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Title: [Effects of glutamine on skeletal muscle membrane repair in severely burned mice and the functional mechanism].
Author: Wu D, Wang C, Wang ZE, Hu JH, Shi Y, Zhu YJ, Peng X.
Journal: Zhonghua Shao Shang Za Zhi; 2019 May 20; 35(5):341-350. PubMed ID: 31154731.
Abstract:
Objective: To observe how glutamine affect the skeletal muscle membrane repair in severely burned mice through promoting the mitsugumin 53 (MG53) dimerization in skeletal muscle and to explore its functional mechanism. Methods: (1) Animal experiments. A total of 179 BALB/c male mice aged 6 to 8 weeks were divided into sham injury group (n=43), burn group (n=73) and burn+ glutamine group (n=63) according to the random number table (the same grouping method below). Mice in sham injury group were sham injured on the back, and mice in burn group and burn+ glutamine group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Mice in burn+ glutamine group were intragastrically administered with glutamine (1 mg/kg), and the other two groups were given the same amount of amino acid solution once per day for 14 days. On post burn hour 12, 10 mice from burn group were taken for preparation of burn serum, which is used in the following cell experiments. Blood samples were collected from the hearts to prepare serum from 10 mice in sham injury group immediately after burn and from 10 mice in burn group and burn+ glutamine group on post burn day (PBD) 5, 10, and 14, respectively. And then the whole gastrocnemius muscle was harvested after the mice were sacrificed. On PBD 10, the whole flexor brevis digitorum was harvested from 6 mice in the 3 groups respectively after the mice were sacrificed. On PBD 5, 10, and 14, the whole gastrocnemius muscle tissue was harvested from another 9 mice in the 3 groups respectively after the mice were sacrificed. The mass of the whole gastrocnemius muscle of mice was weighed. The total protein content of gastrocnemius muscle of mice was detected by coomassie brilliant blue method. The repair function of myolemma of flexor brevis digitorum of mice was detected by two-photon laser fiber membrane perforating. The serum content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of mice was determined with radioimmunoassay. The expressions of MG53 dimer and monomer in gastrocnemius of mice were determined with non-reductive electrophoresis-Western blotting. The protein expressions of endoplasmic reticulum stress sign proteins CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) in gastrocnemius of mice were determined with Western blotting. (2) Cell experiments. Mice skeletal muscle precursor cells C2C12 were cultured in vitro, and cells of the second passage were selected for the experiments. The cells were divided into normal control group, burn serum group, and burn serum+ glutamine group, with 3 dishes in each group and 1×10(3) cells in each dish. Cells in normal control group were cultured with 1 mL Dulbecco's modified Eagle medium (DMEM) with fetal bovine serum of volume fraction 10%, cells in burn serum group were cultured with 1 mL DMEM with burn serum of volume fraction 10%, and cells in burn serum+ glutamine group were cultured with 1 mL DMEM with burn serum of volume fraction 10% and 4 μL glutamine with a final molar concentration of 8 mmol/L. After 24 hours of culturing, the repair function of myocyte membrane after differentiation of skeletal muscle precursor cells in mice was detected with the same method before. Another cells were grouped and cultured as before, with 3 wells in each group and 1×10(5) cells in each well. After 24 hours of culturing, the expressions of MG53 dimer and monomer and endoplasmic reticulum stress marker proteins in the cells were detected as before. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least significant difference t test, and Student Newman Keuls test. Results: Animal experiments. (1) Compared with those in sham injury group, the mass and total protein content of gastrocnemius muscle of mice in burn group were significantly decreased on PBD 5, 10, and 14 (P<0.05). Compared with those in burn group, the mass and total protein content of gastrocnemius muscle of mice in burn+ glutamine group were significantly increased on PBD 5, 10, and 14 (P<0.05). (2) Compared with that in sham injury group (0.9±0.4), the fluorescence intensity of FM1-43 in myofiber of mice in burn group (7.8±0.4) was significantly increased on PBD 10 (t=7.75, P<0.05). Compared with that in burn group, the fluorescence intensity of FM1-43 in myofiber of mice in burn+ glutamine group (4.0±0.4) was significantly decreased on PBD 10 (t=-4.31, P<0.05). (3) Compared with that in sham injury group, the serum content of TNF-α and IL-6 of mice in burn group was significantly increased on PBD 5, 10, and 14 (P<0.05). Compared with that in burn group, the serum content of TNF-α and IL-6 of mice in burn+ glutamine group was significantly decreased on PBD 5, 10, and 14 (P<0.05). (4) Compared with 56.97±2.82, 44.89±4.72, 42.46±1.06, 14.26±0.99, 62.36±2.74, and 29.45±0.84 in sham injury group, the expressions of MG53 dimer and monomer in gastrocnemius of mice were significantly decreased in burn group on PBD 5, 10, and 14 (6.16±0.25, 26.09±1.22, 28.86±1.53, 5.63±0.25, 26.74±0.79, 4.41±0.52, P<0.05). Compared with those in burn group, the expression of MG53 dimer of gastrocnemius of mice in burn+ glutamine group was significantly increased on PBD 10 and 14 (36.79±1.44, 43.96±1.62), and the expression of MG53 monomer of gastrocnemius muscle of mice in burn+ glutamine group was significantly increased on PBD 14 (13.16±2.17, P<0.05). Compared with those in sham injury group, the protein expressions of CHOP and GRP78 in gastrocnemius muscle of mice in burn group were significantly elevated on PBD 5, 10, and 14 (P<0.05). Compared with those in burn group, the protein expressions of CHOP and GRP78 in gastrocnemius of mice in burn+ glutamine group were significantly reduced on PBD 5, 10 (P<0.05). Cell experiments. (1) Compared with that in normal control group (1.76±0.25), the fluorescence intensity of FM1-43 in cells in burn serum group (9.46±1.22) was significantly increased after 24 hours of culturing (t=12.28, P<0.05). Compared with that in burn serum group, the fluorescence intensity of FM1-43 in cells in burn serum+ glutamine group (4.71±0.45) was significantly decreased after 24 hours of culturing (t=-7.59, P<0.05). (2) The expressions of MG53 monomer of cells were similar in normal control group, burn serum group, and burn+ glutamine group after 24 hours of culturing (P>0.05). Compared with 58.5±1.8 in normal control group, the expression of MG53 dimer of cells in burn serum group was significantly decreased after 24 hours of culturing (14.1±1.4, P<0.05). Compared with that in burn serum group, the expression of MG53 dimer of cells in burn serum+ glutamine group was significantly increased after 24 hours of culturing (30.9±0.6, P<0.05). Compared with those in normal control group, the protein expressions of CHOP and GRP78 of cells were significantly elevated in burn serum group after 24 hours of culturing (P<0.05). Compared with those in burn serum group, the protein expressions of CHOP and GRP78 of cells were significantly reduced in burn serum+ glutamine group after 24 hours of culturing (P<0.05). Conclusions: Glutamine can promote MG53 dimerization by alleviating endoplasmic reticulum stress in severely burned mice. Thus it can accelerate skeletal muscle membrane repair, reduce the local inflammatory reaction of skeletal muscle and consumption of skeletal muscle. 目的： 观察谷氨酰胺如何通过促进骨骼肌MG53二聚化，从而对严重烧伤小鼠骨骼肌膜修复产生影响，并探讨其作用机制。 方法： (1)动物实验。按随机数字表法(分组方法下同)将179只6～8周龄BALB/c雄性小鼠分为假伤组(43只)、烧伤组(73只)、烧伤＋谷氨酰胺组(63只)。将假伤组小鼠背部致假伤，将烧伤组和烧伤＋谷氨酰胺组小鼠背部造成Ⅲ度30%体表总面积烫伤(以下称烧伤)。烧伤＋谷氨酰胺组小鼠灌胃谷氨酰胺1 mg/kg，另2组小鼠灌胃等量氨基酸溶液，每天1次，持续14 d。伤后12 h，取10只烧伤组小鼠制备烧伤血清，用于后续细胞实验。假伤组在伤后即刻取10只小鼠，烧伤组和烧伤＋谷氨酰胺组分别在伤后5、10、14 d各取10只小鼠，心脏采血制备血清，然后处死取整块腓肠肌。伤后10 d，3组分别另取6只小鼠处死后取整块趾短屈肌。伤后5、10、14 d，3组分别另取9只小鼠处死后取整块腓肠肌。称取整块小鼠腓肠肌质量，考马斯亮蓝法检测小鼠腓肠肌总蛋白含量；双光子激光肌膜打孔检测小鼠趾短屈肌肌膜修复功能；放射免疫分析检测小鼠血清中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)含量；采用非还原性电泳-蛋白质印迹法检测小鼠腓肠肌中MG53二聚体及单体表达，蛋白质印迹法检测小鼠腓肠肌中内质网应激标志蛋白CCAAT/增强子结合蛋白同源蛋白(CHOP)、葡萄糖调节蛋白78(GRP78)蛋白表达。(2)细胞实验。体外培养小鼠骨骼肌前体细胞C2C12，选第2代细胞进行实验。将细胞分为正常对照组、烧伤血清组、烧伤血清＋谷氨酰胺组，每组3皿，每皿1×10(3)个细胞，正常对照组细胞加入1 mL含有体积分数10%胎牛血清的DMEM培养液，烧伤血清组细胞加入1 mL含有体积分数10%烧伤血清的DMEM培养液，烧伤血清＋谷氨酰胺组细胞加入1 mL含有体积分数10%烧伤血清及4 μL终物质的量浓度为8 mmol/L的谷氨酰胺的DMEM培养液。培养24 h后，同前检测小鼠骨骼肌前体细胞分化后肌细胞膜修复功能。另取细胞同前分组及培养，每组3孔，每孔1×10(5)个细胞，培养24 h后同前检测细胞中MG53二聚体及单体表达及细胞中内质网应激标志蛋白表达。对数据行析因设计方差分析、单因素方差分析、LSD-t检验、SNK检验。 结果： 动物实验。(1)与假伤组比较，烧伤组伤后5、10、14 d小鼠腓肠肌质量和总蛋白含量均明显降低(P<0.05)；与烧伤组比较，烧伤＋谷氨酰胺组伤后5、10、14 d小鼠腓肠肌质量和总蛋白含量均明显增加(P<0.05)。(2)与假伤组的0.9±0.4比较，烧伤组伤后10 d小鼠肌纤维内FM1-43荧光强度(7.8±0.4)明显升高(t＝7.75，P<0.05)；与烧伤组比较，烧伤＋谷氨酰胺组伤后10 d小鼠肌纤维内FM1-43荧光强度(4.0±0.4)明显降低(t＝－4.31，P<0.05)。(3)与假伤组比较，烧伤组伤后5、10、14 d小鼠血清TNF-α、IL-6含量均明显增加(P<0.05)；与烧伤组比较，烧伤＋谷氨酰胺组伤后5、10、14 d小鼠血清TNF-α、IL-6含量均明显减少(P<0.05)。(4)与假伤组(56.97±2.82、44.89±4.72、42.46±1.06，14.26±0.99、62.36±2.74、29.45±0.84)比较，烧伤组伤后5、10、14 d小鼠腓肠肌中MG53二聚体和单体表达均明显降低(6.16±0.25、26.09±1.22、28.86±1.53，5.63±0.25、26.74±0.79、4.41±0.52，P<0.05)；与烧伤组比较，烧伤＋谷氨酰胺组伤后10、14 d小鼠腓肠肌中MG53二聚体表达(36.79±1.44、43.96±1.62)和伤后14 d小鼠腓肠肌中MG53单体表达明显升高(13.16±2.17，P<0.05)。与假伤组比较，烧伤组伤后5、10、14 d小鼠腓肠肌中CHOP、GRP78蛋白表达明显升高(P<0.05)；与烧伤组比较，烧伤＋谷氨酰胺组伤后5、10 d小鼠腓肠肌中CHOP、GRP78蛋白表达均明显降低(P<0.05)。细胞实验。(1)与正常对照组的1.76±0.25比较，烧伤血清组培养24 h细胞FM1-43荧光强度(9.46±1.22)明显升高(t＝12.28，P<0.05)；与烧伤血清组比较，烧伤血清＋谷氨酰胺组培养24 h细胞FM1-43荧光强度(4.71±0.45)明显降低(t＝－7.59，P<0.05)。(2)正常对照组、烧伤血清组、烧伤血清＋谷氨酰胺组细胞培养24 h MG53单体表达相近(P>0.05)。与正常对照组(58.5±1.8)比较，烧伤血清组培养24 h细胞MG53二聚体表达明显降低(14.1±1.4，P<0.05)，与烧伤血清组比较，烧伤血清＋谷氨酰胺组培养24 h细胞MG53二聚体表达明显升高(30.9±0.6，P<0.05)。与正常对照组比较，烧伤血清组培养24 h细胞CHOP、GRP78蛋白表达明显升高(P<0.05)；与烧伤血清组比较，烧伤血清＋谷氨酰胺组培养24 h细胞CHOP、GRP78蛋白表达明显降低(P<0.05)。 结论： 谷氨酰胺能通过减轻严重烧伤小鼠内质网应激促进MG53二聚化，加速骨骼肌膜修复，减轻骨骼肌局部炎症反应，减少骨骼肌消耗。.

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