^MLK3-,（7）TAC+Agomir,（8）TAC+Antagomir.The（1）-（3）group divided 75 mice into 5 time points at 0,1,2,4 and 8 weeks,（4）-（8）group divided 30 mice into 2 time points in 1 and 8 weeks.（3）The second part of experimental study was divided the animals into eight groups:（1）Sham,（2）TAC,（3）Sham+U-099,（4）TAC+U-099,（5）XYT-Low-dose,（6）XYT-Middle-dose,（7）XYT-High-dose,（8）Perindopril,each group consisted of 30 mice,2 time points in1 and 8 weeks.2.Cell experimental study（1）Cell culture:HL1 mouse cardiomyoblast cell cultured in minimum Eagle’s medium（MEM）（Gibco Laboratories,USA）supplemented with 10%fetal bovine serum（FBS）（Gibco Laboratories,USA）and 100?U/ml penicillin/100?mg/ml streptomycin in an atmosphere of 95%air and 5%CO2 at 37°C.（2）Cell treatment and intervention:LPS was dissolved in sterile deionized water and used at a final concentration of 0.5?μg/ml,as an inducer of pyroptosis.The MLK3 inhibitor URMC-099 was dissolved in dimethyl sulfoxide（DMSO）and used at a concentration of200?μM.MCC950,as a NLRP3 inhibitor,was dissolved in DMSO and used at a concentration of 50?μM.Fin56 was dissolved in DMSO and used at a final concentration of 0.5?μg/ml,as an inducer of ferroptosis,and the ferroptosis inhibitor Ferrostatin-1 was used at a concentration of 1μM which dissolved in DMSO.（3）Detection indexes and methodsAfter the intervention of mice or cells in each group,Echocardiography was used to observe the changes of cardiac function,Hematoxylin-eosin（HE）staining,Masson staining and Sirius red staining were used to observe the myocardial fibrosis and collagen deposition,Scanning electron microscope（SEM）was used to observe the pyroptosis of myocardial cells,Transmission electron microscopy（TEM）was used to observe the morphological changes of myocardial mitochondria,apoptosis of myocardial cells was observed by Tunel staining,ROS deposition was observed by frozen section staining,the expression of NLRP3 in myocardial cells was observed by immunofluorescence assay,the expressions of Collagen I,Collagen III,-SMA and Fibronectin proteins were observed by immunohistochemistry assay,Western Blot was used to detect the expression of proteins associated with inflammation,pyroptosis,oxidative stress and ferroptosis,Micro RNA and m RNA expression levels associated with inflammation,myocardial injury and fibrosis were detected by RT-PCR,the levels of MDA,T-SOD and GSH were observed by colorimetric method,Caspase 1 Activity detection kit detects Caspase 1 Activity level in cell lysis buffer,Target Scan Human database was used to predict micro RNA interacting with MLK3,and double luciferase experiment report was used to detect the binding effect of micro RNA and MLK3.The main components of Xinyang tablets were analyzed by high pressure liquid phase-time of flight high resolution mass spectrometry.The effective components of Xinyang tablets were docked with MLK3 by molecular docking method,and the binding mode and affinity were predicted to help determine the regulatory mechanism of Xinyang tablets.Results:（1）MLK3 inhibitor and Xinyang tablets can significantly improve the cardiac function of TAC mice at different stages,reduce myocardial hypertrophy and myocardial fibrosis.（2）At the early stage of TAC,in 1 week.Compared with sham or Sham+AAV^NC group,the cardiac function was significantly reduced,the heart was significantly larger,more inflammatory cell infiltration,the collagen deposition was significantly increased（P<0.01）,the level of apoptosis was significantly increased（P<0.01）,inflammasomes was significantly increased,the expression of p-NF-κB p65、NLRP3、ASC、AIM2、pro IL-1β、cleaved IL-1β、caspase 1、cleaved caspase 1、GSDMD and cleaved GSDMD were significantly increased（P<0.01 or P<0.05）,the expression of IL-1β、IL-18、MCP-1、MIP1α、ICAM1、CXCL1、CXCL2、MMP2、MMP9、collagen I and fibronectin m RNA in ventricular cells were significantly increased（P<0.01）,in TAC or TAC+AAV^NC group;Compared with TAC or TAC+AAV^NC group,the cardiac function of was significantly improved,the heart was significantly smaller,the collagen deposition was significantly decreased（P<0.01）,the level of apoptosis was significantly decreased（P<0.01）,inflammasomes was significantly decreased,the expression of p-NF-κB p65、NLRP3、ASC、AIM2、pro IL-1β、cleaved IL-1β、caspase 1、cleaved caspase 1、GSDMD and cleaved GSDMD were significantly decreased（P<0.01 or P<0.05）,the expression of IL-1β、IL-18、MCP-1、MIP1α、ICAM1、CXCL1、CXCL2、MMP2、MMP9、collagen I and fibronectin m RNA in ventricular cells were significantly decreased（P<0.01）,in U-099,TAC+AAV^MLK3-group and XYT-Low-dose,XYT-Middle-dose,XYT-High-dose groups.（3）At the advanced stages of TAC,in 8 weeks.Compared with sham or Sham+AAV^NCgroup,the cardiac function was significantly reduced,the heart was significantly larger,the cross-sectional area of myocardial cells significantly increased（P<0.01）,the collagen deposition was significantly increased（P<0.01）,the mitochondria were irregularly arranged,smaller,the membrane were thicker,and the color was darker,the level of ROS was significantly increased（P<0.01）,the content of MDA significantly increased（P<0.01）,the content of T-SOD and GSH were significantly decreased（P<0.01）,the levels of Collagen I,Collagen III,α-SMA and Fibronectin proteins were significantly increased（P<0.01）,the expression of COX2,P53 and JNK proteins were significantly increased（P<0.01）,the expression of FTH1,GPX4 and x CT proteins were significantly decreased（P<0.01）,the expression of MMP2、MMP9、collagen I and fibronectin m RNA in ventricular cells were significantly increased（P<0.01）,in TAC or TAC+AAV^NC group;Compared with TAC or TAC+AAVNC group,the cardiac function was significantly improved,the heart was significantly smaller,the cross-sectional area of myocardial cells significantly decreased（P<0.01）,the collagen deposition was significantly decreased（P<0.01）,the level of ROS was significantly decreased（P<0.01）,the content of MDA significantly decreased（P<0.01）,the content of T-SOD and GSH were significantly increased（P<0.01）,the levels of Collagen I,Collagen III,α-SMA and Fibronectin proteins were significantly decreased（P<0.01）,the expression of COX2,P53 and JNK proteins were significantly decreased（P<0.01）,the expression of FTH1,GPX4 and x CT proteins were significantly increased（P<0.01）,the expression of MMP2、MMP9、collagen I and fibronectin m RNA in ventricular cells were significantly decreased（P<0.01）,in U-099,TAC+AAV^MLK3-group and XYT-Low-dose,XYT-Middle-dose,XYT-High-dose groups.（4）After intervention of LPS,the expression of MLK3,NLRP3 and GSDMD proteins were significantly increased（P<0.01）,the expression of IL-18,IL-1β,ANP and BNP m RNA were significantly increased（P<0.01）,the level of caspase 1 activity was significantly increased（P<0.01）;After intervention of URMC-099 and XYP,the expression MLK3,NLRP3,IL-1βand GSDMD proteins were significantly reduced（P<0.01）,the expression of IL-18,IL-1β,ANP and BNP m RNA were significantly decreased（P<0.01）,the level of caspase 1 activity was significantly decreased（P<0.01）;After intervention of MCC950,the expression of NLRP3 and GSDMD proteins were significantly decreased（P<0.01）,the expression of IL-18,IL-1β,ANP and BNP m RNA were significantly decreased（P<0.01）,the level of caspase 1 activity was significantly decreased（P<0.01）.（5）After intervention of FIN56,the expression of MLK3,p-JNK,P53 and COX2proteins were significantly increased（P<0.01）,the expression of FTH1 protein was significantly decreased（P<0.01）,the content of MDA was significantly increased（P<0.01）,the content of T-SOD and GSH were significantly decreased（P<0.01）,the expression of ANP and BNP m RNA were significantly increased（P<0.01）;After intervention of URMC-099and XYP,the expression of MLK3,P-JNK,p53,and COX2 proteins were significantly reduced（P<0.01）,the expression of GPX4 and FTH1 protein was significantly increased（P<0.01）,the content of MDA was significantly decreased（P<0.01）,the content of T-SOD and GSH were significantly increased（P<0.01）,the expression of ANP and BNP m RNA were significantly decreased（P<0.01）;After intervention of Ferrostatin-1,the expression of COX2protein was significantly decreased（P<0.01）,the expression of FTH1 protein was significantly increased（P<0.01）,the content of MDA was significantly decreased（P<0.01）,the content of T-SOD and GSH were significantly increased（P<0.01）,the expression of ANP and BNP m RNA were significantly decreased（P<0.01）.（6）Compared with Sham group,mi R351 significantly decreased in TAC group in doofferent time points at 0,1,2,4 and 8 weeks.The expression MLK3 significantly decreased,and the cardiac function,cardiac hypertrophy,collagen deposition were significantly improved after increasing mi R351 level（P<0.01）.Dual-luciferase report results showed that luciferase expression levels of wild-type MLK3 vector co-transfected with mi R351 Mimics were significantly lower than those of the control group（P<0.01）,and the luciferase expression of mutant MLK3 vector after co-transfection with mi R351 mimics showed no significant difference compared with the control group（P>0.05）.The expression of mi R351was significantly decreased in XYT-Low,Middle and High-dose groups in 1 and 8 weeks.（7）Analysis of main components of Xinyang tablets showed that 52 active ingredients were detected in the positive and negative ion mode of xinyang tablet extract,among which24 had effective affinity with MLK3 and binding energy less than-6.0 kcal/mol.Conclusion:miR-351/MLK3 play an important role in ventricular remodeling in chronic heart failure,it mainly regulates NF-κB/NLRP3 signaling pathway mediated inflammation and that pyroptosis causes myocardial fibrosis in the early stages of chronic heart failure.Similarly,it mainly regulates the JNK/p53 signaling pathway mediated oxidative stress and that ferroptosis causes myocardial fibrosis in the advanced stages of chronic heart failure.The Traditional Chinese medicine Xinyang Tablet of Yiqi Wenyang Huoxue Lishui method can effectively improve ventricular remodeling in chronic heart failure,its mechanism may be through the regulation of mi R-351/MLK3 mediated inflammation and pyroptosis,oxidative stress and ferroptosis.