Experimental Study On Ursolic Acid Treatment Of Autoimmune Myocarditis By Activating Nrf2/HO-1 Signaling Pathway

Part 1 Dissection of pathogenesis of autoimmune myocarditis based on RNA sequencingObjective: Transcriptome sequencing was used to reveal the molecular mechanism of autoimmune myocardial inflammatory injury in mice,so as to better understand the pathophysiological mechanism in the occurrence and development of myocarditis.Methods: Adult male inbred Balb/c mice were used as the main subjects to construct an experimental autoimmune myocarditis(EAM)model.The Balb/c mice were randomly divided into two groups: Sham group and EAM group.On days 0 and 7,mice in EAM group were injected with antigen polypeptide α-My HC polypeptide(Ac-RSLKLMATLFSTYASADR-OH)in subcutaneous lymph nodes to construct a mouse model of EAM.At the experimental endpoint of 21 days,RNA was extracted and transcriptome sequencing was performed after removing the mouse heart.The transcriptome sequencing results were analyzed for differential gene expression,differential gene GO enrichment and KEGG pathway enrichment.Results: A total of 2228 differentially expressed genes were identified,including 1785 up-regulated genes and 443 down-regulated genes.KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in immune-related,inflammation-related and oxidative stress-related pathways and diseases.Conclusion: The pathogenesis of autoimmune myocarditis involves not only immune and inflammatory pathways,but also oxidative stress damage pathways,which may provide theoretical basis for the exploration of new targets for treatment of myocarditis in the future.Part 2 Therapeutic effect of ursolic acid on autoimmune myocarditisObjective: To observe the effect of ursolic acid on cardiac function and myocardial inflammatory injury in mice with experimental autoimmune myocarditis.Methods: The experimental autoimmune myocarditis mice were modeled by the same method as the first part.The mice were randomly divided into three groups: Sham group,EAM group and ursolic acid intervention group(UA group).On days 0 and 7,mice in EAM group and UA group were injected with antigen polypeptide α-My HC polypeptide(Ac-RSLKLMATLFSTYASADR-OH)in subcutaneous lymph nodes to construct a mouse model of EAM.Ursolic acid(100 mg/kg/day)was administered by gavage to mice in the UA group from day 7 to day 21.Echocardiography was performed to measure cardiac function on day 21.After sampling,the whole hearts of mice were observed for gross morphology,weighed and recorded for body weight,heart weight and tibia length,and the ratio of heart weight to body weight and the ratio of heart weight to tibia length were calculated.Masson staining of cardiac histopathological sections was performed to observe the area of myocardial fibrosis.Real-Time Quantitative Polymerase Chain Reaction(RT-q PCR)was performed to detect the transcript levels of Col1a1 and Col3a1,which were indicators related to myocardial fibrosis.The immunofluorescence staining of α-SMA was performed to compare the activation of myofibroblasts in each group.Hematoxylin and eosin staining(HE)was used to observe the inflammatory infiltration in myocardial tissue.Interleukin-6(IL-6)immunofluorescence staining was used to compare the expression of inflammatory cytokines in myocardium.The transcription and secretion levels of IL-6 were detected by RT-q PCR and enzyme-linked immunosorbent assay(ELISA).Myocardial injury was observed by the level of c Tn I and BNP detecting by ELISA in peripheral blood and terminal deoxynucleotidyl transferase d UTP nick-end labeling(TUNEL)staining.Results: Compared with the sham group,systolic function of EAM mice was reduced,heart volume was increased,the ratio of of heart weight to body weight and heart weight to tibia length in EAM group was increased(P<0.001).Aggravated myocardial fibrosis were indicated by the masson staining(P<0.05),increased transcription levels of Col1a1 and Col3a1(P<0.001),and increased expression of α-SMA.The results of HE staining showed that the inflammatory score of myocardial tissue was increased(P<0.001),and the transcription and secretion levels of IL-6 in myocardial tissue were elevated(P<0.001).The expression levels of c Tn I and BNP in peripheral blood were increased(P<0.01).TUNEL assay showed severe myocardial injury in EAM mice(P<0.001).Compared with EAM group,cardiac systolic function was recovered,heart volume was decreased,the ratio of of heart weight to body weight and heart weight to tibia length was reduced in UA group(P<0.001).The reduced myocardial tissue fibrosis was indicated by the masson staining(P<0.05),decreased transcription levels of Col1a1 and Col3a1,and decreased expression of α-SMA.The results of HE staining showed that the inflammatory score of myocardial tissue was decreased,and the transcription(P<0.001)and secretion levels(P<0.05)of IL-6 in myocardial tissue were reduced.The expression levels of c Tn I and BNP in peripheral blood(P<0.05)and TUNEL assay(P<0.001)showed that the myocardial injury was relieved.Conclusion: UA improved myocardial dysfunction,reduced the degree of myocardial hypertrophy,alleviated myocardial inflammatory injury,and decreased the degree of myocardial fibrosis caused by experimental autoimmune myocarditis.Part 3 Study on the mechanism of ursolic acid alleviating oxidative stress injury in autoimmune myocarditisObjective: To investigate the effect of ursolic acid on oxidative stress in mice with autoimmune myocarditis and inflammatory injury of H9c2 cardiomyocytesMethods: The animal modeling and ursolic acid intervention were same as in Part 2.The Balb/c mice were randomly divided into three groups: Sham group,EAM group and UA group.After sampling hearts on 21 days,frozen sections of heart tissues were stained with Dihydroethidium(DHE)to assess the level of reactive oxygen species(ROS)in myocardial tissue.Malondialdehyde(MDA)in peripheral blood was used to evaluate lipid oxidation product in heart.Glutathione(GSH)in peripheral blood was used to detect the status of antioxidant system in myocardium.The expression levels of Nrf2 and HO-1 in total protein of myocardial tissue and Nrf2 in nuclear protein and cytoplasmic protein were detected by Western blot.Inflammatory cytokine IL-6 induced H9c2 cells to construct an inflammatory injury model of cardiomyocytes.They were divided into three groups: Sham group,IL-6 stimulation group(IL-6 group)and ursolic acid intervention group(UA group).MDA was used to evaluate lipid oxidation in cardiomyocytes after inflammatory injury.GSH was used to detect the status of antioxidant system in cardiomyocytes.TUNEL staining was used to detect apoptosis damage in all groups.The expression levels of Nrf2 and HO-1 in total protein of cardiomyocytes and Nrf2 in nuclear protein and cytoplasmic protein were detected by Western blot.The targeted binding of ursolic acid to Nrf2 was verified by simulated molecular docking of computer.Results: Compared with the sham group,the level of ROS in myocardial tissues of EAM group was increased(P<0.001),the expression level of MDA was increased(P<0.001)and the level of GSH was decreased(P<0.001).After UA intervention,the level of ROS in myocardial tissue was decreased(P<0.001),the expression level of MDA was decreased(P<0.001)and the level of GSH was increased(P<0.01).Total protein levels of Nrf2 and HO-1 in myocardial tissue decreased in the EAM group(P<0.05),whereas they increased in the UA group(P<0.05).Additionally,the EAM mice expressed Nrf2 at lower levels in the nucleus and at higher levels in the cytoplasm of cardiac tissues compared to the sham group(P<0.05).After UA intervention,the expression levels of Nrf2 were higher in the nucleus and lower in the cytoplasm of cardiac tissues in the UA group(P<0.05).In IL-6-induced inflammatory injury of H9c2 cardiomyocytes,the expression of MDA was increased in IL-6 group.However,the expression of MDA decreased in a dose-dependent manner with increasing ursolic acid concentration at UA intervention concentrations less than 30 μM(P<0.05).When UA was more than 30μM,the decline trend was stable.The results of DHE staining and GSH assay showed that the level of ROS was increased(P<0.001)and the level of GSH was decreased in IL-6 group(P<0.001).After UA intervention,the increase of ROS and the decrease of GSH could be reversed to a certain extent.TUNEL staining of cardiomyocytes showed that the degree of apoptosis of cardiomyocytes was reduced and cardiomyocytes damage was alleviated.Total protein levels of Nrf2 and HO-1 were decreased in the IL-6 group,while they were increased in the UA group.The expression levels of Nrf2 and HO-1 also showed a dose-dependent increase when UA intervention concentrations less than 30 μM.IL-6 group showed lower Nrf2 expression levels in nucleoproteins than the sham group,whereas UA group showed higher Nrf2 expression levels.In addition,there was no significant difference between pre-and post-stimulated Nrf2 expression level in the cytoplasm.The results of computer simulation of UA and Nrf2 molecular docking showed that UA had a better binding effect with the target protein Nrf2.Conclusion: UA attenuated myocardial inflammatory injury in EAM mice and oxidative stress in IL-6-induced inflammatory injury in H9c2 cells.UA activated Nrf2 into the nucleus to play antioxidant effects and attenuated oxidative stress damage caused by inflammatory lesions.