目的 基于lncRNA NEAT1和IRE1α/XBP1信号通路,探讨透骨消痛胶囊延缓膝骨关节炎(KOA)软骨退变的机制.方法 48只8周龄小鼠适应性喂养1周后,采用随机数字表法分为空白组12只、造模组36只,造模组动物经5%异氟醚吸入麻醉后,采用Hulth法诱导KOA模型,随机数字表法分为模型组12只、透骨消痛胶囊组12只和对照组12只.透骨消痛胶囊组予以透骨消痛胶囊 368 mg/kg进行灌胃;对照组使用牛磺熊去氧胆酸500 mg/kg进行灌胃;空白组和模型组给予等量生理盐水灌胃,连续4周.干预结束后,麻醉状态下处死各组小鼠,分离与收集膝关节软骨组织,Micro-CT观察小鼠膝关节软骨组织形态变化;Western blot检测关节软骨IRE1α/XBP1通路相关蛋白表达;qPCR检测软骨组织中lncRNA NEAT1表达水平.结果 与空白组比较,模型组半月板结构缺失,胫骨平台软骨面呈现凹凸不平状、伴有明显的骨赘增生,软骨组织IRE1α、XBP1、GRP78、CHOP蛋白含量、lncRNA NEAT1表达均显著升高(P＜0.05);透骨消痛胶囊组、对照组小鼠内侧半月板结构缺失,2组软骨组织的IRE1α、XBP1、GRP78、CHOP蛋白含量均显著降低(P＜0.05),lncRNA NEAT1表达水平均显著降低(P＜0.05).结论 透骨消痛胶囊可通过下调KOA小鼠关节软骨组织中lncRNA NEAT1水平,抑制软骨组织与内质网应激有关的IRE1α/XBP1通路蛋白表达发挥作用.

Objective To evaluate the effect of Rongjin Niantong Recipe on knee osteoarthritis model rats, and to explore the mechanism of delaying cartilage degeneration in knee osteoarthritis based on long noncoding RNA (lncRNA) NEAT1 and Nrf2/ARE pathway. Methods After adaptive feeding for one week, a total of 45 SPF SD rats aged two months were randomly divided into blank group and sodium iodoacetate group, with 15,30 cases in each group. Under anesthesia, 0.05 mL sodium iodoacetate at a concentration of 20 mg/mL was injected into the right knee joint to establish the knee osteoarthritis model, and the blank group was injected with the same amount of normal saline. After modeling was assessed to be successful, the sodium iodoacetate group were randomly divided into model group and Rongjin Niantong Recipe group, with 15 cases in each group. The Rongjin Niantong Recipe group were given 1.9 g/(kg·d) the medicine by gavage, and the blank group and the model group were given the same amount of normal saline by gavage for eight weeks. After the intervention, all groups were sacrificed under anesthesia, and the right knee tissues were separated and collected; HE staining was used to observe the pathological changes; Western blot was used to detect the expression levels of Nrf2, heme oxygenase-1 (HO-1), NAD (P) H quinone oxidoreductase-1 (NQO-1), kelch like ECH-associated protein 1 (Keap1) and inducible nitric oxide synthase (iNOS) in cartilage; Real-time PCR was used to detect the expression of lncRNA NEAT1 in cartilage tissue. Results 1) Pathological changes of cartilage tissue: the cartilage matrix was light red and uniform, with complete tissue structure and clear tissue margins of each layer in the blank group; in the model group, the chondrocytes grew in clusters with thinner tangent layer. The surface layer was destroyed and the transitional layer and the radiation layer became disordered and partial calcified. There were obvious calcifications in the calcified layer. In addition, the adhesion line between the calcified layer and the subchondral bone fluctuated greatly and was discontinuous. The tangents of the superficial layer in the Rongjin Niantong Recipe group were completed and basically paralleled to the articular surface, and the transitional layer, radiation layer, calcification layer and other layers were relatively clear. 2) Expressions of Nrf2/ARE pathway related proteins in the cartilage: compared with the blank group, the protein expression levels of Keap1 and iNOS of the model group significantly increased (P<0.05) and the protein expression levels of Nrf2, HO-1 and NQO-1 significantly decreased (P<0.05). Compared with the model group, the protein expression levels of Keap1 and iNOS in the Rongjin Niantong Recipe group significantly decreased (P<0.05), while the protein expression levels of Nrf2, HO-1 and NQO-1 significantly increased (P<0.05). 3) Expressions of lncRNA NEAT1 in cartilage: compared with the blank group, the expression level of lncRNA NEAT1 in the model group was significantly higher (P<0.05); compared with the model group, the expression level of lncRNA NEAT1 in the Rongjin Niantong Recipe group significantly decreased (P<0.05). Conclusion Rongjin Niantong Recipe can delay cartilage degeneration in rats with osteoarthritis. The mechanism may be related to the down-regulation of lncRNA NEAT1 level, up-regulation of Nrf2 signal and antioxidant signal molecules expressions, and down-regulation of pro-oxidation signal molecules and inhibition of oxidative stress level.

骨关节炎病位在体为筋骨,在脏为肝肾,两者相辅相成;肝肾相济则骨坚筋强,肝肾不足则骨痿筋弱.研究表明临床运用补肝肾、强筋骨类中药,可促进软骨细胞增殖,延缓软骨退变,但其具体作用机制尚不明确.故以补肝肾、强筋骨类中药及相关活性成分促进软骨细胞增殖为切入点,查阅文献并综述认为,其作用机制可能与促进细胞外基质合成、调控细胞周期相关蛋白、促进转化生长因子-β相关受体表达、调控细胞外调节激酶、Wnt信号通路及miRNA有关,可为进一步研究中药靶向治疗骨关节炎提供思路.

目的 基于PERK通路探讨荣筋拈痛方(RJNTD)对毒胡萝卜素(TG)诱导的软骨细胞内质网应激反应的抑制作用.方法 将30只4周龄SPF级雄性C57BL/6小鼠处死后,体外分离双膝软骨细胞置于低糖DMEM培养基培养,经Ⅱ型胶原免疫细胞化学染色法鉴定后将软骨细胞分成空白组、对照组、模型组和中药组.空白组常规培养4 h,对照组常规培养4 h后更换300μg/mL RJNTD培养液培养12 h,模型组用25μmol/L TG培养液培养4 h后改为常规培养,中药组用25μmol/L TG培养液培养4 h后更换300μg/mL RJNTD培养液培养12 h.干预后采用Real-time PCR检测各组软骨细胞中miRNA-377-3p相对表达水平;Western blot检测各组软骨细胞中内质网降解增强子(EDEM1)、蛋白激酶R样内质网激酶(PERK)、转录激活子4(ATF4)、免疫球蛋白重链结合蛋白(BIP)与DNA损伤诱导基因153(GADD153)蛋白表达水平.结果 对照组各指标与空白组比较均无统计学意义(P均>0.05);与空白组比较,模型组及中药组miRNA-377-3p相对表达水平显著降低(P<0.05),且中药组较模型组降低更不明显(P<0.05);与空白组比较,模型组及中药组EDEM1、PERK、ATF4、BIP、GADD153蛋白表达水平均升高(P均<0.05),且中药组上述各指标较模型组升高更不明显(P均<0.05).结论 RJNTD可通过PERK通路抑制内质网应激反应,进而发挥延缓TG诱导的软骨细胞退变作用,其潜在作用机制可能与调节miRNA-377-3p相关.

目前,治疗类风湿关节炎的优选策略是控制炎症与其他病理反应,缓解其疼痛不适等临床症状.槲皮素具有抑制炎症介质的表达和分泌、抗氧化、免疫调节、抑制基质金属蛋白酶的生成,以及抑制滑膜细胞增生等多种生物活性,可抑制类风湿关节炎的病理进程.槲皮素在预防和治疗类风湿关节炎上具有广阔的应用和发展前景.

类风湿关节炎的治疗方法颇多,但以改善病情、缓解临床症状为主.山柰酚是从姜科植物山柰的根茎提取的黄酮醇类化合物,具有抗氧化、抗炎及免疫调节等作用,尤其在干预类风湿关节炎的病理进程方面已成为研究热点之一.探讨山柰酚治疗类风湿关节炎的作用机制,以期为山柰酚对类风湿关节炎治疗的进一步研究提供思路.

随着全社会人口老龄化的加剧,绝经后女性罹患骨质疏松性骨关节炎的风险亦呈现高发趋势,严重危害中老年人的健康.研究证实血管形成及功能异常是加剧骨质疏松、骨关节炎病理退变的重要事件.H型血管(内皮细胞)在骨与软骨损伤修复中兼具成骨和成血管偶联功能,其形态与功能异常将影响骨质疏松、骨关节炎的损伤修复.铁死亡作为近年来发现的一种新型细胞死亡模式,其主要由铁依赖性脂质过氧化和活性氧诱导损伤引起细胞膜断裂,线粒体变小等改变.而H型血管铁死亡对骨质疏松性骨关节炎病程的影响途径及调节机制尚需进一步探讨,旨在为今后以H型血管铁死亡为切入点进而防治骨质疏松性骨关节炎开辟新方向.

目的:基于生物信息学和网络药理学方法,筛选荣筋拈痛方君药牛膝相关活性成分,通过动物体内体外实验观察其对骨关节炎相关治疗靶点的影响.方法:通过中药系统药理学数据库与分析平台(TCMSP)数据库筛选中药牛膝主要活性成分和治疗靶点;通过GEO数据库获得与疾病的差异表达基因,并进行交叉分析;采用实时荧光定量聚合酶链式反应(Real-time PCR),蛋白免疫印迹法(Westemblot)检测牛膝主要活性成分对骨关节炎大鼠体内体外富集治疗靶点的影响.结果:牛膝作用骨关节炎相关活性成分共20种,槲皮素为其中重要成分之一;靶基因共3个,其中骨桥蛋白(OPN),纤维蛋白溶解抑制剂-1(PAI-1)等为该网络中的关键基因;基因本体(GO)分析共获得相关条目227条,涉及到的功能包括创伤生理反应调节(GO:1903034),创伤反应负调节(GO:1903035)等.京都基因与基因组百科全书(KEGG)通路分析获得相关通路12条,涉及细胞外基质受体相互作用(hsa04512)等.动物实验中,与正常组比较,模型组OPN,PAI-1 mRNA和蛋白表达量增加与模型组比较,槲皮素组OPN,PAI-1 mRNA和蛋白表达量降低(P＜0.05).细胞实验中,与正常组比较,模型组OPN,PAI-1蛋白表达量增加,Ⅱ型胶原(CollagenⅡ)蛋白表达量降低;与模型组比较,槲皮素组和抑制剂组OPN,PAI-1蛋白表达量降低,CollagenⅡ蛋白表达量明显增加(P＜0.05).结论:牛膝可能主要通过其活性成分(槲皮素)下调软骨细胞OPN,PAI-1靶mRNA表达,上调CollagenⅡ蛋白表达,实现抑制软骨退变,防治骨关节炎.

H型血管作为一种高表达血小板-内皮细胞粘附分子-1(CD31)与内皮黏蛋白(Emcn)的特殊血管类型,常分布于成骨活跃的部位,因其周围聚集大量骨祖细胞,具有成血管与成骨偶联作用,与骨质疏松症病程预后转归关系密切.探讨H型血管与骨形成之间的偶联作用对骨质疏松症病理进程的影响机制,以期为骨质疏松症的防治提供新思路.

细胞焦亡、铁死亡是区别于传统细胞凋亡、坏死、自噬的两种新型细胞程序性死亡方式,两者具有不同的生物学特征,对骨关节炎软骨基质稳态的调控方式、作用特点亦有区别.通过细胞焦亡、铁死亡对骨关节炎软骨基质稳态的影响及可能作用途径进行初步探讨,为进一步防治骨关节炎开辟新方向.

目的:基于核转录因子-κB(NF-κB)炎症相关信号通路,观察乌头汤水提物对脂多糖(LPS)诱导体外培养软骨细胞炎症模型的影响.方法:①制备乌头汤水提物;②分离、培养软骨细胞;③噻唑蓝(MTT)法检测乌头汤对LPS诱导软骨细胞活性的影响;采用Western Blot法检测NF-κB p65、IκB激酶复合物(IKK)、磷酸化核因子 κB抑制蛋白(pIκB)含量表达.结果:①MTT实验结果显示,乌头汤水提物对LPS诱导软骨细胞的最佳干预时间和浓度分别为24 h和150μg?mL-1;②Western Blot检测结果显示,乌头汤水提物100μg?mL-1组、150μg?mL-1组、200μg?mL-1组对LPS诱导的软骨细胞干预24 h后,可调节与炎症相关的NF-κB p65、IKK、pIκB含量表达,尤其以乌头汤水提物150μg?mL-1组变化显著(P<0.01).结论:乌头汤通过降低LPS诱导的软骨细胞中与炎症相关的NF-κB途径调节因子含量表达,发挥抗炎作用.

目的:利用生物信息学方法分析肿瘤坏死因子-α(TNF-α)和白细胞介素-17A(IL-17A)对骨关节炎患者成纤维样滑膜细胞的作用机制.方法:从GEO数据库中下载GSE93720基因芯片数据集,利用GEO2R筛选出差异表达基因.用DAVID 6.8在线数据库对差异表达基因进行GO分析和KEGG信号通路分析,String数据库构建蛋白质间相互网络,导入Cytoscape 3.7.1软件进行可视化编辑,筛选出网络中的核心基因,然后用MCODE插件进行子网络模块分析.结果:共筛选出差异表达基因256个,其中上调基因213个,下调基因43个.差异最显著的5个基因是C15orf48、CXCL8、CCL20、CXCL3、CXCR4.上调的差异基因GO分析主要涉及免疫反应、炎症反应、信号转导、细胞因子活性、趋化因子活性等.上调的差异基因KEGG信号通路分析主要涉及细胞因子-细胞因子受体相互作用、肿瘤坏死因子信号通路、趋化因子信号通路等.结论:利用生物信息学方法分析TNF-α 和IL-17A诱导的骨关节炎成纤维样滑膜细胞的基因表达谱变化,为探究骨关节炎的发病机制提供指导.