Effects of glutamine on skeletal muscle membrane repair in severely burned mice and the functional mechanism / 中华烧伤杂志

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.

The qualitative effects of resveratrol and coenzyme Q10 administration on the gluteus complex muscle morphology of SJL/J mice with dysferlinopathy / Efectos cualitativos de la administración de resveratrol y coenzima Q10 en la morfología del complejo muscular glúteo de ratones SJL/J con disferlinopatía

Dysferlinopathy is a form of muscular dystrophy affecting muscles of the shoulder and pelvic girdles, resulting from inheritance of a mutated dysferlin gene. The encoded dysferlin protein is proposed to be involved in sarcolemmal vesicle fusion with a disrupted plasma membrane; however, with defective protein function these vesicles accumulate beneath the disruption site but are unable to fuse with it and reseal the membrane, thus rendering the membrane repair mechanism defective. The SJL/J mouse model presents with characteristics much like the commonest human condition. Immune modulators have long been under study in the maintenance of muscle health in muscular dystrophies. Such supplementary treatment would ideally suppress inflammation, preventing the immune response toward degenerating muscle from causing additional muscle fiber death, and thus provide a mechanism by which to prolong the life of muscle fibers with inherently defective healing apparatus. For this purpose the anti-inflammatory supplement resveratrol and the membrane-protective supplement coenzyme Q10 were administered separately and in combination to experimental animals to determine their effectiveness in possible therapy of dysferlinopathy. The findings of this study report that low doses of resveratrol and coenzyme Q10 supplementation in exclusivity were unable to afford much protection to muscle fibers at the tissue level. High doses of coenzyme Q10 proved more effective in reducing attenuating inflammation; and combination treatment with resveratrol and coenzyme Q10 provided not only the membrane-protective effects of coenzyme Q10, but also the anti-inflammatory effects of resveratrol which failed to materialize at sufficient levels in exclusive administration.

Disferlinopatía es una forma de distrofia muscular que afecta a los músculos de los hombros y cintura pélvica, resultado de la herencia y mutación del gen de la distrofina. Sugerimos que la proteína codificada distrofina que integra la estructura sarcolemal con una membrana plasmática interrumpida, que al presentar una proteína defectuosa, las estructuras se acumulan debajo del sitio de alteración sin lograr fundirse con éste y cerrar la membrana afectando el mecanismo de reparación. El modelo de ratón SJL / J se presenta con características muy similares a una condición humana común. Los inmunomoduladores han sido objeto de estudio en el mantenimiento de la salud muscular en las distrofias musculares. Este tipo de tratamiento suplementario puede ser ideal para suprimir la inflamación, en la prevención de la respuesta inmune en la degeneración muscular causando la muerte adicional de fibra muscular, y al mismo tiempo proporcionar, un mecanismo con el cual prolongar la vida útil de aquellas fibras musculares con el aparato de sanación comprometido. Para ello, el Resveratrol suplemento anti-inflamatorio y el suplemento protector de membrana coenzima Q10 se administró por separado y en combinación en los animales de laboratorio para determinar su efectividad en el tratamiento de posible disferlinopatía. Los resultados de este estudio indican que el Resveratrol en menor dosis y la coenzima Q 10 administrados como suplementos de manera exclusiva, no demostraron efectos de protección de las fibras musculares a nivel del tejido. Una alta dosis de coenzima Q10 demostró ser más efectiva en la reducción de la inflamación; adicionalmente, el tratamiento combinado de Resveratrol y coenzima Q10 proporcionó efectos protectores de membrana, además de los efectos anti-inflamatorios del Resveratrol cuyo nivel no alcanzó la efectividad suficiente al ser administrado en forma exclusiva.

Clinical and immune pathological characteristics of skeletal muscle in UCMD with sarcolemma-specific collagen Ⅵ deficiency / 复旦学报（医学版）

Objective To investigate the clinical and immune pathological features of Ullrich congenital muscular dystrophy (UCMD) with sarcolemma-specific collagen Ⅵ deficiency (SSCD). Methods The clinical aspects of 2 patients with SSCD were analyzed and the muscle specimens from them were studied by immunofluorescence. Results SSCD patients were clinically characterized by neonatal hypotonia with proximal contractures and distal hyperlaxity at birth or early infancy. Immunofluorescence staining revealed partial deficiency of collagen Ⅵ. Double immunofluorescence staining revealed sarcolemma-specific deficiency of collagen Ⅵ, while collagen Ⅳ intact in thesarcolemma. Conclusions The clinical picture and severity of UCMD with SSCD are similar to the cases with collagen Ⅵ complete deficiency. The proximal contractures and distal hyperlaxity are the clinical hallmarks of both types. Sarcolemma-specific collagen Ⅵ deficiency can be better demonstrated by double immunofluorescence staining.

EFFECTS OF GINSENOSIDES ON THE ACTIVITIES OF Na~+, K~+-ATPase OF DOG HEART SARCOLEMMA / 中国药理学通报

The effects of ginsenosides(including the total ginsenoside extracted from stems and leaves-GNS, ginsenoside extracted from rots-GRS, panaxadiol saPonin-PDS and panaxatriol saponin-PTS) on the activities of Na+, K+-ATPase of dog heart sarcolemma were studied in vivo and in vitro.Both GNS and GRS showed dramatic inhibitory effects on the activities of Na+, K+-ATPase at 10, 20 and 40 mg/kg .The activities of Na+, K+-ATPase were also significantly inhibited by PDS and PTS at 5, 10 and 20 mg/kg. In vitro, all the ginsenosides used in this experiment, at the concentrations of 0.01, 0.10 and 1.00 g/L, Possessed significant inhibitory effects on the activities of dog heart sarcolemmal Na+, K+-ATPase .These results indicated that the enhancement of the contractility of cardiac muscle by ginsenosides is associated with its inhibition on the activities of Na+, K+-ATPase .

Effect of low concentrations of strophanthidin on isolated guinea pig failure hearts / 中国药理学通报

AIM To study the effects of strophanthidin (Str) on cardiac function and Na +,K +-ATPase activity in isolated guinea pig heart failure model. METHODS Langendorff isolated heart failure models made by perfusing heart with K-H solution containing sodium pentobarbital. Eight-channel physiological recording instrument was used to determine cardiac function. Colorimetry method was used to determine cardiac sarcolemmal Na +,K +-ATPase activity. RESULTS Str increased the heart rate, left ventricular systolic pressure and the maximum rise or decline rate of left ventricular pressure in a concentration-dependent manner at 1?10 -9～1?10 -7 mol?L -1. But Str caused first a rise, then a reduction of contractility and arrhythmia accompanied by inhibition of Na +,K +-ATPase when the concentration of Str was higher than 1?10 -6 mol?L -1. Str had no obvious effect on Na +,K +-ATPase activity at 1?10 -7 mol?L -1, but increased cardiac activity at 1?10 -10～1?10 -8 mol?L -1. CONCLUSION The inotropic effect and heart toxicity of Str at higher concentration is due to inhibition of Na +,K +-ATPase, but the inotropic effect of Str at lower concentration is not the result of inhibition of Na +,K +-ATPase activity.

THE EFFECTS OF TAURINE ON LIPID PEROXIDATION OF RABBIT'S MYOCARDIAL SARCOLEMMA AND PHOSPHOLIPID LIPOSOME / 中国药理学通报

In the rabbit's myocardial sarcolemma and artificial biomem-brane-liposomes, the lipid peroxidation caused by free radical gene-arting system ( FeCls & ascorbic acid ) was significantly inhibited by taurine at the concentractions of 5~20 m mol/L, and the inhibition effects of taurine were both dose and time-dependent. The results showed that taurine had an antioxidation effects and served as a scavenger of free radicals at the level of membrane.

A rapid method for preparation of sarcolemma from frog skeletal muscle.

A rapid method for the preparation of sarcolema from frog skeletal muscle has been described. The purified cell segments were transparent and devoid of contractile material. The Na+, Κ+ -ATPase and 5'-nucleotidase activities in sarcolemma purified by this method were comparable to those reported for sarcolemmal preparations purified by density gradient centrifugation. The preparation also possessed acid phosphatase, alkaline phosphatase and Κ+-activated, ouabain-sensitive p-nitrophenyl phosphatase activities. The cholesterol to phospholipid ratio of the sarcolemma was 0.33, indicating its high purity; further, the preparation was free from mitochondria and contractile proteins.

EFFECT OF Se ON Na~+ ,K~+-ATPase AND 5'-NUCLEOTIDASE ACTIVITIES OF CARDIAC SARCOLEMMA IN RATS / 西安交通大学学报(医学版)

Sprague—dawey rats were fed for one to three months with Se—deficient grains from Keshan disease area, Se—supplemented diet and stock diet,respectively. Changes of Na~+ ,K~+ ATPase and 5'—nucleotidase activities in cardiac sarcolemma ,Se Contents in liver and plasma and GSH—px activities in Liver and red blood cells of rats were observed. The results showed that Na~+ ,K~+—ATPase. and 5'—nucleotidase activities in cardiac sarcolemma in Se-deficient group rats were significantly decreased,hepatic and plasma Se contents as well as hepatic and erythrocyte GSH—px activities were also significantly decreased at of feeding 30,60 and 90 d as compared to Se—supplemented or stock diet group. There was no significant difference in these parameters between Se—supplemented or stock diet groups There was no significant difference in these paramefers between Se—supplemented and stock diet groups。