The Effect Of Baicalin On Improving ISO Induced Myocardial Injury By Regulating Myocardial Ferroptosis Through Nrf2/HO-1

This study explores the reversal effect of baicalin(Bai)in myocardial injury,activating the nuclear factor E2 related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway mediated myocardial ferroptosis to explore the molecular mechanism of Bai’s improvement of myocardial injury and broaden our understanding of the pathogenesis of heart injury,And provide new directions and approaches for clinical research.Objective: To establish a model of myocardial hypertrophy by intraperitoneal injection of isoproterenol(ISO),and to investigate the effects of Bai on cardiac function,inflammatory response,fibrosis in the model of myocardial hypertrophy by intraperitoneal injection of Bai.Method: 1.Randomly divide mice into 4 groups(n=6): Con group(Con),model group(ISO),Bai low-dose group(ISO+Bai-25),Bai high-dose group(ISO+Bai-50).After intervention with baicalin,evaluate mouse heart function using echocardiography;Use pathological staining and MDA,CK-MB,LDH kits to evaluate myocardial injury and fibrosis degree;Use WB to detect the expression levels of proteins related to hypertrophy,fibrosis,and inflammation.2.Randomly divide the mice into 4 groups(n=6): Con group(Con),baicalin group(Con+Bai-50),model group(ISO),and Bai high-dose group(ISO+Bai-50).After intervention therapy with baicalin,evaluate the cardiac function of mice using echocardiography;Use pathological staining and MDA,CK-MB,LDH kits to evaluate myocardial injury and fibrosis degree;Use WB to detect the expression levels of myocardial hypertrophy,fibrosis,antioxidant,inflammatory,and iron death related proteins.3.Mice were randomly divided into 5 groups(n=6): Con group(Con),model group(ISO),Bai high-dose group(ISO+Bai),Nrf2 inhibitor group(ISO+Bai+ML385),and iron death activator group(ISO+Bai+Erastin).Evaluate mouse cardiac function,inflammatory changes,and fibrosis using the above methods,and use an iron ion reagent kit to detect the content of iron ions in myocardial tissue;Immunofluorescence assessment of Nrf2 entry into the nucleus;Elisa detected the content of MDA,GSH,and ROS in myocardial tissue;WB detection of Nrf2,HO-1 signaling pathway proteins,and iron death related proteins.result: 1.HE/Masson staining showed that compared with ISO,the Bai group significantly improved myocardial injury and changes in myocardial fibers;Echocardiography showed that compared with ISO,Bai group showed improvements in HW/BW,IVSd,LVPWd,and LVIDd;The content of CK-MB and LDH in the ISO group mice significantly increased,while the Bai group showed a dose-dependent decrease;The activity of GSH in ISO group mice significantly decreased,and the content of MDA increased.In Bai group,GSH increased in a concentration dependent manner,and the content of MDA decreased.The WB results showed that compared with the Con group,the ISO group and Bai group showed downregulation of SOD1 and CAT expression,while NF-κB、TNF-α、The expression of IL-6,p-Smad2,Collagen I,and BNP was upregulated(P<0.05);Compared with the ISO group,the expression of SOD1 and CAT proteins in the Bai group was upregulated in a dose-dependent manner(P<0.05).2.(1)Through echocardiography detection,the LVIDd values of the ISO group mice were significantly lower than those of the Con group,while the LVSd and LVPWd of the ISO group mice hearts were significantly higher than those of the control group(P<0.05);The results of heart sampling showed that the heart mass and heart weight/body weight ratio of ISO group mice were significantly higher than those of the control group,while the heart mass and heart weight/body weight ratio of ISO+Bai-50 group mice were significantly lower than those of ISO group mice(P<0.05);WB analysis revealed that in the ISO group,B-type natriuretic peptide(BNP),a protein associated with myocardial hypertrophy β-myosin heavy chain(β-MHC)was significantly higher than that of the control group,and the myocardial hypertrophy related protein BNP β-The expression level of MHC was lower than that of the ISO group(P<0.05).(2)Masson staining showed that the severity of perivascular and interstitial fibrosis in ISO group mice was significantly higher than that in the control group,and the fibrosis degree in ISO+Bai-50 group mice was reduced;WB analysis results: Fibrotic markers in ISO group mice: Collagen I,Fibronectin(Fib),Transformed Growth Factor-β(TGF-β)、The protein expression level of Smad2 transcription factor was significantly higher than that of the control group,and the ISO+Bai-50 group mice Collagen I,FIB,TGF-β、The expression level of Smad2 protein was significantly lower than that of the ISO group(P<0.05).(3)The detection of myocardial injury markers showed that the activity of CK-MB and LDH in the induction group mice was significantly higher than that in the control group,while the activity of CK-MB and LDH in the ISO+Bai-50 group mice was significantly lower than that in the ISO group(P<0.05).(4)The WB analysis results of oxidative stress indicators show that ISO nuclear factor-κB(NF-κB)Interleukin-6(IL-6),Tumor Necrosis Factor(TNF-α)The protein expression level of the ISO+Bai-50 group was significantly higher than that of the control group-κB.IL-6 and TNF-α The protein expression level was significantly lower than that of the ISO group(P<0.05);The protein expression levels of superoxide dismutase(SOD1)and catalase(CAT)in ISO group were significantly lower than those in control group,and the protein expression levels of SOD1 and CAT in ISO+Bai-50 group were significantly higher than those in ISO group(P<0.05).3.Nrf2 inhibitor(ML385),iron death activator Erastin,and Bai jointly intervened in mice,and the results showed an improvement in myocardial injury caused by Bai.Compared with the ISO+Bai group,the ML385 and Erastin groups showed an upward trend in echocardiography,fibrosis,inflammatory response,and myocardial injury indicators.(1)The iron ion kit detection showed that the iron ion concentration in the ISO group was higher than that in the control group,while the iron ion concentration in the ISO+Bai group mice was significantly lower than that in the ISO group.The iron ion concentration in the ML385 group and the iron death activator Erastin group was higher than that in the significantly ISO+Bai group(P<0.05).(2)The MDA,ROS,and GSH test kits showed that compared with the control group,the MDA and ROS content of the ISO group mice significantly increased,while the GSH content decreased;Compared with the ISO group,the content of MDA and ROS in the ISO+Bai group mice significantly decreased,while the content of GSH increased;Compared with the ISO+Bai group,the ML385 group and the iron death activator Erastin mice showed an increase in MDA and ROS content,while the GSH content decreased.(3)WB analysis showed that after using ML385 and Erastin,the expression levels of Nrf2,HO-1,and NOQ1 proteins in myocardial tissue were significantly inhibited;The expression of GPX4 and FTH1 proteins was downregulated,while the expression of PTGS2 protein was upregulated;The expression of antioxidant proteins SOD1 and CAT was downregulated.Conclusion: Bai can improve cardiac injury by activating Nrf2/HO-1 signaling pathway to inhibit myocardial iron death.