酸枣仁皂苷A通过miR-223-3p/SGK1/NLRP3轴在脓毒症小鼠肺上皮细胞损伤中的保护作用  

Protective effect of Jujuboside A on lung epithelial cell injury in mice with sepsis through miR-223-3p/SGK1/NLRP3 axis

作　　者：黄光明 戴国友[2] 付金铂 HUANG Guangming;DAI Guoyou;FU Jinbo(Department of Pharmacy,Nanjing Pukou District Hospital of Traditional Chinese Medicine,Nanjing Jiangsu 211800,China)

机构地区：[1]南京市浦口区中医院药剂科,江苏南京211800 [2]江苏省中医院药学部,江苏南京210029

出　　处：《中国急救复苏与灾害医学杂志》2024年第4期506-513,共8页China Journal of Emergency Resuscitation and Disaster Medicine

基　　金：江苏省优势学科建设工程项目(编号:YSHL0824-227)。

摘　　要：目的 探讨酸枣仁皂苷A通过miR-223-3p/血清和糖皮质激素诱导激酶1(SGK1)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)轴在脓毒症小鼠肺上皮细胞损伤中的保护作用及潜在机制。方法 通过盲肠结扎和穿刺(CLP)诱导脓毒症动物模型。通过酸枣仁皂苷A治疗实验动物,苏木精和伊红染色分析组织病理学变化。相应试剂盒分析肾损伤指标(血清BUN、SCr和NGAL水平)和血清炎性因子(TNF-α、IL-1β、IL-6)的水平。生信分析miR-223-3p在脓毒症患者中的表达及其与炎症反应因子的相关性,预测miR-223-3p与SGK1的结合位点。双荧光素酶实验验证miR-223-3p与SGK1的结合关系。构建脂多糖(LPS)诱导的HK2细胞的体外模型。荧光实时定量PCR(qPCR)检测miR-223-3p在患者血清或LPS处理的HK-2细胞中的表达。脱氧核苷酸末端转移酶介导的dUTP缺口末端标记(TUNEL)检测细胞凋亡,蛋白质免疫印迹检测中BCL2相关的X(BAX)、B细胞淋巴瘤-2(Bcl-2)和裂解的半胱氨酸蛋白酶(cleaved caspase-3)的表达,酶联免疫吸附试验(ELISA)检测患者血清或细胞上清液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)的水平。qPCR和蛋白质免疫印迹法检测不同处理组HK-2细胞中SGK1的表达,蛋白质免疫印迹检测不同处理组HK-2细胞中NLRP3的表达。结果 酸枣仁皂苷A治疗可以降低脓毒症小鼠急性肾小管坏死评分,减轻肾损伤。血清血尿素氮(BUN)、血肌酐(Scr)和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)水平降低,血清TNF-α、IL-1β、IL-6水平亦下降。酸枣仁皂苷A治疗可以降低脓毒症小鼠肾脏组织中miR-223-3p的表达水平。生信分析和临床样本分析显示,miR-223-3p在脓毒症患者中高表达。细胞实验结果显示,miR-223-3p在LPS处理的HK-2细胞中呈高表达。miR-223-3p促进LPS诱导的HK-2细胞凋亡和炎症反应,这种促进作用是由miR-223-3p通过负调控SGK1/NLRP3介导的。结论 酸�Objective To investigate the protective effect and potential mechanism of jujuboside A on lung epithelial cell injury in septic mice through miR-223-3p/serum and glucocorticoid-regulated kinase 1(SGK1)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)axis.Methods The animal model of sepsis was induced by cal ligation and puncture(CLP).Experimental animals were treated with jujuboside A,and histopathological changes were analyzed by hematoxylin and eosin staining.The corresponding kits were used to analyze the levels of renal injury indicators(serum BUN,SCr and NGAL levels)and serum inflammatory factors(TNF-α,IL-1β,IL-6).The expression of miR-223-3p in sepsis patients and its correlation with inflammatory factors were analyzed,and the binding site of miR-223-3p and SGK1 was predicted.The binding relationship between miR-223-3p and SGK1 was verified by dual luciferase assay.Quantitative real-time PCR(qPCR)was used to detect the expression of miR-223-3p in patients'serum or LPS-treated HK-2 cells.Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL).Western blotting was used to detect the expression of BCL2-associated X(BAX),B-cell lymphoma-2(Bcl-2)and cleaved caspase-3.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor alpha(TNF-α),interleukin-1 beta(IL-1β)and interleukin-6(IL-6)in serum or cell supernatant of patients.The expression of SGK1 in HK-2 cells of different treatment groups was detected by qPCR and Western blotting,and the expression of NLRP3 in HK-2 cells of different treatment groups was detected by Western blotting.Results The treatment of jujuboside A can reduce the score of acute renal tubular necrosis in septic mice,reduce renal injury(decreased serum blood urea nitrogen(BUN),serum creatinine(Scr)and neutrophil gelatinase associated lipocalin(NGAL)levels,and reduce the levels of TNF-α,IL-1βand IL-6 in serum.Jujuboside A treatment can reduce the expression level of miR-223-3p

关 键 词：酸枣仁皂苷A miR-223-3p SGK1 NLRP3 脓毒症 急性肾损伤 

分 类 号：R3[医药卫生—基础医学]