Expression And Significance Of ROS Mediated By NADPH Oxidases In Skeletal Muscle Tissues Of Animal Model Of Idiopathic Inflammatory Myopathy

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:The BALB/c mice that were healthy and female were randomly divided into Control group and EAM group and mice in the EAM group were immunized with skeletal muscle homogenate proteins of guinea pig to induce experimental autoimmune myositis.The clinical manifestations of mice in each group were observed and scored according to Lennon’s scoring method of clinical manifestation.The body weight,serum muscle enzyme levels and muscle strength measurement time were measured.HE staining was used to detect the pathological changes of skeletal muscle tissues in each group,and the pathological scores were made according to the pathological grading standards of Kohyama muscle tissues by HE staining.The expression of NOX2 and NOX4 in skeletal muscle tissues of mice was detected by Immunohistochemistry.The contents of NADPH and ROS in skeletal muscle tissues of mice were determined by spectrophotometry and enzyme-linked immunosorbent assay respectively.Results:(1)The results of HE staining showed that the area size and structural morphology 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.The histopathological scores of skeletal muscle in the control group was 0.42±0.38 points.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 was 2.92±0.74 points,which was significantly higher than that in the control group,and the difference was statistically significant(P < 0.05).(2)The contents of NADPH and ROS in skeletal muscle tissues of the control group were 33.32±3.93 nmol/g and 169.83±9.06U/ml respectively.The contents of NADPH and ROS in skeletal muscle tissues of the EAM group were 17.08±4.00nmol/g and 275.33±10.23 U/ml respectively.Compared with the control group,the content of NADPH decreased and the content of ROS increased in the skeletal muscle tissues of EAM group mice(P < 0.05).(3)The results of Immunohistochemical staining showed that the positive expression rates of NOX2 and NOX4 in the skeletal muscle tissues of mice in control group both were 16.7%,and the Immunohistochemical scores of NOX2 and NOX4 were 2.30±0.68 points and 2.47±0.52 points respectively.The positive expression rates of NOX2 and NOX4 in the skeletal muscle tissues of mice in EAM group both were 100.0%,and the Immunohistochemical scores of NOX2 and NOX4 were5.57±0.75 points and 5.87±0.62 points respectively.The expressions of NOX2 and NOX4 in skeletal muscle tissues of mice in EAM group were higher than those in control group(P < 0.05).(4)The results of Pearson correlation analysis showed that the contents of ROS in skeletal muscle tissues of mice in EAM group were positively correlated with the Immunohistochemical scores of NOX2 and NOX4,and negatively correlated with the contents of NADPH,and the correlation coefficients were r = 0.968,P = 0.002;r =0.911,P = 0.012;r =-0.878,P = 0.021,respectively.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,and negatively correlated with the contents of NADPH,and the correlation coefficients were r = 0.933,P = 0.007;r = 0.942,P = 0.005;r = 0.934,P = 0.006;r =-0.920,P = 0.009,respectively.Conclusion:(1)ROS mediated by NOXs may be involved in the pathogenesis of IIM through ferroptosis pathway,suggesting that NOXs-mediated ROS play a crucial role in the pathogenesis of IIM.(2)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.(3)NADPH,NOX2,NOX4 and ROS may be new indicators to evaluate the degree of muscle tissues injury in IIM.