大鼠严重烧伤后心肌组织溶酶体液泡型腺苷三磷酸酶活性变化对心肌损害的影响及其机制

Effects of change in the activity of vacuolar adenosine triphosphatase of myocardial lysosome on myocardial damage in rats after severe burn and its mechanism

目的探讨大鼠严重烧伤后心肌组织溶酶体液泡型ATP（V－ATP）酶活性变化对心肌损害的影响及其机制。方法取12只SD大鼠心脏，采用超高速梯度密度离心法提取心肌组织溶酶体，蛋白质印迹法和透射电镜观察鉴定。将120只SD大鼠按随机数字表法分为单纯烧伤组、ATP组、正常对照组、巴佛洛霉素组，每组30只。单纯烧伤组和ATP组大鼠造成背部约40%TBSA Ⅲ度烫伤，单纯烧伤组大鼠伤后即刻腹腔注射乳酸林格液4 mL·%TBSA^－1·kg^－1，ATP组大鼠分别于伤前12 h、伤后即刻、伤后12 h腹腔注射ATP 0.4 mg/kg。正常对照组大鼠不行任何处理，巴佛洛霉素组与ATP组大鼠同时相点腹腔注射巴佛洛霉素A1 0.3 mg/kg。于伤后24 h，提取各组大鼠心肌组织溶酶体，采用酶偶联分析法检测V－ATP酶活性，蛋白质印迹法检测心肌自噬相关蛋白微管相关蛋白1轻链3（LC3）和P62的表达。取左心室动脉血检测心肌酶谱5项和心肌肌钙蛋白T（cTnT）的含量。对数据行单因素方差分析、t检验。结果（1）经鉴定，样品溶酶体相关膜蛋白1表达水平最高，溶酶体纯度高、结构完整。（2）伤后24 h，单纯烧伤组、ATP组、正常对照组、巴佛洛霉素组大鼠心肌组织溶酶体V－ATP酶活性分别为（2.03±0.67）、（3.01±0.58）、（4.29±0.26）、（1.83±0.52）μmol·mg－1·h－1。其中单纯烧伤组大鼠心肌组织溶酶体V－ATP酶活性明显低于ATP组和正常对照组（t值分别为3.14和8.87，P值均小于0.01），正常对照组大鼠心肌组织溶酶体V－ATP酶活性明显高于巴佛洛霉素组（t＝11.87，P〈0.01）。伤后24 h，单纯烧伤组大鼠心肌LC3和P62的表达显著高于ATP组和正常对照组（t值为3.73～5.88，P值均小于0.01），正常对照组大鼠心肌LC3和P62的表达明显低于巴佛洛霉素组（t值分别为2.64和3.07，P〈0.05或P〈0.01）。伤后24 h，单纯烧伤组大鼠心肌酶谱5项和cTnT含量明显高于ATP组和正常对照组（t值为3.24～16.72，P值均小于0.01），正常对照组大鼠心肌酶谱5项和cTnT含量明显低于巴佛洛霉素组（t值为2.39～10.70，P值均小于0.01）。结论大鼠严重烧伤后心肌组织溶酶体V－ATP酶活性下降，可通过抑制大鼠心肌自噬流引起心肌损害。

ObjectiveTo explore the effects of change of activity of vacuolar adenosine triphosphatase （V-ATPase） of myocardial lysosome on myocardial damage in rats after severe burn and its mechanism.MethodsThe myocardial lysosomes were extracted from the hearts of 12 SD rats with ultra-high speed gradient density centrifugation, then Western blotting and transmission electron microscope observation were conducted for identification. One hundred and twenty rats were divided into pure burn group, ATP group, normal control group, and bafilomycin group according to the random number table, with 30 rats in each group. Rats in pure burn group and ATP group were inflicted with 40% TBSA full-thickness scald on the back. Immediately after injury, rats in pure burn group were intraperitoneally injected with lactated Ringer′s solution in 4 mL·%TBSA^-1·kg^-1, and rats in ATP group were intraperitoneally injected with ATP in 0.4 mg/kg at 12 h before burn, immediately after burn, and 12 h after burn. Rats in normal control group did not receive any treatment, and rats in bafilomycin group were intraperitoneally injected with bafilomycin A1 in 0.3 mg/kg at the same time points as those of ATP group. At 24 h after burn, 30 rats from each group were collected for determining activity of V-ATPase of myocardial lysosome with coupled-enzyme assay and the expression of myocardium autophagy-related proteins microtubule-associated protein 1 light chain 3 （LC3） and P62 by Western blotting. Left ventricular arterial blood was collected to detect the content of 5 items of myocardial enzyme spectrum and cardiac troponin T （cTnT）. Data were processed with one-way analysis of variance and t test.Results（1） After identification, both the expression level of lysosome-related membrane protein 1 and purity of lysosome in the sample were high, and the structure of lysosome was intact. （2） At 24 h after burn, the activity values of V-ATPase of myocardial lysosome in rats of pure burn group, ATP group, normal control group, and bafilomycin group were （2.03±0.67）, （3.01±0.58）, （4.29±0.26）, and （1.83±0.52） μmol·mg-1·h-1, respectively. The activity value of V-ATPase of myocardial lysosome in rats of pure burn group was significantly lower than the values in ATP group and normal control group （with t values respectively 3.14 and 8.87, P values below 0.01）. The activity values of V-ATPase of rats in normal control group were significantly higher than those in bafilomycin group （t=11.87, P〈0.01）. At 24 h after burn, the expressions of myocardial LC3 and P62 in pure burn group were significantly higher than those in ATP group and normal control group （with t values from 3.73 to 5.88, P values below 0.01）. The expressions of myocardial LC3 and P62 in normal control group were significantly lower than those in bafilomycin group （with t values respectively 2.64 and 3.07, P〈0.05 or P〈0.01）. At 24 h after burn, the content of 5 items of myocardial enzyme spectrum and cTnT in pure burn group was significantly higher than that in ATP group and normal control group （with t values from 3.24 to 16.72, P values below 0.01）. The content of 5 items of myocardial enzyme spectrum and cTnT in normal control group was significantly lower than that in bafilomycin group （with t values from 2.39 to 10. 70, P values below 0.01）.ConclusionsThe activity of V-ATPase of myocardial lysosome decreased in rats after severe burn, which can result in myocardial damage by inhibiting myocardial autophagy flux.