辨证治疗AECOPD呼吸衰竭疗效评价及对单核细胞Toll样受体和外周血细胞因子的影响

【作者】 王海峰；

【导师】 李建生；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的慢性阻塞性肺疾病急性加重(AECOPD)呼吸衰竭是导致严重危害公众健康的重大疾病,致死率高,经济负担重。中医药干预AECOPD呼吸衰竭具有一定的疗效和优势,但缺乏系统研究,机制探讨不深入。炎性细胞介导的炎症反应在其中起着重要的作用,Toll样受体(Toll like receptors, TLRs)信号途径在COPD发病机制中的作用受到广泛关注,但对TLR在COPD患者体内实际作用和表达情况的研究较少。本文目的在于从临床方面进行中医辨证治疗AECOPD呼吸衰竭的临床疗效评价,并从单核细胞免疫功能、外周血细胞因子等方面探索中医辨证治疗方案治疗AECOPD呼吸衰竭的机制。对象与方法按照随机对照原则,将AECOPD呼吸衰竭患者分为试验组(辨证治疗+西医常规治疗)和对照组(西医常规治疗),试验组入选52例,对照组52例,试验组在常规治疗基础上进行辨证治疗,评价病死率、气管插管率、撤机率、VAP发生率,于治疗第0,4,7,14天,评测临床症状体征积分,APACHEⅡ评分,血常规,血气分析,MMRC评分等；应用流式细胞仪全血免疫荧光直标法检测单核细胞TLR4蛋白和外周血中T细胞亚群CD4+.CD8+及其比值；应用RT-PCR去测定单核细胞TLR4mRNA、CD 14mRNA、NF-κBp65mRNA水平；酶连免疫吸附法(ELISA)测定外周血CRP,细胞因子β-EP.IL-lb. IL-6、IL-8、TNF-α、IFN-y,安全性评价采用报告表记录患者不良反应,并观察治疗前后血常规,尿常规,大便常规,肝肾功能,心电图进行评价。结果本项目共完成104例病例,其中试验组52例,对照组52例,试验组脱落3例,对照组脱落4例,临床观察结果表明：1试验组死亡2例(3.85%),气管插管8例(15.38%),拔管撤机6例(75.0%),与对照组的3例(5.77%),11例(21.15%),4例(36.36%)比较无统计学意义。试验组患者中2例出现机械通气相关肺炎(VAP),与对照组8例比较差异有统计学意义(P=0.0397)。2试验组治疗后症状体征总积分和症状评分明显低,与对照组差异有统计学意义(P<0.05),体征总积分与对照组比较无统计学意义(F=0.989,P=-0.322)；咳嗽、咯痰、喘息、气短、乏力、心悸、烦燥、腹胀、浮肿等单项积分试验组显著低于对照组(P＜0.05),胸闷、汗出、头痛、语言错乱、食少、哮鸣音、紫绀、精神状态积分两组间差异无统计学意义(P>0.05)。3两组治疗后APACHEⅡ评分均较治疗前降低,试验组下降幅度较对照组明显,差异有统计学意义(F=4.325,P=0.040)。4两组治疗后白细胞总数及中性粒细胞百分比渐下降,组间比较(P=0.336；P=0.517)无显著差异。5试验组治疗后血氧分压与对照组比升高显著(P=0.002)；试验组二氧化碳降低与对照组比较差异无统计学意义(P=0.988)。6治疗后试验组CD4~+、CD4~+/CD8+轻度上升,CD8+与治疗前比较明显回升(P＜0.01)。对照组CD4~+,CD8+台疗后较治疗前轻度升高,CD4~+/CD8+下降,无统计学差异(P＞0.05)。各指标试验组与对照组差异无统计学意义(P>0.05)。7两组治疗后MMRC评分均较治疗前下降,试验组呼吸困难评分下降与对照组差异有统计学意义(P=0.007)。8 AECOPD呼吸衰竭患者外周血单核细胞CD14~+低于正常人组,与稳定期患者比较显著下降(P<0.05)；AECOPD呼吸衰竭患者CD14蛋白(荧光强度)表达低于稳定期组和正常人组,差异无统计学意义(P>0.05)；AECOPD呼吸衰竭患者TLR4阳性细胞相对百分率高于稳定期组和正常人组,两组与正常人组间差异有统计学意义(P<0.05)；AECOPD呼吸衰竭患者TLR4平均荧光强度高于稳定期组和正常人组,各组间差异无统计学意义(P>0.05)。9治疗后试验组CD14阳性细胞数与治疗前比较轻度上升(P>0.05),CD14蛋白(荧光强度)轻度下降(P＞0.05),与对照组比较差异无统计学意义(P>0.05)；治疗后试验组TLR4阳性细胞数和TLR4蛋白(荧光强度)轻度下降(P>0.05),与对照组比较差异无统计学意义(P>0.05)。10治疗后试验组血单核细胞TLR4mRNA.CD14mRNA. NF-κBp65mRNA水平均较对照组下降趋势显著,其中CD14mRNA与对照组比较有显著差异(P=0.020)。11治疗后试验组血清CRP下降明显,与对照组有显著差异(P=0.045)。12治疗后试验组血清β-EP, IL-1b、IL-6、IL-8、TNF-α水平均出现下降,对照组大都变化不明显或持续上升,试验组与对照组在IL-8(P>=0.047)、TNF-α(P=0.032)比较有显著性差异。试验组IFN-γ轻度上升,对照组第7天上升,第14天下降到治疗前水平，两组比较无显著差异(P>0.05)。13安全性评价方面,辨证治疗AECOPD安全有效,未出现严重不良反应,最常见的不良事件是腹泻,腹胀。两组间无显著差异(P>0.05)。结论1辨证治疗AECOPD呼吸衰竭临床效果显著。能够降低咳嗽、咯痰、喘息、气短、乏力、心悸、烦燥、腹胀等症状积分,降低浮肿的体征积分；提高动脉血氧分压(PaO2)；降低呼吸困难评分(MMRC);降低APACHEⅡ评分。2外周血单核细胞免疫功能与AECOPD呼吸衰竭的发病有关。3中医辨证治疗AECOPD呼吸衰竭的机制可能与下调单核细胞表面TLR4的表达,减轻TLR4和NF-kB介导的炎症反应有关。CRP、IL-8、TNF-α水平与病情严重程度有关,可能可作为判断AECOPD病情严重程度的候选指标。4辨证治疗在减少VAP发生率方面有一定优势；在插管率、拔管率方面显示了较好的趋势,但无统计学意义,可能与样本量较少有关；在降低病死率方面与对照组比较无显著差异。更多还原

【Abstract】 Objectivechronic obstructive pulmonary disease （COPD） combined with acute respiratory failure are key drivers of morbidity and mortality, which results in an economic and social burden that is both substantial and increasing. It is reported that treatment with Traditional Chinese Medicine（TCM） may do some benefit to the disease, but the quality of evidence is poor, and mechanism about TCM treatment is not so clear. Inflamation induced by inflammatory cells appears to be amplified in patients who develop COPD. TLRs （Toll-like receptors） comprise a family of proteins whose function is principally to facilitate the detection of, and response to pathogens, viruses, bacteria and fungi can all activate TLR signaling pathway, and these signals have important roles in the activation of COPD host defence. But most of our knowledge of TLRs has emerged from studies of animals. The contribution of TLRs function to human COPD is less advanced.To investigate clinical therapeutic effect of patients with AECOPD and respiratory failure by treatment of syndromes differentiation of TCM, and the pathogenesis of treatment of syndromes differentiation of TCM, their cellular and molecular mechanisms, and how these underlie physiologic abnormalities and symptoms characteristic of the disease.MethodsOf a total of 104 patients, we randomly assigned 52 to the control group and 52 to the experimental group. The control group were treated by conventional medicine treatment, the experimental group were treated by TCM besides conventional medicine treatment. The primary efficacy endpoint was mortality, intubation and extubation rate and VAP rate. Secondary endpoints included health status measured by symptomes scores, APACHEⅡscores, blood rutine examination, blood gas analysis and MMRC scores （measured the 0,4,7,14 day after study medication）. The expression of TLR4 surface molecules on human CD 14⁺ monocytes was assessed using FACS analysis by flow cytometry （measured the 0 and 14 day）, so is CD4⁺、CD8+T cells. The TLR4mRNA、CD14mRNA、NF-κBp65 mRNA of human CD 14⁺ monocytes was assessed by Reverse Transcriptase Polymerase Chain Reaction method（measured the 0,7,14 day）..β-EP、IL-1b、IL-6、IL-8、TNF-α、IFN-γin peripheral blood of patients were assessed by means of enzyme linked immunosorbent assay method（measured the 0,7,14 day）. Safety was assessed by documenting all adverse events. Routine examination of blood, urine, stools, liver and renal function, electrocardiograms were performed at days 0 and 14.ResultsOf a total of 104 patients,52 were assigned to the control group and 52 to the experimental group.3 in experimental group and 4 in the control group drop out. 1 2（3.85%） of patients in experimental group died compared with 3 （5.77%） in the control group （P=0.6467）.8 （15.38%） of patients in experimental group intubated compared with 11 （21.15%） in the control group（P=0.6118）.6（75.0%） extubated in experimental group compared with 4（36.36%） in the control group（P=0.0959）. More ventilator-associated pneumonia were reported in the control group relative to experimental group （P=0.0397）.2 The clinical status in experimental groups decreased significantly compared to the control group during the study period（P=0.041）, and so was the symptomes scores（P=0.016）. There was no significant difference about scores of signs between the two groups （P=0.322）.Score of the independent item such as cough, expectoration, gasping, short breath, lack of power, palpitation, upset, abdominal distension and edema decreased significantly compared to the control group during the study period. Dyspnea, sweating, headache, paraphasia, anorexia, wheezing, cyanosis and mental status scores of TCM patients showed no difference to the control group.3 APACHEⅡscore were decreased in both the two groups at days 14, it was lower in the experimental treatment group than the control group during the study period （P=0.040）4 Total white cell count in peripheral blood and the neutrophil percentage decreased in the two groups at days 14, it was lower in the experimental treatment group during the study period, there was no major difference between the two groups（P=0.336, P=0.517）.5 Arterial oxygen tension（PaO₂） increased in both the two groups during the study period, it was higher in the experimental treatment group, and there was significant difference between the two groups（P=0.002）. Partial pressure of carbon dioxide （PaCO2） in the experimental treatment group showed no significant difference to the conventional treated patients （P=0.988）.6 CD4（+） T cells and ratio of CD4⁺/CD8⁺ increased in AECOPD patients, CD8（+） T cells in experimental group increased over the study period（P<0.01）, CD4（+）, CD8（+） T cells in control group increased too ratio of CD4⁺/CD8+ decreaced. there were no significant difference between the two groups（P>0.05）.7 The MMRC dyspnoea scale scores decreased in both group during the study period （P=0.000）, it was lower in the experimental treatment group than the control group during the study period （P=0.007）8 CD 14 on AECOPD patients peripheral blood monouclear cell were little lower compared to normal using FACS analysis, and decreased significantly compared to the stable COPD patients （P<0.05）. the CD 14 X-mean expressed elevated in stable COPD patients and decreased in severe AECOPD patients, there were no significant difference between them. TLR4 was elevated in stable and AECOPD patients as compared to normal ones, there was significant difference between COPD patients and normals （P<0.05）. TLR4 X-mean expressed elevated in stable and AECOPD patients, there was no significant difference between three groups. 9 CD 14 expression were elevated in the experimental（P>0.05） and down-regulated in control group（P>0.05） after treatment. CD14 X-mean expression were not changed much in both groups, and there was no significant difference between them. Expression of TLR4 on monouclear cell were similar in the two groups, and reduced over the treatment period, there was no significant difference as compared to before treatment. TLR4 X-mean expression down-regulated in experimental group and control group, there was no significant difference as compared to before treatment and between them（P>0.05）.10 Compared with the control group, TLR4mRNA、CD14mRNA. NF-κBp65mRNA in monocyte of the experimental group decreased after treatment, The expression levels of CD14mRNA decreased significantly（P=0.020）11 C-reactive protein in serum was reduced in both the two groups after treatment, CRP in the experimental group reduced more compare to the control group （P=0.045）.12 The level ofβ-EP、IL-1b、IL-6、IL-8、TNF-αin serum was reduced in the experimental group after treatment, all the cytokines in the experimental group reduced more than the control group, especially the IL-8 （P=0.047） and TNF-α（P=0.032）. IFN-γin serum was increased gently in the experimental group, but no more than the control group （P<0.05）.13 Safety evaluation, treatment by syndromes differentiation of TCM is safe and effective, no severe adverse reaction reported. Only a few reporting some adverse event, the most frequent of which were diarrhea and abdominal distension.Conclusion1 Treatment by syndromes differentiation of TCM to patients with COPD exacerbations and respiratory failure have obvious therapeutic effect. It can relieve clinical symptoms, decrease APACHEⅡscore and the MMRC dyspnea scale outcome. The mechanism of action may related to the following reason.2 CD 14 and TLR4 expression on monouclear cell increaced in patients with AECOPD and respiratory failure, so peripheral blood monouclear cell may related to the disease.3 TCM may have the role cf inhibiting activation of TLR4 and nuclear factor-kappaB signaling pathway on peripheral blood monoeytes in patients with AECOPD with respiratory failure, which may associated with reduced cytokine concentration, then relieving the sytemic inflammatory responses. The concentration of CRP、IL-8、TNF-αrelevant to severity of the disease, and maybe it can be take as a biomarker for AECOPD.4 The experimental group have some advantage in VAP rate. The experimental group have less in intubation and more extubation patients, given more sample, it might be a difference conclusion. There were no significant difference in death rate in the two groups,更多还原