【作者】 梁江；

【导师】 林昌松；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2012， 博士

【摘要】 目的本研究分为两部分：临床部分通过采取随机、阳性平行对照的试验设计,旨在观察、评价断藤益母汤治疗类风湿关节炎(RA)的临床疗效与安全性。实验部分是通过体外培养类风湿关节患者滑膜成纤维细胞原代细胞(RA-FLS),以含药血清为干预手段初步探讨断藤益母汤对RA-FLS异常增殖的抑制作用,同时探讨该药含药血清对RA-FLS中Toll样受体4(TLR4)人类髓样分化因子(MyD88)、核转录因子(NF-κ B)三个信号通路元件的影响,旨在从炎症信号转导通路及相关细胞因子的角度探索断藤益母汤这一昆明山海棠中药复方配伍抗RA的作用机制,为该药的临床应用提供理论和实验依据。方法一、临床部分按1987年美国风湿病学学会(ACR) RA分类标准为诊断依据,共入选RA患者70例,简单数字随机化等分为两组：试验组：口服断藤益母汤(由川续断15g,益母草30g,桂林产昆明山海棠45g组成,其中昆明山海棠先煎3.5h,再加入其他两味药同煎0.5h)每日一剂,分早晚两次服用,每次150ml。对照组：口服来氟米特片20mg qd,(购自江苏新凯-长征制药公司)治疗,治疗12周。在治疗的第0、2、6、12周记录两组的观察指标,计算两组患者DAS28、 ACR20、 ACR50变化情况,评价疗效。主要观察指标包括：症状体征指标(压痛关节数、肿胀关节数、晨僵时间、患者及研究者病情总体情况VAS评分、疼痛VAS评分、HAQ评分)、炎症活动相关实验室指标(血沉、C反应蛋白、类风湿因子、血清TNF-α含量)、安全性观察指标：(血常规、肝功能、肾功能及女性患者月经情况及其他不良反应事件)。二、体外细胞实验部分1.制取含药血清清洁级环境中饲养12只清洁级健康雄性家兔,随机等分为3组按如下方法灌胃：西药组：来氟米特1.92mg·kg-1；中药组：3.45g·kg-1断藤益母草汤,对照组予等量生理盐水。给药一周后采血分离含药血清,冻存备用。2.细胞培养和鉴定：组织块法体外培养原代RA-FLS细胞和正常FLS,光镜鉴定,实验用的RA-FLS3至5代处于对数生长期的细胞。细胞爬片苏木伊红染色作细胞形态学鉴定。免疫组化染色TLR4细胞爬片及滑膜组织切片。3.各组RA-FLS抑制情况检测取RA-FLS,分成3组按以下方法干预：对照组(含10%对照组家兔血清的RPMI1640培养液),断藤益母汤组(含10%中药组家兔血清的RPMI1640培养液)和来氟米特组含10%西药组家兔血清的RPMI1640培养液)。分别常规培养24h、48h、72h后,用MTT法检测各组细胞的抑制率。4. RT-PCR法检测各组细胞TLR4、MyD88的uRNA表达取RA-FLS,分组如下：.LPS组、.空白组、断藤益母汤组、来氟米特组。对照组(含10%对照组家兔血清的RPMI1640培养液),其余各组均加入LPS20μg· mL-1,含药血清干预方法同按MTT检测。培养24h后RT-PCR法检测TLR4、 MyD88mRNA表达。5. ELISA法检测上一步处理后各组细胞培养上清中TNF-αa含量及细胞总蛋白中的核转录因子(NF-κ B)含量。结果一、临床部分1.临床疗效评估共计64例患者(试验组33例、对照组31例)完成试验全程,6例因依从性不良退出。在治疗12周后,试验组ACR20, ACR50达标率分别为57.14%,20%；对照组ACR20、 ACR50达标率分别为62.86%,22.86%,此时两组间比较无明显差异(P>0.05)；按DAS28疗效评价,治疗12周后试验组总有效率为51.42%,对照组总有效率为57.14%,两组总有效率差异无明显统计意义(P>0.05)。较之基线水平,试验组在治疗2周、6周、12周ACR20达标例数均明显增多(P<0.01),在治疗6周、12周后ACR50达标例数均明显增多(P<0.01)；且该组治疗6周后ACR50达标例数高于2周后(P<0.05)；此外,治疗2周后试验组ACR20达标例数明显多于对照组(P<0.05),而对照组较基线水平明显起效是在6周以后(P<0.05)。2.主要临床症状体征比较：试验组治疗2周后在压痛关节数、肿胀关节数、疼痛VAS评分、患者、研究者病情状况VAS评分、HAQ评分6方而较基线水平均有明显降低(P<0.05)。且此时试验组的肿胀关节数、HAQ评分、疼痛VAS评分、患者及研究者对病情状况VAS评分方面的改善程度明显优于对照组(P<0.05)；6周时试验组肿胀关节数、受试者对病情状况VAS评分明显低于对照组(P<0.05)。两组在所有7项指标在6周、12周后较基线均明显降低(P<0.05),治疗12周后,对照组在研究对患者者病情状况VAS评分、HAQ评分2项指标方面改善程度优于试验组(P<0.05)。两组在其余各项指标改善程度无明显差异(P>0.05)。3.炎症活动相关指标两组风湿三项(血沉、C反应蛋白、类风湿因子)基线水平无明显差异(P>0.05),具备可比性。试验组在治疗2周后C反应蛋白水平较基线显著降低(P<0.05)；治疗6周、12周后,患者的风湿三项较基线水平进行性显著下降(P<0.05)；治疗2周后试验组C反应蛋白较对照组明显降低(P<0.05)；而对照组在治疗6周后、12周后风湿三项均较基线水平明显下降(P<0.05)；但治疗6、12周末两组的风湿三项数值无明显差异(P>0.05)。12周后,较各自基线水平两组TNF-α含量显著下降(P<0.01)但在0、12周两个时间点的组间比较均无明显差异(P>0.05)。4.安全性指标两组中6个安全性指标(红细胞数、白细胞数、血红蛋白数、血肌酐、尿素氮、谷丙转氨酶、谷草转氨酶)在试验的4个时间点(0周,2周、6周、12周)中无明显差异,但12周末试验组在血小板数低于治疗前(P<0.05)。5.不良反应试验组的35例患者中,服药后出现瘙痒皮疹的有1例,自行缓解；出现月经量变少3例,出现不良反应的患者均自愿坚持用药参加完实验全程；肾功能、血液分析未见明显异常。对照组的35例患者中,服药后10例出现轻度脱发,2例出现谷丙转氨酶一过性升高至正常2倍,加用护肝治疗2周后恢复正常,治疗过程中未停药,该组出现不良反应患者参加全程实验。二、实验部分1.成功建立RA-FLS细胞株,该细胞免疫组化染色示TLR4表达量较正常FLS增高。2.MTT结果较之同一时点的对照组,在干预48h,72h后,中、西药含药血清均能抑制RA-FLS增殖,差异有显著统计意义(P<0.05),72h后,西药组细胞抑制程度明显强于中药组(P<0.05)。中、西药组细胞抑制率分别为25.7%、35.9%。3.RT-PCR结果在干预24h后,LPS组细胞TLR4mRNA表达量明显高于对照组(P<0.05),相对于LPS组,而两种含药血清均可明显抑制TLR4mRNA表达量(P<0.05),但中药组、西药组、对照组3组的组间差异不具备显著统计意义(P>0.05)。各组间MyD88mRNA表达量含量无明显差异(P>0.05)。4.ELISA检测结果干预24h后,LPS组与对照组细胞培养上清TNF-α含量相比较无显著差异(P>0.05)。较之LPS组,中药组细胞培养上清TNF-α含量明显降低(P<0.05)。LPS组总蛋白NF-κ B含量明显高于对照组(P<0.05),较之LPS组,两个含药血清组细胞总蛋白NF-κB含量均明显降低(P<0.05),来氟米特组NF-κ B含量明显低于对照组(P<0.05),中西药组间差异不明显(P>0.05)。结论1.经过12周治疗,断藤益母汤、来氟米特均均能有效减轻RA患者临床症状、降低炎症活动实验室指标和DAS28评分,在ACR20、 ACR50达标例数方面疗效相当。但断藤益母汤起效时间明显先于来氟米特,且能一定程度上降低血小板,可能与该方中益母草具有的抗血小板作用有关,但尚不能确定3例患者出现月经异常与服用该方相关。治疗前后两种药物在肝肾功、血常规方面无明显差异,无严重不良反应事件。2.体外实验证明断藤益母汤、来氟米特含药血清能等效抑制RA-FLS的异常增殖,下调LPS刺激后RA-FLS中TLR4mRNA表达量和细胞总蛋白中的NF-κ B含量,断藤益母汤含药血清还能一定程度上降低LPS刺激引起RA-FLS分泌TNF-α,未见LPS刺激、含药血清对MyD88mRNA有调节作用,以上研究提示两种药物下调天然免疫蛋白TLR4和一定程度上影响下游信号元件NF-κ B合成是可能是它们抗RA作用机制之一,但尚不能确定这种机制是否同MyD88信号依赖性通路活化相关,具体途径尚待进一步研究。更多还原

【Abstract】 Objective:This subject include three parts. The first part is Literature Review, mainly introduced three aspects:illustrated the relation between pathogenesis of rheumatoid arthritis(RA) and Rheumatoid fibroblast-like synoiocytes (RA-FLS)activation, research summary about oral TCM therapy for RA, previous correlation study about Duanteng Yimu Decoction(DYD). The second part is a12-weeks trial to assess the efficacy and safety of DYD and Leflunomide for RA patients in active period. The third part is experiments to observe effects of DVD medicinal serum on RA-FLS and TLR4/NF-κ B signal transduction pathway, all above work is to provide to provide experimental and clinical basis for Using DYD to treat RA.Methods1. Clinical part:Accord RA classification criteria from American College of Rheumatology (ACR) Promulgated in1987for diagnosis,70RA cases were divided into test and control group according to the time sequence of seeing doctor.,35cases of test group, to DYD, a does one day,150ml Bid,35cases of control group,to LEF,20mg Qd.Observed clinical indexes of4time points (0w,2w,6w,12w) such as tender joint count,swollen joint count, morning stiffness duration,patient global assessment, overall pain VAS from patients and researcher, HAQ. Lab indexes of4time points (0w,2w,6w,12w) include RF, CRP, ESR. And serum TNF-α of2time points (0w,12w)is also measured and recorded. DAS28change, numbers of cases reached ACR20, ACR50were caculated according to above informations, index of security included routine blood test, renal (BUN,Cr), Liver function (ALT,AST).2. Experimental parts:Synovial specimens obtained from the joint of patients were minced into small pieces of RA-FLS.The morphological features of RA-FLS subcultured for3-5generations were observed under inverted microscope.Our study was a two-stage procedure:evaluation of DYD effects on the the prolifration of RA-FLS at first, and then analysis of its effects on TLR4mRNA, MyD88mRNA,. NF-κ B of cells total protein, TNF-a from culture supernatants. In evaluation of DYD effects on RA-FLS, RA-FLS cells were divided into three groups:the control, LEFgroup(10%serum containing LEF) and DYD group(10%serum containing DYD) and then evaluated by MTT at three time points (24,48,72h). In evaluation of DYD effects on TLR4mRNA, MyD88mRNA. RA-FLS cells were divided into four groups:the control, model control(LPS20μg/ml), LEFgroup(LPS20μg/ml+10%serum containing LEF) and DYD groups(LPS20u g/ml+10%serum containingDYD), after24h, TLR4mRNA and MyD88mRNA of cells RNA from each groups were evaluated by RT-PCR, NF-κ B of cells total protein from each groups were evaluated by Elisa, TNF-α from culture supernatants of different groups were tested by Elisa.Result1. Clinical part:(1)At last,31cases in control and33cases in test finished whole trail, cases shedded because of bad compliance.Accord to standard of DAS28, the total effective rate of test group is51.42%, this rate in the control is57.1%, no significant difference in total effective rate was found between the two groups (P>0.05). After treatments, DAS28, number of cases reach ACR20, ACR50in the two groups were improved obviously than12weeks before.(P<0.01).In test group, the rate reach ACR20is57.14%, the rate reach ACR50is20.00%, In the control group, the rate reach ACR20is62.86%, the rate reach ACR50is22.86%。 There is not significance between the rate reach ACR20, ACR50from the two groups (P>0.05).After12weeks, symptoms of patients from both groups were improved in tender joint number, swollen joint number, morning stiffness duration, patient global assessment, overall pain VAS from patients and researcher, HAQ.(P<0.01). At the end of2rd week, test group has obviously improved in the aforementioned aspects except morni ng stiffness (P<0.05), and compare to the control, the test group improved more significant in swollen and tender joint number, patient global assessment, overall pain VAS from patients and researcher, HAQ (P<0.05), until he end of6th week, in the aforementioned6aspects had been improved obviously in the control.(P<0.05),In the end, compare to the test group, overall pain VAS from patients and researcher, HAQ in the control group had been improved more obviously (P<0.05), no significant difference was found in any other Symptoms observation index.(P>0.05)(2)Lab indexes:after treating for6wand12w, ESR,CRP and RF reduced significantly than baseline data in both groups (P<0.01), in the test group, CRP reduced significantly than baseline at the end of2rd week,(P <0.05) in the control, all the three indexes reduced obviously after6week (P<0.05).At the end of6and12week, TNF-α reduced obviously compare to baseline in both groups (P<0.01), but no difference between the two groups at the same time point (P>0.05)(4) safety indexes:after12weeks, PLT in test group reduced compared to the baseline (P<0.05), no obvious difference was found in other indexes at any time point in the two groups (P>0.05)(5) adverse reaction, lpatient got skin rash in the test group, but soon passed,3women had got few menstrual than before, but all of them adhere to the end. In the control,10patients lost their hair, there were2cases got AST increased to double than before, but released with treatments.2. Experimental parts:At the time point of48h,72h, compare to the control group, LEF and DYD serum have obviously inhibitive effects on the prolifration of RA-FLS from patients (P<0.05) Inhibition rate in LEF is higher than DYD groups after72h.(P <0.05), Similar efficacy is found in reduce of the expression of TLR4mRNA after intervene of LPS (P<0.05), but the effect has not a significant difference between the LEF and DYD group (P>0.05), expression of MyD88mRNA has not a significant difference between all the groups (P>0.05). Test result of TNF-α from control and LPS groups were not different particularly (P>0.05).while TNF-α in DYD group is less than LPS group (P<0.05). NF-κ B in LPS group is more than the control (P<0.05), compare to the control group, NF-κ B reduced more significantly in the two serum groups (P<0.05), LEF group is less than control group (P<0.05), no significant difference were found between LEF and DYD groups (P>0.05).Conclusion1. after12weeks, DYD and LEF effectively improved clinical RA patients’ symptoms, joint function and quality of life, reduced ESR, CRP, and RF, TNF-α in serum, but action of DYD occurs more faster than LEF. DYD reduced PLT partly may be explained as the leonurus in DYD has a effect of antiplatelet.Though3female patients in test group had got irregular menstruation, it remained to be seen in further clinical trail because climacterium may also result the same symptoms. While increasing levels of transaminases in LEF group were in agreement with references,No Serious adverse reaction happened in both groups meant the two drugs were safe withinl2weeks of treatment.2. Serum contained DYD or LEF can inhibit RA-FLS proliferation in regular doses dose, and the does can reduce the expression of TLR4mRNA, NF-κB of cells total protein, but has no effects on expression of MyD88mRNA.The result suggested the therapeutic mechanism of DYD for RA may included two aspests, first DYD could restrain proliferation of RA-FLS, second DYD may downregulated the expression of components from TLR4/NF-κ B pathway, but it is not clear wether MyD88dependent pathway is activated be no association with MyD88.更多还原