黄芪复方治疗重症肌无力的临床与实验研究

【作者】 牛广华；

【导师】 张静生；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 重症肌无力（myasthenia gravis,MG）是一种主要累及神经肌肉接头处（neuromuscular junction,NMJ）突触后膜（postsynaptic）上乙酰胆碱受体（acetylchol ine receptor,AchR）,主要由乙酰胆碱受体抗体（acetylcholinereceptorantibody,AchRab）介导、细胞免疫（cell-mediated immunity,CMI）依赖性、补体（complement）参与的自身免疫性疾病（autoimmune disease）。其特点包括:1.临床上表现为活动后加重、休息后减轻、朝轻暮重的骨骼肌无力。2.电生理上表现为低频重复电刺激波幅递减,微小终板电位降低、单纤维肌电图上颤抖增宽、甚至阻滞。3.药理学上表现为胆碱酯酶抑制剂治疗有效、对箭毒类药物的过度敏感性。4.免疫学上表现为血清乙酰胆碱受体抗体增高。5.免疫病理学上表现为神经肌肉接头处突触后膜的皱褶减少,变平坦,突触后膜上乙酰胆碱受体减少。重症肌无力是一种世界性疑难病,目前西医治疗方法均有不同程度的副作用,因此,发掘减少西药副作用且疗效确切的中药成为当务之急。黄芪复方颗粒剂是导师张静生教授在脾肾学说指导下研制成功的治疗重症肌无力的纯中药制剂,在临床和实验研究中均取得了明显疗效。目的:观察黄芪复方治疗重症肌无力的临床疗效;观察黄芪复方对重症肌无力患者外周血淋巴细胞亚群:辅助性淋巴细胞亚群Th1/Th2、抑制性淋巴细胞亚群Tc1/Tc2;细胞因子IFN-γ、IL-4及TNF-α水平;免疫球蛋白IgG、IgA、IgM、补体C3、C4和AChRAb水平的影响,从细胞水平揭示中药黄芪复方治疗MG的免疫机理。方法:1.根据诊治先后,将符合纳入标准的重症肌无力患者随机分为治疗组和对照组,两组各30例。治疗组服用黄芪复方颗粒剂和强的松;对照组服用强的松和模拟剂。90天为一疗程。观察治疗前后的临床疗效积分和中医证候积分变化。2.应用流式细胞仪检测治疗前后的重症肌无力患者外周血淋巴细胞亚群分布的变化情况。3.应用流式细胞仪检测黄芪复方颗粒剂及模拟剂和强的松治疗前后重症肌无力患者外周血Th1、Tc1、Th2、Tc2细胞的变化情况。4.采用酶联免疫吸附实验技术检测重症肌无力患者治疗前后外周血血清细胞因子IFN-γ、IL-4及TNF-α水平的变化。5.采用免疫比浊及酶联免疫吸附实验技术检测重症肌无力患者治疗前后外周血IgG、IgA、IgM、C3、C4、AChRAb水平的变化。结果:1.临床研究结果显示:黄芪复方对重症肌无力具有明显疗效。从临床疗效积分变化统计:治疗组显效率70.0%,对照组显效率56.7%,二者相比较,治疗组疗效优于对照组,具有显著性差异（P＜0.05）;从中医证候积分变化统计:治疗组有效率73.33%,对照组有效率仅为56.67%,两组中医证候改善情况有显著差异,P＜0.01,提示治疗组在中医证候改善方面明显优于对照组。研究还发现,对照组治疗后出现轻度肝功能异常、空腹血糖增高、向心性肥胖等副作用;而中药黄芪复方具有确切的治疗作用,且未发现任何毒副作用,明显优于对照组。2.两组患者治疗前外周血CD4⁺细胞及CD4⁺/CD8⁺比值均明显高于健康组,差异均有统计学意义（P＜0.01）。CD8⁺细胞、NK细胞（CD16⁺/56⁺）水平较健康组显著降低,差异有统计学意义（P＜0.01）,两组CD3⁺、总B细胞（CD19⁺）比较无统计学差异（P＞0.05）。治疗后,两组CD3⁺细胞无明显变化（P＞0.05）,而CD4⁺细胞、CD4⁺/CD8⁺比值及总B细胞（CD19⁺）与治疗前比较均有明显下降,CD8⁺及NK细胞（CD16⁺/56⁺）均明显增加,差异均有统计学意义（P＜0.05）。与对照组治疗后相比较治疗组CD4⁺、CD8⁺、CD4⁺/CD8⁺、CD19⁺、CD16⁺/56⁺均有显著差异（P＜0.05）。3.与健康组相比较,两MG组外周血中Th1、Th2、Tc1和Tc2细胞百分数显著增高,差异有统计学意义（P＜0.01）,Th1/Th2、Tc1/Tc2比值同健康组相比差异有统计学意义（P＜0.05）。治疗后,治疗组Th1型、Tc1型细胞明显减少（P＜0.01）,与对照组治疗后比较有显著性差异（P＜0.05）。4.两MG组外周血IFN-γ、IL-4、TNF-α水平均明显高于健康组,差异均有统计学意义（P＜0.01）。与治疗前相比较,两MG组经治疗后IFN-γ、IL-4、TNF-α水平均显著降低,差异均有统计学意义（P＜0.01）。两组治疗后比较,治疗组各项指标下降显著,与对照组比较,差异均有统计学意义（P＜0.05）。5.与健康组比较,两MG组AchRab水平显著增高,差异有统计学意义（P＜0.01）。两MG组IgA、IgM水平均显著低于健康组,差异有统计学意义（P＜0.01）。两MG组C₃、C₄水平显著降低,与健康组比较差异有统计学意义（P＜0.05）。经治疗后,治疗组补体C₃、C₄水平与治疗前相比显著增高,差异均有统计学意义（P＜0.05）。治疗组AchRab水平与治疗前比较有明显下降,差异有统计学意义（P＜0.01）。治疗组补体C₃、C₄及AchRab水平与对照组治疗后比较,差异有统计学意义（P＜0.05）。结论:1.黄芪复方+基础西药能明显改善重症肌无力患者的临床症状和中医证候,在中医证候改善方面明显优于单独应用强的松治疗,而且无任何毒副作用。2.黄芪复方可能通过抑制Th细胞增殖,促进T细胞活化,降低CD4⁺/CD8⁺,使重症肌无力患者体内T淋巴细胞亚群比例重新分布,纠正重症肌无力异常的免疫应答过程,最终抑制B细胞产生以乙酰胆碱受体抗体为主的自身抗体。3.黄芪复方通过抑制MG患者Th1型细胞和Tc1型细胞的分化,减少Th1/Tc1型细胞因子如IFN-γ的分泌而起到抗炎和改变病程的作用。4.经过黄芪复方治疗后,血清中IFN-γ、IL-4、TNF-α均有不同程度的降低,可以反映中药治疗MG的免疫效应。在免疫抑制方面,黄芪复方+免疫抑制剂强的松治疗组比单用免疫抑制剂强的松效果明显,说明黄芪复方对调节免疫功能是双向的、多靶点的,而强的松对病人的免疫功能是过度抑制的,这显示出中药制剂黄芪复方的整体调节特征和优势。5.黄芪复方规范化治疗以后MG患者血清中的补体C3、C4明显增高提示对MG患者测定血清补体,有助于进行动态观察,并可作为判断病情严重程度（活动性）的客观指标。更多还原

【Abstract】 Myasthenia gravis is a kind of autoimmune disease,it mainly involves the Acetylcholine receptor（AchR） on the postsynaptic membrane of neuromuscular junction,mediated by the specific nicotinic acetylcholine receptor（N₂AchRab）, and the complement participates its process.Its characteristics involve:1.Clinical symptoms:weakness of skeletat muscle,aggravated after action,and relieved after rest,slight in daytime and gravis at night.2.Electromyography:the amplitude decreased after echo-low frequency transduction of electric stimulation,at the same time,the miniature end-plate potential decreased,and the jitter of single fiber EMG widened,even blocked.3.Pharmacology:treated with cholinesterase inhibitor is effective, and over anaphylaxis to tubocurarine.4.Immunology:the increasing of acetylcholine receptor antibody in blood serum.5,Immunopathology:the wrinkles of postsynaptic membrane at neuromuscular junction decreased,flattened,and the AchR on the postsynaptic membrane decreased,myasthenia gravis is a cosmopolitan stubborn disease,the western medical treatment has its side effect, therefore,develop the effective Chinese patent medicine is the urgent mission. the complex recipe of Astragalus membranaceus is a pure Chinese drugs pharmaceutics,developed by Pro.Zhang Jingsheng under the directions of PISHEN theory,and obtained manifest therapeutic effect on the clinical and experimental study till now.Purpose:Observe the complex recipe of Astragalus membranaceus’s clinical effect in treating Myasthenia graves,provide its experimental basement;Observe the influence of the complex recipe of Astragalus membranaceus to T lymphocyte、Tc1/Tc2、Th1/Th2、IFN-γ、IL-4、TNF-α、IgG、IgA、IgM、C3、C4、AchRAb in peripheral blood of myasthenia gravis patients,reveal its immunological regulation effect on the cellulax level.Methods:1.According to the hospitalization date and the random-double blind-control principle,the myasthenia gravis patients was randomly divided into 2 groups, the curing group and the control group,30 cases per group.Patients of curing group administered the complex recipe of Astragalus membranaceus and prednisone placebo,Patients of control group administered prednisone tablet and Zhongjiling tablet placebo.Course of treatment is 90 days.Compare the pre-treatment and post-treatment clinical curative effect scores and Chinese medicals symptom scores.2.Detect the pre-treatment and post-treatment distribution of T lymphocyte subgroup of myasthenia gravis patients with flow cytometry.3.Detect the pre-treatment and post-treatment distribution of T lymphocyte subgroup Th1、Tc1、Th2、Tc2 of myasthenia gravis patients with flow cytometry.4.Detect the the pre-treatment and post-treatment level of IFN-γ、IL-4、TNF-αof myasthenia gravis patients with ELISA assay technology。5.Using the immunoturbidimetric and ELISA assay technology to detect the level of IgG、IgA、IgM、C3、C4、AChRAb in patients’ peripheral blood pre-and post-administration.Results:1.Clinical research:The complex recipe of Astragalus membranaceus has significant effect to myasthenia gravis,the clinical syndrome score statistic result shows that the effective ratio of curing group is 70.0%,and that if control group is 56.7%,manifest difference exit between these two groups, P＜0.05;the change of Chinese medical symptom score statistic result shows that the effective ratio of curing group is 73.33%,and that if control group is 56.67%,manifest difference exit between these two groups,P＜0.01,it indicate that the curing group is better than that control group in improving Chinese medical symptoms.In this study,the patients in control group appeared side-effects such as sight hepatic dysfunction,increasing fasting serum glucose,femoral head necrosis and central obesity,on the contrary,The complex recipe of Astragalus membranaceus has exact curing effect without any side-effect,these shows that it is better than control group.2.Before treatment,the CD4⁺ cell percentage and CD4⁺/CD8⁺ of these two groups are higher than that of normal group,P＜0.01,the CD8⁺ and NK（CD16⁺/56⁺）cell percentage of these two groups are lower than that of normal group,P＜0.01.The CD3⁺ and B（CD19⁺） cell percentage of these two groups have no difference with that of normal group,P＞0.05.After treatment,there is no change on the CD3⁺ cell percentage of these two groups,P＞0.05,CD4⁺ and CD19⁺ cell percentage and the ratio of CD4⁺/CD8⁺ decreased significantly,CD8⁺ and NK（CD16⁺/56⁺） cell increased significantly,P＜0.05.Compare with that of control group,there is significant difference for the CD4⁺,CD8⁺,CD4⁺/CD8⁺,CD19⁺ and CD16⁺/56⁺ cell percentage,P＜0.05.3.The Th1,Th2,Tc1 and Tc2 cell percentage of these two groups are higher than that of normal group,P＜0.01,Th1/Th2 and Tc1/Tc2 have difference with that of normal group,P＜0.05.After treatment,The Th1 and Tc1 cell percentage of the curing group decreased significantly,P＜0.01,there is obvious difference with that of control group.4.The IFN-γ,IL-4 and TNF-αlevel of these two groups are significant higher than that of normal group,P＜0.01.After treatment,the IFN-γ,IL-4 and TNF-αlevel of two groups depressed,compared with that of pre-treatment, significant difference exited,P＜0.01.While in control group,the IFN-γ,IL-4 and TNF-αlevel decreased significantly,there is obvious difference with that of control group,P＜0.05.5.The AchRab level of these two groups are higher than that of normal group, P＜0.01.The IgA and IgM level of these two groups are lower than that of normal group,P＜0.01.The C₃ and C₄ level of these two groups decreased significantly, there is obvious difference with that of normal group,P＜0.05.After treatment, the C₃ and C₄ level of curing group increased,compared with that of pre-treatment,significant difference exited,P＜0.05,the AChRAb level of the curing group decreased significantly,P＜0.01,while in control group,the C₃, C₄ and AchRab level are obvious difference with that of control group,P＜0.05. Conclusion:1.The complex recipe of Astragalus membranaceus can significantly improve the myasthenia gravis pationts’ clinical symptoms and Chinese medicine syndromes,Its effect of improving Chinese medicine symptoms is better than that of only using prednisone,and no adverse reaction exited in this study.2.The complex recipe of Astragalus membranaceus caused the redistribution of T lymphocyte subgroup ratio,this effect maybe due to its inhibition of Th cell proliferation,promoting Ts cell’s activation and depress CD4⁺/CD8⁺ ratio, then rectified the abnormal immune response of myasthenia gravis patients, as a result,inhibited the production of auto antibody,mainly AchRab.3.Th1 and Tc1 subsets decreased in the peripheral blood after MG patients were treated with leflunomide.The effect of leflunomide might be related to the prevention of Th1 and Tc1 differentiation and the decrease of Th1 and Tc1 cytokines such as IFN-γ.4.After treatment with the complex recipe of Astragalus membranaceus,the IFN-γ, IL-4 and TNF-αlevel depressed,compared with that of pre-treatment,this indicated its immunological regulation effect.The complex recipe of Astragalus membranaceus+ prednisone has exact curing effect,it is better than that of only using prednisone.These indicate that The complex recipe of Astragalus membranaceus’s immunological regulation is multi-targets and tow-way regulation.It is different with that of western medicine,which is overall inhibition to patients’ immune function,and this manifest The complex recipe of Astragalus membranaceus’s holo-regulation characteristic and predominance.5.After treatment,the C₃ and C₄ level of curing group increased,it suggests that defecting of the levels of the complements in MG patients has attributions to mobile MG and maybe an objective index in determing the disease severity.更多还原