The Role And Mechanism Of Interleukin-38 In Chronic Heart Failure

Part Ⅰ.Expression of interleukin 38 in chronic heart failure and analysis of its clinical relevance Objective:Interleukin 38(IL-38),also known as IL-1F10,is a newly discovered IL-1family factor in recent years and is widely recognized as an anti-inflammatory factor,which is involved in the development of numerous chronic inflammatory diseases in vivo and influences disease regression.Previous studies by the group have demonstrated that IL-38 exerts protective effects through anti-inflammation in diseases such as acute myocardial infarction,improves post-infarction cardiac function,and inhibits excessive ventricular remodeling.It has been shown that the levels of several inflammatory cytokines are significantly higher in the peripheral blood of patients with chronic heart failure(CHF)compared with controls,suggesting that there may be a systemic inflammatory response in CHF.The aim of this study was to investigate whether IL-38 plays a role in the progression of chronic heart failure and to find new targets for the treatment of chronic heart failure.Methods:1.30 patients with clinical diagnosis of chronic heart failure were included between October 2020 and October 2021,and 20 other patients without chronic heart failure diagnosis and symptoms were selected from the control group,all patients were from Wuhan Union Medical College Hospital,and all patients enrolled were free of other chronic inflammatory diseases,such as diabetes,chronic kidney disease,and neoplastic diseases.ELISA was used to detect the expression of IL-38 in the peripheral blood of the patients.The expression of IL-38 in peripheral blood was measured by ELISA and correlated with other clinical indicators(NT-pro BNP,Ang II)and NYHA cardiac function classification.2.8-week-old male C57BL/6J mice were used to establish a mouse heart failure model by implanting a subcutaneous pump to continuously pump Ang II,and the expression of IL-38 in heart and serum was measured by WESTERN BLOT and ELISA,respectively.3.Cardiomyocytes in the hearts of 2-3 days C57BL/6J mammary mice were isolated to explore the IL-38 expression in cardiomyocytes induced by different concentrations of Ang II in vitro.Results:1.IL-38 expression was significantly elevated in the peripheral blood serum of CHF patients.In CHF patients,the expression of IL-38 was negatively correlated with Ntpro-BNP and Ang II,and also negatively correlated with NYHA cardiac function classification values.2.A mouse model of heart failure was constructed using subcutaneous pumping of AngⅡ.After 8 weeks,the mice showed a decrease in cardiac ejection fraction and cardiac output,and showed heart failure changes;the expression of IL-38 in cardiac tissues and peripheral blood of CHF mice was increased compared with that of the sham-operated(Sham)group.3.IL-38 expression increased after Ang II stimulation of cardiomyocytes in mammary mice,and the increase was most obvious at the Ang II concentration of 100 n M.Conclusion:This part of the experiment found elevated expression of IL-38 in animals and cells of chronic heart failure patients and heart failure models and correlated with the severity of clinical chronic heart failure,suggesting that targeting IL-38 may provide new diagnostic indicators and therapeutic approaches for chronic heart failure.Part Ⅱ The Role of Interleukin 38 in Chronic Heart FailureObjective:In chronic heart failure,ventricular remodeling characterized by myocardial hypertrophy and cardiac fibrosis is an important part of its pathophysiology,and the persistent progression of both is a direct cause of the eventual end-stage,refractory nature of chronic heart failure in clinical practice.In this part of the experiment,we explored the possible role of IL-38 on myocardial hypertrophy and fibrosis in chronic heart failure and verified whether it could be used as a new therapeutic target.Methods:WT group,WT+r IL-38 group,IL-38 KO group were used to establish a mouse heart failure model as described previously.Cardiac function was weighed,and ultrasound was performed at the end of the 8th week after modeling,heart and lung weights were measured,ventricular sections were taken,myocardial hypertrophy was assessed by WGA staining,the degree of fibrosis was assessed by MASSON staining and Sirius red staining,and the expression of genes related to hypertrophy and fibrosis was detected by RT-PCR.In in vitro cell experiments,cardiomyocytes were isolated from 2-3 day old WT C57BL/6J and IL-38 KO mammary rat hearts,and the cells were grouped and stimulated with Ang II to simulate an in vitro heart failure model,and the activity of the cells was detected by CCK-8 assay after 72 h.The effect of IL-38 on Ang II-stimulated mammary rat cardiomyocytes in vitro was observed.Results:Animal experiments confirmed that exogenous injection of r IL-38 could improve cardiac function,reduce myocardial hypertrophy,and decrease fibrosis in a mouse heart failure model.While IL-38 KO mice exhibited more severe cardiac function impairment,myocardial hypertrophy and fibrosis compared to WT group.In in vitro experiments,IL-38 attenuated Ang II-induced cardiomyocyte injury,improved cardiomyocyte activity,and inhibited cardiomyocyte hypertrophy in mastitic rats.Conclusion:It was confirmed from both IL-38 KO and exogenous administration of r IL-38 that IL-38 exhibited potential protective properties against CHF in animal and cellular experiments and significantly inhibited myocardial hypertrophy and cardiac fibrosis.Part Ⅲ Mechanistic study of the role of interleukin 38 in chronic heart failureObjective:In previous studies,IL-38 has been shown to exert protective effects in animal and cellular models of chronic heart failure,specifically by ameliorating myocardial hypertrophy,cardiac fibrosis,and protecting cardiomyocytes,but the specific mechanisms need to be further elucidated.This part of the study aimed to investigate the specific mechanism of the role of IL-38 in the development of chronic heart failureMethods:TUNEL staining was used to measure apoptosis in the hearts of different subgroups of CHF mice,while in vitro experiments were performed to verify whether IL-38 has an ameliorative effect on Ang II-stimulated cardiomyocytes in mammary mice.A mouse heart failure model was established in WT and IL-38 KO mice as described previously,and myocardial tissue was taken from the left ventricle of mice after 8 weeks for transcriptome sequencing to investigate the mechanisms associated with the role of IL-38 in chronic heart failure.The results suggested that activation of the PERK pathway of endoplasmic reticulum stress was significantly elevated in IL-38 KO murine cardiomyocytes.Based on the sequencing results,it was verified in animal models and in vitro cellular experiments that IL-38 could affect endoplasmic reticulum stress and ultimately cardiomyocyte apoptosis in chronic heart failure models.Finally,Ang II-stimulated primary cardiomyocytes were treated with selective PERK inhibitors in in vitro experiments to observe the effects on the downstream pathways of endoplasmic reticulum stress and apoptosis.In vivo revert experiments,whether inflammatory oral administration of selective PERK inhibitor reverses cardiac function,myocardial hypertrophy,cardiomyocyte apoptosis and myocardial fibrosis in IL-38 KO heart failure model.Results:Sequencing results showed that the pathway molecules associated with endoplasmic reticulum stress were significantly elevated in IL-38 knockout murine cardiomyocytes,and subsequent western blot experiments also confirmed that IL-38 regulates PERK phosphorylation and its downstream molecules ATF4 and CHOP expression.In both animal experiments and in vitro cellular experiments,modulation of endoplasmic reticulum stress reduced apoptosis in cardiomyocytes in a heart failure model,and IL-38 may have achieved the effects of protecting cardiomyocytes,reducing apoptosis,and improving cardiac function in this way.Conclusion:IL-38 regulates the changes of PERK,CHOP,and BCL2 in the heart of mice with heart failure model,and reduces cardiomyocyte apoptosis by inhibiting endoplasmic reticulum stress,thus achieving the effect of improving cardiac function.