腺苷一磷酸活化蛋白激酶在严重烫伤大鼠骨骼肌萎缩中的作用

Roles of adenosine monophosphate activated protein kinase in skeletal muscle atrophy in rats with severe scald

目的探讨严重烫伤大鼠骨骼肌中腺苷一磷酸活化蛋白激酶(AMPK)的表达和磷酸化水平变化及其在严重烫伤大鼠骨骼肌萎缩中的作用。方法采用实验研究方法。将100只6周龄雄性Wistar大鼠按随机数字表法分为假伤组和烫伤组,每组50只。测量2组大鼠体重后,烫伤组背部造成30%体表总面积Ⅲ度烫伤,假伤组模拟致烫伤。伤后6 h及1、3、5、7 d,每组各取10只大鼠,测量体重及趾长伸肌和比目鱼肌重量。伤后6 h及1、3、5、7 d,收集2组大鼠胫骨前肌,采用实时荧光定量反转录PCR法检测肌肉萎缩盒F蛋白(MAFbx)和肌肉特异性环指蛋白1(MuRF1)mRNA表达;采用高效液相色谱法检测腺苷一磷酸(AMP)、腺苷二磷酸和ATP含量,并计算AMP/ATP比值及能荷;采用蛋白质印迹法检测AMPK-α及磷酸化AMPK-α(p-AMPK-α)蛋白表达,并计算p-AMPK-α/AMPK-α比值;2组各时间点样本数均为4。对数据行析因设计方差分析及LSD检验。结果伤前及伤后各时间点,2组大鼠体重相近(P>0.05)。伤后6 h,烫伤组大鼠趾长伸肌重量为(0.107±0.007)g,明显重于假伤组的(0.086±0.007)g(P<0.01);伤后3 d,烫伤组大鼠趾长伸肌重量为(0.083±0.016)g,明显轻于假伤组的(0.102±0.005)g(P<0.01)。2组大鼠伤后各时间点比目鱼肌重量相近(P>0.05)。与假伤组比较,烫伤组大鼠伤后6 h胫骨前肌MAFbx mRNA及伤后6 h和1 d胫骨前肌MuRF1 mRNA表达明显上调(P<0.01)。与假伤组比较,烫伤组大鼠伤后6 h和7 d胫骨前肌AMP/ATP比值明显升高(P<0.05或P<0.01),能荷明显下降(P<0.01)。伤后各时间点,2组大鼠胫骨前肌AMPK-α蛋白表达相近(P>0.05)。伤后6 h和7 d,烫伤组大鼠胫骨前肌p-AMPK-α/AMPK-α比值明显高于假伤组(P<0.05或P<0.01)。结论严重烫伤后大鼠骨骼肌能荷下降,AMP/ATP比值升高,激活AMPK;伤后早期AMPK的活化与MAFbx和MuRF1表达上调和骨骼肌重量下降一致,可能是严重烫伤大鼠骨骼肌萎缩发生的分子机制之一。

Objective To investigate the expression and phosphorylation level change of adenosine monophosphate activated protein kinase(AMPK)in skeletal muscle of severely scald rats and its roles in skeletal muscle atrophy in severely scalded rats.Methods The experimental research method was applied.Totally 1006-week-old male Wistar rats were divided into sham injury group and scald group according to the random number table,with 50 rats in each group.After weighing the body weight,rats in scald group were inflicted with full-thickness scald of 30%total body surface area on the back,and rats in sham injury group were simulated with scald.At 6 h and on 1,3,5,and 7 d post injury,10 rats in each group were taken to measure their body weights and weights of extensor digitorum longus and soleus muscle.At 6 h and on 1,3,5,and 7 d post injury,the tibialis anterior muscles were collected,the mRNA expressions of muscle atrophy F-box protein(MAFbx)and muscle-specific RING finger protein 1(MuRF1)were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction;the content of adenosine monophosphate(AMP),adenosine diphosphate,and adenosine triphosphate(ATP)were detected by high performance liquid chromatography,and AMP/ATP ratio and energy charge were calculated;the protein expressions of AMPK-αand phosphorylated AMPK-α(p-AMPK-α)were detected by Western blotting,and the p-AMPK-α/AMPK-αratio was calculated,with sample number of 4 in each time point of each group.Data were statistically analyzed with analysis of variance for factorial design and least significant difference test.Results The body weights of rats in 2 groups before injury and at each time point post injury were close(P>0.05).At 6 h post injury,the weight of extensor digitorum longus of rats in scald group was(0.107±0.007)g,which was significantly heavier than(0.086±0.0607)g of sham injury group(P<0.01).On 3 d post injury,the weight of extensor digitorum longus of rats in scald group was(0.083±0.016)g,which was significantly lighter than(0.102±0.005)g of sham injury group(P<0.01).The weight of soleus of rats in 2 groups were close at each time point post injury(P>0.05).Compared with those of sham injury group,the mRNA expression of MAFbx in tibialis anterior muscle of rats in scald group was significantly up-regulated at 6 h post injury(P<0.01),and the mRNA expressions of MuRF1 in tibial anterior muscle of rats in scald group were significantly up-regulated at 6 h and on 1 d post injury(P<0.01).At 6 h and on 7 d post injury,compared with those of false injury group,the AMP/ATP ratios of the tibial anterior muscle of rats in scald group were significantly increased(P<0.05 or P<0.01),and energy charges of the tibial anterior muscle of rats in scald group were significantly decreased(P<0.01).At each time point post injury,the protein expressions of AMPK-αof the tibial anterior muscle of rats in 2 groups were close(P>0.05).The p-AMPK-α/AMPK-αratios of the tibial anterior muscle of rats in scald group at 6 h and on 7 d post injury were significantly higher than those in sham injury group(P<0.05 or P<0.01).Conclusions The decrease in energy charge and increase in AMP/ATP ratio of skeletal muscle of rats after severe scald activate AMPK.The activation of AMPK in the early stage of injury is consistent with the up-regulation of MAFbx and MuRF1 expressions and down-regulation of skeletal muscle weight.The above-mentioned changes may be one of the molecular mechanisms of skeletal muscle atrophy in rats with severe scald.