胰岛素对烧伤后心肌细胞损伤保护的初步研究  

Protective effect of insulin on burn serum-challenged cardiomyocytes in vitro

作　　者：吕根法 郑宏智 石宏伟 任罗达 Lyu Genfa;Zheng Hongzhi;Shi Hongwei;Ren Luoda(Department of Burn,Hospital of PLA Unit 96606,Luoyang 471003,Henan,China)

机构地区：[1]解放军96606部队医院烧伤科,河南洛阳471003

出　　处：《中华危重病急救医学》2020年第7期824-827,共4页Chinese Critical Care Medicine

基　　金：国家自然科学基金(30772250)。

摘　　要：目的观察胰岛素对烧伤后乳鼠心肌细胞的保护作用。方法原代培养出生2 d SD乳鼠心肌细胞,并分为假伤组(Sham组)、烧伤组、胰岛素组和胰岛素活化抑制剂LY294002预处理组(LY组)。制备清洁级3月龄SD大鼠烧伤血清〔30%总体表面积(TBSA)Ⅲ度烫伤大鼠,术后6 h取腹主动脉血清〕诱导的SD乳鼠心肌细胞损伤模型;胰岛素组在细胞培养基中同时加入10%烧伤大鼠血清和10 U/L胰岛素;LY组先加入50μmol/L的LY294002后30 min,再加入烧伤血清和胰岛素。Sham组仅给予10%假伤大鼠血清(假伤大鼠仅置于37℃温水中)。各组细胞继续培养12 h后,采用酶联免疫吸附试验(ELISA)测定心肌细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和肌酸激酶(CK)含量;采用蛋白质免疫印迹试验(Western Blot)检测心肌细胞肌钙蛋白T(cTnT)蛋白表达;采用Hoechst 33258染色法检测心肌细胞凋亡情况。结果与Sham组比较,经烧伤血清诱导后,心肌细胞发生炎症反应及损伤,表现为TNF-α、IL-6、CK含量升高〔TNF-α(ng/L):273±48比21±6,IL-6(ng/L):416±83比44±11,CK(U/L):1.44±0.24比0.14±0.08,均P<0.01〕,cTnT蛋白表达下调(cTnT/β-actin:0.12±0.04比0.86±0.34,P<0.01),凋亡细胞增多〔(19.1±5.6)%比(5.2±1.3)%,P<0.01〕;胰岛素能够明显减轻烧伤血清诱导的心肌细胞损伤,TNF-α、IL-6和CK含量均较烧伤组明显降低〔TNF-α(ng/L):105±37比273±48,IL-6(ng/L):176±77比416±83,CK(U/L):0.82±0.26比1.44±0.24,均P<0.05〕,且cTnT蛋白表达明显上调(cTnT/β-actin:0.41±0.16比0.12±0.04,P<0.05),凋亡细胞明显减少〔(10.7±3.2)%比(19.1±5.6)%,P<0.05〕;而胰岛素活化抑制剂LY294002能够明显抑制胰岛素对损伤心肌的保护作用。结论在烧伤血清诱导的乳鼠心肌细胞损伤模型中,胰岛素可能通过抗炎、抗损伤和抑制凋亡等发挥细胞保护作用。Objective To investigate the protective effect of insulin on burn serum-challenged cardiomyocytes in vitro.Methods Primary culture of cardiomyocytes from Sprague-Dawley (SD) 2-day-old neonate rats were divided into Sham group, burn group, insulin group, and insulin activation inhibitor LY294002 pretreatment group (LY group). The model of cardiomyocytes injury induced by burn serum of 3-month-old SD rats [the serum of abdominal aortic was collected at 6 hours after modelling 30% total surface area (TBSA) Ⅲ degree scald rat] was reproduced. In the insulin group, 10% burn serum and insulin (10 U/L) were added into cell culture medium, and in the LY group, LY294002 (50 μmol/L) was pretreated for 30 minutes before the addition of burn serum and insulin. Sham group was only given 10% serum of sham injured rats (sham rats were only placed in 37 ℃ warm water). After the cells were cultured for 12 hours, the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and creatine kinase (CK) were determined by enzyme-linked immunosorbent assay (ELISA). The cardiac troponin T (cTnT) protein expression was examined by Western Blot. Apoptosis of cardiomyocytes was observed after Hoechst 33258 staining.Results Compared with the Sham group, the cardiomyocytes were damaged and released inflammatory cytokines after burn serum-challenged. The levels of TNF-α, IL-6 and CK increased [TNF-α (ng/L): 273±48 vs. 21±6, IL-6 (ng/L): 416±83 vs. 44±11, CK (U/L): 1.44±0.24 vs. 0.14±0.08, all P < 0.01], while the expression of cTnT protein decreased (cTnT/β-actin: 0.12±0.04 vs. 0.86±0.34, P < 0.01), and the cardiomyocyte apoptosis increased [(19.1±5.6)% vs. (5.2±1.3)%, P < 0.01]. Insulin could significantly reduce the damage of cardiomyocytes, decrease the release of TNF-α, IL-6 and CK induced by burn serum [TNF-α (ng/L): 105±37 vs. 273±48, IL-6 (ng/L): 176±77 vs. 416±83, CK (U/L): 0.82±0.26 vs. 1.44±0.24, all P < 0.05], the expression of cTnT protein significantly increased (cTnT/β-actin: 0.41±0.16 vs. 0.1

关 键 词：烧伤血清 心肌细胞 胰岛素 

分 类 号：R644[医药卫生—外科学] R542.2[医药卫生—临床医学]