丹曲林对严重烧伤大鼠骨骼肌损害的治疗作用及机制

Effect and mechanism of dantrolene on skeletal muscle of rats with severe scald injury

目的：探讨丹曲林对严重烧伤大鼠骨骼肌损害的治疗作用及可能机制。方法选取清洁级Wistar雄性大鼠56只，按照随机数字表法分为对照组8只、致伤组及丹曲林组各24只（两组3个时相点各8只）。致伤组和丹曲林组大鼠在麻醉后与其背部及腹部行50％总体表面积（ TBSA ）Ⅲ度烫伤，伤后腹腔注射林格平衡液抗休克，致伤组尾静脉注射5％甘露醇，丹曲林组尾静脉注射丹曲林钠2 mg／kg（溶于5％甘露醇）。对照组行假烫伤，其余操作同致伤组。于伤后第1、4、7天收集各组大鼠胫骨前肌组织，透射电镜观察组织超微结构，电子探针X射线显微分析法检测肌细胞微区Ca2＋含量，蛋白印迹方法检测胫骨前肌总蛋白中钙蛋白酶1、2表达，酶法检测胫骨前肌中钙蛋白酶的活性。结果致伤组胫骨前肌肌丝伤后第1天出现排列混杂，第7天出现局部Z线消失。而丹曲林组第1、4天肌组织超微结构与对照相比无显著变化，仅在第7天肌丝存现排列卷曲、轻度断裂。伤后第1、4天致伤组胫骨前肌细胞质Ca2＋水平显著高于对照组[（0.964±0.060）、（0.639±0.067）比（0.266±0.029）μmol／L]，肌浆网Ca2＋水平显著低于对照组[（0.368±0.060）、（0.814±0.089）比（1.337±0.112）μmol／L]（均P＜0.05）；而丹曲林组细胞质和肌浆网Ca2＋水平[（0.310±0.069）、（0.490±0.039）和（1.241±0.073）、（1.161±0.094）μmol／L]与对照组差异均无统计学意义（均P＞0.05）。伤后第1、4天致伤组胫骨前肌钙蛋白酶1和钙蛋白酶2相对表达量均显著高于对照组（1.371±0.034、1.214±0.030比0.838±0.017和1.464±0.015、1.390±0.023比0.806±0.026）（均P＜0.05），而丹曲林组钙蛋白酶1和钙蛋白酶2相对表达量（0.984±0.031、0.935±0.023和0.836±0.014与0.741±0.020）均显著低于致伤组（均P＜0.05）。伤后第1、4、7天致伤组和丹曲林组钙蛋白酶活性分别为（8.33±0.21）、（9.33±0.21）、（10.59±0.18）和（7.76±0.28）、（7.86±0.20）、（7.91±0.22）μmol／L，对照组为（7.62±0.19）μmol／L，致伤组均显著高于丹曲林组及对照组（均P＜0.05），但丹曲林组与对照组差异均无统计学意义（均P＞0.05）。结论丹曲林能够明显减弱严重烧伤引起的骨骼肌超微结构变化，减轻肌组织损伤。其可能是通过抑制严重烧伤后肌细胞肌浆网Ca2＋的过度释放，降低严重烧伤后肌细胞质内钙蛋白酶1、2的表达及其活性而发挥作用。

Objective To explore the effect and mechanism of ryanodine receptor antagonist dantrolene on skeletal muscle of rats with severe scald injury.Methods A total of 56 Wistar rats were divided into control , scald and dantrolene treatment groups according to a random digital table.Rats in scald and dantrolene treatment groups were subject to 50%total body surface area （ TBSA） full-thickness scald by a 12-second immersion of back and a 6-second immersion of abdomen in 94 ℃water and then received an intraperitoneal injection of Ringer′s solution.At the same time , the rats in scald group received 5%mannitol through caudal vein while those in dantrolene treatment group received dantrolene 2 mg/kg （ dissolved in 5%mannitol ）.Rats in control group were sham-injured through an immersion of back and abdomen into 37 ℃warm water.Tibialis anterior muscle samples were harvested at Days 1, 4 and 7 post-scalding.Changes of skeletal muscle ultrastructure were observed by transmission electron microscope , subcellular calcium ion （ Ca2＋） contents of skeletal muscle （ including cytoplasm , mitochondria ＆amp; sarcoplasm reticulum ） were detected by electron probe X-ray microanalysis （ EPMA） and the levels of calpain-1 and calpain-2 protein were determined by Western blot.And the activities of calpain were detected by enzyme-linked immunosorbent assay.Results In scald group , assorted arrangement appeared immediately at Day 1 post-injury and partial disappearance of Z lines at Day 7 post-injury.There were no significant ultrastructure changes in dantrolene treatment group at Day 1 and 4 post-injury.Curled filament and mild fracture occurred merely in dantrolene treatment group at Day 7 post-injury.The cytoplasmic contents of Ca 2＋ were significantly higher in scald group than those in control group at Day 1 and 4 （（0.964 ±0.060）, （0.639 ± 0.067） vs （0.266 ±0.029） μmol/L respectively, all P〈0.05） while the contents of Ca2＋within sarcoplasm reticulum were obviously lower in scald group than those in control group at Day 1 and 4 （（0.368 ±0.060）, （0.814 ±0.089） vs （1.337 ±0.112） μmol/L respectively, all P〈0.05）.However, those subcellular regions in dantrolene treatment group （ （ 0.310 ±0.069 ） , （ 0.490 ±0.039 ） and （ 1.241 ±0.073 ） , （1.161 ±0.094） μmol/L） had no significant difference with control group （ all P〉0.05）.Caplain-1 and calpain-2 protein levels in scald group increased significantly at Day 1 and 4 post-injury versus control group （1.371 ±0.034, 1.214 ±0.030 vs 0.838 ±0.017 ＆amp; 1.464 ±0.015, 1.390 ±0.023 vs 0.806 ±0.026 respectively , all P 〈0.05 ） , whereas caplain-1 and calpain-2 protein levels in dantrolene treatment （0.984 ±0.031, 0.935 ±0.023 and 0.836 ±0.014, 0.741 ±0.020） obviously were lower than those in scald group respectively （ all P〈0.05）.The activities of calpain in scald and dantrolene treatment groups at Day 1, 4 and 7 post-injury were （8.33 ±0.21）, （9.33 ±0.21）, （10.59 ±0.18） and （7.76 ±0.28）, （7.86 ±0.20）, （7.91 ±0.22） μmol/L respectively while the activity of calpain in control group was （7.62 ±0.19） μmol/L.The activities of calpain in scald group were significantly higher than those in dantrolene treatment and control groups （ all P 〈0.05 ） whereas the activities of calpain in dantrolene treatment group had no obvious change versus control group （ all P〉0.05 ） .Conclusions Dantrolene offers significant protection from skeletal muscle tissue damage and minimizes the ultrastructural change of tibialis anterior muscle induced by severe scald injury.The mechanism is probably through inhibiting an excessive release of Ca 2＋within sarcoplasm reticulum and down-regulated cytoplasmic expression and activity of calpain-1 and calpain-2.