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 1×103 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 1×105 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.
ABSTRACT
Objective@#To explore the effects of different doses of dopamine on organ function of rats at early stage of severe scald.@*Methods@#Thirty-two male Wistar rats aged 8 to 12 weeks were divided into sham injury (SI) group, simple resuscitation (SR) group, small dose (SD) group, and moderate dose (MD) group according to the random number table, with 8 rats in each group. After rats in the 4 groups were performed cardiac catheterization, rats in group SI were sham injured on the back by immersing in 37 ℃ warm water for 18 s, and rats in the other 3 groups were inflicted with 30% total body surface area (TBSA) full-thickness scald on the back by immersing in 97 ℃ hot water for 18 s. Rats in group SI were not treated after the injury, while rats in the other 3 groups were performed fluid resuscitation for 24 h through jugular vein catheter with micro syringe pump according to the Parkland formula. They were given 4.0 mL·kg-1·% TBSA-1 normal saline during the first 24 h, of which they were given half of the total amount for the first 8 h, and they were given half of the total amount for the second and third 8 h. Rats in group SR were infused normal saline only, while rats in group SD and group MD were infused normal saline+ 1.25 μg·kg-1·min-1dopamine and normal saline+ 6.00 μg·kg-1·min-1 dopamine respectively. Volume of 0.5 mL venous blood of all rats were taken through the cardiac catheter with serum separated at post injury hour (PIH) 1, 3, 6, 12, and 24. Serum content of cardiac troponin I (cTnI) was determined by enzyme-linked immunosorbent assay; serum content of diamine oxidase (DAO) was detected by ultraviolet spectrophotometer; serum content of β2-microglobulin (β2-MG) was determined by latex-enhanced immunoturbidimetric assay; serum content of total bile acid (TBA) was determined by enzyme colorimetry; serum content of lactic acid, malondialdehyde, and myeloperoxidase (MPO) was determined by ultraviolet spectrophotometer. Data were processed with analysis of variance for repeated measurement, one-way analysis of variance, least significant difference test, and Bonferroni correction.@*Results@#(1) At PIH 1, 3, 6, 12, and 24, serum content of cTnI of rats in group SR, group SD, and group MD [(2.69±0.19), (3.04±0.19), (4.96±0.25), (6.88±0.28), (4.75±0.31) μg/L, (2.70±0.14), (3.08±0.13), (5.06±0.19), (7.11±0.21), (4.89±0.16) μg/L, (2.18±0.14), (2.54±0.09), (3.97±0.14), (5.46±0.34), (3.32±0.33) μg/L] were higher than that in group SI [(1.70±0.08), (1.70±0.08), (1.69±0.11), (1.69±0.08), (1.70±0.08) μg/L, P<0.05], serum content of cTnI of rats in group SR and group SD was similar (P>0.05), and serum content of cTnI of rats in group MD was lower than that in group SR and group SD (P<0.05). (2) At PIH 1 to 24, serum content of DAO of rats in group SR, group SD, and group MD was higher than that in group SI (P<0.05), serum content of DAO of rats in group SR and group MD was similar (P>0.05), and serum content of DAO of rats in group SD was lower than that in group SR and group MD (P<0.05). (3) At PIH 1 to 24, serum content of β2-MG of rats in group SR, group SD, and group MD was higher than that in group SI (P<0.05), serum content of β2-MG of rats in group SR and group MD was similar (P>0.05), and serum content of β2-MG of rats in group SD was lower than that in group SR and group MD (P<0.05). (4) At PIH 1 to 24, serum content of TBA of rats in group SR, group SD, and group MD was similar (P>0.05) and higher than that in group SI (P<0.05). (5) At PIH 1 to 24, serum content of lactic acid of rats in group SR, group SD, and group MD was higher than that in group SI (P<0.05), serum content of lactic acid of rats in group SR and group MD was similar (P>0.05), and serum content of lactic acid of rats in group SD was lower than that in group SR and group MD (P<0.05). (6) At PIH 1 to 24, serum content of malondialdehyde and MPO of rats in group SR, group SD, and group MD was higher than that in group SI (P<0.05), serum content of malondialdehyde and MPO of rats in group SR and group MD was similar (P>0.05), and serum content of malondialdehyde and MPO of rats in group SD was significantly lower than that in group SR and group MD (P<0.05).@*Conclusions@#With effective liquid recovery, dopamine of MD can improve early cardiac function of rats with severe scald, while dopamine of SD can alleviate tissue ischemia and hypoxia, reduce oxygen free radical damage in internal organs, and improve functions of intestine and kidney.