胰岛素治疗对严重烫伤大鼠骨骼肌消耗的作用及相关机制

Effects of insulin therapy on skeletal muscle wasting in severely scalded rats and its related mechanism

目的探讨胰岛素治疗对严重烫伤大鼠骨骼肌消耗的作用及相关机制。方法将48只雄性7~8周龄Wistar大鼠按随机数字表法分为单纯烫伤组和胰岛素治疗组,每组24只。2组大鼠称取体质量、用血糖仪检测尾部末梢血糖水平后,均在94℃热水中浸烫12s制成30%体表总面积Ⅲ度背部烫伤模型。伤后1~7d每天8:00,胰岛素治疗组大鼠皮下注射甘精胰岛素1U/kg,单纯烫伤组大鼠给予等量生理盐水;伤后1~7d每天8:00(注射胰岛素后)、13:00、18:00,2组大鼠均给予肠内营养乳剂灌胃各1次,每次10mL/kg。伤后1~4、6、7d每天注射胰岛素前及伤后8、9、11、12、14d晨,用血糖仪检测2组大鼠尾部末梢血糖水平。称取2组大鼠伤后14d未行任何处理时的体质量。伤后4、7、14d,每组分别取8只大鼠,采集胫骨前肌标本,称取伤后14d胫骨前肌质量,透射电镜观察伤后7d胫骨前肌肌细胞超微结构变化,末端脱氧核苷酸转移酶介导的脱氧尿三磷酸缺口末端标记法测定伤后4、7、14d胫骨前肌肌细胞凋亡率,免疫组织化学法检测伤后4、7、14d胫骨前肌半胱氨酸天冬氨酸蛋白酶3(caspase-3)表达,蛋白质印迹法检测伤后4、7、14d胫骨前肌内质网应激反应(ERS)相关蛋白葡萄糖调节蛋白78(GRP78)、caspase-12活性体、CCAAT/增强子结合蛋白同源蛋白(CHOP)蛋白表达。对数据行完全随机设计t检验、重复测量方差分析、析因设计方差分析、t检验及Bonferroni校正。结果伤前2组大鼠血糖水平、体质量相近(t=0.204、0.405,P>0.05)。伤后1、6、9、11、12、14d,2组大鼠血糖水平相近(t=0.229、3.339、1.610、0.178、0.181、0.079,P>0.05);伤后2、3、4、7、8d,胰岛素治疗组大鼠血糖水平显著低于单纯烫伤组(t=7.245、4.165、4.609、4.018、3.995,P<0.05或P<0.01)。伤后14d,胰岛素治疗组大鼠体质量和胫骨前肌质量分别为(271±19)、(0.47±0.05)g,均明显高于单纯烫伤组的(254±12)、(0.43±0.04)g(t=2.159、2.375,P<0.05)。伤后7d,单纯烫伤组大鼠胫骨前肌肌细胞部分细胞核固缩、变形,染色质分布不均,内质网肿胀;胰岛素治疗组大鼠胫骨前肌肌细胞凋亡改变和内质网肿胀较单纯烫伤组减轻。伤后4、7、14d,胰岛素治疗组大鼠胫骨前肌肌细胞凋亡率均较单纯烫伤组明显降低(t=4.262、9.153、9.799,P<0.01)。伤后7、14d,胰岛素治疗组大鼠胫骨前肌caspase-3表达均较单纯烫伤组明显减少(t=10.429、7.617,P<0.01)。与单纯烫伤组比较,胰岛素治疗组大鼠伤后4、14d胫骨前肌GRP78蛋白表达明显增加(t=4.172、4.437,P<0.05),伤后7、14d胫骨前肌caspase-12活性体蛋白表达明显减少(t=11.049、11.181,P<0.01),伤后4、7、14d胫骨前肌CHOP蛋白表达明显减少(t=13.837、9.572、6.930,P<0.01)。结论胰岛素治疗可能通过缓解ERS,减少严重烫伤大鼠骨骼肌肌细胞凋亡和骨骼肌消耗。

Objective To explore the effects of insulin therapy on skeletal muscle wasting(SMW)in severely scalded rats and its related mechanism.Methods Totally 48 male Wistar rats aged 7-8 weeks were divided into simple scald(SS)group and insulin therapy(IT)group according to the random number table,with 24 rats in each group.After weighing the body mass and measuring the blood glycemic level of the tail end with a glucometer,the rats in the two groups were immersed in hot water at 94℃for 12 seconds to make a full-thickness dorsal scald model involving 30% total body surface area.Rats in group IT were subcutaneously injected with 1 U/kg insulin glargine at 8:00 a day from post injury day(PID)1 to 7,whilst rats in group SS were given the same amount of normal saline.Rats in the two groups were given 10 mL/kg enteral nutritional emulsion by intragastric infusion at 8:00(after insulin administration),13:00,and 18:00 a day respectively from PID 1 to 7.The blood glycemic levels of tail end of rats in the two groups were measured by glucometer before insulin administration on PID 1-4,6,and 7 and on every morning of PID 8,9,11,12,and 14.The body mass of rats in the two groups on PID 14 without any treatment was weighed.Eight rats from each group were collected respectively on PID 4,7,and 14 to harvest tibialis anterior muscle(TAM)samples.The mass of TAM on PID 14 was weighed.The ultrastructural changes of TAM myocytes on PID 7 were observed with transmission electron microscope.The apoptotic rates of TAM myocytes on PID 4,7,and 14 were assessed by the assay of terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphate-biotin nick end labeling,the expressions of cysteine-aspartic protease-3(caspase-3)of TAM on PID 4,7,and 14 were detected with immunohistochemistry,and protein expressions of endoplasmic reticulum(ER)stress(ERS)associated proteins glucose-regulated protein 78(GRP78),CCAAT/enhancer binding protein-homologous protein(CHOP),and activated caspase-12 of TAM on PID 4,7,and 14 were detected with Western blotting.Data were processed with completely random design t test,analysis of variance for repeated measurement,analysis of variance for factorial design,t test,and Bonferroni correction.Results The blood glycemic level and body mass of rats in the two groups before injury were similar(t=0.204,0.405,P>0.05).There were no statistically significant differences in blood glycemic levels of rats between the two groups on PID 1,6,9,11,12,and 14(t=0.229,3.339,1.610,0.178,0.181,0.079,P>0.05).Compared with those of group SS,blood glycemic levels of rats in group IT were significantly lower on PID 2,3,4,7,and 8(t=7.245,4.165,4.609,4.018,3.995,P<0.05 or P<0.01).On PID 14,the body mass and TAM mass of rats in group IT were(271±19)g and(0.47±0.05)g respectively,both obviously higher than(254±12)g and(0.43±0.04)g of group SS(t=2.159,2.375,P<0.05).On PID 7,nuclear pyknosis and deformation,chromosome misdistribution,and ER swelling in TAM myocytes of rats in group SS were observed;the apoptotic alterations and ER swelling of TAM myocytes were alleviated in rats of group IT as compared with those of group SS.The apoptotic rates of TAM myocytes of rats in group IT were obviously lower than those of group SS on PID 4,7,and 14(t=4.262,9.153,9.799,P<0.01).The expressions of caspase-3 in TAM of rats in group IT were obviously lower than those of group SS on PID 7 and 14(t=10.429,7.617,P<0.01).Compared with those of group SS,the protein expressions of GRP78 were obviously increased on PID 4 and 14(t=4.172,4.437,P<0.05),the protein expressions of activated caspase-12 were obviously decreased on PID 7 and 14(t=11.049,11.181,P<0.01),and the protein expressions of CHOP were obviously decreased on PID 4,7,and 14(t=13.837,9.572,6.930,P<0.01)in TAM of rats in group IT.Conclusions Insulin therapy may reduce skeletal muscle myocytes apoptosis and SMW by alleviating ERS in rats with severe scald.