补肾通督胶囊对胶原诱导关节炎大鼠的骨破坏与免疫调节机制研究

【作者】 林琳；

【导师】 侯丽萍；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2014， 博士

【摘要】 第一节前言类风湿性关节炎(Rheumatoid arthritis, RA)为一种慢性、全身性的自身免疫性疾病,以对称性关节滑膜炎为主要特征。中医以旭痹为名,“尪”字与“尫”、“尩”通用,此字意为足跛不能行走,胫曲不能伸展,而关节、骨骼受到损害,伴见身体赢弱的废用性疾病。传统中医药在治疗RA方面有着疗效确切、耐受性好的独特优势,以往的临床研究提示补肾通督胶囊(Bu Shen Tong Du Capsule, BS)对RA和骨关节炎皆有显著疗效,故本课题希望通过观察补肾通督胶囊对胶原诱导性关节炎(Collagen induced arthritis, CIA)大鼠的免疫调控和骨破坏作用,探讨中医药治疗RA的作用机制,初步阐明补肾通督胶囊对CIA的调控及其延缓骨破坏的免疫学机制,为中医药治疗RA及中医药调节内环境的优势提供理论和科学依据,开辟新的治疗思路。第二节文献综述综述一类风湿性关节炎的中医文献综述主要论述中医对RA的定义、病因病机和治疗进展。综述二类风湿性关节炎的西医文献综述主要论述西医对RA的定义、发病机制和治疗进展,提出RA的特异性免疫反应和骨和软骨破坏的病理机制。综述三类风湿性关节炎动物模型的文献综述主要论述多个被用于RA病因、病理和药物研究的动物模型,说明选用胶原诱导关节炎大鼠模型的理由。综述四中医治疗类风湿性关节炎的作用机理主要论述单味和复方中药治疗RA的作用机理,提出中药的免疫调控和对骨和软骨的保护作用为药理研究的关键。第三节实验研究研究一补肾通督胶囊对CIA大鼠的疗效评价目的：观察及评价补肾通督胶囊对CIA大鼠的疗效。方法：将42只大鼠,分为六组,每组7只,正常对照(C)组、空白模型(M)组、雷公藤(TG)组、补肾通督胶囊的低剂量(BSL)组、中剂量组(BSM)组和高剂量(BSH)组。CIA大鼠于第1天和第7天注射Ⅱ型胶原蛋白(Type II Collagen, CII)/不完全弗氏佐剂(Incomplete Freund’s adjuvant, IFA)乳化剂进行造模,从13天开始灌胃18天。造模后,每隔2-3天,测量大鼠体重、后脚掌的厚度和进行关节评分。结果：BS可显著降低CIA大鼠后脚掌厚度和关节评分(p<0.05),同时BS可缓解关节炎症对体重的影响。结论：BS可显著缓解CIA大鼠关节炎症。研究二补肾通督胶囊对CIA大鼠Th1、2细胞的调控作用目的：研究BS对CIA大鼠脾脏、Thl和Th2细胞及相关RNA和细胞因子的调控作用方法：将42只大鼠,分为六组,C组、M组、TG组、BSL组、BSM组和BSH组。CIA大鼠于第1天和第7天注射CII/IFA乳化剂进行造模,从第13天开始灌胃18天。于第31天取材,进行(1)脾脏HE病理切片；(2)流式细胞术检测外周血Th1和Th2细胞;(3)RT-PCR检测IFN-γ和IL-4RNA表达；(4)ELISA检测血清IFN-γ、TNF-α、IL-4和IL-10水平。结果：(1)随BS剂量的提高,白髓和主要生发中心的增生有所缓解；(2)BS各组外周血Th1细胞的百分比随剂量的提高有下调的趋势,Th2细胞的百分比显著上调(p<0.01),而Th1/Th2的比例显著下降(p<0.01)；(3)随着BS剂量的提高,IFN-γ的RNA表达呈现下降的趋势,而IL-4的RNA表达则有上升的趋势(p<0.05)；(4)随着BS剂量的提高,IFN-γ和TNF-α呈现下降的趋势,其水平与C组比较未见明显差异(p>0.05)。BS各组的IL-4和IL-10水平均较M组上调。结论：BS可抑制CIA大鼠脾脏的白髓增生、恢复Th1和Th2细胞之间的平衡和抑制炎症细胞因子的表达,以缓解关节炎症和骨破坏。研究三补肾通督胶囊对CIA大鼠Treg和Th17细胞的调控作用目的：研究BS对CIA大鼠Treg细胞及Treg和Th17细胞相关RNA和细胞因子的调控作用方法：将42只大鼠,分为六组,C组、M组、TG组、BSL组、BSM组和BSH组。CIA大鼠于第1天和第7天注射CII/IFA乳化剂进行造模,从第13天开始灌胃18天。于第31天取材,进行(1)流式细胞术检测外周血Treg细胞；(2)RT-PCR检测Foxp-3、TGF-β、IL-6和IL-17RNA表达；(3)ELISA检测血清IL-6和IL-17水平。结果：(1)BS的CD4+CD25+Treg细胞和CD4+CD25+Foxp-3+活化Treg细胞百分比显著上调(p<0.05)；(2)BS可上调Foxp-3和TGF-β的RNA表达,同时下调IL-6和IL-17的RNA表达；(3)随着BS剂量的提高,IL-6和IL-17的血清水平呈现下调的趋势,与C组比较未见显著差异(p>0.05)。结论：BS可上调和激活Treg细胞,同时下调Th17细胞的增殖和活化,以下调促炎症细胞因子的表达,从而达到缓解关节炎症作用。研究四补肾通督胶囊对CIA大鼠Wnt信号通路的调控作用目的：研究补肾通督胶囊对CIA大鼠Wnt信号通路的调控作用方法：将42只大鼠,分为六组,C组、M组、TG组、BSL组、BSM组和BSH组。CIA大鼠于第1天和第7天注射CII/IFA乳化剂进行造模,从第13天开始灌胃18天。于第31天取材,进行ELISA检测血清DKK1水平。结果：随着BS剂量的提高,DKK1水平呈现下调的趋势,其水平较M组显著下调(p<0.01)。结论：BS可能通过调控Wnt信号通路以缓解CIA大鼠的关节炎症和骨破坏。研究五补肾通督胶囊对CIA大鼠骨和软骨破坏的调控作用目的：研究BS对CIA大鼠踝关节X-光、HE切片和骨和软骨破坏血清指标的调控作用方法：将42只大鼠,分为六组,C组、M组、TG组、BSL组、BSM组和BSH组。CIA大鼠于第1天和第7天注射CII/IFA乳化剂进行造模,从第13天开始灌胃18天。于30天拍摄(1)踝关节X-光片。于第31天取材,进行(2)踝关节HE病理切片；(3)ELISA检测血清IL-1、Anti-Coll Ⅱ, COMP、MMP-3、ICTP、Cath-K、PINP和OC水平。结果：(1)BS各组的踝关节X-光软组织肿胀和骨破坏评分随着剂量的提高有下降的趋势,与M组比较显著下降(p<0.05)；(2)BS组的HE切片的细胞浸润、滑膜增生和骨破坏程度随着剂量的提高有下降的趋势,与M组比较显著下降(p<0.05)；(3)BS可下调血清促炎症细胞因子(IL-1和TNF-α)、工型(MMP-3、ICTP和Cath-K)和Ⅱ型胶原蛋白降解(anti-Coll Ⅱ和COMP)和骨重塑指标(OC和PINP)的水平。结论：BS通过下调炎症细胞因子和骨和软骨破坏指标的效果,证明BS可缓解关节炎症、骨破坏、抑制Ⅰ型和Ⅱ型胶原蛋白降解和调控骨重塑以预防骨和软骨破坏的发生。第四节总结据临床观察和研究,RA患者多表现为肾虚,故侯丽萍主任医师根据自身经验结合清末民初老中医石广济先生的经验以阳和汤为基础加马钱子制成补肾通督胶囊,方中阳和汤和马钱子均为治疗RA的有效药物,互相配伍可达到增效减毒的作用。全方辛散滋补同伍,温而不燥,滋而不腻,通中有补,扶正与祛邪兼顾,以达到补肾通督、化瘀散结消肿的作用。此实验显示补肾通督胶囊对Ⅱ型胶原蛋白诱导的关节炎症,从免疫调控和骨和软骨代谢两方面,均见疗效,可有助缓解CIA大鼠的关节炎症和缓解骨和软骨破坏,从而阐明其临床疗效。临床研究亦显示雷公藤多甙片联合阳和汤治疗RA疗效较常规治疗显著,故临床上可以考虑运用补肾通督胶囊联合雷公藤多甙片治疗RA。更多还原

【Abstract】 Chapter1IntroductionRheumatoid Arthritis is a autoimmune diseases that results in a chronic, systemic inflammatory disorder that presents with joint inflammation and synovitis. Chinese Medicine name RA as WangBi and "Wang" is referred as weakening of hind limb and body, disability to walk and damage to joint and bone. Traditional Chinese Medicine is effective in the treatment of RA. Previous studies also showed that Bu Shen Tong Du Capsule (BS) is effective in treating RA and osteoarthritis. Therefore, our study aimed to investigate the therapeutic mechanism of Bu Shen Tong Du Capsule on RA.Chapter2Literature ReviewReview1The literature review of RA in Chinese Medicine mainly introduced the definition, etiology, pathology and treatment progress of RA in Chinese Medicine.Review2The literature review of RA in Western Medicine mainly introduced the definition, etiology and treatment progress of RA in Western Medicine and provided the pathology of specific immunity and osteoclasia in RAReview3The literature review of animal model used in RA mainly introduced several commonly used animal model involved in the study of etiology, pathology and drug mechanism of RA.Review4The literature review of Chinese Medicine mechanism in treating RA mainly introduced the action mechanism of single herbs or herbal remedy and suggested immune modulation and bone protection as the research key.Chapter3Laboratory ResearchResearch1The Therapeutic Effect of BS on CIA rat modelAim:To investigate the effect of BS on CIA rat modelMethod:42female SD rats were randomly assigned to six groups, normal control group(C), model group(M), low dose of BS group(BSL), medium dose group(BSM), high dose group (BSH) and tripterygium glycosides group (TG). Arthritis would be induced on day1and7by injection of incomplete freund’s adjuvant and bovine type Ⅱ collagen. After induction of arthritis on day1, the weight,foot thickness and arthritis scores were measured every2to3days. From Day13, the rat was administrated with BSL120mg/d/kg, BSM240mg/d/kg, BSH480mg/d/kg, TG24mg/d/kg and saline for18days. Results:BS could significantly reduced foot thickness and arthritis scores of CIA rat(p<0.05). Also, BS is effective in relieving the effect of arthritis on weight.Conclusion:BS could significantly relieve arthritis. Research2The Modulation Effect of BS on Thl and Th2cells of CIA rat modelAim:To investigate the effect of BS on spleen pathology, Thl cell and Th2cell related cytokines of CIA rat modelMethod:42female SD rats were randomly assigned to six groups, C group, M group, BSL group, BSM group, BSH group and TG group. Arthritis would be induced on day1. From Day13, the rat was administrated with BSL120mg/d/kg, BSM240mg/d/kg, BSH480mg/d/kg, TG24mg/d/kg and saline for18days. At day31, rats were sacrificed to perform(1)HE stain of spleen;(2)Flow cytometry to analyze peripheral percentage of Thl and Th2cell;(3)RT-PCR to analyze RNA quantity of IFN-γ and IL-4;(4)ELISA to detect serum IFN-y, TNF-a,IL-4and IL-10.Results:(1)BS could relieve white pulp hyperplasia of spleen.(2)BS could down regulate Th1%and up regulate Th2%(p<0.01) to restore balance of Th cell.(3)BS could down regulate IFN-Y RNA presentation and up regulate IL-4RNA presentation.(4) Serum levels of IFN-γ、TNF-α presented downward trend and IL-4and IL-10presented upward trend while the dose of BS was raised.Conclusion:BS could suppress humoral immune response, restore the balance of Th cells and suppress production and action of inflammatory cytokines to relieve joint inflammation and osteoclasia. Research3The Modulation Effect of BS on Treg and Th17cells of CIA rat modelAim:To investigate the effect of BS on Treg cell, Treg cell related cytokines and Th17cell related cytokines of CIA rat modelMethod:42female SD rats were randomly assigned to six groups, C group, M group, BSL group, BSM group, BSH group and TG group. Arthritis would be induced on day1. From Day13, the rat was administrated with BSL120mg/d/kg, BSM240mg/d/kg, BSH480mg/d/kg, TG24mg/d/kg and saline for18days. At day31, rats were sacrificed to perform (1) Flow cytometry to analyze peripheral percentage of Treg cell;(2) RT-PCR to analyze RNA quantity of Foxp-3, TGF-0, IL-6and IL-17RNA;(3) ELISA to detect serum IL-6and IL-17.Results:(1) BS could up regulate presentation of CD4+CD25+Treg cells and CD4+CD25+Foxp3+activated Treg cells in peripheral circulation;(2)BS could up regulate Foxp3, TGF-βRNA presentation and down regulate IL-6, IL-17RNA presentation;(3) Serum levels of IL-6and IL-17presented downward trend while the dose of BS was raised.Conclusion:The up regulation of Treg cell presentation would result in suppression of Th17cell which would lower the production of inflammatory cytokines to relieve joint inflammation. Research4The Modulation Effect of BS on Wnt Signaling pathway of CIA rat modelAim:To investigate the effect of BS on Wnt Signaling pathway of CIA rat model Method:42female SD rats were randomly assigned to six groups, C group, M group, BSL group, BSM group, BSH group and TG group. Arthritis would be induced on day1. From Day13, the rat was administrated with BSL120mg/d/kg, BSM240mg/d/kg, BSH480mg/d/kg, TG24mg/d/kg and saline for18days. At day31, rats were sacrificed and serum was obtained for ELISA to detect serum DKK1.Results:Serum levels of DKK1presented downward trend while the dose of BS was raised. The level is significantly lower than M group (p<0.01). Conclusion:BS could have modulation effect on Wnt signaling pathway to relieve joint inflammation and osteoclasia. Research5The Modulation Effect of BS on osteoclasia of CIA rat modelAim:To investigate the effect of BS on osteoclasia of CIA rat model Method:42female SD rats were randomly assigned to six groups, C group, M group, BSL group, BSM group, BSH group and TG group. Arthritis would be induced on day1. From Day13, the rat was administrated with BSL120mg/d/kg, BSM240mg/d/kg, BSH480mg/d/kg, TG24mg/d/kg and saline for18days. At day30, X-ray was performed. At day31, rats were sacrificed to perform (1)HE stain for ankle joint;(2) ELISA to detect serum IL-1, type II Collagen antibodies, cartilage oligomeric matrix protein, matrix metalloproteinase-3, C-telopeptide of type I collagen、Cathepsin K, propeptide of type-I procollagen and osteocalcin. Results:(1) BS could relieve radiological score of soft tissue edema and osteoclasia(p<0.05);(2) BS could relieve HE score of cellular infiltration, synovial hyperplasia and osteoclasia(p<0.05);(3)BS could down regulate serum level of pro-inflammatory cytokines(IL-1, TNF-a), type I collage degradation markers(MMP-3, ICTP, Cath-K), type Ⅱ collagen degradation markers(anti-Coll II, COMP)and bone remodeling markers(OC, PINP).Conclusion:BS could relieve joint inflammation, osteoclasia, suppress degradation of type I and type II collagen and modulate bone remodeling to prevent the occur of osteoclasia. Chapter4ConclusionBased on clinical observation and study, RA patients often present with deficiency of kidney. Therefore, Prof. Hou Liping formulated Bu Shen Tong Du capsule based on personal experience and experience of Qing famous Chinese Medicine practitioner Shi Guangji. Bu Shen Tong Du capsule is constituted of Yang He Tang and Semen Strychni which are effective in treating RA. The major compositions of the formula are Rehmannia Glutinosa, Colla Cornus Cervi, Cinnamomum Cassia Presl, Semen Sinapis, Semen Strychni and Ephedra Sinica Stapf. The formula applied pathogens dispersing herbs with enhancing and nourishing herbs to expel pathogens and enhance deficiency at the same time. The experiment showed that BS could relieve collagen-induced arthritis through modulation of immunity and bone and cartilage metabolism. The results elaborated and explained the therapeutic mechanism of BS. Since clinical study indicated that the combination of TG and Yang He Tang could effectively relieve clinical symptoms, we could also consider the combination of TG and BS in the future treatment of RA.更多还原