生长激素释放相关肽GHRL-12在免疫检查点抑制剂相关心肌炎小鼠中的作用与机制研究

Role and mechanism of growth hormone release related peptide GHRL-12 in mice with immune checkpoint inhibitor-related myocarditis

目的探讨生长激素释放相关肽GHRL-12在免疫检查点抑制剂相关心肌炎中的作用与机制,为其临床应用提供理论依据。方法6~8周龄BALB/c小鼠,采用随机数生成器,随机分为对照组、心肌炎组、GHRL-12组和GHRL-12+心肌炎组。GHRL-12组和GHRL-12+心肌炎组小鼠尾静脉注射10 mg/kg GHRL-12连续1周,同时对照组和心肌炎组小鼠尾静脉注射同等体积的无功能scramble肽。在第7天和第14天分别给予心肌炎组和GHRL-12+心肌炎组小鼠250μg心肌肌钙蛋白I(cTnI),从第14天开始,每间隔1天给予小鼠腹腔注射5 mg/kg anti-PD-1,共5次。每7天称量小鼠体重,测量小鼠心体比和心胫比。超声心动图检测心功能指标。HE染色检测心肌组织炎症细胞浸润程度,Masson染色检测心肌组织纤维化程度,并用ImageJ对炎症细胞浸润和纤维化程度进行量化。取小鼠眼球血,检测血清中肌酸激酶(CK)、CK同工酶(CK-MB)和乳酸脱氢酶1(LDH-1)变化。酶联免疫吸附试验(ELISA)检测小鼠血清中炎症因子cTnI、cTnT、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)释放情况。蛋白质印迹技术测定小鼠心肌组织中自噬相关蛋白高迁移率族蛋白B1(HMGB1)、酵母Atg6同源物(Beclin-1)、自噬基因-相关蛋白5(ATG5)和微管相关蛋白轻链3B(LC3B)的表达,同时测定小鼠心肌组织中凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶3(Caspase-3)和多聚腺苷二磷酸核糖聚合酶(PARP)的表达。对心肌细胞核用DAPI染成蓝色,凋亡细胞被TUNEL染成绿色,检测小鼠心肌组织发生凋亡的情况。结果(1)与对照组相比,GHRL-12组小鼠体重无显著差异,心肌炎组小鼠体重增长不明显;与心肌炎组比较,给药第19天起GHRL-12+心肌炎组小鼠体重增长较为明显。与对照组和GHRL-12组相比,心肌炎组小鼠心体比、心胫比显著增加,而与心肌炎组小鼠相比,GHRL-12+心肌炎组小鼠心体比、心胫比下降。(2)超声心动图显示,单纯给予GHRL-12干预对小鼠心功能无显著影响,与对照组相比,心肌炎组小鼠左心室射血分数(LVEF)、缩短分数(FS)显著下降,左心室收缩末期内径(LVEDs)、左心室舒张末期内径(LVEDd)显著增加;而与心肌炎组小鼠相比,GHRL-12+心肌炎组小鼠LVEF、FS升高,LVEDs、LVEDd减少。(3)HE染色结果显示,与对照组小鼠相比,心肌炎组小鼠炎症细胞浸润显著增加,而与心肌炎组小鼠相比,GHRL-12+心肌炎组小鼠炎症细胞浸润减少。(4)Masson染色结果表明,与对照组小鼠相比,心肌炎组小鼠心肌组织纤维化程度增加,而与心肌炎组小鼠相比,GHRL-12+心肌炎组小鼠心肌组织纤维化程度降低。(5)与对照组小鼠相比,心肌炎组小鼠血清中CK、CK-MB、LDH-1、cTnI和cTnT均显著上升,而与心肌炎组小鼠相比,GHRL-12+心肌炎组均显著下降。(6)蛋白质印迹结果显示,与对照组小鼠相比,心肌炎组小鼠心肌组织HMGB1、Beclin-1、ATG5和LC3B蛋白表达均显著增加,而与心肌炎组小鼠相比,GHRL-12+心肌炎组均显著下降;同时,心肌炎组Caspase-3和PARP蛋白表达均显著增加,GHRL-12+心肌炎组均显著减少。(7)TUNEL结果显示,与对照组小鼠相比,心肌炎组小鼠心肌组织凋亡显著增加,与心肌炎组小鼠相比,GHRL-12+心肌炎组小鼠心肌组织凋亡显著下降。结论多肽GHRL-12能够改善免疫检查点抑制剂诱导的心功能损伤,减少心肌组织中炎症细胞浸润和纤维化,抑制心肌组织凋亡和自噬,为未来免疫检查点抑制剂相关心肌炎的治疗提供潜在可能。

Objective To elucidate the scope mechanism of ghrelin and obestatin prepropeptide-12(GHRL-12)in immune checkpoint inhibitor(ICI)-related myocarditis,providing a theoretical foundation for its future clinical application.Methods BALB/c mice aged 6 to 8 weeks were randomly divided into control group,myocarditis group,GHRL-12 group and GHRL-12+myocarditis group by random number generator.Mice in GHRL-12 group and GHRL-12+myocarditis group were injected with 10 mg/kg GHRL-12 through tail vein for 1 week,while mice in control group and myocarditis group were injected with the same volume of non-functional scramble peptide through tail vein.Mice in myocarditis group and GHRL-12+myocarditis group were given 250μg cardiac troponin I(cTnI)on day 7 and day 14,respectively,and intraperitoneal injection of 5 mg/kg anti-PD-1 was given every day from day 14,for a total of 5 times.The mice were weighed every 7 days and the heart-body ratio and heart-shin ratio were measured.Echocardiography was used to detect cardiac function.HE staining was used to detect the degree of inflammatory cell infiltration in myocardial tissue,Masson staining was used to detect the degree of myocardial fibrosis.ImageJ was used to quantify the degree of inflammatory cell infiltration and fibrosis,respectively.The changes of creatine kinase(CK),CK isoenzyme(CK-MB)and lactate dehydrogenase 1(LDH-1)in serum were detected.Enzyme-linked immunosorbent assay(ELISA)was used to detect cTnI,cTnT,interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factorɑ(TNF-α)in serum of mice.The expression of autophagy associated protein high mobility group B1(HMGB1),yeast Atg6 homology(Beclin-1),autophagy gene-associated protein 5(ATG5)and microtubule associated protein light chain 3B(LC3B)in mouse myocardial tissue was determined by western blotting.The expressions of cysteine aspartate protease 3(Caspase-3)and polyadenosine diphosphoribose polymerase(PARP)in myocardial tissue of mice were also measured.The myocardial nuclei were dyed blue by DAPI and the apoptotic cells were dyed green by TUNEL.Results(1)Compared with the control group,the body weight of mice in GHRL-12 group was not significantly different,and the body weight of mice in myocarditis group was not significantly increased;Compared with myocarditis group,the weight of mice in GHRL-12+myocarditis group increased significantly from the 19th day of administration.Compared with the control group and GHRL-12 group,the heart-body ratio and heart-shin ratio of mice in myocarditis group were significantly increased,while compared with myocarditis group,the heart-body ratio and heart-shin ratio of mice in GHRL-12+myocarditis group were decreased.(2)Echocardiography showed that the intervention of GHRL-12 alone had no significant effect on the cardiac function of mice.Compared with the control group,the left ventricular ejection fraction(LVEF)and shortening fraction(FS)of mice in myocarditis group were significantly decreased,and the left ventricular end-systolic diameter(LVEDs)and left ventricular end-diastolic diameter(LVEDd)were significantly increased.In GHRL-12+myocarditis group,LVEF and FS were increased,while LVEDs and LVEDd were decreased compared with the myocarditis group.(3)HE staining showed that inflammatory cell infiltration increased significantly in the myocarditis group compared with the control group,but decreased in the GHRL-12+myocarditis group compared with the myocarditis group.(4)Masson staining showed that the degree of myocardial fibrosis increased in the myocarditis group compared with the control group,but decreased in the GHRL-12+myocarditis group compared with the myocarditis group.(5)Serum CK,CK-MB,LDH-1,cTnI and cTnT in myocarditis group were significantly increased compared with the control group,while those in GHRL-12+myocarditis group were significantly decreased compared with the myocarditis group.(6)Western blot results showed that the protein expressions of HMGB1,Beclin-1,ATG5 and LC3B in myocarditis group were significantly increased compared with the control group,while those of GHRL-12+myocarditis group were significantly decreased compared with the myocarditis group;Meanwhile,the protein expressions of Caspase-3 and PARP were significantly increased in myocarditis group compared with the control group,and significantly decreased in GHRL-12+myocarditis group compared with the myocarditis group.(7)TUNEL results showed that myocardial apoptosis was significantly increased in myocarditis group compared with the control group and significantly decreased in GHRL-12+myocarditis group compared with the control group.Conclusions GHRL-12 peptide can improve the cardiac dysfunction induced by immune checkpoint inhibitors,reduce inflammatory cell infiltration and fibrosis in myocardial tissue,inhibit apoptosis and autophagy in myocardial tissue,and provide potential for the future treatment of immune checkpoint inhibitors-related myocarditis.