Effects of Triptolide on the Expression and Activity of NF-κB in Synovium of Collagen-induced Arthritis Rats

Summary： The expression and activity of NF-kB in the synovium of collagen-induced arthritis （CIA） rats was detected in order to investigate the possible therapeutic effects of triptolide on rheumatoid arthritis （RA）. The experimental Wistar rat model of CIA was set up by intradermal injection of emulsion of bovine collagen 11 and the successful rate of setting-up models was evaluated by arthritis index （AI）. Rats were grouped randomly into three groups： normal, model and treatment group. The expression of TNF-α and IL-6 in synovial fluid was detected by ELISA, and the expression and activity of NF kB in synovium by immunohistochemistry method and by electrophoretic mobility shift assay （EMSA） respectively. As compared with normal group, the expression of TNF a and IL-6 in synovia （P〈0. 05）, and the expression and activity of NF-kB （P〈0.05） in synovium were increased in model group. There was statistical difference in above-mentioned indexes between model group and treatment group. Triptolide may play a protective role in IRA via downregulating the expression and activity of NF-kB in synovium.

Prediction of Response of Collagen-induced Arthritis Rats to Methotrexate： An ~1H-NMR-based Urine Metabolomic Analysis

Over one half the patients with rheumatoid arthritis （RA） are being treated with methotrexate （MTX）. Although well proven, the efficacy of MTX varies in individual patients. This study examined the metabolic biomarkers that can be used to predict the therapeutic effect of MTX by using metabolomic analysis. Rats were immunized with collagen to rapidly cause collagen-induced arthritis （CIA） and then treated with 0.1 mg/kg MTX for 4 weeks. The clinical signs and the histopathological features of CIA were observed to evaluate the therapeutic effects. Urine samples of CIA rats were collected, and analyzed by using 600 M 1H-nuclear magnetic resonance （1H-NMR） for spectral binning after the therapy. The urine spectra were divided into spectral bins, and 20 endogenous metabolites were assigned by Chenomx Suite. Multivariate analyses were performed to identify the spectral pattern of endogenous metabolites related to MTX therapy. The results showed that the clustering of the spectra of the urine samples from the responsive rats （n=20） was different from that from the non-responsive rats （n=11）. Multivariate analysis showed difference in metabolic profiles between the responsive and non-responsive rats by using partial least squares-discrimination analysis （PLS-DA） （R2=0.812, Q2=0.604）. In targeted profiling, 13 endogenous metabolites （uric acid, taurine, histidine, methionine, glycine, etc.） were selected as putative biomarkers for predicting therapeutic response to MTX. It was suggested that 1H-NMR-based metabolomic analysis can be used to predict the therapeutic effect of MTX, and several metabolites were found to be related to the therapeutic effects of MTX.

A novel anti-inflammatory and immunomodulatory drug CP-25 alleviated collagen induced arthritis by down-regulating BAFF-NF-κκB signaling pathway

OBJECTIVE To investigated the regulatory effect of paeoniflorin-6′-O-benzene sulfonate(CP-25) on B cell activating factor(BAFF)/BAFF receptor-nuclear factor of kappa B(NF-κB) signaling in B cell of collagen induced-arthritis(CIA) mice.METHODS Mice CIA was induced by injection of typeⅡcollagen(CⅡ).The arthritis index(AI) and swollen joint count(SJC) were assessed,and histopathology of spleen and joints were observed.The percentage of B cells subsets,BAFF receptor expressions were analyzed by flow cytometry.BAFF and immunoglobulin(Ig) levels were measured by protein antibody array.The expressions of TRAF2,MKK3,MKK6,p-P38,and p-NF-κB65 in NF-κB signaling mediated by BAFF were analyzed by western blot.RESULTS CP-25 decreased AI and SJC,restored abnormal weights,reduced thymus index and spleen index,inhibited T/B cells proliferation,alleviated the histopathology of spleen and joints in CIA mice.CP-25 also reduced high levels of serum BAFF and immunoglobulin,decreased CD19+B cells,CD19+CD27+B cells,and CD19-CD27+CD138+plasma cells,inhibited BAFFR and TACI expressions,decreased the expressions of TRAF2,MKK3,MKK6,p-P38,and p-NF-κB65.Compared with biological agents etanercept and rituximab,CP-25 restored high T cells proliferation and percentages of B subsets to normal level,and recovered the high levels of IgA,IgD,IgG1,IgG2 a and high expressions molecules in NF-κB signaling to normal levels.The action intensity of rituximab and etanercept was more strong than CP-25.The inhibitor effects of rituximab and etanercept on AI and SJC,thymus index,proliferation of T cells and B cells subsets were strong,and down-regulated the indexes to under normal levels.CONCLUSION CP-25 might be a promising anti-inflammatory immune and regulation drug,which alleviated CIA and regulated the functions of B cells through BAFF/BAFF receptor-NF-κB signaling.

Treatment of a mouse model of collagen antibody-induced arthritis with human adipose-derived secretions

The use of adipose-derived cells as a treatment for a variety of diseases is becoming increasingly common. These therapies include the use of cultured mesenchymal stem cells (MSCs) and freshly isolated stromal vascular fraction (SVF) alone, or in conjunction with other cells such as adipocytes. There is a substantial amount of literature published on the therapeutic properties of MSCs and their secretions as the main driver of their therapeutic effect. However, there is little data available on the therapeutic potential of secretions from SVF, either with or without adipocytes. We investigated the ability of secretions from human adipose SVF alone and the SVF co-cultured with adipocytes as a proxy for cell therapy, to ameliorate an inflammatory disorder. This ethics approved study involved the treatment of collagen antibody-induced arthritis (CAIA) in mice with secretions from SVF, SVF co-cultured with adipocytes, or a vehicle control via both intravenous (IV) and intramuscular (IM) routes. Treatment outcome was assessed by paw volume, ankle size and clinical arthritis score measurements. Serum samples were obtained following euthanasia and analysed for a panel of 32 mouse cytokines and growth factors. The dose and timing regime used for the IM administration of both human secretion mixtures did not significantly ameliorate arthritis in this model. The IV administration of SVF adipocyte co-culture secretions reduced the paw volume, and significantly reduced the ankle size and clinical arthritis score when compared to the IV vehicle control mice. This was a superior therapeutic effect than treatment with SVF secretions. Furthermore, treatment with SVF adipocyte coculture secretions resulted in a significant reduction in serum levels of key cytokines, IL-2 and VEGF, involved in the pathogenesis of rheumatoid arthritis. Therefore, the SVF cocultured with adipocytes is an attractive therapeutic for inflammatory conditions.

Attenuation of Collagen Induced Arthritis by Centella asiatica Methanol Fraction via Modulation of Cytokines and Oxidative Stress

Objective To investigate the anti-inflammatory, antioxidant and anti-arthritic effects of Centello asiatica methanolfraction （CAME） on collagen-induced arthritis （ClA）, an animal model of rheumatoid arthritis. Methods Arthritis was induced in female wistar rats by immunization with porcine type II collagen. The CIA rats were treated orally with CaME （50, 150, and 250 mg/kg/day） for 15 d （beginning on day 21 of the experimental period）. The clinical, histological, biochemical, and immunological parameters were assessed. Results CaME treatment （150 and 250 mg/kg） significantly attenuated the severity of CIA and reduced the synovial inflammation, cartilage erosion, and bone erosion as evident from both histological and radiographic data. The escalated plasma levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-12 alongwith nitric oxide in CIA rats decreased significantly on CaME treatment. The serum levels of type-Ⅱ collagen antibody were significantly lower in rats of CaME （150 and 250 mg/kg） treated group than those in the arthritic group. Furthermore, by inhibiting the above mediators, CaME also contributed towards the reversal of the disturbed antioxidant levels and peroxidative damage. Conclusion Our results clearly indicate that oral administration of CaME suppresses joint inflammation, cytokine expression as well as antioxidant imbalance, thereby contributing to an amelioration of arthritis severity in CIA rats.

REGULAR EXPRESSION OF DISCOIDIN DOMAIN RECEPTOR 2 IN THE IMPROVED ADJUVANT-INDUCED ANIMAL MODEL FOR RHEUMATOID ARTHRITIS

Objective To investigate the expression of discoidin domain receptor 2 (DDR2) of fibroblast-like synovial cells in im- proved adjuvant-induced animal (AIA) model for rheumatoid arthritis (RA) and to provide evidence for DDR2’s antagonist use clinically. Methods AIA was modified by administrating 0.1 mL of complete Freund’s adjuvant (CFA, mixed with 5 mg Bacillus Calmette-Guerin vaccine/mL) into rats’ right hind paws and 0.125 mL tumor necrosis factor-α (2 U/mL) into right ankles and subpatellar fatty tissue. The expression of DDR2 in fibroblast-like synovial cells was assessed using immunohistochemistry, immunofluorescence histochemistry, and in situ hybridization methods. Levels of anti-collagen II antibody were measured using enzyme-linked immunosorbent assay. Results Given the terms mentioned above, we found a more practical rat model, apparently decreasing immunization time (average 3-5 days). DDR2 can be detected upon the 15th day of immunization; expression gradually increased with time going on, and reaching a peak 35 days after immunization before gradually decreasing. Serum anti-collagen II antibody showed similar expression patterns as DDR2, but reached peak later than DDR2, about 40 days after immunization. Conclusion Regular expression of DDR2 in animal models infers its important role in the pathological process of RA.

穿石通痹汤通过调节GABRB1-SOCS1/JAK1/STAT3信号途径对CIA模型大鼠关节炎症损伤的影响

目的 探讨穿石通痹汤对胶原诱导性关节炎(Collagen II induced arthritis,CIA)模型大鼠关节滑膜免疫炎性损伤的疗效及作用机制。方法 SPF级雄性SD大鼠,体质量(200±10)g,采用大鼠尾根部注射牛CⅡ方法制备CIA模型,将成模大鼠按随机数字表法分为模型组、阳性药组、穿石通痹汤组,每组各6只,另取6只未造模大鼠作为正常组。正常组与模型组隔日予去离子水10 ml/kg灌胃,阳性药组予甲氨蝶呤注射液每周0.5 mg/kg腹腔注射,穿石通痹汤组按生药量11.2 g/kg隔日灌胃给药,连续干预4周后处死大鼠,采集血浆、膝关节滑膜及踝关节标本。以HE染色观察大鼠踝关节病理变化;采用Westernblot检测大鼠膝关节滑膜pJAK1、JAK1、p38、GABRB1的蛋白表达水平;ELISA检测血浆IL-6,STAT3,SOCS1表达水平;并采用UHPLC-Q-Exactive Orbitrap MS分析血浆代谢物成分。结果 与正常组比较,模型组大鼠踝关节滑膜明显增生、严重骨质破坏以及炎性细胞浸润;与模型组比较,穿石通痹汤组大鼠关节骨质破坏、关节病变整体评分明显改善。与正常组比较,模型组GABRB1、SOCS1蛋白表达水平显著降低(P<0.01),p38、pJAK1、IL-6,STAT3蛋白表达水平明显升高(P<0.01,P<0.05);与模型组比较,穿石通痹汤组及阳性药组GABRB1、SOCS1蛋白表达水平均显著上调(P<0.01,P<0.05),并明显抑制p38、pJAK1、IL-6,STAT3的蛋白表达(P<0.01),且穿石通痹汤组对GABRB1的上调作用及pJAK1抑制作用显著优于阳性药组(P<0.01);血浆代谢物分析发现甘油磷脂代谢、花生四烯酸代谢、鞘脂代谢等代谢途径及其中50个代谢产物与本病发生密切相关。结论 穿石通痹汤能有效改善CIA模型大鼠关节滑膜免疫炎性代谢性损伤,其机制可能与调节GABRB1-SOCS1/JAK1/STAT3信号途径,调控脂质代谢途径相关。

诃子水提物通过上调PD-1/PD-L1表达调节胶原蛋白诱发关节炎(CIA)大鼠脾脏中细胞的细胞免疫减轻关节炎症

目的研究诃子水提取物(TCWE)对胶原蛋白诱发关节炎(CIA)大鼠细胞免疫及程序性死亡蛋白1/程序性死亡蛋白1配体1(PD-1/PD-L1)通路的影响。方法SD大鼠随机分为对照组、CIA组、TCWE处理组、甲氨蝶呤(MTX)处理组,每组15只。除对照组外,其余各组SD大鼠皮下注射Ⅱ型胶原蛋白制备胶原蛋白诱发关节炎(CIA)模型,TCWE组大鼠采用[20 mg/(kg·d)]TCWE处理,MTX组大鼠采用[1.67 mg/(kg·d)]MTX处理。处理14 d后,通过HE染色检查软骨形态,流式细胞术检测脾脏T淋巴细胞凋亡和调节性T细胞(Treg)/Th17细胞比率,反转录PCR检测脾脏中的维甲酸相关孤核受体γt(RORγt)和叉头盒转录因子P3(FOXP3)、PD-1和PD-L1的mRNA表达,免疫组织化学染色检测RORγt、FOXP3的表达和定位。Western blot法检测脾脏淋巴细胞的PD-1和PD-L1的蛋白表达,ELISA检测大鼠血清白细胞介素17(IL-17)和转化生长因子β(TGF-β)水平。结果与CIA组相比,TCWE组和MTX组的软骨和滑膜病变明显减轻,脾脏中的T淋巴细胞凋亡率增加,Treg/Th17细胞比率上调,RORγt的表达降低,FOXP3、PD-1和PD-L1的表达增加。血清IL-17水平降低,血清TGF-β水平升高。结论TCWE处理可能激活脾脏细胞的PD-1/PD-L1通路调节CIA大鼠脾脏中细胞的细胞免疫,减轻软骨损伤。

补骨脂-淫羊藿对类风湿关节炎抗炎机制的分子对接分析:动物实验验证

背景:临床上补骨脂-淫羊藿治疗类风湿关节炎疗效明显,但两者所含有效成分复杂,在分子水平上治疗类风湿关节炎的作用机制仍不明确。目的:基于网络药理学和分子对接技术建立胶原诱导型关节炎模型,验证补骨脂-淫羊藿治疗类风湿关节炎可能的作用靶点及通路,为以补骨脂-淫羊藿为主的临床方剂使用提供可靠的实验依据。方法:借助中医药研究平台、中医百科全书和上海有机所的中药与化学成分数据库等检索并筛选有效成分,从PubChem平台获取3D分子式,通过PharmMapper和SwissTargetPrediction平台进行靶标预测;结合DrugBank、GeneCards、OMIM等基因数据库完成类风湿关节炎的疾病靶点获取,经Uniport数据库校准靶标,借助VENNY 2.1获取补骨脂-淫羊藿与疾病交集靶点并绘制韦恩图;采用STRING平台构建蛋白质互作网络图;使用Metascape平台进行基因本体论功能分析及京都基因与基因组百科全书分析,进行数据可视化,利用Cytoscape 3.9.0构建中药-成分-靶点-疾病-通路四重网络模型;运用AutoDock-Vina软件将主要有效成分与核心靶点进行分子对接验证,探索最佳结合靶点。建立Ⅱ型胶原+佐剂诱导型关节大鼠模型,用补骨脂-淫羊藿干预21 d后,观察其对相关通路靶点及炎性细胞因子的影响。结果与结论:①筛选补骨脂与淫羊藿活性成分28个,与类风湿关节炎交集靶点共288个,主要成分有异补骨脂素、补骨脂定、淫羊藿苷等;交集靶点主要有丝氨酸/苏氨酸蛋白激酶1(AKT1)、肿瘤坏死因子、血管内皮生长因子A等;②基因本体论分析获得生物过程2232条,主要与丝氨酸蛋白磷酸化、AKT正调控、活性氧代谢过程等功能有关;③京都基因与基因组百科全书富集分析结果202条,主要有PI3K/AKT信号通路和表皮生长因子受体信号通路等,可能通过调节滑膜细胞凋亡与增殖、抑制炎性因子等发挥治疗作用;④分子对接结果表明补骨脂-淫羊藿主要与AKT1及雌激素受体转录因子1结合活性最强,并形成稳定结构,与PI3K/AKT等凋亡增殖、炎性介导等调控信号通路密切相关;⑤补骨脂-淫羊藿可降低胶原诱导型关节炎大鼠模型血清中白细胞介素1β、白细胞介素6、肿瘤坏死因子α的表达;⑥补骨脂-淫羊藿可调低胶原诱导型关节炎大鼠模型关节滑膜中p-PI3K、p-AKT、p-FOXO1蛋白的表达;⑦结果证明,补骨脂-淫羊藿可能经PI3K/AKT/FOXO1信号通路抑制关节滑膜细胞增殖和抑制炎性因子表达等发挥治疗作用,这可能与类风湿关节炎关节炎症和骨破坏的发生密切相关,同时为临床的合理使用及新药开发提供了参考依据。

铁死亡诱导剂咪唑酮埃拉斯汀(IKE)通过抑制IL-6、CCL5及CXCL9分泌缓解CIA小鼠的肺纤维化

目的 探索铁死亡诱导剂咪唑酮埃拉斯汀(IKE)对胶原蛋白诱发关节炎(CIA)小鼠肺纤维化的作用及潜在机制。方法 选取8周龄~10周龄DBA/1小鼠,按照完全Freund佐剂(CFA)与鸡Ⅱ型胶原蛋白混合乳化方法建立CIA小鼠模型,设置对照组、 CIA组、 CIA联合IKE组。每2 d进行关节评分与爪垫厚度测量,39 d后收集小鼠各脏器组织。HE染色、番红-固绿染色、甲苯胺蓝染色评价关节处组织病理改变;Masson染色评价肺部组织病理改变。免疫组织化学染色法检测肺组织α平滑肌肌动蛋白(α-SMA)、成纤维细胞活化蛋白α(FAPα)、转化生长因子β(TGF-β)及Ⅰ型胶原蛋白(Col1)、白细胞介素1(IL-1)、 IL-6、 IL-17及肿瘤坏死因子α(TNF-α)的表达水平;Olink mouse exploratory panel检测小鼠血清细胞因子IL-17α、 IL-17F、 TGF-β1、整合素亚基β6(ITG-β6)、 TNF受体超家族成员11B(TNFRSF11b)、 TNF受体超家族成员12A (TNFRSF12a)、 IL-6、 IL-1α、 IL-1β、 IL-10、 TNF-α、 CC趋化因子配体5(CCL5)、 CCL2、 CXC趋化因子配体9(CXCL9)、 CXCL1、烟酰胺腺嘌呤二核苷酸激酶(NADK)、促红细胞生成素(EPO)、集落刺激因子2(CSF2)、 TGF-α、 CCL20、 CCL3水平。结果 与CIA组相比,CIA联合IKE组治疗后关节炎症及关节损伤明显缓解;肺组织α-SMA、 FAPα、 TGF-β和Col1表达水平均呈下降趋势,表明CIA小鼠肺部胶原蛋白聚集减少,组织病变明显缓解;IL-6、 CCL5、 CXCL9、 IL-6水平显著下降表明CIA小鼠肺部炎症显著缓解。结论 IKE除缓解CIA小鼠关节炎症及关节损伤外,还可通过抑制IL-6、 CCL5及CXCL9表达缓解CIA伴发的肺纤维化。

基于IL-6/STAT3通路研究苗药方剂“四大血”对胶原诱导性关节炎小鼠的保护作用

目的基于白细胞介素-6(IL-6)/STAT3通路研究苗药方剂“四大血”(SX)对胶原诱导性关节炎(CIA)小鼠的保护作用机制。方法取40只C57BL/6雄性小鼠随机均分为4组[正常组、模型组、SX组和甲氨蝶呤(MTX)组],采用鸡Ⅱ型胶原和完全弗氏佐剂混合乳化剂诱导类风湿性关节炎(RA)小鼠模型。造模成功后按照实验分组(每组6只)进行灌胃给药。分别检测各组小鼠的足肿胀程度、关节病理改变、关节炎指数(AI)评分、肿瘤坏死因子-α(TNF-α)和IL-6水平、关节滑膜组织中p-STAT3蛋白表达情况。结果SX对RA疾病相关指标有显著改善作用,可以降低小鼠足肿胀程度、关节炎滑膜炎症、AI评分、TNF-α和IL-6水平,抑制p-STAT3蛋白的表达。结论SX可以发挥明显的抗RA作用,其可通过抑制IL-6/STAT3信号通路蛋白(p-STAT3)表达发挥对CIA小鼠的保护作用。

黑骨藤追风活络胶囊调节NF-κB/STAT3/mPGES-1信号通路改善胶原诱导性关节炎的作用研究

目的:研究黑骨藤追风活络胶囊(Heiguteng Zhuifenghuoluo cupsule,HZC)调节NF-κB/STAT3/mPGES-1信号通路改善胶原诱导性关节炎(Collagen induced arthritis,CIA)大鼠的作用机制。方法:取24只SD大鼠,随机分组为正常组、模型组、HZC组和甲氨蝶呤(methotrexate,MTX)组。除正常组外,其余三组采用牛Ⅱ型胶原和完全弗氏佐剂混合乳化剂构建类风湿性关节(rheumatoid arthritis,RA)大鼠模型。按照实验分组分别给予生理盐水、MTX(1 mg/kg)和HZC(243 mg/kg)灌胃,共给药21天。每周测量大鼠足肿胀程度、观察并进行足肿胀评分、检测大鼠血清中前列腺素E 2(Prostaglandin E2,PGE 2),检测大鼠滑膜组织中微粒体前列腺素E2合酶-1(microsomal prostaglandin E synthase 1,mPGES-1)以及p-p65、p-STAT3蛋白表达情况。结果:模型组大鼠足肿胀程度、肿胀程度评分、大鼠血清PGE 2明显增加,HZC和MTX组大鼠足肿胀程度、肿胀程度评分和大鼠血清PGE 2明显降低。进一步机制研究发现模型组大鼠关节滑膜组织中mPGES-1、p-p65、p-STAT3蛋白的表达增加,HZC和MTX组大鼠关节滑膜组织中mPGES-1、p-p65、p-STAT3蛋白的表达明显降低。结论:HZC通过抑制NF-κB/STAT3/mPGES-1信号通路,发挥其对胶原诱导性关节炎(CIA)大鼠的治疗作用。

基于HIF-1α/VEGF-α信号通路探讨南续益母颗粒抑制胶原诱导型关节炎小鼠滑膜血管翳生成的作用机制

目的基于缺氧诱导因子1α(HIF-1α)/血管内皮生长因子α(VEGF-α)信号通路探讨南续益母颗粒(南蛇藤、续断、益母草、红景天等)抑制胶原诱导型关节炎(CIA)小鼠滑膜血管翳生成的作用机制。方法将40只DBA/1雄性小鼠随机分为正常组、模型组、甲氨蝶呤组(1 mg·kg^(-1))和南续益母颗粒组(12.33 g·kg^(-1)),每组10只。除正常组外,其余小鼠采用二次免疫法复制CIA小鼠模型。二次免疫后,南续益母颗粒组灌胃给药,每日1次,连续30 d;甲氨蝶呤组每3 d灌胃1次,连续30 d。采用关节炎指数(AI)评分评估小鼠关节炎症程度;采用HE染色法观察小鼠踝关节滑膜病理变化;番红固绿染色法观察小鼠踝关节软骨及骨质结构变化;Micro-CT三维重建观察小鼠踝关节骨皮质表面变化;采用全自动生化分析仪检测小鼠血清C反应蛋白(CRP)、类风湿因子(RF)、天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、尿素氮(BUN)、肌酐(CR)水平;免疫组化法检测小鼠踝关节滑膜中血小板-内皮细胞黏附分子1(CD31)、HIF-1α、VEGF-α的表达。结果各组小鼠之间的血清AST、ALT、BUN、CR、RF水平无明显变化(P>0.05)。与正常组比较,模型组小鼠的踝关节及足面明显肿胀,活动度明显受限,AI评分显著升高(P<0.001),血清中的炎症指标CRP水平显著升高(P<0.001);踝关节可见显著滑膜增生(P<0.001)和炎性浸润(P<0.001),血管数量显著增加(P<0.001),踝关节可见显著软骨侵袭和骨破坏(P<0.001);踝关节骨质出现明显局灶性侵蚀,骨表面失去光滑及完整性,部分关节融合呈强直状;踝关节滑膜CD31、HIF-1α、VEGF-α蛋白表达显著上调(P<0.001)。与模型组比较,甲氨蝶呤组和南续益母颗粒组小鼠的踝关节及足面红肿均有改善,小鼠关节活动度基本正常,AI评分显著下降(P<0.01);甲氨蝶呤组小鼠血清CRP水平明显下降(P<0.05),南续益母颗粒组小鼠血清CRP水平有一定程度下降,但差异无统计学意义(P>0.05);甲氨蝶呤组和南续益母颗粒组小鼠踝关节的滑膜增生和炎性浸润均明显减轻(P<0.05),血管数量显著减少(P<0.01,P<0.001),软骨侵袭明显改善(P<0.05),骨破坏程度明显减轻(P<0.05,P<0.01);踝关节骨质破坏程度明显减轻,骨表面轻度粗糙,小部分存在局灶性侵蚀;踝关节滑膜CD31、HIF-1α、VEGF-α蛋白表达明显下调(P<0.05,P<0.01)。结论南续益母颗粒能够显著改善CIA模型小鼠关节肿胀症状及其病理变化,抑制踝关节滑膜血管翳形成,其作用机制可能与抑制HIF-1α/VEGF-α信号通路激活相关。

Effect of Wenhua Juanbi Recipe(温化蠲痹方) on Proliferation and Apoptosis of Synoviocytes in Rats with Collagen-Inducing Arthritis

Objective： To study the effect of Wenhua Juanbi Recipe （温化蠲痹方, W JR） on proliferation and apoptosis of synoviocytes in rats with collagen-inducing arthritis （CIA）. Methods： A CIA model was induced by intradermal injection of bovine collagen type 1[ emulsion at the base of rat tails. Thirty modeled healthy Wistar rats were randomly assigned to one of three groups （10 per group）： the model group, the methotrexate （MTX）- treated group （0.78 mg/kg） and the WJR-treated group （22.9 g/kg）. A group of 10 healthy rats was used as normal control. Treatments or normal saline for the control group were administered by oral gavage once daily. Rats were sacrificed after 30-day treatment and subjected to the following examinations： arthritis index （AI） was estimated, inflammatory cell infiltration and proliferation in synovial membrane were evaluated by microscopy, the synoviocyte apoptosis was determined by TUNEL assay, and the cell apoptosis index was calculated. Results： AI was lowered significantly in the WJR group compared to the model group （P〈0.01）. The pathological findings observed in the model group were reversed in the WJR group, including increase in inflammatory cell infiltration and synoviocyte proliferation in synovial membrane and reduction in cell apoptosis index （all P〈0.01）. Conclusions： Synoviocyte proliferation and apoptosis reduction were present in CIA rats. WJR was effective in treating the rat model of CIA. The therapeutic effect might be exerted through inducing apoptosis and suppressing proliferation of synoviocytes.

Effect of Yangqixue Qufengshi Recipe (养气血祛风湿方) on Rheumatoid Arthritis Model Mice under Different Genetic Backgrounds

Objective： To study the effect of Yangqixue Qufengshi Recipe （养气血祛风湿方, YQXQFS） on rheumatoid arthritis （RA） model mice under different genetic backgrounds. Methods： Collagen Induced Arthritis （CIA） were established on HLA-DR4 transgenic （TG） mice and non-transgenic （NTG） mice, which partly were raised with YQXQFS, and the onset day of CIA, the level of type Ⅱ collagen （C Ⅱ ）-reactive antibodies and the pathological scores of CIA were assessed. Results： Under HLA-DR4 TG background （compared with NTG mice）, the earlier onset day of CIA （ 11.22±3.35 days vs 16.56 ±4.75 days, P〈0.05） and higher level of C Ⅱ-reactive antibodies （0. 2274±0. 1390μg/ml vs 0.1101±0. 0560μg/ml, P〈0.05） were observed, but the pathological scores of CIA remained unchange. YQXQFS could not influence the onset day of CIA and the level of C Ⅱ-reactive antibodies, but had a certain effect on the total pathological scores （6.56±3.43 scores vs 11.11±5.64 scores） and bone erosion （0.22±0.44 scores vs 1.67±1.50 scores） of CIA on NTG mice （P〈0.05）, NTG YQXQFS group compared with NTG experimental group. Conclusion： YQXQFS had a certain effect on RA model, but had no significant effect on HLA-DR4 related CIA.

防己黄芪汤对CIA大鼠关节PI3K-Akt通路的调控作用研究

目的:研究防己黄芪汤(FHD)治疗胶原诱导性关节炎(CIA)大鼠的药理学机制及其对PI3K-Akt信号通路的调控作用。方法:60只雌性Wistar大鼠随机分为健康对照组(Control)、CIA组、不同剂量FHD[2.2、4.4、8.8 g(/kg·d)]干预组。除对照组外,其余各组采用牛Ⅱ型胶原构建CIA模型,不同剂量FHD干预,持续给药6周。HE染色检测大鼠关节和脾脏病理变化,ELISA检测血清TNF-α、IL-1、IL-6表达,网络药理学分析FHD治疗类风湿关节炎(RA)的核心信号通路,Western blot检测核心通路蛋白表达。结果:与Control组相比,CIA组大鼠关节炎症细胞浸润和骨质破坏明显,脾脏内白髓增生显著,血清TNF-α、IL-1、IL-6表达显著升高(P<0.05,P<0.01);与CIA组相比,不同剂量FHD组大鼠关节炎症及骨破坏显著改善,脾脏白髓增生减轻,低、中剂量FHD组大鼠血清TNF-α、IL-1、IL-6表达显著降低(P<0.05,P<0.01),高剂量FHD组TNF-α和IL-1表达差异无统计学意义(P>0.05);网络药理学分析显示FHD治疗RA的核心通路为PI3K-Akt通路;CIA组大鼠关节p-PI3K、p-Akt表达较Control组显著升高(P<0.01),不同剂量FHD组p-PI3K、p-Akt表达较CIA组显著降低(P<0.01),各组大鼠PI3K、Akt蛋白表达差异无统计学意义(P>0.05)。结论:成功构建CIA大鼠模型;FHD对CIA大鼠具有良好治疗作用,其安全用药剂量为2.2~4.4 g(/kg·d);FHD可能通过抑制PI3K-Akt通路激活和炎症因子生成发挥RA治疗作用。

低氧诱导因子-1α对胶原诱导关节炎小鼠脾细胞中T细胞亚群的调控作用

目的:探讨低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)的小干扰RNA(small interfering RNA,siRNA)载体对胶原诱导关节炎(collagen induced arthritis,CIA)小鼠脾细胞中T细胞亚群的调控作用。方法:DBA/1小鼠采用牛Ⅱ型胶原建立类风湿关节炎的动物模型CIA小鼠,尾静脉注射HIF-1α-siRNA腺病毒和阴性对照腺病毒,每周1次,共4周,实验分为空白组、模型组、阴性对照组和HIF-1α-siRNA组。实验结束后处死小鼠。流式细胞术检测小鼠脾脏中Th1和Th2、Th17和Treg比例的变化;HE染色观察小鼠踝关节的病理变化。结果:与空白组比较,模型组小鼠脾细胞中Th1细胞和Th17细胞的比例升高,差异有统计学意义(P<0.05);Treg的比例是下降的,但没有明显的统计学差异;而Th2细胞的变化并不明显。与模型组和阴性对照组比较,HIF-1α-siRNA组小鼠脾细胞中Th1细胞和Th17细胞的比例降低,差异有统计学意义(P<0.05),Th2细胞和Treg虽然呈上升的趋势,但差异没有统计学意义;模型组小鼠踝关节出现滑膜增生及大量的淋巴细胞浸润,HIF-1α-siRNA作用后,CIA小鼠踝关节的滑膜增生和淋巴细胞浸润的情况明显减轻。结论:HIF-1α-siRNA可以降低脾细胞中Th1细胞和Th17细胞的比例,同时上调Th2细胞和Treg的比例,并减轻CIA小鼠滑膜增生和淋巴细胞浸润情况,提示HIF-1α-siRNA可以通过调控脾细胞中的T细胞亚群,进而发挥治疗CIA小鼠的作用。

CPI-203通过抑制p38 MAPK/AKT通路改善类风湿关节炎

目的探讨CPI-203对类风湿关节炎影响及作用机制。方法将生长至5~7代的RA滑膜成纤维细胞(RASF)分为正常对照组,白介素1β(IL-1β,10 ng/ml)诱导组,CPI-203(1μmol/L)用药组,CPI-203+IL-1β组。采用Transwell和划痕实验评估RASF侵袭和迁移能力,血管形成实验检测血管形成能力,qRT-PCR和ELISA测定表型相关分子表达水平。Western blot检测丝裂原活化蛋白激酶(MAPK)和蛋白激酶B(AKT)通路相关蛋白表达。使用II型胶原及佐剂混合而成的乳浊液构建胶原诱导关节炎(CIA)模型。实验组CIA模型诱导成功后腹腔注射CPI-203,200 mg/kg,每两天注射1次;阴性对照组不进行任何处理;阳性对照组构建CIA模型后腹腔注射等量DMSO,每两天注射1次。CPI-203治疗30 d后,观察鼠爪临床病理特征变化,ELISA检测各组小鼠血清中炎性因子表达水平,Mircro-CT分析鼠爪骨侵蚀变化,苏木精-伊红(HE)染色评估滑膜增生和软骨降解情况。结果CPI-203显著抑制了IL-1β诱导的RASF侵袭和迁移,并降低血管形成数(P<0.05)。与阳性对照组相比,实验组小鼠关节临床指标得分、滑膜增生、软骨降解及骨损伤减少,血清中炎性因子IL-6、IL-8水平降低(P<0.033)。结论CPI-203可抑制RASF中炎性因子的表达,减弱RASF的侵袭、迁移及血管形成能力,缓解CIA模型小鼠关节炎症,对类风湿关节炎具备一定治疗潜力。

苗药金乌健骨胶囊对胶原诱导关节炎模型大鼠肠道、滑膜炎症及IL-17α/TRAF6/NF-κB信号通路的影响

目的探讨苗药金乌健骨胶囊对胶原诱导关节炎(collagen-induced arthritis,CIA)模型大鼠肠道、滑膜炎症及参与NF-κB信号通路基因IL-17受体信号分子ACT1(nuclear factor kappa B activator 1,ACT1)、泛素化TRAF6(TNF receptor associated factor 6,TRAF6)、转化生长因子β激活激酶1(transforming growth factor-β-activated kinase 1,TAK1)、核因子-κB上游激酶(IκB kinaseα,IKKα)、核因子κB/p65、核因子κB/p50表达的影响。方法将70只SPF级6~8周龄雌性Wistar大鼠随机分为7组,分别为空白对照组、模型组、金乌健骨胶囊低剂量组、金乌健骨胶囊中剂量组、金乌健骨胶囊高剂量组、甲氨蝶呤组及益生菌组,每组10只。适应性喂养后除空白对照组外,其余6组构建CIA模型,两次免疫造模成功后分别进行药物金乌健骨胶囊、甲氨蝶呤、益生菌灌胃治疗,治疗4周后静脉取血,处死大鼠,分离大鼠滑膜组织及肠道组织。利用HE染色法检测大鼠滑膜及大肠组织病理情况;ELISA法检测大鼠血清TGF-β1、IL-1α、IL-17α、IL-21、IL-23的含量;Western blot法检测大鼠肠道组织ACT1、TRAF6、TAK1、IKKα、P65、P50蛋白的表达水平;RT-qPCR法检测大鼠肠道ACT1、TRAF6、TAK1、IKKα、P65、P50基因的表达情况。结果滑膜病理结果显示,与模型组相比,金乌健骨胶囊中、高剂量组和甲氨蝶呤组炎性浸润、增生、血管新生组织有明显改善;肠道病理结果显示,与模型组相比,金乌健骨胶囊低、中、高剂量组及甲氨蝶呤组、益生菌组肠道组织细胞可见少量炎性细胞浸润,肠道组织细胞病变情况较模型组有不同程度的改善;ELISA结果显示,与模型组相比,各给药组血清含量显著降低(P<0.05或P<0.01);Western blot结果显示,各给药组大鼠肠道组织ACT1、TRAF6、TAK1、IKKα、P65、P50的蛋白表达显著降低(P<0.05或P<0.01);RT-qPCR结果显示,各给药组ACT1、TRAF6、TAK1、IKKα、P65、P50基因的表达显著降低(P<0.05或P<0.01);金乌健骨胶囊高剂量组的作用最佳。结论苗药金乌健骨胶囊能够减轻CIA大鼠肠道、滑膜炎症,同时能够影响肠道IL-17α/TRAF6/NF-κB信号途径相关基因的表达情况。

松萝酸经HMGB1-RAGE信号通路治疗胶原诱导性关节炎大鼠关节炎机制研究

目的:探讨松萝酸经高迁移率族蛋白B1(HMGB1)-晚期糖基化终产物受体(RAGE信号通路治疗胶原诱导性关节炎(CIA)大鼠关节炎的机制。方法:选取SPF级5周龄Wistar雌鼠54只,随机分为空白对照组,模型对照组,松萝酸低、中、高剂量组和羟氯喹组,每组9只。除空白对照组外,其余各组采用Ⅱ型胶原乳剂建立CIA模型。松萝酸低、中、高剂量组分别给予2mg·kg^(-1)·d^(-1)、6 mg·kg^(-1)·d^(-1)、54 mg·kg^(-1)·d^(-1)松萝酸灌胃,羟氯喹组给予36 mg·kg^(-1)·d^(-1)羟氯喹灌胃,空白对照组及模型对照组则予以等量生理盐水灌胃。连续灌胃4周后,处死大鼠,收集血清,取滑膜组织,分别行HE染色病理检测,ELISA法检测白细胞介素-1β(IL-1β)、基质金属蛋白酶(MMP)、血管内皮生长因子(VEGF)含量,Western blot法检测HMGB1、RAGE mRNA表达水平;PCR法检测HMGB1、RAGE蛋白表达量。结果:与模型对照组比较,松萝酸中、高剂量组滑膜增生程度减低,炎性细胞浸润减少,血清HMGB1、IL-1β、MMP、VEGF表达显著下降(P<0.05),且HMGB1、RAGE蛋白活性、m RNA含量明显降低(P<0.05)。结论:松萝酸能减少炎性细胞浸润及滑膜细胞增殖,其机制可能与抑制HMGB-RAGE信号通路有关。