NADPH氧化酶介导的ROS在特发性炎性肌病动物模型骨骼肌组织中的表达及意义

Expression and significance of ROS mediated by NADPH oxidases in skeletal muscle tissues of animal model of idiopathic inflammatory myopathy

目的探讨NADPH氧化酶中的NOX2和NOX4介导的活性氧(ROS)在特发性炎性肌病(IIM)发病机制中的作用及意义。方法将14只健康雌性BALB/c小鼠随机分为对照组(n=6)和EAM组(n=8),EAM组小鼠采用豚鼠骨骼肌匀浆蛋白免疫诱导实验性自身免疫性肌炎动物模型。观察小鼠的体重变化、临床表现,测定其血清肌酶水平、四肢肌力及骨骼肌组织病理改变;并采用免疫组织化学法、分光光度法和ELISA法分别检测各组小鼠骨骼肌组织中NOX2、NOX4、NADPH及ROS的表达情况或含量。结果与对照组相比,末次免疫后1周EAM组小鼠体重下降,差异有统计学意义(P<0.05)。HE染色结果显示,对照组小鼠骨骼肌纤维大小、形态、结构基本正常,有少量炎性细胞浸润,未见肌纤维萎缩、变性、坏死;EAM组小鼠骨骼肌纤维大小不一、粗细不等,有大量炎性细胞浸润,可见不同程度的萎缩、变性、坏死;EAM组小鼠骨骼肌组织HE染色病理学评分显著高于对照组(P<0.05)。与对照组相比,EAM组小鼠骨骼肌组织中NADPH含量降低、ROS含量升高(P<0.05)。免疫组化染色结果显示,NOX2和NOX4在EAM组小鼠骨骼肌组织中的表达均高于对照组(P<0.05)。Pearson相关分析结果显示,EAM组小鼠骨骼肌组织中ROS的含量与NOX2、NOX4的免疫组化评分均呈正相关(P<0.05),而与NADPH的含量呈负相关(P<0.05);EAM组小鼠骨骼肌组织HE染色病理学评分与NOX2、NOX4的免疫组化评分及ROS的含量均呈正相关(P<0.05),而与NADPH的含量呈负相关(P<0.05)。结论NADPH氧化酶中的NOX2和NOX4可能通过消耗NADPH产生大量的ROS,导致组织细胞氧化性损伤并诱导铁死亡反应来参与IIM的发病;NADPH、NOX2、NOX4和ROS有望成为新的评价IIM肌肉组织损伤程度的指标。

Objective This study was performed to detect the expression of NADPH,ROS,NOX2 and NOX4 in the skeletal muscle tissues of experimental autoimmune myositis mice and investigate the role and significance of reactive oxygen species mediated by NADPH oxidases in the pathogenesis of Idiopathic Inflammatory Myopathy.Methods Fourteen healthy female BALB/c mice were randomly divided into control group and EAM group(6 in control group;8 in EAM group),and guinea pig skeletal muscle homogenate was used to induce experimental autoimmune myositis in EAM group.We should observe the changes of weight and clinical manifestations and measure the serum creatinase level,limb muscle strength and skeletal muscle pathology of mice in each group.Then the contents of NADPH and ROS in mice skeletal muscle tissues of each group were detected by spectrophotometry and ELISA respectively,and the expressions of NOX2 and NOX4 were measured by immunohistochemistry.Results The results of HE staining showed that the size,morphology and structure of skeletal muscle fibers in the control group were basically normal,with a small amount of inflammatory cell infiltration,and no muscle fibers atrophy,degeneration or necrosis.Skeletal muscle fibers of mice in the EAM group were different in size and thickness,with a large number of inflammatory cell infiltration and different degrees of atrophy,degeneration and necrosis.The histopathological scores of skeletal muscle of mice in the EAM group were significantly higher than that in the control group(P<0.05).Compared with the control group,the levels of NADPH were decreased(P<0.05)and levels of ROS were increased(P<0.05)in the skeletal muscle tissues of the EAM group.The expressions of NOX2 and NOX4 in skeletal muscle tissues of EAM group mice were higher than that of control group mice(P<0.05).The contents of ROS in skeletal muscle tissues of mice in EAM group were positively correlated with the Immunohistochemical scores of NOX2 and NOX4(P<0.05),and negatively correlated with the contents of NADPH(P<0.05).The pathological scores of HE staining in skeletal muscle tissues of mice in EAM group were positively correlated with the Immunohistochemical scores of NOX2 and NOX4 and the contents of ROS(P<0.05),and negatively correlated with the contents of NADPH(P<0.05).Conclusion NOX2 and NOX4 may induce ferroptosis by consuming NADPH and producing a large amount of ROS,and ferroptosis may be involved in the pathogenesis of IIM by promoting oxidative damage and accelerating inflammatory response.NADPH,NOX2,NOX4 and ROS may be new indicators to evaluate the degree of muscle tissues injury in IIM.