祛风清热、活血通络法对大鼠胶原诱导关节炎的作用机制研究
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摘要
目的:确立祛风清热、活血通络法。制备Ⅱ型胶原诱导大鼠关节炎模型(CIA),评价金边祛风饮对CIA大鼠关节炎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)理论探讨祛风清热、活血通络法。(2)采取Wistar大鼠60只,建立大鼠胶原诱导的关节炎模型,随机分为6组,对CIA大灌服不同剂量金边祛风饮,观察其对大鼠关节炎指数、病理改变的影响,同时检测治疗前后CIA大鼠血清TNF-α、IL-1β、MMPs-3水平变化;培养CIA大鼠滑膜细胞,MTT法检测金边祛风饮对滑膜细胞增殖的影响,流式细胞仪测定滑膜细胞凋亡,观察金边祛风饮对培养的CIA大鼠滑膜细胞和TNF-α、IL-1β、MMPs-3生成的影响。
结果:(1)CIA模型结果:足内注射注射Ⅱ型胶原蛋白、弗氏不完全佐剂、乙酸混合物后18 h,左后足爪开始出现红、肿、热,持续3 d后逐渐减轻。10～14 d,出现以多发性关节炎为特征的全身迟发性变态反应,50只大鼠的四肢关节中98%出现不同程度的红肿,以左后肢为主,约占98%,前肢的肿胀占10%,炎症此起彼伏,迁延达2月余,部分出现关节畸形。另外,多数大鼠的耳和尾部出现炎性小结,内脏风湿结节明显。部分大鼠出现变应性角膜翳等关节外表现。(2)金边祛风饮对CIA的治疗效果:各组口服金边祛风饮后,大鼠关节疼痛指数均有所改善,以高、中剂量组明显,病程明显缩短(P<0.01),关节外表现较少、病变较轻。给药第2周时,各组关节肿胀指数均低于模型对照组,有显著性差异( P<0.05) ,给药第3周时,各组关节肿胀指数低于对照组,以高剂量组明显,与对照组均有显著性差异(P<0.01)。(3)CIA大鼠关节病理检查:病变关节软组织高度肿胀,滑膜炎症反应表现突出,具有衬里细胞层增厚,间质水肿并有大量以淋巴细胞为主的炎细胞浸润,有血管翳形成并侵袭软骨和软骨下骨组织造成骨和软骨的破坏。经药物治疗后关节滑膜病理检查,模型对照组:关节面腔隙内软组织可见大量的梭形成纤维细胞,组织稀疏、水肿,并见大量中性粒细胞和一些淋巴细胞、单核细胞浸润,毛细血管充血明显,并有红血球渗出。金边祛风饮中剂量组关节面上皮细胞多呈梭形,上皮下可见到少量单核细胞和淋巴细胞浸润,纤维细胞轻度增生,毛细血管有增生和充血,部分可见到浆细胞浸润。正常对照组:组织和细胞排列规则,无明显炎症反应。金边祛风饮能减轻大鼠关节皮下软组织及滑膜组织的充血和水肿程度,抑制滑膜细胞的增生,缓解炎性细胞的浸润,表明金边祛风饮在缓解CIA关节炎症方面疗效确切,值得做进一步的研究。(4)金边祛风饮治疗28天后,血清、细胞上清液TNF-α、IL-1β、MMPs-3水平均降低,以高、中剂量最明显(P<0.05)。各剂量金边祛风饮均有抑制滑膜细胞生长的趋势(P<0.05),诱导滑膜细胞凋亡(P<0.05),以高剂量明显(P<0.01),细胞上清液中TNF-α、IL-1β、MMPs-3水平均亦明显降低(P<0.01)。各病变关节从大体上观察均恢复至造摸前。
结论:以祛风清热、活血通络立法指导而组成的金边祛风饮治疗大鼠CIA有效,本方能减轻关节滑膜的炎症,其治疗机制与调节炎症细胞分泌炎性因子IL-1β、TNF-α和调控MMPs-3表达及抑制滑膜细胞增殖、诱导滑膜细胞凋亡有关,为金边祛风饮治疗类风关提供了一定的理论依据。
Objective: Establishment of expelling wind and removing heat and promoting blood circulation to remove meridian obstruction method.To evaluated the influence of JBY on pathological changes and inflammatory cytokines and metalloproteinase (MMP) levels in CIA Rats, exploring the possible mechanism for JBY in therapy of arthritis.
Methods: (1)Theory study of expelling wind and removing heat and promoting blood circulation to remove meridian obstruction method. Chose 60 Wistar Rats to prepare type II collagen-induced arthritis (CIA) model, randomly divided into 6 groups, feed by different doses of JBY for 28 weeks. The arthritis scores and pathological arthritic changes were observed in both JBY-treated CIA rats and control ones. The levels of inflammatory cytokines interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and metalloproteinase MMP3 in CIA rat sera as well as in supernatant of cultured CIA rat synoviocytes treated with or without JBY were examined by ELISA. The influence of JBY on CIA synoviocyte proliferation were evaluated by MTT.
Results:(1)CIA model results; the incidence of arthritis: 90% appears naked eye, joint swelling, mainly in the left hind legs, accounting for about 9/10, forelimb swelling accounted for 3/10. Serum ELISA test; a typeⅡcollagen antibody exists.(2)CIA rat joints pathological examination: a high degree of soft tissue lesions joint swelling, synovitis outstanding inflammatory response with a lining cell layer thickening, interstitial edema, and there is an abundance of inflammatory cell infiltration consisting mainly of lymphocytes, with pannus formation and invasion of cartilage and subchondral bone tissue caused by bone and cartilage destruction. Saline group: articular surface of the soft tissue inside the compartment can be seen the formation of a large number of spindle fiber cells. Organization sparse edema. And to see a large number of neutrophils and some lymphocytes, mononuclear cell infiltration. Capillary congestion significantly. And a red blood cell leakage. Medium dose Duocheng articular surface skin cells, spindle-shaped. The skin can be seen on a number of monocytes and lymphocytes. Some fibroblast proliferation. There capillary hyperplasia and congestion. Part of the plasma cell infiltration can be seen. Control group: tissue and cell arrangement rules, no significant inflammatory response.(3) serum inflammatory cytokines and metalloproteinases changes: After JBY treatment of serum TNF-α, IL-1β, MMP-3 an average drop of water to high and middle dose of the most significant (P <0.05)(4) synovial cell culture and stimulation: Each dose of JBY, were inhibited JBY trends in the growth of synovial cells.(5)JBY on the therapeutic effect of RA in rats: injection injection of typeⅡcollagen, Vladimir style is not entirely complete adjuvant, a mixture of acetic acid after 18 h, the right hind feet appear red, swollen, hot, continuous 3 d After gradually reducing. 10-14d,appearance of the multiple systemic arthritis, characterized by delayed hypersensitivity, 10 rat limb joints in 90% (56/60) there are different degrees of swelling, inflammation are numerous, persistent up to 2 more than a month Some developing joint deformity. In addition the majority of the ears and tail appeared in rat inflammatory nodules, some rats exhibited allergic corneal nebula and other extra-articular manifestations. Oral JBY rats in each group, joint lesions were improved, high-, medium-dose group was, significantly shorter duration (P <0.01). Fewer extra-articular manifestations, disease less. 28 days after treatment, the lesions from the general observation of both joints, such as pre-immune restoration, but joints slices on microscopic observation, the visible part of the joint organization of collagen in varying degrees of proliferation and periosteal plane. Conclusion: JBY was proved to be an efficient mixture in treatment of CIA rats. And the roles of inhibiting production of inflammatory cytokines and proliferation synoviocytes may be the therapeutic mechanism on treating CIA rats.
方法:(1)理论探讨祛风清热、活血通络法。(2)采取Wistar大鼠60只,建立大鼠胶原诱导的关节炎模型,随机分为6组,对CIA大灌服不同剂量金边祛风饮,观察其对大鼠关节炎指数、病理改变的影响,同时检测治疗前后CIA大鼠血清TNF-α、IL-1β、MMPs-3水平变化;培养CIA大鼠滑膜细胞,MTT法检测金边祛风饮对滑膜细胞增殖的影响,流式细胞仪测定滑膜细胞凋亡,观察金边祛风饮对培养的CIA大鼠滑膜细胞和TNF-α、IL-1β、MMPs-3生成的影响。
结果:(1)CIA模型结果:足内注射注射Ⅱ型胶原蛋白、弗氏不完全佐剂、乙酸混合物后18 h,左后足爪开始出现红、肿、热,持续3 d后逐渐减轻。10～14 d,出现以多发性关节炎为特征的全身迟发性变态反应,50只大鼠的四肢关节中98%出现不同程度的红肿,以左后肢为主,约占98%,前肢的肿胀占10%,炎症此起彼伏,迁延达2月余,部分出现关节畸形。另外,多数大鼠的耳和尾部出现炎性小结,内脏风湿结节明显。部分大鼠出现变应性角膜翳等关节外表现。(2)金边祛风饮对CIA的治疗效果:各组口服金边祛风饮后,大鼠关节疼痛指数均有所改善,以高、中剂量组明显,病程明显缩短(P<0.01),关节外表现较少、病变较轻。给药第2周时,各组关节肿胀指数均低于模型对照组,有显著性差异( P<0.05) ,给药第3周时,各组关节肿胀指数低于对照组,以高剂量组明显,与对照组均有显著性差异(P<0.01)。(3)CIA大鼠关节病理检查:病变关节软组织高度肿胀,滑膜炎症反应表现突出,具有衬里细胞层增厚,间质水肿并有大量以淋巴细胞为主的炎细胞浸润,有血管翳形成并侵袭软骨和软骨下骨组织造成骨和软骨的破坏。经药物治疗后关节滑膜病理检查,模型对照组:关节面腔隙内软组织可见大量的梭形成纤维细胞,组织稀疏、水肿,并见大量中性粒细胞和一些淋巴细胞、单核细胞浸润,毛细血管充血明显,并有红血球渗出。金边祛风饮中剂量组关节面上皮细胞多呈梭形,上皮下可见到少量单核细胞和淋巴细胞浸润,纤维细胞轻度增生,毛细血管有增生和充血,部分可见到浆细胞浸润。正常对照组:组织和细胞排列规则,无明显炎症反应。金边祛风饮能减轻大鼠关节皮下软组织及滑膜组织的充血和水肿程度,抑制滑膜细胞的增生,缓解炎性细胞的浸润,表明金边祛风饮在缓解CIA关节炎症方面疗效确切,值得做进一步的研究。(4)金边祛风饮治疗28天后,血清、细胞上清液TNF-α、IL-1β、MMPs-3水平均降低,以高、中剂量最明显(P<0.05)。各剂量金边祛风饮均有抑制滑膜细胞生长的趋势(P<0.05),诱导滑膜细胞凋亡(P<0.05),以高剂量明显(P<0.01),细胞上清液中TNF-α、IL-1β、MMPs-3水平均亦明显降低(P<0.01)。各病变关节从大体上观察均恢复至造摸前。
结论:以祛风清热、活血通络立法指导而组成的金边祛风饮治疗大鼠CIA有效,本方能减轻关节滑膜的炎症,其治疗机制与调节炎症细胞分泌炎性因子IL-1β、TNF-α和调控MMPs-3表达及抑制滑膜细胞增殖、诱导滑膜细胞凋亡有关,为金边祛风饮治疗类风关提供了一定的理论依据。
Objective: Establishment of expelling wind and removing heat and promoting blood circulation to remove meridian obstruction method.To evaluated the influence of JBY on pathological changes and inflammatory cytokines and metalloproteinase (MMP) levels in CIA Rats, exploring the possible mechanism for JBY in therapy of arthritis.
Methods: (1)Theory study of expelling wind and removing heat and promoting blood circulation to remove meridian obstruction method. Chose 60 Wistar Rats to prepare type II collagen-induced arthritis (CIA) model, randomly divided into 6 groups, feed by different doses of JBY for 28 weeks. The arthritis scores and pathological arthritic changes were observed in both JBY-treated CIA rats and control ones. The levels of inflammatory cytokines interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and metalloproteinase MMP3 in CIA rat sera as well as in supernatant of cultured CIA rat synoviocytes treated with or without JBY were examined by ELISA. The influence of JBY on CIA synoviocyte proliferation were evaluated by MTT.
Results:(1)CIA model results; the incidence of arthritis: 90% appears naked eye, joint swelling, mainly in the left hind legs, accounting for about 9/10, forelimb swelling accounted for 3/10. Serum ELISA test; a typeⅡcollagen antibody exists.(2)CIA rat joints pathological examination: a high degree of soft tissue lesions joint swelling, synovitis outstanding inflammatory response with a lining cell layer thickening, interstitial edema, and there is an abundance of inflammatory cell infiltration consisting mainly of lymphocytes, with pannus formation and invasion of cartilage and subchondral bone tissue caused by bone and cartilage destruction. Saline group: articular surface of the soft tissue inside the compartment can be seen the formation of a large number of spindle fiber cells. Organization sparse edema. And to see a large number of neutrophils and some lymphocytes, mononuclear cell infiltration. Capillary congestion significantly. And a red blood cell leakage. Medium dose Duocheng articular surface skin cells, spindle-shaped. The skin can be seen on a number of monocytes and lymphocytes. Some fibroblast proliferation. There capillary hyperplasia and congestion. Part of the plasma cell infiltration can be seen. Control group: tissue and cell arrangement rules, no significant inflammatory response.(3) serum inflammatory cytokines and metalloproteinases changes: After JBY treatment of serum TNF-α, IL-1β, MMP-3 an average drop of water to high and middle dose of the most significant (P <0.05)(4) synovial cell culture and stimulation: Each dose of JBY, were inhibited JBY trends in the growth of synovial cells.(5)JBY on the therapeutic effect of RA in rats: injection injection of typeⅡcollagen, Vladimir style is not entirely complete adjuvant, a mixture of acetic acid after 18 h, the right hind feet appear red, swollen, hot, continuous 3 d After gradually reducing. 10-14d,appearance of the multiple systemic arthritis, characterized by delayed hypersensitivity, 10 rat limb joints in 90% (56/60) there are different degrees of swelling, inflammation are numerous, persistent up to 2 more than a month Some developing joint deformity. In addition the majority of the ears and tail appeared in rat inflammatory nodules, some rats exhibited allergic corneal nebula and other extra-articular manifestations. Oral JBY rats in each group, joint lesions were improved, high-, medium-dose group was, significantly shorter duration (P <0.01). Fewer extra-articular manifestations, disease less. 28 days after treatment, the lesions from the general observation of both joints, such as pre-immune restoration, but joints slices on microscopic observation, the visible part of the joint organization of collagen in varying degrees of proliferation and periosteal plane. Conclusion: JBY was proved to be an efficient mixture in treatment of CIA rats. And the roles of inhibiting production of inflammatory cytokines and proliferation synoviocytes may be the therapeutic mechanism on treating CIA rats.
引文
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44王斌,陈敏珠,徐叔云.白芍总甙对佐剂性关节炎滑膜细胞功能和脾细胞增殖反应的影响[J].中国药理学与毒理学杂志,1994,8(2):128-131.
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50何金华,梁清华,张花先,等.痹肿消汤对实验性关节炎大鼠血浆TNF-α的影响[J].湖南医科大学学报,2002 ,27(5) :425.
51 Abramson S B,Amin A. Blocking the effect of IL - 1 in rheumatoid arthritis protects bone and cartiage[J].Rheumatology,2002,41(9) :972.
52赵欣华.基质金属蛋白酶-3在类风湿关节炎中的研究[J].医学理论与实践,2009,22(6):121 .
53 KimYJ,SongM,Ryu JC.Inflammation nationinmethotrexate-indueed Pulmonarytoxicity occursvia the P38MAPK Pathway Toxicology[J]. 2009,256(3):183-90.
54姜彤,中药血清药理学方法初探.黑龙江医药.2007.17(6):430-433.
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49王文君,王培训.青藤碱对环氧化酶2活性的选择性抑制作用[J].广州中医药大学学报,2002,19(1):46-47.
50何金华,梁清华,张花先,等.痹肿消汤对实验性关节炎大鼠血浆TNF-α的影响[J].湖南医科大学学报,2002 ,27(5) :425.
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52赵欣华.基质金属蛋白酶-3在类风湿关节炎中的研究[J].医学理论与实践,2009,22(6):121 .
53 KimYJ,SongM,Ryu JC.Inflammation nationinmethotrexate-indueed Pulmonarytoxicity occursvia the P38MAPK Pathway Toxicology[J]. 2009,256(3):183-90.
54姜彤,中药血清药理学方法初探.黑龙江医药.2007.17(6):430-433.
1聂英坤,张凤山,孙凤琴.类风湿性关节炎的治疗现状与进展[J].临床荟萃.
2 Reuss-Borst MA. Rheumatic and hemato-/oneological disorders[J].
3吴祖群,许以平.调节性T细胞的研究进展[J].现代免疫学,2004,24(1):77-79.
4 Inazuka M, Ttahira, T Hofiuchi,et a1.Analysis ofo53 tumour suppr~ssor gene somatic mutations in rheumatoid arthritis synovium[j].Rheumatology,2000,39(3):262-266.
5 Aupperle KR,Bolye DL,Hendrix M, et a1. Regu lation of syno—viocyte proliferation,apotosis,and invasion by the r63 tumor suppressor gene[J]. Am J Pathol, 1998,152(4): 1091-1098.
6 Murrell GA,Jang D,Williams RJ Nitric oxide activates metal—loprotease enzymes in articular cartilage[J].Biochem Biophhys Res Commun.1995.206(1):15-21.
7 Okada Y,Nagase H,Harris ED Jr.Matrix matelloproteinases l,2.and 3 from rhe umatoid synoveal cells are suffeient to destroy joints~J3.Rheumatol,1987,14(5):41.
8 YeS,Patodi N,Walker-Bone K.et a1.Variation in the ma-trix metalloproteinase -3-7,-12and- 13 genes is associated with functional status in rheumatoid arthritis.Int [J] Immuno–genet,2007,34 (2 ):81 - 85.
9 Matsuno H,Yudoh K, Katayama R,et al. The ro le of TN f2al2 pha in the pathogenesis of inflammation and jo int destruction in rheumato id arth ritis (RA ):a study using a human RA? SC ID mouse ch imera[J ]. Rheumato logy (Oxfo rd),2002,41 (3) : 329- 337.
10成胜权,张国成,李琦,等.类风湿关节炎患者血清和关节液细胞因子水平的变化及临床意义[J ].细胞与分子免疫学杂志,2001,17 (4) : 353-355.
11 Carter SD,Bames A,Gilmone WH.[J] Vet 1999.69(2-4):201-214.
12 Nearth MF.Hildner K,Becker C,et al [J].Chin Exp Inoumol.1999.115(1):42-55.
13 Palmer G,M ezin F,Juge2A ubry CE,et al. Interferon beta stim2 ulates interleuk in 1 recep to r antagonist p roduction in humanarticular chondrocytes and synovial fibroblasts [ J ]. A nn Rheum D is,2004,63 (1) : 43-49.