Zhao Y N, Li Z Y, Kan K, Su H T
Department of Burns, the Fifth Hospital of Harbin, Harbin 150040, China.
Zhonghua Shao Shang Za Zhi. 2018 Feb 20;34(2):102-106. doi: 10.3760/cma.j.issn.1009-2587.2018.02.008.
To investigate the mechanism of cell autophagy for regulating skeletal muscle wasting of rats after severe burns. Seventy-two Sprague-Dawley rats were collected and divided into sham injury group, simple burn group, burn+ phosphate buffer solution (PBS) group, and burn+ 3-methyladenine (3-MA) group according to the random number table, with 18 rats in each group. Rats in simple burn group, burn+ PBS group, and burn+ 3-MA group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns). Rats in sham injury group were sham injured. Immediately after burns and fluid resuscitation, rats in burn+ PBS group were intraperitoneally injected with 1 mL PBS, and rats in burn+ 3-MA group were intraperitoneally injected with 1 mL 3-MA (125 g/L). On post injury day 3 and 7, the weights of anterior tibial muscle of right hind limbs and body of rats were measured to calculate percentage of anterior tibial muscle of right hind limbs weight. Protein expressions of microtubule related protein 1 light chain 3A (LC3A) and Beclin-1 of anterior tibial muscle were observed by immunofluorescence method and detected by Western blotting, and ratio of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ was calculated. Data were processed with analysis of variance of factorial design, one-way analysis of variance, -test and Bonferroni correction. On post injury day 3 and 7, percentages of anterior tibial muscle of right hind limbs weight of rats in simple burn group were (0.148±0.009)% and (0.134±0.018)%, respectively, which were significantly lower than those in sham injury group [(0.203±0.009)%, (0.181±0.015)%, =10.585, 4.913, <0.01]. Percentages of anterior tibial muscle of right hind limbs weight of rats in burn+ 3-MA group were (0.187±0.004)% and (0.192±0.009)%, respectively, which were obviously higher than those in burn+ PBS group [(0.162±0.005)%, (0.167±0.005)%, =9.564, 5.948, <0.01]. On post injury day 3 and 7, protein expressions of Beclin-1 and microtubule related protein 1 LC3A of anterior tibial muscle of rats in simple burn group were significantly higher than those in sham injury group, while protein expressions of Beclin-1 and microtubule related protein 1 LC3A of anterior tibial muscle of rats in burn+ 3-MA group were significantly lower than those in burn+ PBS group. Ratios of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ of anterior tibial muscle of rats in simple burn group were significantly higher than those in sham injury group (=3.461, 3.353, <0.05), while ratios of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ of anterior tibial muscle of rats in burn+ 3-MA group were significantly lower than those in burn+ PBS group (=3.129, 3.977, <0.05). Cell autophagy induced by severe burns is involved in the process of skeletal muscle wasting of rats, and inhibition of cell autophagy may contribute to the remission of skeletal muscle wasting of rats induced by burns.
探讨细胞自噬调控严重烧伤后大鼠骨骼肌萎缩的机制。选取72只Sprague-Dawley大鼠,按随机数字表法分为假伤组、单纯烧伤组、烧伤+磷酸盐缓冲液(PBS)组、烧伤+3-甲基腺嘌呤(3-MA)组,每组18只。单纯烧伤组、烧伤+PBS组、烧伤+3-MA组大鼠行30%总体表面积全层烫伤(以下简称烧伤)。假伤组大鼠行假伤处理。烧伤及液体复苏后即刻,烧伤+PBS组大鼠腹腔注射1 mL PBS,烧伤+3-MA组大鼠腹腔注射1 mL 3-MA(125 g/L)。于伤后第3天和第7天,测量大鼠右后肢胫骨前肌重量及体质量,计算右后肢胫骨前肌重量占体质量的百分比。采用免疫荧光法观察并通过蛋白质印迹法检测大鼠胫骨前肌微管相关蛋白1轻链3A(LC3A)和Beclin-1的蛋白表达,并计算微管相关蛋白1 LC3A-Ⅱ与LC3A-Ⅰ的比值。数据采用析因设计的方差分析、单因素方差分析、t检验及Bonferroni校正进行处理。伤后第3天和第7天,单纯烧伤组大鼠右后肢胫骨前肌重量占体质量的百分比分别为(0.148±0.009)%和(0.134±0.018)%,显著低于假伤组[(0.203±0.009)%,(0.181±0.015)%,F=10.585,4.913,P<0.01]。烧伤+3-MA组大鼠右后肢胫骨前肌重量占体质量的百分比分别为(0.187±0.004)%和(0.192±0.009)%,明显高于烧伤+PBS组[(0.162±0.005)%,(0.167±0.005)%,F=9.564,5.948,P<0.01]。伤后第3天和第7天,单纯烧伤组大鼠胫骨前肌Beclin-1和微管相关蛋白1 LC3A的蛋白表达显著高于假伤组,而烧伤+3-MA组大鼠胫骨前肌Beclin-1和微管相关蛋白1 LC3A的蛋白表达显著低于烧伤+PBS组。单纯烧伤组大鼠胫骨前肌微管相关蛋白1 LC3A-Ⅱ与LC3A-Ⅰ的比值显著高于假伤组(t=3.461,3.353,P<0.05),而烧伤+3-MA组大鼠胫骨前肌微管相关蛋白1 LC3A-Ⅱ与LC3A-Ⅰ的比值显著低于烧伤+PBS组(t=3.129,3.977,P<0.05)。严重烧伤诱导的细胞自噬参与大鼠骨骼肌萎缩过程,抑制细胞自噬可能有助于缓解烧伤诱导的大鼠骨骼肌萎缩。