氯沙坦减少细胞凋亡延缓失神经骨骼肌萎缩的实验研究

EXPERIMENTAL STUDY ON LOSARTAN DECREASING DENERVATED SKELETAL MUSCLE ATROPHY THROUGH REDUCING CELL APOPTOSIS

目的失神经骨骼肌萎缩是临床亟待解决的难题,研究旨在探讨氯沙坦能否减少细胞凋亡,以期寻找延缓失神经骨骼肌萎缩的新途径。方法雄性SD大鼠42只,随机分成3组(n=14),分别为Ⅰ组(对照组)、Ⅱ组(失神经对照组)和Ⅲ组(氯沙坦治疗组)。Ⅰ组不作处理,Ⅱ组和Ⅲ组制备失神经腓肠肌(gastrocnemius,GAS)动物模型。术后Ⅲ组大鼠采用氯沙坦以10mg/kg·d空腹灌胃4周;Ⅰ、Ⅱ组大鼠分别以等剂量生理盐水灌胃。术后4周处死大鼠,以GAS和体重(bod ymass,BM)之比(GAS/BM)作为骨骼肌萎缩指标;TUNEL法检测腓肠肌细胞凋亡;免疫组织化学和Western blot检测GASBcl-2及Bax的表达。结果术后4周Ⅰ组腓肠肌正常粗细;Ⅱ、Ⅲ组腓肠肌明显萎缩,肌肉变细,与周围组织粘连明显。GAS/BMⅡ组为11.68±1.98,与Ⅰ组(12.86±0.74)比较,差异有统计学意义(P<0.05);Ⅲ组GAS/BM为12.11±0.65;与Ⅱ组比较,差异无统计学意义(P>0.05)。TUNEL法检测:Ⅰ组罕见凋亡细胞核,凋亡率为0.56%±0.21%;Ⅱ组凋亡细胞核较Ⅰ组明显增多,凋亡率为11.32%±4.51%,两组比较差异有统计学意义(P<0.05);Ⅲ组可见凋亡细胞核,凋亡率为7.21%±2.05%,与Ⅱ组比较差异有统计学意义(P<0.05)。免疫组织化学染色观察:Bcl-2阳性率Ⅱ组为18.3%±4.9%,较Ⅰ组(27.5%±2.8%)和Ⅲ组(25.5%±3.5%)低,差异均有统计学意义(P<0.05);Ⅲ组与Ⅰ比较,差异无统计学意义(P>0.05);Bax阳性率:Ⅱ组为24.1%±3.1%,较Ⅰ组(22.1%±3.6%)和Ⅲ组(21.7%±2.3%)高,差异均有统计学意义(P<0.05);Ⅲ组与Ⅰ组比较差异无统计学意义(P>0.05)。Ⅰ、Ⅱ及Ⅲ组Bax/Bcl-2平均分别为0.8、1.3及0.8。Westernblot检测:Bcl-2蛋白表达Ⅱ组为122.5±14.6,较Ⅰ组(135.3±6.2)下降(P<0.05),Ⅲ组为139.2±16.2,较Ⅱ组增加(P<0.05);Bax蛋白表达Ⅱ组为107.1±15.8,Ⅰ组为89.3±8.4,差异有统计学意义(P<0.05),Ⅲ组为94.2±9.5,较Ⅱ组下降(P<0.05);Bcl-2蛋白和Bax蛋白表达Ⅲ组与Ⅰ组比较,差异均无统计学意义(P>0.05)。结论细胞凋亡在失神经骨骼肌萎缩中发挥重要作用,可能是骨骼肌萎缩的原因之一;氯沙坦可通过抑制肌细胞凋亡延缓大鼠失神经骨骼肌的萎缩。

Objective To explore the role of cell apoptosis in denervated skeletal muscle atrophy in rats and the effect of iosartan on it. Methods Forty-two Sprague Dawley rats were randomly divided into 3 groups： group Ⅰ （n = 14, normal control group）, group Ⅱ （n =14, denervated group） and group Ⅲ （n =14, losartan group）. The rats were not treated in group Ⅰ, and were made denervated gastrocnemius models in groups Ⅱ and Ⅲ. Ⅰn group Ⅲ, the rats were treated with losartan 10 mg/kg.d by garage and with normal saline in groups Ⅰ and Ⅱ. After 4 weeks, gastrocnemius mass to body mass ratio （GAS/BM） served as the degree of muscle atrophy. Apoptotic cells in gastrocnemius were stained in situ by using TUNEL. Gastrocnemius Bcl-2 and Bax protein were quantified by immunohistochemistry and Western blot. Bax/Bcl-2 served as the degree of apoptosis. Results The ratio of apoptosis was higher in group Ⅱ than that in group Ⅰ （11.32% ± 4.51% vs 0.56% ±0.21%, P 〈 0.05）. The ratio of apoptosis was lower in group Ⅲ than that in group Ⅱ （7.21% ± 2.05% vs 11.32% ± 4.51%, P 〈 0.05）. The atrophy of skeletal muscle （GAS/BM） in group Ⅱ was more serious than that in group Ⅰ （11.68 ± 1.98 vs 12.86 ± 0.74, P 〈 0.05）, there was no significant difference between group Ⅲ and group Ⅱ （12.11 ±0.65 vs 11.68±1.98, P 〉 0.05）. The expression of Bcl-2 in group Ⅱ （18.3%±4.9%） was significantly lower than that in group Ⅰ （27.5% ± 2.8%） and group Ⅲ （25.5% ± 3.5%）; there was no significant difference between group Ⅲ and group Ⅰ （P 〉 0.05）. The expression of Bax in group Ⅱ （24.1% ±3.1%） was significantly higher than that in group Ⅰ （22.1% ±3.6%） and group Ⅲ （21.7% ± 2.3%）; there was no significant difference between group Ⅲ and group Ⅰ （P 〉 0.05）. Western blot results showed that： the expressions of Bcl-2 were 122.5± 14.6 in group Ⅱ, 135.3 ± 6.2 in group Ⅰ and 139.2± 16.2 in group Ⅲ; showing significant diffeerences between group Ⅱ and group Ⅰ, between group Ⅲ and group Ⅱ （P 〈0.05）. The expressions of Bax were 107.1 ±15.8 in group Ⅱ, 89.3 ± 8.4 in group Ⅰ, and 94.2 ±9.5 in group Ⅲ; showing significant diffeerences between group Ⅱ and group Ⅰ, between group Ⅲ and group Ⅱ （P 〈 0.05）. There was no significant difference in the expression of Bcl-2 and Bax between group Ⅲand group Ⅰ （P 〉 0.05）. Conclusion Cell apoptosis plays an important role in denervated skeletal muscle atrophy in rats and may be one of the factors causing skeletal muscle atrophy. Losarton can decrease skeletal muscle cell apoptosis through regulating the ratio of Bax / Bcl-2.