Chu W L, Chai J K, Wang X T, Han S F, Liu L Y
Department of Burns and Plastic Surgery, the Fourth Medical Center of PLA General Hospital, Beijing 100048, China.
Zhonghua Shao Shang Za Zhi. 2019 May 20;35(5):333-340. doi: 10.3760/cma.j.issn.1009-2587.2019.05.003.
To explore the effects of insulin therapy on skeletal muscle wasting (SMW) in severely scalded rats and its related mechanism. 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 test, analysis of variance for repeated measurement, analysis of variance for factorial design, test, and Bonferroni correction. The blood glycemic level and body mass of rats in the two groups before injury were similar (=0.204, 0.405, >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 (=0.229, 3.339, 1.610, 0.178, 0.181, 0.079, >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 (=7.245, 4.165, 4.609, 4.018, 3.995, <0.05 or <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 (=2.159, 2.375, <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 (=4.262, 9.153, 9.799, <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 (=10.429, 7.617, <0.01). Compared with those of group SS, the protein expressions of GRP78 were obviously increased on PID 4 and 14 (=4.172, 4.437, <0.05), the protein expressions of activated caspase-12 were obviously decreased on PID 7 and 14 (=11.049, 11.181, <0.01), and the protein expressions of CHOP were obviously decreased on PID 4, 7, and 14 (=13.837, 9.572, 6.930, <0.01) in TAM of rats in group IT. Insulin therapy may reduce skeletal muscle myocytes apoptosis and SMW by alleviating ERS in rats with severe scald.
探讨胰岛素治疗对严重烫伤大鼠骨骼肌萎缩（SMW）的影响及其相关机制。将48只7-8周龄雄性Wistar大鼠按随机数字表法分为单纯烫伤（SS）组和胰岛素治疗（IT）组，每组24只。两组大鼠经称重及用血糖仪测量尾端血糖水平后，于94℃热水中浸浴12秒，制成30%体表面积的全层背部烫伤模型。IT组大鼠于伤后第1天至第7天每天8:00皮下注射1 U/kg甘精胰岛素，SS组大鼠给予等量生理盐水。两组大鼠于伤后第1天至第7天每天8:00（胰岛素注射后）、13:00及18:00分别经胃内输注给予10 mL/kg肠内营养乳剂。于伤后第1-4天、6天、7天及伤后第8天、9天、11天、12天和14天每天上午用血糖仪测量两组大鼠尾端血糖水平。于伤后第14天对两组大鼠未作任何处理时称重。每组分别于伤后第4天、7天和14天取8只大鼠，采集胫前肌（TAM）样本。称取伤后第14天TAM质量。用透射电子显微镜观察伤后第7天TAM肌细胞超微结构变化。采用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸生物素缺口末端标记法检测伤后第4天、7天和14天TAM肌细胞凋亡率，用免疫组织化学法检测伤后第4天、7天和14天TAM中半胱氨酸天冬氨酸蛋白酶-3（caspase-3）的表达，用蛋白质印迹法检测伤后第4天、7天和14天TAM中内质网（ER）应激（ERS）相关蛋白葡萄糖调节蛋白78（GRP78）、CCAAT/增强子结合蛋白同源蛋白（CHOP）及活化的caspase-12的蛋白表达。数据采用完全随机设计检验、重复测量方差分析、析因设计方差分析、检验及Bonferroni校正处理。两组大鼠伤前血糖水平和体质量相近（=0.204，0.405，>0.05）。伤后第1天、6天、9天、11天、12天和14天两组大鼠血糖水平差异无统计学意义（=0.229，3.339，1.610，0.178，0.181，0.079，>0.05）。与SS组相比，IT组大鼠伤后第2天、3天、4天、7天和8天血糖水平显著降低（=7.245，4.165，4.609，4.018，3.995，<0.05或<0.01）。伤后第14天，IT组大鼠体质量和TAM质量分别为（271±19）g和（0.47±0.05）g，均明显高于SS组的（254±12）g和（0.43±0.04）g（=2.159，2.375，<0.05）。伤后第7天，观察到SS组大鼠TAM肌细胞核固缩、变形，染色体分布异常，内质网肿胀；与SS组相比，IT组大鼠TAM肌细胞凋亡改变及内质网肿胀减轻。伤后第4天、7天和14天，IT组大鼠TAM肌细胞凋亡率明显低于SS组（=4.262，9.153，9.799，<0.01）。伤后第7天和14天，IT组大鼠TAM中caspase-3表达明显低于SS组（=10.429，7.617，<0.01）。与SS组相比，IT组大鼠伤后第4天和14天TAM中GRP78蛋白表达明显升高（=4.172，4.437，<0.05），伤后第7天和14天活化的caspase-12蛋白表达明显降低（=11.049，11.181，<0.01），伤后第4天、7天和14天CHOP蛋白表达明显降低（=13.837，9.572，6.930，<0.01）。胰岛素治疗可能通过减轻严重烫伤大鼠的ERS来减少骨骼肌肌细胞凋亡和SMW。
