肝硬化大鼠膈肌骨架蛋白和肌浆网钙泵基因表达的变化

Changes of sarcoplasmic reticulum calcium ATPase, titin, and nebulin expressions in the diaphragm of rats with liver cirrhosis

目的探讨肝硬化大鼠膈肌损伤及其发生机制。方法成年雄性Sprague-Dawley大鼠30只,随机分成对照组(CON组,n=10)和肝硬化组(LC组,n=20)。对照组给予正常饲料和自来水;肝硬化组采用CCl4复合因素建立肝硬化大鼠模型。第9周,测定两组大鼠体重和膈肌重/体重比;体外灌流大鼠膈肌条,测定其单收缩力(Pt)、最大强直力(Po)、峰值收缩时间(CT)、半舒张时间(1/2RT)和张力-频率曲线的变化,同时测定膈肌组织超氧化物歧化酶(SOD)和琥珀酸脱氢酶(SDH)的活性及丙二醛(MDA)和髓过氧化物酶(MPO)的含量;电镜观察膈肌超微结构的变化;RT-PCR检测膈肌组织中肌浆网钙泵(SERCA)及骨架蛋白titin,nebulin的基因表达。结果 (1)与CON组相比,LC组大鼠体重和膈肌/体重比明显降低(P<0.01);Pt和Po明显降低(P<0.01),CT和1/2RT明显延长(P<0.01);在10、20、40、60、100 Hz刺激下,膈肌条张力明显降低(P<0.01);(2)与CON组比较,LC组大鼠膈肌组织的SOD、SDH的活性明显降低(P<0.01),MDA、MPO的含量明显增高(P<0.01);(3)与CON组比较,LC组的titin、nebulin和SERCA mRNA表达均明显降低(P<0.01);(4)电镜显示LC组大鼠膈肌肌丝紊乱、断裂,Z线模糊或消失;线粒体数量减少,水肿。结论肝硬化引起大鼠膈肌自由基生成增多,炎症反应和脂质过氧化增强,损伤膈肌线粒体,骨架蛋白titin,nebulin和SERCA表达降低,导致膈肌功能障碍。

Objective To investigate the molecular mechanisms of diaphragm injury in rats with liver cirrhosis. Methods Thirty adult male Sprague-Dawley rats were randomized into control group （n=10） and carbon tetrachloride-induced liver cirrhosis group （LC group, n=20）. In the 9th week, the rat body weight and diaphragm to body weight ratio were measured, and the parameters of diaphragm contractility including peak twitch tension （Pt）, maximum tetanic tension （Po）, time to peak contraction （CT）, half relaxation time （1/2RT）, and force-frequency curve were assessed using a Medlab-U/4C biological signal collecting system. The activities of superoxide dismutase （SOD）, succinic dehydrogenase （SDH） and myeloperoxidase （MPO） and malondiadehyde （MDA） content in the diaphragm were detected. The mRNA expression levels of sarcoplasmic reticulum calcium ATPase （SERCA） and cytoskeletal proteins （titin and nebulin） in the diaphragm were detected by RT-PCR, and the diaphragm ultrastructure was examined with electron microscopy. Results Compared with those in the control group, body weight, diaphragm to body weight ratio, Pt, Po, and tetanic force under the stimulus frequency of 10, 20, 40, 60, 100 Hz were all significantly decreased （P〈0.01）, while CT and 1/2RT were significantly prolonged in LC group （P〈0.01）. SOD and SDH activities were significantly lowered （P〈0.01） while the contents of MDA and MPO activity were significantly increased in LC group （P〈0.01） with significantly decreased SERCA, titin and nebulin mRNA expressions in the diaphragm （P〈0.01）. Electron microscopy of the diaphragm in LC group revealed myofibrillar degeneration, absence of the Z line, and mitochondria swelling and edema. Conclusions Liver cirrhosis increases free radicals and aggravates inflammatory response and lipid peroxidation in the diaphragm, thus leading to mitochondrial damages and decreased expressions of cytoskeletal proteins and SERCA to cause diaphragmatic dysfunction.