Deng H P, Cai J H, Chai J K, Shen Z A, Li L G, Sun T J, Chen J J, Li D J, Dong N, Liu L Y
Department of Burns and Plastic Surgery, Burns Institute of PLA, the Fourth Medical Center of PLA General Hospital, Beijing 100048, China.
Department of Dermatology, Aerospace Center Hospital, Beijing 100049, China.
Zhonghua Shao Shang Za Zhi. 2021 Jul 20;37(7):640-646. doi: 10.3760/cma.j.cn501120-20200416-00227.
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. The experimental research method was applied. Totally 100 6-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-α and phosphorylated AMPK-α (p-AMPK-α) were detected by Western blotting, and the p-AMPK-α/AMPK-α ratio 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. The body weights of rats in 2 groups before injury and at each time point post injury were close (>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 (<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 (<0.01). The weight of soleus of rats in 2 groups were close at each time point post injury (>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 (<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 (<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 (<0.05 or <0.01), and energy charges of the tibial anterior muscle of rats in scald group were significantly decreased (<0.01). At each time point post injury, the protein expressions of AMPK-α of the tibial anterior muscle of rats in 2 groups were close (>0.05). The p-AMPK-α/AMPK-α ratios 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 (<0.05 or <0.01). 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.
探讨严重烫伤大鼠骨骼肌中腺苷单磷酸活化蛋白激酶(AMPK)的表达及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。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。称重后,烫伤组大鼠背部给予30%体表面积的全层烫伤,假伤组大鼠进行烫伤模拟。于伤后6 h及伤后1、3、5、7 d,每组取10只大鼠测量体重及趾长伸肌和比目鱼肌重量。于伤后6 h及伤后1、3、5、7 d,取胫骨前肌,采用实时荧光定量逆转录聚合酶链反应检测肌肉萎缩F盒蛋白(MAFbx)和肌肉特异性E3泛素连接酶1(MuRF1)的mRNA表达;采用高效液相色谱法检测腺苷一磷酸(AMP)、腺苷二磷酸和腺苷三磷酸(ATP)含量,并计算AMP/ATP比值和能荷;采用蛋白质免疫印迹法检测AMPK-α及磷酸化AMPK-α(p-AMPK-α)的蛋白表达,并计算p-AMPK-α/AMPK-α比值,每组每个时间点样本数为4。数据采用析因设计方差分析和最小显著差法进行统计学分析。两组大鼠伤前及伤后各时间点体重相近(>0.05)。伤后6 h,烫伤组大鼠趾长伸肌重量为(0.107±0.007)g,显著重于假伤组的(0.086±0.0607)g(<0.01)。伤后3 d,烫伤组大鼠趾长伸肌重量为(0.083±0.016)g,显著轻于假伤组的(0.102±0.005)g(<0.01)。两组大鼠比目鱼肌重量在伤后各时间点相近(>0.05)。与假伤组相比,烫伤组大鼠伤后6 h胫骨前肌MAFbx的mRNA表达显著上调(<0.01),烫伤组大鼠伤后6 h及1 d胫骨前肌MuRF1的mRNA表达显著上调(<0.01)。伤后6 h及7 d,与假伤组相比,烫伤组大鼠胫骨前肌AMP/ATP比值显著升高(<0.05或<0.01),烫伤组大鼠胫骨前肌能荷显著降低(<0.01)。伤后各时间点,两组大鼠胫骨前肌AMPK-α的蛋白表达相近(>0.05)。烫伤组大鼠伤后6 h及7 d胫骨前肌p-AMPK-α/AMPK-α比值显著高于假伤组(<0.05或<0.01)。严重烫伤后大鼠骨骼肌能荷降低及AMP/ATP比值升高激活AMPK。伤后早期AMPK的激活与MAFbx和MuRF1表达上调及骨骼肌重量下降一致。上述变化可能是严重烫伤大鼠骨骼肌萎缩的分子机制之一。