不同程度哮喘患者线粒体及精氨酸代谢的差异分析

Differential analysis of mitochondria and arginine metabolism in patients with different degrees of asthma

目的探讨不同病情严重程度的哮喘患者气道炎症程度、线粒体功能、精氨酸水平的变化趋势及规律。方法选择2016年11月至2017年5月新疆医科大学附属中医医院收治的122例哮喘急性发作期患者为研究对象,按照病情严重程度分级标准将患者分为间歇状态组(n=45)、轻度哮喘组(n=23)、中度哮喘组(n=30)、重度哮喘组(n=24)。另选择2016年11月至2017年5月于新疆医科大学附属中医医院体检中心体检的44例健康人为健康对照组。分别抽取各组受试者静脉血18~20 mL,采用高效液相联合质谱法检测受试者血浆中左旋精氨酸(L-Arg)及不对称二甲基精氨酸(ADMA)水平,酶联免疫吸附法检测受试者血浆中1型蛋白精氨酸甲基转移酶(PRMT1)、二甲基精氨酸二甲氨基水解酶1(DDAH1)、活性氧(ROS)、硫代巴比妥酸反应产物(TBARS)、8-异前列腺素(8-iso)、过氧化脂质(LPO)、丙二醛(MDA)水平;提取各组受试者的血小板及线粒体,应用JC-1流式细胞法测定血小板线粒体膜电位(MMP),并检测线粒体中三磷酸腺苷(ATP)及细胞色素C氧化酶(COX)活性。结果间歇状态组、轻度哮喘组、中度哮喘组和重度哮喘组患者血浆中8-iso水平显著高于健康对照组,重度哮喘组患者血浆中8-iso水平显著高于间歇状态组、轻度哮喘组和中度哮喘组(P<0.05);间歇状态组、轻度哮喘组和中度哮喘组患者血浆中8-iso水平呈增高趋势,但两两比较差异无统计学意义(P>0.05)。5组受试者血浆中TBARS水平比较差异无统计学意义(P>0.05)。间歇状态组、轻度哮喘组、中度哮喘组和重度哮喘组患者血浆中ROS、MDA水平显著高于健康对照组,中度哮喘组和重度哮喘组患者血浆中ROS、MDA水平显著高于间歇状态组和轻度哮喘组(P<0.05);间歇状态组与轻度哮喘组患者血浆中ROS、MDA水平比较差异无统计学意义(P>0.05),中度哮喘组与重度哮喘组患者血浆中ROS、MDA水平比较差异无统计学意义(P>0.05)。间歇状态组、轻度哮喘组、中度哮喘组和重度哮喘组患者血浆中LPO水平显著高于健康对照组,中度哮喘组和重度哮喘组患者血浆中LPO水平显著高于间歇状态组(P<0.05);轻度哮喘组、中度哮喘组和重度哮喘组3组间患者血浆中LPO水平比较差异无统计学意义(P>0.05)。间歇状态组、轻度哮喘组、中度哮喘组和重度哮喘组患者线粒体MMP、ATP、COX活性显著低于健康对照组(P<0.05)。间歇状态组、轻度哮喘组、中度哮喘组和重度哮喘组患者间线粒体MMP、ATP、COX活性比较差异无统计学意义(P>0.05)。间歇状态组、轻度哮喘组、中度哮喘组和重度哮喘组患者血浆L-Arg、L-Arg/ADMA比值、DDAH1水平显著低于健康对照组(P<0.05);间歇状态组、轻度哮喘组、中度哮喘组和重度哮喘组患者血浆L-Arg、L-Arg/ADMA比值、DDAH1水平比较差异无统计学意义(P>0.05)。5组间患者血浆ADMA、PRMT1水平比较差异无统计学意义(P>0.05)。结论不同病情严重程度哮喘患者都存在线粒体功能障碍和精氨酸代谢异常,尽管不同病情严重程度患者间在细胞微观层面的变化趋势上统计学差异不显著,但机体内氧化损伤程度的变化趋势与病情的加重呈现一致性。

Objective To explore the trends and patterns of changes in airway inflammation,mitochondrial function,and arginine levels in patients with different degrees of asthma.Methods A total of 122 patients with acute asthma admitted to the Affiliated Traditional Chinese Medicine Hospital of Xinjiang Medical University from November 2016 to May 2017 were selected as the research subjects.According to the severity grading criteria,the patients were divided into the intermittent state group(n=45),mild asthma group(n=23),moderate asthma group(n=30),and severe asthma group(n=24).Another 44 healthy individuals who underwent physical examinations at the Medical Examination Center of the Affiliated Traditional Chinese Medicine Hospital of Xinjiang Medical University from November 2016 to May 2017 were selected as the healthy control group.About 18-20 mL of venous blood was extracted from each group of subjects.The levels of L-arginine(L-Arg)and asymmetric dimethylarginine(ADMA)were detected by high-performance liquid chromatography-tandem mass spectrometry,and the levels of type 1 protein arginine methyltransferase(PRMT1),dimethylarginine dimethylaminohydrolase 1(DDAH1),reactive oxygen species(ROS),thiobarbituric acid reactive substances(TBARS),8-isoprostaglandin(8-iso),lipid peroxide(LPO),and malondialdehyde(MDA)were detected by enzyme-linked immunosorbent assay.Platelets and mitochondria were extracted from each group of subjects.The platelet mitochondrial membrane potential(MMP)was measured using JC-1 flow cytometry,and the mitochondrial adenosine triphosphate(ATP)and cytochrome C oxidase(COX)activity were detected.Results The level of 8-iso in plasma of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group were significantly higher than that in the healthy control group(P<0.05);the level of 8-iso in plasma of patients in the severe asthma group was significantly higher than that in the intermittent state group,mild asthma group,and moderate asthma group(P<0.05);the level of 8-iso in plasma of patients in the intermittent state group,mild asthma group,and moderate asthma group showed an increasing trend,but there was no statistically significant difference(P>0.05).There was no statistically significant difference in the TBARS level in the plasma of subjects in the five groups(P>0.05).The levels of ROS and MDA in plasma of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group were significantly higher than those in the healthy control group(P<0.05);the levels of ROS and MDA in plasma of patients in the moderate asthma group and severe asthma group were significantly higher than those in the intermittent state group and mild asthma group(P<0.05);there was no statistically significant difference in plasma ROS and MDA levels between the intermittent state group and the mild asthma group(P>0.05),and there was no statistically significant difference in plasma ROS and MDA levels between the moderate asthma group and the severe asthma group(P>0.05).The plasma LPO level of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group was significantly higher than that in the healthy control group(P<0.05);the plasma LPO level of patients in the moderate asthma group and severe asthma group was significantly higher than that in the intermittent state group(P<0.05);there was no statistically significant difference in plasma LPO level of patients in the mild asthma group,moderate asthma group,and severe asthma group(P>0.05).The mitochondrial MMP,ATP,and COX activity in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group were significantly lower than those in the healthy control group(P<0.05);there was no statistically significant difference in mitochondrial MMP,ATP,and COX activity of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group(P>0.05).The levels of plasma L-Arg,the ratio of L-Arg/ADMA,and DDAH1 in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group were significantly lower than those in the healthy control group(P<0.05);there was no statistically significant difference in plasma L-Arg,the ratio of L-Arg/ADMA,and DDAH1 levels of patients in the intermittent state group,mild asthma group,moderate asthma group,and severe asthma group(P>0.05).The plasma ADMA and PRMT1 levels showed no statistically significant difference among the five groups of patients(P>0.05).Conclusion Asthma patients exhibit mitochondrial dysfunction and abnormal arginine metabolism at any stage of the disease.Although there is no statistically significant difference in the change trend at the cellular micro level among patients with different stages of asthma,the trend of changes in oxidative damage in the body is consistent with the aggravation of the disease.