祛风清热、活血通络法在类风湿关节炎中的应用
|
摘要
目的：确立祛风清热、活血通络法。培养类风湿关节炎及骨关节炎关节滑液滑膜细胞,评价金边祛风饮对RA滑膜细胞的治疗作用和对炎性细胞因子及金属蛋白酶水平的影响,探讨可能的作用机制,为金边祛风饮在临床上的应用提供实验依据。
方法：(1)通过对文献资料的整理,总结及归纳祛风清热、活血通络法运用于类风湿关节炎的理论基础。(2)选择类风湿关节炎(RA)6例及骨关节炎(OA)6例,分为治疗组(RA)与对照组(OA)。患者在无菌条件下行膝关节穿刺抽取关节液,计数细胞数量,加关节液的量按每25cm2培养瓶接种1×107个细胞计算,并加无血清DMEM培养液(含100U/ml青霉素和100mg/L链霉素5-6ml),混匀后置37℃、5%二氧化碳细胞培养箱培养,于倒置显微镜下观察细胞生长情况,用ELISA.PT-PCR法检测祛风清热、活血通络法对类风湿关节炎患者滑膜细胞炎症因子IL-1β,TNF-α和MMP-3水平的表达,MTT法测定滑膜细胞增殖率。
结果：(1)滑膜细胞的形态学改变：经反复贴壁及传代后,滑膜细胞逐渐纯化,多以成纤维样滑膜细胞为主。给予金边祛风饮处理72h后,光镜下可见中浓度治疗组及高浓度治疗组RA滑膜细胞细胞密度均一定程度降低,细胞体积明显缩小,在低浓度治疗组,细胞体积变化不明显；(2)ELISA法检测培养上清液中各炎性因子表达：不同浓度金边祛风饮刺激滑膜细胞72h后,培养上清液中的TNF-α.IL-1β、MMP-3均降低,与OA对照组比较有显著差异(P<0.05),与空白对照组相比具有极显著性差异(P<0.01)。组间比较,高剂量组在降低TNF-α、IL-1β和MMP-3水平中有显著性差异(P<0.05)；(3)MTT法检测RA及0A滑膜细胞增殖：不同浓度的金边祛风饮刺激RA、OA滑膜细胞72h后,各浓度治疗组的RA滑膜细胞与空白对照组相比,增殖均受到明显抑制(P<0.05),以高剂量组的抑制率最为明显(P<0.01),RA各治疗组与OA各治疗组比,细胞增殖抑制显著(P<0.05),而OA滑膜细胞在药物各浓度刺激下,细胞增殖没有显著改变(P>0.05)；(4)RT-PCR检测RA滑膜细胞TNF-α、IL-1β及MMP-3表达：结果显示随着金边祛风饮作用浓度的增加,滑膜细胞TNF-α、IL-1β及MMP-3的mRNA表达明显减少,表明金边祛风饮能抑制炎性细胞因子及滑膜细胞基质金属蛋白酶的产生。
结论：以祛风清热、活血通络立法指导而组成的金边祛风饮能明显抑制RA滑膜细胞增殖,并呈剂量依赖性减少炎症细胞炎性因子IL-1β、TNF-α、MMP-3分泌,提示本方具有减轻类风湿关节滑膜增殖及炎症反应的理论依据。
Objective: To establish a formula Jin Bian Qu Feng Yin (JBY) in treatment ofrheumatoid arthritis based on the theory of traditional Chinense medicine “Qu FengQing Re”-expelling wind and clearing away heat and “Huo Xue Tong Luo”-promoting blood circulation to remove meridian obstruction. To observe the influenceof JBY liquid on production of proinflammatory cytokines and metalloproteinases incultured synovial fibroblasts from patients with rheumatoid arthritis, evaluatingpossible mechanisms of JBY in treatment of rheumatoid arthritis.
Methods: The synovial fluid is collected from6patients with rheumatoid arthritis(RA), as well as from6patients with knee osteoarthritis (OA) as control. The cells insynovial fluid are cultured in25cm2bottles and stimulated with low, middle, highdose of JBY, and the level of proinflammatory cytokines IL-1β, TNF-α andmetalloproteinase MMP-3are detected by ELISA and RT-PCR. The proliferation ofthe cells is evaluated by MTT.
Results:(1) Synovial cell morphological change: after repeated attached wall andpassaged, synovial cells gradually purified as fibroblast-like cells. After stimulated byJBY72hours, the cells treated with middle or high dose of JBY became small.However, the cells treated with low dose JBY slightly changed as multi-form. Theproliferation of synovial fibroblasts was significantly interfered by JBY with the dosedependent manner；(2) Measured by ELISA the culture supernatant of InflammatoryCytokines: Stimulated72h with different concentration of JBY, culture supernatantsof TNF-ɑ, IL-1β MMP-3were lower, with the OA control group compared significantdifferences (P <0.05), has a very significant difference (P <0.01) compared with thecontrol group,between the two groups, the high dose group in reducing TNF-α, IL-1βand MMP-3level, a significant difference (P <0.05)；(3) MTT assay of RA and OAsynovial cell proliferation:Stimulated72h with different concentration of JBY, thelevel of TNF-α, IL-1β MMP-3in supernatant of synovial cells reduced significantlywith the dose dependent manner, lower in middle-and high-dose JBY-stimulated cellsthan in low-dose stimulated cells.(P <0.05)；(4) RT-PCR show RA synovial cells ofTNF-α, IL-1β and MMP-3expression:In the cells the mRNA expression level ofTNF-α, IL-1β and MMP-3also reduced after co-cultured by JBY. These results weresuggested that JBY could inhibit production of proinflammatory cytokines and MMPsin synovial cells.
Conclusion: Based on the theory of traditional Chinese medicine “expelling wind and removing heat and promoting blood circulation to remove meridian obstruction”, theformula JBY significantly suppress RA synovial cell proliferation and reduce IL-1β,TNF-α and MMPs-3production, suggesting JBY may play important role in treatmentof rheumatoid arthritis. The mechanism of the traditional Chinense medical formulashould be further studied.
方法：(1)通过对文献资料的整理,总结及归纳祛风清热、活血通络法运用于类风湿关节炎的理论基础。(2)选择类风湿关节炎(RA)6例及骨关节炎(OA)6例,分为治疗组(RA)与对照组(OA)。患者在无菌条件下行膝关节穿刺抽取关节液,计数细胞数量,加关节液的量按每25cm2培养瓶接种1×107个细胞计算,并加无血清DMEM培养液(含100U/ml青霉素和100mg/L链霉素5-6ml),混匀后置37℃、5%二氧化碳细胞培养箱培养,于倒置显微镜下观察细胞生长情况,用ELISA.PT-PCR法检测祛风清热、活血通络法对类风湿关节炎患者滑膜细胞炎症因子IL-1β,TNF-α和MMP-3水平的表达,MTT法测定滑膜细胞增殖率。
结果：(1)滑膜细胞的形态学改变：经反复贴壁及传代后,滑膜细胞逐渐纯化,多以成纤维样滑膜细胞为主。给予金边祛风饮处理72h后,光镜下可见中浓度治疗组及高浓度治疗组RA滑膜细胞细胞密度均一定程度降低,细胞体积明显缩小,在低浓度治疗组,细胞体积变化不明显；(2)ELISA法检测培养上清液中各炎性因子表达：不同浓度金边祛风饮刺激滑膜细胞72h后,培养上清液中的TNF-α.IL-1β、MMP-3均降低,与OA对照组比较有显著差异(P<0.05),与空白对照组相比具有极显著性差异(P<0.01)。组间比较,高剂量组在降低TNF-α、IL-1β和MMP-3水平中有显著性差异(P<0.05)；(3)MTT法检测RA及0A滑膜细胞增殖：不同浓度的金边祛风饮刺激RA、OA滑膜细胞72h后,各浓度治疗组的RA滑膜细胞与空白对照组相比,增殖均受到明显抑制(P<0.05),以高剂量组的抑制率最为明显(P<0.01),RA各治疗组与OA各治疗组比,细胞增殖抑制显著(P<0.05),而OA滑膜细胞在药物各浓度刺激下,细胞增殖没有显著改变(P>0.05)；(4)RT-PCR检测RA滑膜细胞TNF-α、IL-1β及MMP-3表达：结果显示随着金边祛风饮作用浓度的增加,滑膜细胞TNF-α、IL-1β及MMP-3的mRNA表达明显减少,表明金边祛风饮能抑制炎性细胞因子及滑膜细胞基质金属蛋白酶的产生。
结论：以祛风清热、活血通络立法指导而组成的金边祛风饮能明显抑制RA滑膜细胞增殖,并呈剂量依赖性减少炎症细胞炎性因子IL-1β、TNF-α、MMP-3分泌,提示本方具有减轻类风湿关节滑膜增殖及炎症反应的理论依据。
Objective: To establish a formula Jin Bian Qu Feng Yin (JBY) in treatment ofrheumatoid arthritis based on the theory of traditional Chinense medicine “Qu FengQing Re”-expelling wind and clearing away heat and “Huo Xue Tong Luo”-promoting blood circulation to remove meridian obstruction. To observe the influenceof JBY liquid on production of proinflammatory cytokines and metalloproteinases incultured synovial fibroblasts from patients with rheumatoid arthritis, evaluatingpossible mechanisms of JBY in treatment of rheumatoid arthritis.
Methods: The synovial fluid is collected from6patients with rheumatoid arthritis(RA), as well as from6patients with knee osteoarthritis (OA) as control. The cells insynovial fluid are cultured in25cm2bottles and stimulated with low, middle, highdose of JBY, and the level of proinflammatory cytokines IL-1β, TNF-α andmetalloproteinase MMP-3are detected by ELISA and RT-PCR. The proliferation ofthe cells is evaluated by MTT.
Results:(1) Synovial cell morphological change: after repeated attached wall andpassaged, synovial cells gradually purified as fibroblast-like cells. After stimulated byJBY72hours, the cells treated with middle or high dose of JBY became small.However, the cells treated with low dose JBY slightly changed as multi-form. Theproliferation of synovial fibroblasts was significantly interfered by JBY with the dosedependent manner；(2) Measured by ELISA the culture supernatant of InflammatoryCytokines: Stimulated72h with different concentration of JBY, culture supernatantsof TNF-ɑ, IL-1β MMP-3were lower, with the OA control group compared significantdifferences (P <0.05), has a very significant difference (P <0.01) compared with thecontrol group,between the two groups, the high dose group in reducing TNF-α, IL-1βand MMP-3level, a significant difference (P <0.05)；(3) MTT assay of RA and OAsynovial cell proliferation:Stimulated72h with different concentration of JBY, thelevel of TNF-α, IL-1β MMP-3in supernatant of synovial cells reduced significantlywith the dose dependent manner, lower in middle-and high-dose JBY-stimulated cellsthan in low-dose stimulated cells.(P <0.05)；(4) RT-PCR show RA synovial cells ofTNF-α, IL-1β and MMP-3expression:In the cells the mRNA expression level ofTNF-α, IL-1β and MMP-3also reduced after co-cultured by JBY. These results weresuggested that JBY could inhibit production of proinflammatory cytokines and MMPsin synovial cells.
Conclusion: Based on the theory of traditional Chinese medicine “expelling wind and removing heat and promoting blood circulation to remove meridian obstruction”, theformula JBY significantly suppress RA synovial cell proliferation and reduce IL-1β,TNF-α and MMPs-3production, suggesting JBY may play important role in treatmentof rheumatoid arthritis. The mechanism of the traditional Chinense medical formulashould be further studied.
引文
1Rannou F，Francois MCorvol MT，et a1．Cartilage breakdown in rheumatoid arthritis[J]．Joint BoneSine，2006，73(1)：29—36。
2李凤菊.抗环瓜氨酸肽抗体等4种抗体联合检测在早期类风湿性关节炎诊断中的意义[J]．《临床荟萃》，2005；20(14)：821—822．
3姚航平，李敏伟，张立煌，等.滑膜成纤维细胞环氧化酶-2和诱导型一氧化氮合酶在类风湿性关节炎中的表达[J].中华检验医学杂志，2002，25(6):1-34Pugner KM，Scott DI，Hoimes JW，et al. The costs of rheumatoid arthritis：an intermational long-term view．Semin Arthritis Rheum2000；39：305-20。
5蒋明，朱立平，林孝义.风湿病学（上册）[M].北京：科学出版社，1995-818
6杨丽萍，张江华,杨剑,等.痹证的病因病机及证型研究现状[J].辽宁中医药大学学报，2008，10（8）:68-70
7刘云.痹证探微[A].中华中医药学会首届学术流派研讨会论文集[C].2009年
8娄高峰．中国名老中医药专家学术经验集·立虚邪癣说的治痹名家娄多峰[M]．贵州科技出版社，1995，(2)：398．
9周学平，周仲瑛，金妙文，等．益肾蠲痹、消痰化瘀法治疗中、晚期类风湿性关节炎的临床研究[J]．中医杂志，1998；39(10)：606-609．
10黄颖．中医对类风湿性关节炎病因病机的认识[J]．辽宁中医药大学学报，2007(7)：36．
11楼宇舫．从肾论治话痹证[J]．浙江中医药大学学报，2007(3)：272—273．
12陈湘君．扶正法为主辨治类风湿性关节炎[J]．上海中医药大学学报，2007(5)：l一2．
13张栓平．从瘀论治类风湿性关节炎的探讨[J]．陕西中医学院学报，2005(7)：3．
14国家药典委员会．中国药典，I部[S]．北京：化学工业出版社，2005：93。
15朱海涛,陈吉炎,安志斌,等.江边一碗水的生药鉴别[J].中药材，2005，28（11）：894
16李德清.复方雪里见提取物对小鼠的镇痛作用及对肝功能影响的初步研究[J].湖北民族学院学报·医学版.2003，20(3)：24-26
17张国安，刘哨兵，韩定献.复方雪里见胶囊治疗风湿类疾病的疗效观察[J].中国医院药学杂志，2006,26（8）：847-848.
18全国中草药汇编编写组.全国中草药汇编[M].北京：人民卫生出版社，1990：903.
19郝小燕，曹晓红，梁妍，等.高效液相色谱法测定飞龙掌血中异茴芹香豆素含量[J].中国中药杂志，2004，29（8）：768.
20方志先,赵晖,赵敬华.土家族医药志[M].2005.
21江苏新医学院编.中药大辞典[M].上海:上海科学技术出版社,1992:1
22韩定献,倪芳,周志彬,等.铁箍散有效成分研究及其抗病毒作用[J].中药材,2005,28(12):253.
23王宝庆、李学彬、田志发等.穿山龙现代研究概况[J].中国林福特产，2010,108（5）:96-97
24王本祥．现代中药药理学[M]．天津：天津科学技术出版社，l997．472-473
25高巍，赵铁华．穿山龙总皂苷对小鼠免疫功能的影响[J]．承德中医学院学报，2001，18(1)：9-10。
26熊传榘,吴桂华,黎芸,曾定伦,等.苗药“血藤”五味子科品种考辨[J].中国民族医药杂志：1006－6810（2004）05－0140－06
27邓虹珠,侯连兵,孙士勇等．红毛五加镇痛解热作用及毒性的实验研究[J]．中国中药杂志，1994，19(3)：171-174.
28朱海涛，王彦祥，陈黎,等.人血七药材的生药学鉴别[J].华西药学杂志，2010,25（2）：138-143
29中华人民共和国药典(一部)[M].2005;93
30钱丽娜,陈平,陈新等.竹节参地上部分中总皂苷成分抗炎镇痛活性[J].中国医院药学杂志，2008,28（20）:1731.
31常新金，丁丽霞．中药活性成分分析手册(上)[M]．北京：学苑f出版社，2002：886．
32国家药典委员会．中华人民共和国药典[S]．北京：化学工业出版社，2005：79，125．
33周强，栗占国．白芍总苷的药理作用及其在自身免疫性疾病中的应用[J]．中国新药与临床杂志，2003，22(11)：687—691
34戴自英主编．实用内科学(上册)[M].北京：人民卫生出版社，1994，(9)：780—783．
35潘琳.类风湿关节炎病因及其发病机制[J].2002年第42卷第18期山东医药。
36董怡.类风湿关节炎的发病机制及生物制品的应用[J].中华内科杂志1993年第31卷第7期。
37古洁若.类风湿关节炎研究进展[J].实用医学杂志，2005,21（13）:136738Gary S．Fires lein．Mlchele Yec．eta1．Apoplosis in rheumatoid arthll is synovvium．J Clin Invest．199596：1631。
39李鹏，方剑乔.雷公藤制剂治疗类风湿性关节炎的临床概况[J].内蒙古中医药。
40Barrera P, Boerbooms AMT, Janssen E, et al. Circulating soluble tumornecrosisfactor recetors, interleukin-2receptors, tumor necrosis factor alpha, andinterleukin-6levels in rheumatoid arthritis[J]. Arthritis Rheum,1993,36:1070
41Danis VA, March LM, Nelson DS, et al. Interleukin-1secretion by peripheralblood monocytes and synovial macrophages from patients with rheumato id arthritis[J]. JRheum,1987,14:33
42Yocum DE．Esparzal，Dubry S，et a1．Characteristics of tumornecrosis factorproduction in rheumatoid arthritis[J].Cell Immunol.1989,199：70
43张进玉．类风湿性关节炎[M]．北京：人民卫生出版社，1998
44Smeets TJ，Barg EC,Kraan MC, et al.Analysis of the cell infiltrate andexpression of proinflammatory cytokines and matrix metalloproteinases inarthroscopic synovial biopsies: comparison with synovial samples from patients withend stage, destructive rheumatoid arthritis[J].Ann Rheum Dis.2003Jul;62(7):635-638.
45Holt I，Cooper RG，Denton J,et al. Lytokin inter-relationships and theirassociation with disease activity in arthritis[J].Br J Rbcumatol,1992.31：725—733．
46Catania JM，Chen G，Parrish AR．Role of matrix metalloproteinases inrenal pathophysiologies[J]．Am J Pbysiol Renal Physiol，2007，292(3)：905—911．
47Verstappen J,Von den Hoff JW．Tissue inhibitors of metalloprotein-asses(TIMPs)：their biological functions and involvement in oral disease[J]．J DentRes，2006，85(12)：1074—1084．
48胡伟，荣超，陈飞虎，吴繁荣，刘丽萍.基质金属蛋白酶在类风湿关节炎发病机制中的作用研究进展[J].安徽医学，2001,32(5):671-672
49Ribbens C，Andre B，Jaspar J，el a1．Matrix metalloproteinase-3sertumlevels are correlated with disease activity and predict clinical response in rheumatoidarthritis[J]．J Rheumatol，2000，27(4)：888—893．
50Calguneri M,Pay S,Caliskaner Z,et al. Combination therapy versus monotherapyfor the treatment of patients with rheumatoid arthritis. Clin Exp REUMATOL1999,17:699-704。
51张宁，类风湿关节炎的治疗进展[J].用药物与临床2006年第9卷第3期
52叶任高,陆再英.《内科学》第6版[M].第九篇，第二章
53徐秋萍,等.药理学1998年04月第1版[M].第五篇，第二十九章
54孙琳,刘湘源,赵金霞,等.生物制剂治疗类风湿关节炎至严重感染的状况及预防[J].临床药物治疗杂志，2010，8（1）
1Yamannishi Y,Firestein GS.Pathogenesis of rheumatoid arthritis:The role ofsynoviocytes.Rheuma Arthritis,2001;27(2):355-68
2Erlman H,pagliari LJ,Liu H,Koch Ae et al.Rheumatoid arthritis synovialmacrophages express the Fas-assciated death domain-like interleukin-1β-convertingenzyme-inhibitory protein and are refractory to Fas-mediated apoptosis.Arthritis&pheuma,2001;44(1):21-30
3Lafyatis R,Remmers EF,Roberts AB,et al.Anchorage-independent growth ofsynoviocytes from arthritic and normal joints:stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta andretinoids.J Clin Invest,1989,83:1267-1276.
4Szent-Gyorgyi,A.G,the early history of the biochemistry of muscle cont-raction.JGen Physiol,2004.123(6):p.631-41.
5kyriakis J M,Banerjee P,Niklokaki E et al.The stress-acti-vated protein kinasesubfamily of c-jun kinases. Nature,1994;369(6476):156-60
6Menghshol JA，VineentiMP，Coon Cl，et al. Interleukin-1induetion ofcollagenase-3(matrix metalloproteinase13)gene expression in chondroeytes requiresP38,c-Jun N-terminal kinase，and nuelear factor kappaB: differential regulation ofcollagenase1and collagenase3[J].Arthritis Rheum2000，43(4):801一811.
7孙铁铮，吕厚山，药立波et al.酪氨酸激酶在TNF-α诱导类风湿关节炎成纤维样滑膜细胞MAPKs活化中的作用.中华微生物学和免疫学杂志，2001；21（1）：70-4
8Guy GR, Philp R, Tan YH. Activation of protein kinases and the inactivation ofprotin phosphatase2A in tumour necrosis factor and interleukin-1signal-transductionpathway. Eur J Biochem,1995:229(2):503-11
9Auron PE. The interleukin-1receptor: ligand interaction and signal transduction.Cyt-o Grow Fact Rev,1998,314:221-2371
10Fumio TI, Kenji, Oki, et al. Effect of mitogen-activated protein kinase inhibitorsor phosphodiesterase inhibitors on IL-1induced cytokines production in synoviumderived cells[J]. Immunol Lett,1999;68:275–9
11Lu H, Sun T, Yao L et al. Role of protein tyrosine kinase in IL-1beta inducedactivation of mitogen-activated protein kinase in fibroblast-like synoviocytes ofrheumatoid arthritis. Chin Med J Engl，2000;113(10):872一6
12Dinarello CA.The interleukin-1familly:10years of discovery.Faseb J,1994,8:1313-1325.
13Guesdon F,Freshney N,Waller R,et al.Interleukin-1and tumor necrosis factorsimulate two novel protein kinases that phosphorylate the heat shock protein hsp27and β-casein. J Biol Chem,1993,268:4236-4243.
14Auron PE.The interleukin-1receptor:ligand interaction and signal trans du-ction.Cyto Grow Fact Rev,1998,314:221-237.
15Guy GR,Chua SP,Wong NS,et al.Interleukin-1and tumor necrosis factor activatecommon multiple protein kinases in human fibroblast.J Biol Chem,1991,266:14343-14352.
16Guy GR,Tan YH.Activation of protein kinases and the inactivation of proteinphosphatase2A in tumor necrosis factor and interleukin-1signal-transductionpathway.Eur J Biochem,1995,229:503-511.
17Bird TA,Saklatvala J.IL-1and TNF transmodulate epidermal gro-wth factorreceptors by a protein kinase C-independent machanism.J Immunol,1989,142:126-133.
18Clark JW,Santos-Moore A,Stevenson LE,et al.Effects of tyrosine kinase inhibitorson the proliferation of human breast cancer cell lines and proteins important in the rassignalling pathway.J Cancer,1996,65:186-191.
19Tetuka T,Stivastava SK,Morrison AR,et al.Tyrosine kinase inhibitors,genistein andherbimycin A,do not block interleukin-1-induced activation of NF-κB in rat mesangialcells.Biochem Biophy Com,1996,218:808-812.
2李凤菊.抗环瓜氨酸肽抗体等4种抗体联合检测在早期类风湿性关节炎诊断中的意义[J]．《临床荟萃》，2005；20(14)：821—822．
3姚航平，李敏伟，张立煌，等.滑膜成纤维细胞环氧化酶-2和诱导型一氧化氮合酶在类风湿性关节炎中的表达[J].中华检验医学杂志，2002，25(6):1-34Pugner KM，Scott DI，Hoimes JW，et al. The costs of rheumatoid arthritis：an intermational long-term view．Semin Arthritis Rheum2000；39：305-20。
5蒋明，朱立平，林孝义.风湿病学（上册）[M].北京：科学出版社，1995-818
6杨丽萍，张江华,杨剑,等.痹证的病因病机及证型研究现状[J].辽宁中医药大学学报，2008，10（8）:68-70
7刘云.痹证探微[A].中华中医药学会首届学术流派研讨会论文集[C].2009年
8娄高峰．中国名老中医药专家学术经验集·立虚邪癣说的治痹名家娄多峰[M]．贵州科技出版社，1995，(2)：398．
9周学平，周仲瑛，金妙文，等．益肾蠲痹、消痰化瘀法治疗中、晚期类风湿性关节炎的临床研究[J]．中医杂志，1998；39(10)：606-609．
10黄颖．中医对类风湿性关节炎病因病机的认识[J]．辽宁中医药大学学报，2007(7)：36．
11楼宇舫．从肾论治话痹证[J]．浙江中医药大学学报，2007(3)：272—273．
12陈湘君．扶正法为主辨治类风湿性关节炎[J]．上海中医药大学学报，2007(5)：l一2．
13张栓平．从瘀论治类风湿性关节炎的探讨[J]．陕西中医学院学报，2005(7)：3．
14国家药典委员会．中国药典，I部[S]．北京：化学工业出版社，2005：93。
15朱海涛,陈吉炎,安志斌,等.江边一碗水的生药鉴别[J].中药材，2005，28（11）：894
16李德清.复方雪里见提取物对小鼠的镇痛作用及对肝功能影响的初步研究[J].湖北民族学院学报·医学版.2003，20(3)：24-26
17张国安，刘哨兵，韩定献.复方雪里见胶囊治疗风湿类疾病的疗效观察[J].中国医院药学杂志，2006,26（8）：847-848.
18全国中草药汇编编写组.全国中草药汇编[M].北京：人民卫生出版社，1990：903.
19郝小燕，曹晓红，梁妍，等.高效液相色谱法测定飞龙掌血中异茴芹香豆素含量[J].中国中药杂志，2004，29（8）：768.
20方志先,赵晖,赵敬华.土家族医药志[M].2005.
21江苏新医学院编.中药大辞典[M].上海:上海科学技术出版社,1992:1
22韩定献,倪芳,周志彬,等.铁箍散有效成分研究及其抗病毒作用[J].中药材,2005,28(12):253.
23王宝庆、李学彬、田志发等.穿山龙现代研究概况[J].中国林福特产，2010,108（5）:96-97
24王本祥．现代中药药理学[M]．天津：天津科学技术出版社，l997．472-473
25高巍，赵铁华．穿山龙总皂苷对小鼠免疫功能的影响[J]．承德中医学院学报，2001，18(1)：9-10。
26熊传榘,吴桂华,黎芸,曾定伦,等.苗药“血藤”五味子科品种考辨[J].中国民族医药杂志：1006－6810（2004）05－0140－06
27邓虹珠,侯连兵,孙士勇等．红毛五加镇痛解热作用及毒性的实验研究[J]．中国中药杂志，1994，19(3)：171-174.
28朱海涛，王彦祥，陈黎,等.人血七药材的生药学鉴别[J].华西药学杂志，2010,25（2）：138-143
29中华人民共和国药典(一部)[M].2005;93
30钱丽娜,陈平,陈新等.竹节参地上部分中总皂苷成分抗炎镇痛活性[J].中国医院药学杂志，2008,28（20）:1731.
31常新金，丁丽霞．中药活性成分分析手册(上)[M]．北京：学苑f出版社，2002：886．
32国家药典委员会．中华人民共和国药典[S]．北京：化学工业出版社，2005：79，125．
33周强，栗占国．白芍总苷的药理作用及其在自身免疫性疾病中的应用[J]．中国新药与临床杂志，2003，22(11)：687—691
34戴自英主编．实用内科学(上册)[M].北京：人民卫生出版社，1994，(9)：780—783．
35潘琳.类风湿关节炎病因及其发病机制[J].2002年第42卷第18期山东医药。
36董怡.类风湿关节炎的发病机制及生物制品的应用[J].中华内科杂志1993年第31卷第7期。
37古洁若.类风湿关节炎研究进展[J].实用医学杂志，2005,21（13）:136738Gary S．Fires lein．Mlchele Yec．eta1．Apoplosis in rheumatoid arthll is synovvium．J Clin Invest．199596：1631。
39李鹏，方剑乔.雷公藤制剂治疗类风湿性关节炎的临床概况[J].内蒙古中医药。
40Barrera P, Boerbooms AMT, Janssen E, et al. Circulating soluble tumornecrosisfactor recetors, interleukin-2receptors, tumor necrosis factor alpha, andinterleukin-6levels in rheumatoid arthritis[J]. Arthritis Rheum,1993,36:1070
41Danis VA, March LM, Nelson DS, et al. Interleukin-1secretion by peripheralblood monocytes and synovial macrophages from patients with rheumato id arthritis[J]. JRheum,1987,14:33
42Yocum DE．Esparzal，Dubry S，et a1．Characteristics of tumornecrosis factorproduction in rheumatoid arthritis[J].Cell Immunol.1989,199：70
43张进玉．类风湿性关节炎[M]．北京：人民卫生出版社，1998
44Smeets TJ，Barg EC,Kraan MC, et al.Analysis of the cell infiltrate andexpression of proinflammatory cytokines and matrix metalloproteinases inarthroscopic synovial biopsies: comparison with synovial samples from patients withend stage, destructive rheumatoid arthritis[J].Ann Rheum Dis.2003Jul;62(7):635-638.
45Holt I，Cooper RG，Denton J,et al. Lytokin inter-relationships and theirassociation with disease activity in arthritis[J].Br J Rbcumatol,1992.31：725—733．
46Catania JM，Chen G，Parrish AR．Role of matrix metalloproteinases inrenal pathophysiologies[J]．Am J Pbysiol Renal Physiol，2007，292(3)：905—911．
47Verstappen J,Von den Hoff JW．Tissue inhibitors of metalloprotein-asses(TIMPs)：their biological functions and involvement in oral disease[J]．J DentRes，2006，85(12)：1074—1084．
48胡伟，荣超，陈飞虎，吴繁荣，刘丽萍.基质金属蛋白酶在类风湿关节炎发病机制中的作用研究进展[J].安徽医学，2001,32(5):671-672
49Ribbens C，Andre B，Jaspar J，el a1．Matrix metalloproteinase-3sertumlevels are correlated with disease activity and predict clinical response in rheumatoidarthritis[J]．J Rheumatol，2000，27(4)：888—893．
50Calguneri M,Pay S,Caliskaner Z,et al. Combination therapy versus monotherapyfor the treatment of patients with rheumatoid arthritis. Clin Exp REUMATOL1999,17:699-704。
51张宁，类风湿关节炎的治疗进展[J].用药物与临床2006年第9卷第3期
52叶任高,陆再英.《内科学》第6版[M].第九篇，第二章
53徐秋萍,等.药理学1998年04月第1版[M].第五篇，第二十九章
54孙琳,刘湘源,赵金霞,等.生物制剂治疗类风湿关节炎至严重感染的状况及预防[J].临床药物治疗杂志，2010，8（1）
1Yamannishi Y,Firestein GS.Pathogenesis of rheumatoid arthritis:The role ofsynoviocytes.Rheuma Arthritis,2001;27(2):355-68
2Erlman H,pagliari LJ,Liu H,Koch Ae et al.Rheumatoid arthritis synovialmacrophages express the Fas-assciated death domain-like interleukin-1β-convertingenzyme-inhibitory protein and are refractory to Fas-mediated apoptosis.Arthritis&pheuma,2001;44(1):21-30
3Lafyatis R,Remmers EF,Roberts AB,et al.Anchorage-independent growth ofsynoviocytes from arthritic and normal joints:stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta andretinoids.J Clin Invest,1989,83:1267-1276.
4Szent-Gyorgyi,A.G,the early history of the biochemistry of muscle cont-raction.JGen Physiol,2004.123(6):p.631-41.
5kyriakis J M,Banerjee P,Niklokaki E et al.The stress-acti-vated protein kinasesubfamily of c-jun kinases. Nature,1994;369(6476):156-60
6Menghshol JA，VineentiMP，Coon Cl，et al. Interleukin-1induetion ofcollagenase-3(matrix metalloproteinase13)gene expression in chondroeytes requiresP38,c-Jun N-terminal kinase，and nuelear factor kappaB: differential regulation ofcollagenase1and collagenase3[J].Arthritis Rheum2000，43(4):801一811.
7孙铁铮，吕厚山，药立波et al.酪氨酸激酶在TNF-α诱导类风湿关节炎成纤维样滑膜细胞MAPKs活化中的作用.中华微生物学和免疫学杂志，2001；21（1）：70-4
8Guy GR, Philp R, Tan YH. Activation of protein kinases and the inactivation ofprotin phosphatase2A in tumour necrosis factor and interleukin-1signal-transductionpathway. Eur J Biochem,1995:229(2):503-11
9Auron PE. The interleukin-1receptor: ligand interaction and signal transduction.Cyt-o Grow Fact Rev,1998,314:221-2371
10Fumio TI, Kenji, Oki, et al. Effect of mitogen-activated protein kinase inhibitorsor phosphodiesterase inhibitors on IL-1induced cytokines production in synoviumderived cells[J]. Immunol Lett,1999;68:275–9
11Lu H, Sun T, Yao L et al. Role of protein tyrosine kinase in IL-1beta inducedactivation of mitogen-activated protein kinase in fibroblast-like synoviocytes ofrheumatoid arthritis. Chin Med J Engl，2000;113(10):872一6
12Dinarello CA.The interleukin-1familly:10years of discovery.Faseb J,1994,8:1313-1325.
13Guesdon F,Freshney N,Waller R,et al.Interleukin-1and tumor necrosis factorsimulate two novel protein kinases that phosphorylate the heat shock protein hsp27and β-casein. J Biol Chem,1993,268:4236-4243.
14Auron PE.The interleukin-1receptor:ligand interaction and signal trans du-ction.Cyto Grow Fact Rev,1998,314:221-237.
15Guy GR,Chua SP,Wong NS,et al.Interleukin-1and tumor necrosis factor activatecommon multiple protein kinases in human fibroblast.J Biol Chem,1991,266:14343-14352.
16Guy GR,Tan YH.Activation of protein kinases and the inactivation of proteinphosphatase2A in tumor necrosis factor and interleukin-1signal-transductionpathway.Eur J Biochem,1995,229:503-511.
17Bird TA,Saklatvala J.IL-1and TNF transmodulate epidermal gro-wth factorreceptors by a protein kinase C-independent machanism.J Immunol,1989,142:126-133.
18Clark JW,Santos-Moore A,Stevenson LE,et al.Effects of tyrosine kinase inhibitorson the proliferation of human breast cancer cell lines and proteins important in the rassignalling pathway.J Cancer,1996,65:186-191.
19Tetuka T,Stivastava SK,Morrison AR,et al.Tyrosine kinase inhibitors,genistein andherbimycin A,do not block interleukin-1-induced activation of NF-κB in rat mesangialcells.Biochem Biophy Com,1996,218:808-812.