【作者】 王玥；

【导师】 曾秋棠；

【作者基本信息】 华中科技大学 ， 内科学（心血管病学）， 2023， 博士

【摘要】 第一部分白细胞介素38在慢性心力衰竭中表达情况及临床相关性分析目的:白细胞介素38(Interleukin 38,简称IL-38)又称IL-1F10,是近年新发现的IL-1家族因子,并被普遍认为是一种抗炎因子,其在体内参与众多慢性炎症性疾病的发生发展,影响疾病的转归。课题组前期研究已经证实,IL-38在急性心肌梗死等疾病中通过抗炎发挥保护作用,改善心梗后心功能,抑制过度心室重塑。研究表明,与对照组相比,临床上慢性心力衰竭(chronic heart failure,CHF)患者外周血中多种炎性细胞因子水平明显升高,这提示CHF可能存在着系统性炎症反应。本研究的目的在于探究IL-38是否在慢性心力衰竭的进展中发挥作用,为寻找治疗慢性心力衰竭寻找新的靶点。方法:1.在2020年10月至2021年10月期间,收录30例临床诊断为慢性心力衰竭(均为超声心动图中有EF值相对下降的射血分数下降型心力衰竭,HFr EF)的患者,对照组选取20例无慢性心力衰竭诊断、症状的其他病人,患者均来自武汉协和医院,入选的所有病人无其他慢性炎症性疾病,如糖尿病,慢性肾病,肿瘤性疾病等。ELISA法检测患者外周血中IL-38的表达表达情况,并与患者的其他临床指标(NT-pro BNP,AngⅡ)、NYHA心功能分级行相关性分析。2.选用8周龄雄性C57BL/6J小鼠,通过植入皮下泵持续泵入AngⅡ的方式建立小鼠心衰模型,利用WESTERN BLOT和ELISA分别测量心脏和血清的IL-38表达3.分离2-3天C57BL/6J乳鼠心脏中的心肌细胞,探索不同浓度的AngⅡ在体外诱导心肌细胞的IL-38表达情况。结果:1.IL-38在CHF患者外周血血清中的表达量明显升高。在CHF患者中,IL-38的表达量和Ntpro-BNP、AngⅡ呈负相关,同时与NYHA心功能分级数值呈负相关。2.利用皮下泵泵入AngⅡ构建了心衰小鼠模型,8周后小鼠心脏超声射血分数、心输出量等指标下降,呈现心衰改变;CHF小鼠的心脏组织及外周血中的IL-38表达均相较于假手术(Sham)组升高。3.AngⅡ刺激乳鼠心肌细胞后IL-38表达上升,且在AngⅡ浓度为100n M时升高最明显。结论:本部分实验发现IL-38慢性心力衰竭患者和心衰模型的动物、细胞中表达升高,且与临床慢性心力衰竭的严重程度相关,这表明以IL-38为靶点提供对于慢性心力衰竭新的诊断指标和治疗方法。第二部分白细胞介素38在慢性心力衰竭中的作用目的:慢性心力衰竭中,以心肌肥厚和心脏纤维化为特点的心室重塑是其病理生理的重要部分,这两者的持续性进展也是临床上慢性心衰最终走向终末、难治性的直接原因。这部分实验我们探索IL-38对慢性心力衰竭中心肌肥厚和纤维化的作用,验证其是否能作为新的治疗靶点。方法:构建IL-38 KO小鼠,将WT组、WT+r IL-38组和IL-38 KO小鼠用前述方法建立小鼠心衰模型。在造模后第8周末称重并进行超声检测心功能,测量心脏、肺重量,取心室部分切片,WGA染色评估心肌肥大,MASSON染色及天狼星红染色评估纤维化程度,RT-PCR检测肥大与纤维化相关基因的表达。体外细胞实验中,从2-3天的WT C57BL/6J和IL-38 KO乳鼠心脏分离心肌细胞,将细胞分组后用AngⅡ刺激模拟体外心衰模型,72h后CCK-8实验检测细胞的活性,观察IL-38对体外AngⅡ刺激的乳鼠心肌细胞的作用。结果:动物实验证实,外源性注射r IL-38能够改善小鼠心衰模型的心功能,降低心肌肥厚,减少纤维化。而IL-38KO鼠相较于WT组则表现出了更严重的心功能损伤、心肌肥厚及纤维化。在体外实验中,IL-38减轻AngⅡ诱导的乳鼠心肌细胞损伤,改善心肌细胞活性,抑制心肌细胞肥大。结论:从IL-38 KO和外源性给予r IL-38两个角度证实,IL-38在动物、细胞实验中都表现出了对CHF潜在的保护性,能显著抑制心肌肥厚及心脏纤维化。第三部分白细胞介素38在慢性心力衰竭中作用的机制研究目的:在之前的研究中,IL-38被证实能在慢性心力衰竭的动物和细胞模型中发挥保护作用,具体表现为改善心肌肥厚,减轻心脏纤维化,保护心肌细胞活力下降,但其具体机制有待进一步阐明。这部分研究旨在探究IL-38在慢性心力衰竭发生发展中作用的具体机制方法:用TUNEL染色法衡量不同分组的CHF小鼠心脏中心肌细胞的凋亡情况,同时在体外实验验证IL-38对AngⅡ刺激的乳鼠心肌细胞是否具有改善凋亡的作用。在WT和IL-38KO小鼠中按前述方法建立小鼠心衰模型,8周后从小鼠左心室取心肌组织进行转录组测序,以探究IL-38在慢性心力衰竭中发挥作用的相关机制。结果提示,IL-38KO鼠心肌细胞中内质网应激的PERK通路激活显著升高。根据测序结果,在动物模型和体外细胞实验验证IL-38可影响慢性心力衰竭模型中的内质网应激,并最终影响心肌细胞凋亡。最后,体外实验中用选择性PERK抑制剂处理AngⅡ刺激的原代心肌细胞,观察其对内质网应激下游通路及细胞凋亡的影响。体内回复实验,炎症口服选择性PERK抑制剂是否可以逆转IL-38KO心衰模型的心功能、心肌肥厚、心肌细胞凋亡和心肌纤维化。结果:测序结果显示,IL-38敲基因鼠心肌细胞中内质网应激的相关通路分子显著升高,后续western blot实验也证实IL-38能调控PERK磷酸化及其下游分子ATF4、CHOP表达。在动物实验和体外细胞实验中,调控内质网应激都能减少心衰模型中心肌细胞的凋亡,IL-38可能是通过这一方式达到了保护心肌细胞、减少凋亡、改善心功能的作用。结论:IL-38调控了心衰模型小鼠心脏中PERK、CHOP、BCL2的变化,通过抑制内质网应激减少心肌细胞凋亡,从而达到改善心功能的作用。更多还原

【Abstract】 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.更多还原