线粒体功能障碍及其影响因素在不同脏腑虚损哮喘患者中的变化趋势分析

Mitochondrial Dysfunction and Its Influencing Factors in Asthma Patientswith Deficiency of Various Viscera:A Trend Analysis

【目的】通过对不同脏腑虚损哮喘患者线粒体功能及其相关受损因素的分析,探寻不同脏腑虚损哮喘患者的病例特征,并通过对不同脏腑虚损组哮喘患者的线粒体功能变化及影响因素的差异性分析,试图发现不同脏腑虚损哮喘患者DDAH1/L-Arg/ADMA通路以及线粒体功能、氧代谢的变化趋势及规律。【方法】选取符合纳入标准的支气管哮喘急性发作期患者共122例,对入组患者进行临床资料采集,按脏腑虚损辨证分型标准分为肺气虚组47例、肺脾气虚组39例、肺脾肾虚组36例;并于同期选取正常对照组44例。观察不同脏腑虚损哮喘患者的第1秒用力呼气量(FEV1)、第1秒用力呼气量与用力肺活量的比值(FEV1/FVC)、呼气峰值流速(PEF)等肺功能指标和病情严重程度情况;同时,抽取患者静脉血,采用高效液相联合质谱法(HPLC-MS/MS)检测左旋精氨酸(L-Arg)及不对称二甲基精氨酸(ADMA)水平,采用酶联免疫吸附法(ELASA)检测蛋白精氨酸甲基转移酶1(PRMT1)、二甲基精氨酸二甲氨基水解酶1(DDAH1)等精氨酸代谢相关指标及活性氧自由基(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)等氧化损伤标记物水平;提取哮喘患者及正常对照组的血小板及线粒体,JC-1流式细胞法测定血小板线粒体膜电位(MMP)、三磷酸腺苷(ATP)和细胞色素C氧化酶(COX)的活性,以此评估线粒体功能;分析不同脏腑虚损组哮喘患者的线粒体功能变化及影响因素的差异性,并与正常对照组比较。【结果】(1)不同脏腑虚损哮喘患者的年龄和肺功能(FEV1、FEV1/FVC、PEF)比较,差异均有统计学意义(P<0.01)。表现为肺脾肾虚组的年龄明显高于肺气虚组和肺脾气虚组(P<0.05),以及随脏腑虚损情况的加重,肺功能(FEV1、FEV1/FVC、PEF)呈显著下降趋势(P<0.05)。(2)不同脏腑虚损患者的病情严重程度分布比较,差异有统计学意义(P<0.01)。其中,肺气虚组以间歇状态(1级)为主(65.96%),其次为轻度哮喘(2级)(25.53%);肺脾气虚组以中度哮喘(3级)为主(41.03%),其次为间歇状态(1级)(30.77%);肺脾肾虚组以重度哮喘(4级)为主(52.78%),其次为中度哮喘(3级)(33.33%)。即随着脏腑虚损情况的加重,哮喘的病情严重程度也表现出随之加重的趋势。(3)不同脏腑虚损组哮喘患者与正常对照组的血浆8-Iso、ROS、LPO、MDA水平比较,差异均有统计学意义(P<0.05)。其中,不同脏腑虚损组哮喘患者的血浆8-Iso、LPO水平和肺脾气虚组、肺脾肾虚组患者的血浆MDA、ROS水平均明显高于正常对照组(P<0.05),且肺脾气虚组、肺脾肾虚组患者的血浆ROS水平均明显高于肺气虚组(P<0.05);而不同脏腑虚损组哮喘患者的血浆TBARS水平有高于正常对照组趋势,但差异无统计学意义(P>0.05)。表明不同脏腑虚损哮喘患者机体内均有活性氧及过氧化脂质类物质的蓄积,且随脏腑虚损情况的加重其水平均呈增高趋势。(4)不同脏腑虚损哮喘患者与正常对照组的MMP、ATP、COX比较,差异均有统计学意义(P<0.05或P<0.01),表现为不同脏腑虚损哮喘患者的MMP、ATP、COX水平均较正常对照组明显降低(P<0.05),且随着脏腑虚损的加重,COX活性有下降趋势。(5)不同脏腑虚损哮喘患者与正常对照组的血浆L-Arg、L-Arg/ADMA、DDAH1水平比较,差异均有统计学意义(P<0.05)。表现为不同脏腑虚损哮喘患者的血浆L-Arg、L-Arg/ADMA、DDAH1水平均较正常对照组明显降低(P<0.05),且随脏腑虚损的加重,上述指标均有逐渐降低的趋势;而不同脏腑虚损哮喘患者与正常对照组的血浆ADMA、PRMT1比较,差异均无统计学意义(P>0.05)。【结论】该研究从机体代谢角度论证了中医脏腑虚损理论对病情变化趋势判断的正确性,证实了哮喘患者的脏腑虚损状况与临床病情进展、氧代谢及其影响因素和机体氧化损伤程度均表现出一致的变化趋势。

Objective To investigate the clinical characteristics of asthma patients with deficiency of various viscera through the analysis of mitochondrial dysfunction and related influencing factors,and to explore the DDAH1/L-Arg/ADMA pathway as well as the trend of changes in the mitochondrial function and oxygen metabolism of the asthma patients through the analysis.Methods A total of 122 patients with acute exacerbation of bronchial asthma who met the inclusion criteria were selected,and their clinical data were collected.The patients were divided into 3 groups based on the criteria of visceral deficiency syndrome differentiation,namely lung qi deficiency group(47 cases),lung-spleen qi deficiency group(39 cases),and lung-spleen-kidney deficiency group(36 cases).Meanwhile,the normal control group including 44 cases was set up.For the asthma patients with visceral deficiency,an observation was conducted in the lung function indicators of forced expiratory volume in the first second(FEV1),the ratio of FEV1 to forced vital capacity(FEV1/FVC)and peak expiratory flow(PEF)as well as the severity of the disease.Moreover,venous blood was sampled from the patients for the detection of L-arginine(L-Arg)and asymmetric dimethylarginine(ADMA)levels with high-performance liquid chromatography coupled with mass spectrometry(HPLC-MS/MS),and for the detection of arginine metabolism related indicators of protein arginine methyltransferase 1(PRMT1)and dimethylarginine dimethylammonium hydrolysate 1(DDAH1)as well as the oxidative damage parameters of reactive oxygen species(ROS),thiobarbituric acid reactive substance(TBARS),8-iso-prostaglandins(8-iso),lipid peroxide(LPO),and malondialdehyde(MDA)with enzyme-linked immunosorbent assay(ELISA).The platelet mitochondria were extracted from asthma patients and normal controls,and then the mitochondrial membrane potential(MMP),adenosine triphosphate(ATP),and activities of cytochrome C oxidase(COX)in the platelet were determined by JC-1 flow cytometric assay in order to assess the mitochondrial function.The changes in the mitochondrial function and their influence on the mitochondrial function of asthma patients with deficiency of various viscera were analyzed and then compared with those of the normal control group.Results(1)There were statistically significant differences in the age and lung function(FEV1,FEV1/FVC,PEF)among asthma patients with deficiency of various viscera(P<0.01):the age of the lung-spleen-kidney deficiency group was significantly older than that of the lung qi deficiency group and the lung-spleen qi deficiency group(P<0.05),and the lung function(FEV1,FEV1/FVC,PEF)showed a significant downward trend with the aggravation of viscera deficiency(P<0.05).(2)There was a statistically significant difference in the illness grading of patients with deficiency of various viscera(P<0.01).In the lung qi deficiency group,the illness status was predominated by intermittent state of asthma(grade 1),accounting for 65.96%,and then followed by mild asthma(grade 2),accounting for 25.53%.In the lung-spleen qi deficiency group,the illness status was predominated by moderate asthma(grade 3),accounting for 41.03%,and then followed by intermittent state of asthma(grade 1),accounting for 30.77%.In the lungspleen-kidney deficiency group,the illness status was predominated by severe asthma(grade 4),accounting for 52.78%,and then followed by moderate asthma(grade 3),accounting for 33.33%.It is indicated that with the aggravation of viscera deficiency,the severity of asthma tended to be aggravated.(3)There were significant differences in plasma 8-iso,ROS,LPO and MDA levels between asthma patients with deficiency of various viscera and normal control group(P<0.05).The plasma 8-iso and LPO levels of the asthma patients with deficiency of various viscera and the plasma MDA and ROS levels of asthma patients with lung-spleen qi deficiency and lung-spleen-kidney deficiency were significantly higher than those in normal control group(P<0.05),and the plasma ROS level of asthma patients with lung-spleen qi deficiency and lung-spleen-kidney deficiency was significantly higher than that in asthma patients with lung qi deficiency(P<0.05).The plasma TBARS level of asthma patients with deficiency of various viscera tended to be higher than that of normal control group,but the difference was not statistically significant(P>0.05).It is indicated that reactive oxygen species and peroxide lipids accumulated in the body of asthma patients with deficiency of various viscera,and their levels increased with the aggravation of visceral deficiency.(4)The levels of MMP,ATP and COX in asthma patients with deficiency of various viscera were significantly lower than those in normal control group(P<0.05),and the activity of COX decreased with the aggravation of visceral deficiency.(5)The levels of plasma L-Arg,L-Arg/ADMA and DDAH1 in asthma patients with deficiency of various viscera were significantly lower than those in normal control group(P<0.05),and their levels decreased gradually with the aggravation of visceral deficiency.There was no significant difference in the plasma ADMA and PRMT1 levels between asthma patients with deficiency of various viscera and normal control group(P>0.05).Conclusion It is indicated that the visceral deficiency in asthma patients is consistent with the progression of illness status,oxygen metabolism and its influencing factors,and the degree of oxidative damage.It is suggested that the progression of illness status can be judged by visceral deficiency in traditional Chinese medicine according to the parameters of body metabolism.