基于网络药理学探究防风中生物活性成分及对类风湿关节炎的作用机制

目的 通过以网络药理学为基础的策略研究防风治疗类风湿关节炎（rheumatoid arthritis,RA）的分子生物学机制。方法 采用网络药理学方法收集防风活性成分和治疗RA的潜在靶点,并评估活性成分的药理和毒理学等相关参数;构建蛋白质相互作用网络筛选核心靶点,并通过生物信息学方法进一步验证核心靶点和疾病的关联;对核心成分和相应靶点进行分子对接。体外通过CCK-8实验、细胞迁移和侵袭、细胞凋亡、qRT-PCR和Western blotting分析,阐明别欧前胡素对MH7A细胞磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase,PI3K）/蛋白激酶B（protein kinase B,Akt）通路的调控作用。结果 从防风中共鉴定出18种活性成分和66个与筛选出的RA疾病靶基因相交的潜在靶基因,最终获得了汉黄芩素、β-谷甾醇、5-O-甲基维斯阿米醇和别欧前胡素等核心成分。防风治疗RA的潜在机制可能是通过调控PI3K/Akt、白细胞介素-17（interleukin-17,IL-17）、凋亡等信号通路和多种生物过程来实现,以发挥抗炎和免疫调节作用。分子对接证实了所有的核心成分和关键靶点均具有很好的对接活性。别欧前胡素抑制MH7A细胞的活力、迁移和侵袭（P＜0.05、0.01）,诱导细胞凋亡（P＜0.01）,并显著下调IL-1β、IL-6、IL-8、基质金属蛋白酶-1（matrix metalloproteinase-1,MMP-1）和MMP-3的基因表达（P＜0.01）。分子分析表明别欧前胡素通过抑制PI3K/Akt通路发挥对MH7A的调控作用。结论 成功预测了防风治疗RA的有效成分和潜在靶点,为进一步探究其分子机制提供了新的理论基础。揭示了别欧前胡素通过PI3K/Akt通路抑制RA成纤维样滑膜细胞的活力、迁移、侵袭及细胞因子和MMPs的表达,并诱导细胞凋亡。     

佐剂诱导关节炎大鼠模型的建立及成纤维样滑膜细胞的分离

目的建立类风湿性关节炎(RA)动物模型；从炎性关节滑膜中分离成纤维样滑膜细胞(FLS)。方法应用热灭活结核杆菌H37Ra菌株与矿物油混合制备改良的佐剂,尾根部皮内注射Lewis大鼠诱导关节炎；剪取成功诱导关节炎大鼠的病变踝关节,从中剥离滑膜组织,充分剪碎后采用胶原酶消化法分离成纤维样滑膜细胞。结果成功诱导了大鼠关节炎,发病率为100%,发病时间有规律,组织学表现与RA相似；成功在体外培养了FLS并对其进行了鉴定,掌握了其生长形态和特征。结论成功制备RA动物模型并获得FLS,为今后RA发病机制探索和药物评价提供了良好的体内动物模型和体外细胞模型。     

罗格列酮通过抑制RANKL及p-ERK活化抑制类风湿关节炎破骨细胞的分化及功能

目的:探讨罗格列酮(RSG)对类风湿关节炎(RA)成纤维样滑膜细胞(FLS)介导的破骨细胞(Oc)分化及功能的影响及其可能机制。方法:活动期RA患者滑膜体外分离培养FLS,与健康人外周血单核细胞(MNC)共培养,不同浓度RSG(0、5、10和15μmol/L)干预,抗酒石酸酸性磷酸酶(TRAP)染色鉴定Oc并计数;甲苯胺蓝染色和图像分析系统计算骨吸收陷窝面积;Real-time PCR检测共培养体系RANKL和OPG的mRNA表达,Western blot检测RANKL、OPG、p-ERK、p-p38和p-JNK的蛋白含量。结果:与不加RSG组比较,15μmol/L RSG干预后Oc的数量明显减少(P0.01),骨吸收陷窝面积也减少(P0.05);共培养体系RANKL的mRNA及蛋白表达明显降低,OPG的mRNA及蛋白表达明显升高(P0.01);p-ERK的蛋白含量明显降低(P0.05),p-p38及p-JNK的蛋白含量则不受影响。结论:RSG通过抑制RANKL及p-ERK活化影响RA关节微环境中组织细胞与免疫细胞的相互作用,从而抑制Oc分化及骨吸收功能。     

三氧化二砷诱导类风湿关节炎滑膜细胞凋亡的作用及机制

目的研究三氧化二砷(ATO)对人类风湿关节炎成纤维样滑膜细胞(HFLS-RA)凋亡的作用及机制。方法将HFLS-RA分为单纯培养基空白对照组和0．5、2、8μ,mol/L ATO实验组．分别观察ATO作用72 h电镜下细胞的病理改变以及TUNEL法检测细胞的凋亡；四唑盐(MTT)法检测ATO对HFLS-RA生长的影响；反转录聚合酶链反应(RT-PCR)方法检测ATO作用24 h对核转录因子(NF)-κB的mRNA表达影响。结果电镜下,ATO可以诱导HFLS-RA的凋亡,TUNEL法检测ATO组凋亡指数呈剂量依赖性增加,与对照组相比,2、8μ,mol/L ATO组凋亡指数明显增加(P＜0．05)；ATO呈现时间和剂量依赖性抑制HFLS-RA生长的作用；RT-PCR法表明ATO组NF-κB的mRNA表达剂量依赖性减少,2μmol/L以上ATO可以明显下调NF-KB的mRNA表达(P＜0．05),结论ATO可以抑制HFLS-RA的生长,并且可能通过抑制NF-KB的mRNA表达促进HFLS-RA的凋亡。     

Comparative Study of Normal and Rheumatoid Arthritis Fibroblast-Like Synoviocytes Proliferation under Cyclic Mechanical Stretch: Role of Prostaglandin E2

Fibroblast-like synoviocytes (FLSs) are one of the main contributors of prostaglandin E₂ (PGE₂) in the hyperplastic synovium of rheumatoid arthritis (RA) patients. cyclooxygenase-2 (COX-2)/PGE₂ pathway is involved in the proliferation of several cell types. We have previously shown that mechanical stretch affects COX-2 and PGE₂ production in human RA FLSs; however, its role in cell proliferation remains to be elucidated. In this study, a comparison is drawn between human RA and normal FLSs to understand the role of mechanical stretch and PGE₂ on the proliferation of FLSs. The results showed that physiological level (6%, 1 Hz) of cyclic mechanical stretch significantly decreased the proliferation of RA FLSs but not normal FLSs, while the induction of apoptosis was not observed by stretch in either RA or normal FLSs. IL-1β (5 ng/ml)-induced COX-2/PGE₂ levels are downregulated by stretch in RA FLSs only. Further investigation showed that high concentration (100 and 500 ng/ml) of PGE₂ significantly induced cell proliferation only in RA FLSs, and this induction failed to be suppressed by stretch. In conclusion, this study demonstrated that elevated levels of PGE₂ in the synovial cavity are involved in the proliferation of RA FLSs, and cyclic mechanical stretch regulates the RA synovial hyperplasia.     

选择性β肾上腺素受体激动剂对人滑膜细胞增殖能力的影响

目的：观察β-肾上腺素受体( AR)激动药对人成纤维样滑膜细胞( FLS)增殖的作用,探讨β-AR信号对人FLS功能的影响。方法组织块培养法培养人FLS;使用肿瘤坏死因子α( TNF-α)作为刺激剂,MTT法分别检测β1-AR选择性激动剂多巴酚丁胺和β2-AR选择性激动剂沙丁胺醇对人FLS增殖作用的影响,并计算增殖抑制率;使用沙丁胺醇+β2-AR选择性拮抗剂ICI 118551观察其对FLS增殖功能的影响;Western blot法检测人FLSβ-AR、G蛋白偶联受体激酶2(GRK2)和β抑制蛋白2(β-arrestin2)胞膜蛋白的表达水平。结果 TNF-α可以显著促进人FLS的增殖;β2-AR激动剂沙丁胺醇能明显抑制FLS的增殖,其拮抗剂ICI 118551能显著拮抗沙丁胺醇的作用;β1-AR选择性激动剂多巴酚丁胺对FLS的增殖无显著影响;TNF-α和沙丁胺醇均能显著降低β2-AR胞膜表达,上调 GRK2、β-arrestin2的胞膜表达。结论β2-AR信号的激活可以抑制FLS增殖;TNF-α和沙丁胺醇可以使GRK2、β-arrestin2的胞膜表达增加,β2-AR胸膜表达下降。     

A directly GP130-targeting small molecule ameliorates collagen-induced arthritis (CIA) by inhibiting IL-6/GP130 signalling and Th17 differentiation

Rheumatoid arthritis is a chronic inflammatory disease associated with joint inflammation and destruction driven by T helper 17 (Th17) cells. Interleukin-6 (IL-6) is secreted by many cell types, including macrophages and synovial fibroblasts. It induces the differentiation and function of Th17 cells that can increase lymphocytic infiltration in the joint. LMT-28 can suppress IL-6 signalling through direct binding to glycoprotein-130 and alleviate inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. The purpose of this study was to assess whether LMT-28 could potently inhibit Th17 differentiation and to determine the mechanism involved in the attenuating effect of LMT-28 on rheumatoid arthritis through the IL-6 signalling pathway. LMT-28 reduced the arthritis score and showed protective effects against bone and cartilage destruction in collagen-induced arthritis (CIA) mice. In mice with CIA, LMT-28 markedly decreased serum levels of IL-6, TNF and IL-1β compared to vehicle control. Moreover, LMT-28 attenuated Th17 cell activation in lymph nodes of CIA mice. We demonstrated that LMT-28 suppressed differentiation of Th17 in mouse splenocytes and human peripheral blood mononuclear cells (PBMCs). Additionally, LMT-28 inhibited phosphorylation of GP130, STAT3 and ERK induced by Hyper-IL-6 in human fibroblast-like synoviocytes (FLS). Collectively, these results suggest that LMT-28 can inhibit differentiated/activated-Th17 cells in rheumatoid arthritis by blocking activation of the STAT3 pathway. LMT-28 can attenuate rheumatoid arthritis by inhibiting differentiation/activation of Th17 cells and suppressing the proliferation and signalling activation of the IL-6/solubleIL-6 receptor complex stimulated FLS.     

PCGEM1 stimulates proliferation of osteoarthritic synoviocytes by acting as a sponge for miR‐770

Long non‐coding RNAs (lncRNAs) have been reported to play important roles in cellular metabolism and development. Various diseases have been associated with aberrant expression of lncRNAs and the related dysregulation of mRNAs. An lncRNA profiling assay was carried out to identify the key lncRNA in osteoarthritic human synoviocytes; the results revealed that prostate cancer gene expression marker 1 (PCGEM1) was significantly overexpressed in osteoarthritic synoviocytes. Exogenous overexpression of PCGEM1 inhibited apoptosis, induced autophagy, and stimulated the proliferation of human synoviocytes. The increased expression of PCGEM1 in human synoviocytes also suppressed the expression of miR‐770. Transfection of the miR‐770 precursor resulted in reduced proliferation, and induced apoptosis of human synoviocytes. This effect of miR‐770 expression was reversed by co‐introduction of PCGEM1. Target validation showed a direct binding between PCGEM1 and miR‐770. We demonstrate that PCGEM1 act as sponge lncRNA for miR‐770 that regulates proliferation/apoptosis and autophagy, and suggest PCGEM1 as possible target for OA therapy. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:412–418, 2016.     

Lysophosphatidic acid-induced IL-8 secretion involves MSK1 and MSK2 mediated activation of CREB1 in human fibroblast-like synoviocytes

Lysophosphatidic acid (LPA) is a pleiotropic lipid mediator that promotes motility, survival, and the synthesis of chemokines/cytokines such as interleukin-8 (IL-8) and interleukin-6 by human fibroblast-like synoviocytes from patients with rheumatoid arthritis (RAFLS). In those cells LPA was reported to induce IL-8 secretion through activation of various signaling pathways including p38 mitogen-activated protein kinase (p38 MAPK), p42/44 MAPK, and Rho kinase. In addition to those pathways we report that mitogen- and stress-activated protein kinases (MSKs) known to be activated downstream of the ERK1/2 and p38 MAPK cascades and CREB are phosphorylated in response to LPA. The silencing of MSKs with small-interfering RNAs and the pharmacological inhibitor of MSKs SB747651A shows a role for both MSK1 and MSK2 in LPA-mediated phosphorylation of CREB at Ser-133 and secretion of IL-8 and MCP-1. Whereas CREB inhibitors have off target effects and increased LPA-mediated IL-8 secretion, the silencing of CREB1 with short hairpin RNA significantly reduced LPA-induced chemokine production in RAFLS. Taken together the data clearly suggest that MSK1 and MSK2 are the major CREB kinases in RAFLS stimulated with LPA and that phosphorylation of CREB1 at Ser-133 downstream of MSKs plays a significant role in chemokine production.     

TNF-α、IL-1β通过PKA通路诱导成纤维样滑膜细胞中力生长因子表达

目的 探讨炎症因子TNF-α、IL-1β、IL-6对力生长因子（mechano growth factor, MGF）表达的影响。方法 实验组细胞用浓度为25、50、100 ng/mL的TNF-α、IL-6或者是2.5、5.0、10 ng/mL的IL-1β处理12 h,抑制剂组在因子处理前用1.0 mmol KT5720预处理1 h,然后添加炎症因子刺激12 h,对照组细胞保持与实验组培养条件一致的情况下不添加因子刺激。因子刺激后,用定量PCR方法检测细胞中MGF表达量。结果25 ng/mL TNF-α和10 ng/mL IL-1β因子刺激滑膜成纤维细胞诱导MGF表达显著增加（P＜0.05）。IL-6对MGF表达无影响。1.0 mmol PKA通路抑制剂KT5720处理显著降低TNF-α和IL-1β诱导的MGF表达（P＜0.05）。结论 炎症因子TNF-α和IL-1β在浓度分别为25和10 ng/mL时能显著诱导滑膜成纤维细胞中MGF表达,其表达是通过PKA通路介导的。本研究对于促进MGF用于改善膝关节相关组织修复中的应力刺激不足有一定的现实意义。