硫酸镁启动自噬对兔软骨保护作用机制研究北大核心CSCD

目的探讨硫酸镁通过启动自噬保护兔软骨的作用机制。方法取24只成年雌性新西兰兔,采用前交叉韧带切断方法制备创伤性关节炎(post-traumatic osteoarthritis,PTOA)模型,随机分为PTOA组、蒸馏水组和硫酸镁组,每组8只。术后即刻开始,蒸馏水组及硫酸镁组分别于关节腔内注射0.5 mL蒸馏水及20 mmol/L硫酸镁溶液,每周3次,连续4周;PTOA组不作处理。术后观察动物一般情况;术后4周取材,ELISA检测关节腔积液TNF-α和Ⅱ型胶原及静脉血中Ⅱ型胶原表达量,Western blot检测股骨软骨组织中瞬时感受电位V5(transient receptor potential channel vanilloid 5,TRPV5)及微管相关蛋白1轻链3(microtubule associated protein1 light chain 3,LC3)蛋白(LC3-Ⅱ/LC3-Ⅰ)表达,实时荧光定量PCR检测滑膜组织中IL-1β、TNF-α、基质金属蛋白酶3(matrix metalloproteinases 3,MMP-3)及软骨组织中Ⅱ型胶原、蛋白聚糖(Aggrecan,AGN)、SOX9 mRNA水平,软骨组织切片行HE、Masson、阿利新蓝染色并参照改良组织学骨关节炎(osteoarthritis,OA)评分标准评分。结果各组动物均存活至实验完成。与其他两组比较,硫酸镁组关节腔积液中TNF-α以及关节腔积液及静脉血中Ⅱ型胶原表达量均降低,TRPV5蛋白表达明显降低、LC3-Ⅱ/LC3-Ⅰ比值明显升高,滑膜组织中IL-1β、TNF-α、MMP-3 mRNA表达降低,软骨组织中Ⅱ型胶原、AGN、SOX9 mRNA表达升高,OA评分亦显著降低,差异均有统计学意义(P<0.05)。PTOA组及蒸馏水组以上指标比较,差异均无统计学意义(P>0.05)。结论关节腔内注射硫酸镁可减轻兔关节内炎症,减少Ⅱ型胶原及AGN丢失,有利于软骨再生,其作用机制可能是通过抑制钙离子通道TRPV5,进而启动软骨自噬。     

硫酸镁启动自噬对兔软骨保护作用机制研究

目的探讨硫酸镁通过启动自噬保护兔软骨的作用机制。方法取24只成年雌性新西兰兔,采用前交叉韧带切断方法制备创伤性关节炎(post-traumatic osteoarthritis,PTOA)模型,随机分为PTOA组、蒸馏水组和硫酸镁组,每组8只。术后即刻开始,蒸馏水组及硫酸镁组分别于关节腔内注射0.5 mL蒸馏水及20 mmol/L硫酸镁溶液,每周3次,连续4周;PTOA组不作处理。术后观察动物一般情况;术后4周取材,ELISA检测关节腔积液TNF-α和Ⅱ型胶原及静脉血中Ⅱ型胶原表达量,Western blot检测股骨软骨组织中瞬时感受电位V5(transient receptor potential channel vanilloid 5,TRPV5)及微管相关蛋白1轻链3(microtubule associated protein1 light chain 3,LC3)蛋白(LC3-Ⅱ/LC3-Ⅰ)表达,实时荧光定量PCR检测滑膜组织中IL-1β、TNF-α、基质金属蛋白酶3(matrix metalloproteinases 3,MMP-3)及软骨组织中Ⅱ型胶原、蛋白聚糖(Aggrecan,AGN)、SOX9 mRNA水平,软骨组织切片行HE、Masson、阿利新蓝染色并参照改良组织学骨关节炎(osteoarthritis,OA)评分标准评分。结果各组动物均存活至实验完成。与其他两组比较,硫酸镁组关节腔积液中TNF-α以及关节腔积液及静脉血中Ⅱ型胶原表达量均降低,TRPV5蛋白表达明显降低、LC3-Ⅱ/LC3-Ⅰ比值明显升高,滑膜组织中IL-1β、TNF-α、MMP-3 mRNA表达降低,软骨组织中Ⅱ型胶原、AGN、SOX9 mRNA表达升高,OA评分亦显著降低,差异均有统计学意义(P0.05)。PTOA组及蒸馏水组以上指标比较,差异均无统计学意义(P0.05)。结论关节腔内注射硫酸镁可减轻兔关节内炎症,减少Ⅱ型胶原及AGN丢失,有利于软骨再生,其作用机制可能是通过抑制钙离子通道TRPV5,进而启动软骨自噬。     

跳骨片抑制大鼠膝骨关节炎Wnt/β-catenin信号转导通路介导炎症反应的机制研究

目的探讨跳骨片对大鼠膝骨关节炎(OA)Wnt/β-catenin信号转导通路介导炎症反应的机制。方法将30只8周龄雄性SD大鼠随机分为空白组、模型组、跳骨片组,每组10只。空白组不予任何处理,模型组、跳骨片组均采用改良Hulth法建立OA动物模型。建模成功后跳骨片组每天予跳骨片0.32 g/kg灌胃,空白组和模型组每天予等量生理盐水灌胃,干预12周后分别取3组大鼠双侧膝关节股骨髁、关节软骨和滑膜,将股骨髁常规固定、脱钙、包埋及切片,对切片分别进行苏木精-伊红(HE)、甲苯胺蓝染色,光镜下观察关节软骨组织形态学变化;酶联免疫吸附测定(ELISA)检测滑膜中白细胞介素(IL)-1β、基质金属蛋白酶(MMP)-13、肿瘤坏死因子(TNF)-α含量;蛋白质免疫印迹检测关节软骨β-catenin、Wnt-4蛋白表达。结果模型组滑膜中,IL-1β表达量明显高于空白组和跳骨片组(P0.01),MMP-13表达量高于空白组和跳骨片组(P0.05),TNF-α表达量明显高于空白组(P0.01)、高于跳骨片组(P0.05);模型组关节软骨中,β-catenin蛋白表达量明显高于空白组和跳骨片组(P0.01),Wnt-4蛋白表达量高于空白组和跳骨片组(P0.05)。HE染色显示跳骨片组软骨破坏程度低于模型组,甲苯胺蓝染色显示跳骨片组软骨细胞蛋白多糖含量高于模型组(P0.01)。结论跳骨片能抑制关节软骨Wnt/β-catenin信号转导通路,降低滑膜中IL-1β、MMP-13、TNF-α表达,抑制炎症反应介导的软骨基质降解,延缓关节软骨退变。     

髋关节骨性关节炎组织学计量与炎性因子表达

目的 探讨骨性关节炎各组织成分中细胞冈子、金属基质蛋白酶等炎性物质表达水平与疾病的关系.方法 获取行OA组关节置换及创伤性股骨颈骨折组患者手术时的软骨、滑膜组织和软骨下骨,苏木素-伊红(HE)染色行软骨下骨组织形态计量分析,应用放射免疫测定肿瘤坏死因子(TNF)-α与门细胞介素(IL)-1β,以酶联免疫吸附试验(ELISA)测金属基质蛋白酶(MMP)-9蛋白表达水平,并将两组结果进行t检验,软骨下骨组织计量值与细胞因子水平进行相关分析.结果 OA组较非OA对照组软骨下骨骨形成增加,骨质硬化骨小梁数目增加并错乱;滑膜组织中MMP-9表达是调;软骨组织中MMP-9及IL-1 β较对照组提高;而在软骨下骨,MMP-9、TNF-α及IL-β均有增高,并且与软骨下骨组织学改变相关联.结论 证实了OA促炎症条件的病理学基础,并且炎性改变与软骨硬化及关节软骨退变相关.     

治疗型关节炎护膝干预膝骨性关节炎患者滑液细胞因子的研究

目的探讨治疗型关节炎护膝(OA护膝)干预膝骨性关节炎的作用机制。方法选择膝骨性关节炎患者60例,随机分为OA护膝组和微波组各30例,分别采用OA护膝和微波治疗。分别测定两组关节滑液白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(INF-α)、基质金属蛋白酶-3(MMP-3)、透明质酸(HA)浓度。结果护膝组X线Ⅰ、Ⅱ、Ⅲ级患者治疗后IL-1β、IL-6、INF-α、MMP-3含量比治疗前降低,而HA含量升高,结果比较差异有显著统计学意义(P0.01)。OA护膝组与微波组治疗后X线Ⅰ、Ⅱ级患者膝关节滑液IL-1β、IL-6、TNF-a、MMP-3、HA含量比较差异有统计学意义(P0.01,P0.05);两组X线Ⅲ级患者治疗后膝关节滑液IL-1β含量比较也差异有统计学意义(P0.05),而IL-6、INF-α、MMP-3、HA含量比较差异无统计学意义(P0.05)。结论 OA护膝可以有效调节细胞因子、MMP-3、HA的生物效应,从而延缓关节软骨的退变。     

负载miRNA-27b的BMSCs来源的外泌体治疗实验性骨关节炎

[目的]探讨负载miRNA-27b的间充质干细胞来源的外泌体(MSC-27b-Exos)对炎症环境下的软骨细胞的保护作用。[方法]体外利用10 ng/ml白介素-1β(IL-1β)建立软骨细胞炎症环境模型,采用超速离心法收集外泌体,分别以80μg/ml的MSC-27b-Exos,普通外泌体MSC-Exos、inhibitor沉默的MSC-27boff-Exos作用炎症环境下的软骨细胞,并设PBS处理为对照组,检测各处理组软骨细胞凋亡水平,Western blot检测软骨细胞中MMP-13,及凋亡蛋白Caspase的表达水平。体内实验:20只雄性SD大鼠随机分为4组,建立创伤性骨关节炎(post-traumatic osteoarthritis, PTOA)模型,分别注射等量的MSC-miR27b-Exos、MSC-miR27b~(off)-Exos,并设立单纯PBS组和假手术组,6周后取材,观察关节软骨形态;ELISA检测关节液中IL-1β、TNF-α的含量;免疫组化对比各组Ⅱ型胶原表达。[结果]体外实验中,MSC-27b-Exos组的软骨细胞增殖能力较MSC-miR27b~(off)-Exos组增强,较空白组明显旺盛(P0.05),在炎症环境下软骨细胞凋亡明显减弱(P0.05),MSC-27b-Exos组软骨细胞中cleave-Caspase-9水平降低(P0.05),cleave-Caspase-3水平降低(P0.05),MMP-13相对含量减少。SD大鼠体内实验显示, MSC-27bExos组对软骨缺损修复效果优于空白组和MSC-miR27b~(off)-Exos组。MSC-27b-Exos组关节液中IL-1β、TNF-α的含量低于空白组,明显低于MSC-27b~(off)-Exos组。[结论]在实验型SD大鼠PTOA模型中,利用MSC-27b-Exos关节腔内注射具有明显抗炎的作用和减缓关节软骨退行性变。     

兔骨关节炎中基质金属蛋白酶-1、3与白细胞介素-1β表达及关节软骨细胞凋亡的研究

目的 观察骨关节炎(OA)中基质金属蛋白酶(MMP)-1、3与白细胞介素-1β(IL-1β)的表达及关节软骨细胞凋亡,探讨其在OA发病机制中的作用.方法 将20只中国大白兔随机分成正常组(A组)、实验组(B组),每组10只,A组未造模,B组采用Hulth法制成OA模型,4周后取胫骨平台关节软骨进行组织病理学检查,采用免疫组织化学和原位末端标记细胞凋亡检测法分别检测关节软骨和滑膜中的MMP-1、3、IL-1β及软骨细胞凋亡指数(AI)的水平.结果 组织病理学检查可见,B组关节软骨退变程度明显重于A组,符合OA关节软骨退变特征;通过检测MMP-1、3,IL-1β和AI,A组的结果分别是21.005±9.406、18.697±8.225、0.100±0.040和14.900±3.400,B组的结果分别是56.147±22.340、46.182±20.561、0.180±0.060和25.400 ±5.200;B组MMP-1、3和AI的水平均明显高于A组(P＜0.01),B组IL-1β水平高于A组(P＜0.05).结论 MMP-1、MMP-3、IL-1β的表达及关节软骨细胞凋亡在OA发病机制中作用重要,其异常升高与OA关节软骨退变和炎症反应密切相关.     

七叶皂苷A在小鼠软骨细胞及骨关节炎中的作用及相关分子机制

目的:探讨七叶皂苷A(EsA)在小鼠骨关节炎(OA)中的作用及相关分子机制。方法:分离培养C57BL/6小鼠原代软骨细胞,将细胞用10 mg/L白细胞介素(IL)-1β及不同浓度(10、50、100μmol/L)EsA进行处理后,CCK-8检测细胞活性,ELISA法检测细胞培养液上清中IL-6和肿瘤坏死因子-α(TNF-α)含量,实时定量PCR检测基质金属蛋白酶-13(MMP-13)和聚蛋白多糖酶(ADAMTS5)mRNA表达水平,Western blotting检测II型胶原蛋白(Collagen II)、基质金属蛋白酶-9(MMP-9)及缺氧诱导因子(HIF)-2α和核因子-κB(NF-κB)途径相关蛋白表达,免疫荧光染色检测p65表达,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及丙二醛(MDA)试剂盒检测氧化活性水平。将60只C57BL/6雄性小鼠随机分为假手术组、OA组、OA+低剂量EsA组和OA+高剂量EsA组,每组15只,建立了小鼠OA模型后,OA+低剂量EsA组、OA+高剂量EsA组小鼠分别按照5 mg/kg、10 mg/kg进行腹膜注射EsA,每隔一天注射1次,持续6周后处死小鼠,骨密度测量仪检测小鼠股骨近端骨密度,HE染色和番红O固绿染色检测骨组织病理形态学改变。结果:EsA能够抑制IL-1β刺激后引起的细胞存活率下降与TNF-α、IL-6含量升高,使MMP-13和ADAMTS-5 mRNA表达下降,Collagen II蛋白表达升高且MMP-9蛋白表达降低,并抑制了IL-1β诱导的HIF-2α与NF-κB信号通路的激活,使MDA水平下降、SOD与CAT活性升高,差异均有统计学意义(P<0.05)。OA小鼠经低、高剂量EsA治疗后,小鼠股骨骨密度值均升高(P<0.05),骨组织病变现象得到改善。结论:EsA能够抑制小鼠骨关节炎,其机制可能与HIF-2α和NF-κB途径及其发挥抗氧化活性有关。     

5氟脲嘧啶关节腔注射治疗兔膝骨性关节炎的实验研究

[目的]评价5氟脲嘧啶（5-Fluorouracil，5Fu）关节腔注射治疗兔膝骨性关节炎（osteoarthritis，OA）的疗效。[方法]24只兔子制成骨关节炎模型随机分成OA组、5Fu组和对照组，OA组立即处死，5Fu组按5Fu2ms／kg关节腔注射，每周1次连续4次，对照组注射等量生理盐水，最后1次治疗后1周处死。观察3组滑膜组织的光镜、电镜改变及软骨的光镜、MMP-1免疫组化改变，比较软骨Mankin＇s评分及关节液中IL-1的浓度。[结果]5Fu组可见软骨破坏减轻，滑膜炎症明显抑制，Mankin’s评分明显改善（P〈0．01）；关节液IL-1浓度降低（P〈0．05），关节软骨中MMP-1表达减弱。[结论]5Fu关节腔内注射能抑制滑膜炎症，缓解软骨的破坏。     

TLR4/NF-κB通路参与大鼠膝骨关节炎滑膜早期病变的研究

目的:探讨TLR4/NF-κB通路在大鼠膝骨关节炎(OA)滑膜早期病变的表达情况。方法:将8周龄体重为(200±20) g的18只雄性SD大鼠按照随机数字表法分为模型组和对照组,每组9只。模型组按照改良Hulth法构建膝OA模型,对照组不做手术。分别于术后4、 21 d提取滑膜组织和血清,采用PCR法检测CD14、TLR-4、IL-1β、TNF-α、MMP-13、ADAMTS-4表达;采用Western blot法检测NF-κB p65蛋白表达;采用Elisa法检测血清中透明质酸(HA)及Ⅲ型前胶原氨基端(PⅢNP)浓度。结果:术后4、21 d时模型组CD14、ADAMTS-4及NF-κB p65表达均较对照组升高,术后21 d时模型组TLR-4、Ⅱ-1β、TNF-α及MMP-13表达较对照组升高(P0.01)。模型组术后4 d时血清PⅢNP和HA浓度高于对照组,术后21 d时两组比较差异无统计学意义。结论:NF-κB通路可通过CD14/TLR-4早期激活进而触发滑膜分泌炎性因子IL-1β、TNF-a、MMP-13、ADAMTS-4,PⅢNP和HA增加介导膝OA发生。     

A2M inhibits inflammatory mediators of chondrocytes by blocking IL-1β/NF-κB pathway

A hallmark of osteoarthritis (OA) is cartilage degeneration, which has been previously correlated with dramatic increases in inflammatory enzymes. Specifically, interleukin-1β (IL-1β) and subsequent upregulation of nuclear factor kappa B (NF-κB) is implicated as an important player in the development of posttraumatic osteoarthritis (PTOA). Alpha 2-macroglobulin (A2M) can inhibit this inflammatory pathway, making it a promising therapy for PTOA. Herein, we demonstrate that A2M binds and neutralizes IL-1β, blocking downstream NF-κB-induced catabolism seen in in vitro. Human chondrocytes (cell line C28) were incubated with A2M protein and then treated with IL-1β. A2M was labeled with VivoTag™ 680 to localize the protein postincubation. The degree of binding between A2M and IL-1β was evaluated through immunoprecipitation (IP). Catabolic proteins, including IL-1β and NF-kB, were detected by Western blot. Pro-inflammatory and chondrocyte-related gene expression was examined by qRT-PCR. VivoTag™ 680-labeled A2M was observed in the cytoplasm of C28 human chondrocytes by fluorescence microscopy. IP experiments demonstrated that A2M could bind IL-1β. Additionally, western blot analysis revealed that A2M neutralized IL-1β and NF-κB in a dose-dependent manner. Moreover, A2M decreased levels of MMPs and TNF-α and increased the expression of cartilage protective genes Col2, Type2, Smad4, and aggrecan. Mostly importantly, A2M was shown to directly neutralize IL-1β to downregulate the pro-inflammatory responses mediated by the NF-kB pathway. These results demonstrate a mechanism by which A2M reduces inflammatory catabolic activity and protects cartilage after joint injury. Further in vivo studies are needed to fully understand the potential of A2M as a novel PTOA therapy.     

长期低温环境对大鼠膝骨关节炎进展的影响

目的探索低温环境对大鼠膝骨关节炎进展的影响。 方法2019年7月至2019年10月将20只8周龄雄性SD-大鼠根据体重大小编号,将所得编号通过随机数字表法随机分为对照组[n ＝10,饲养温度(25±2)℃]和低温组[n ＝10,饲养温度(15±2)℃],定期剪除两组大鼠双侧膝关节表面毛发。分别在饲养4周和12周后拍摄左膝关节侧位片观察大鼠膝关节影像学情况。饲养12周后,采用颈椎脱臼法处死大鼠,使用酶联免疫吸附(ELISA)法检测大鼠膝关节关节液中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、血管内皮生长因子(VEGF)与基质金属蛋白酶-13(MMP-13)蛋白表达;苏木精-伊红(HE)染色观察软骨组织情况,并结合Mankin’s评分进行对比;免疫组化检测软骨细胞MMP-13蛋白表达情况。IL-1β、TNF-α、VEGF、MMP-13蛋白表达与Mankin’s评分比较均采用独立样本t检验分析。 结果在低温环境下饲养4周后,SD-大鼠的膝关节影像学表现与对照组并无明显差异。在低温环境下饲养12周后,相对于对照组,膝关节侧位片显示低温组大鼠膝关节间隙明显变窄,关节面欠光滑,可见部分关节软骨面塌陷、关节周围骨赘形成;HE染色显示低温组大鼠膝关节软骨面塌陷,小血管侵入软骨层,表层和深层软骨细胞坏死,软骨细胞排列紊乱,两组大鼠的软骨Mankin’s评分差异具有统计学意义(t＝8.01,P<0.01);ELISA与免疫组化结果显示,低温组大鼠膝关节关节液中IL-1β、TNF-α、VEGF、MMP-13蛋白表达增加,差异具有统计学意义(t＝3.70、3.57、4.47、16.11,均为P<0.01)。 结论长期低温环境能够促进IL-1β、TNF-α、VEGF、MMP-13蛋白的表达增加,并最终造成关节软骨的丢失,促进骨关节炎的发生。     

Immunolocalization of selected cytokines and proteases in canine articular cartilage after transarticular loading

Cytokines and proteases are thought to play a role in the destruction of cartilage and the development of osteoarthritis. The purpose of this study was to document chronological involvement of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), stromelysin (MMP-3), fibronectin, and alteration in the chondroitin sulphate sulfation pattern. Canine patellae underwent a closed-joint impact to induce the development of osteoarthritis. The animals were killed at 2, 12, 24, and 52 weeks. The patellar damage included cracks in the superficial zone of cartilage and the zone of the calcified cartilage-bone interface, vertical step-off fractures in the zone of calcified cartilage, and loss of proteoglycan around the cracks in the deep and superficial zones of cartilage. With avidin-biotin immunohistochemistry, these specimens were stained with antibodies to IL-1β, TNF-α, MMP-3, fibronectin, and altered proteoglycan sulfate with the monoclonal antibody 3-B-3. Three of the four specimens obtained at 2 weeks demonstrated a strong cellular and weak matrix staining-pattern for IL-1β, TNF-α, MMP-3, and fibronectin around the cracks in the superficial and transitional zones of cartilage. No consistent staining pattern was noted in the cracks in the deep zone. None of the specimens obtained at 12, 24, or 52 weeks stained for these antibodies. No staining for the abnormal sulfation with the 3-B-3 antibody was evident in any specimen. The specimens obtained at 52 weeks showed healing of the step-off fractures and a filling-in of the proteoglycan loss. This model probably reflects the short-term cartilaginous changes in the patella after trauma; thus, only transient elevations in the cytokines and proteases were evident.     

当归通痹汤联合中药热敷治疗膝关节骨性关节炎的疗效及对IL-1β、MMP-3和TNF-α水平的影响

目的:探讨当归通痹汤联合中药热敷治疗膝关节骨性关节炎的疗效及对白细胞介素(IL)-1β、基质金属酶(MMP)-3和肿瘤坏死因子(TNF)-α水平的影响。方法:选取我院收治的膝关节骨性关节炎患者140例,随机分为两组各70例。对照组给予中药热敷治疗,治疗组在对照组基础上给予当归通痹汤,对比两组治疗后的临床疗效、VAS评分、WOMAC指数和ADL评分,以及IL-1β、MMP-3和TNF-α水平。结果:治疗后,治疗组的总有效率为94.29%,高于对照组的81.43%,差异有统计学意义(P<0.05);两组ADL评分明显升高,WOMAC指数、VAS评分明显降低(P<0.05);且治疗组比对照组明显改善(P<0.05);两组患者治疗后的IL-1β、MMP-3和TNF-α水平均显著低于治疗前(P<0.05);且治疗组低于对照组,差异有统计学意义(P<0.05)。两组患者不良反应发生率差异无统计学意义(P>0.05)。结论:膝关节骨性关节炎患者采用当归通痹汤联合中药热敷治疗,具有较好的临床疗效,能够改善患者膝关节功能,值得在临床上推广应用。     

肿瘤坏死因子α对人腹膜间皮细胞基质金属蛋白酶及其组织抑制物表达的影响

目的 观察肿瘤坏死因子α（TNF-α）对人腹膜间皮细胞(HPMC) 基质金属蛋白酶（MMP）2、MMP-9及其组织抑制物（TIMP）2、TIMP-1 mRNA和Ⅰ型胶原表达的影响，同时观察TNF-α、TGF-β、IL-1单独或协同作用对HPMC的MMP-9活性的影响。 方法 采用半定量RT-PCR法测定细胞MMP-2、MMP-9、TIMP-2及TIMP-1 mRNA 的表达。采用Biotrak MMP-9 活性检测系统来精确定量测定MMP-9活性及MMP-9原的含量。ELISA法检测Ⅰ型胶原蛋白的表达。 结果 TNF-α(1~10 μg/L)分别刺激HPMC 4、16、24及48 h后， HPMC的MMP-9 mRNA表达显著上调，为基础的2.3～4.9倍（P ＜ 0.05），呈时间依赖性。TNF-α（1 μg/L）作用48 h后显著下调TIMP-1、TIMP-2 mRNA 表达，为基础的77.2％、61.3％（P ＜ 0.05），而MMP-2 mRNA表达没有显著变化。TNF-α+TGF-β１ （1~10 μg/L）、 TNF-α+TGF-β1+IL-1(1~10 μg/L)和TNF-α(5~10 μg/L)刺激HPMC 24 h后，促其分泌MMP-9 的作用最为明显。同时，TNF-α明显上调Ⅰ型胶原蛋白表达（P ＜ 0.05）。 结论 TNF-α 明显上调HPMC的MMP-9 mRNA的表达和MMP-9 的活性；联合其他细胞因子后促MMP-9分泌的作用更明显。TNF-α单独或协同其他因子在腹膜纤维化的过程中可能发挥了重要作用。     

miR-204-5p inhibits inflammation of synovial fibroblasts in osteoarthritis by suppressing FOXC1

BackgroundThe paper is aimed at uncovering the mechanism of miR-204-5p in regulating inflammatory responses of human osteoarthritic synovial fibroblasts (SFs).MethodsIL-1β-induced osteoarthritic SFs were established as an osteoarthritis (OA) cell model. The osteoarthritic SFs were accordingly transfected with mimics-miR-204-5p, inhibitors-miR-204-5 or FOXC1 siRNA. MTT tested the vitality of osteoarthritic SFs by analyzing the cell optical density. The expressions of miR-204-5p, FOXC1, TNF-α, IL-6, PGE2, MMP-1, MMP-13 and COX-2 in osteoarthritic SFs were measured by qRT-PCR, Western blotting and/or ELISA. The binding of miR-204-5p to FOXC1 was verified through luciferase reporter assay. The regulatory effect of miR-204-5p on FOXC1 was also tested in normal SFs.ResultsmiR-204-5p was under-expressed and FOXC1 was over-expressed in osteoarthritic SFs. The expressions of FOXC1, TNF-α, IL-6, PGE2, MMP-1, MMP-13 and COX-2 were up-regulated in IL-1β-treated SFs. Up-regulation of miR-204-5p or down-regulation of FOXC1 suppressed the inflammatory responses of osteoarthritic SFs. miR-204-5p negatively regulated FOXC1 by being a sponge in osteoarthritic SFs as well as in normal SFs.ConclusionmiR-204-5p down-regulates FOXC1 to ameliorate inflammation of SFs in OA.     

Early inhibition of subchondral bone remodeling slows load-induced posttraumatic osteoarthritis development in mice

Posttraumatic osteoarthritis (PTOA) is associated with abnormal and increased subchondral bone remodeling. Inhibiting altered remodeling immediately following joint damage can slow PTOA progression. Clinically, however, inhibiting remodeling when significant joint damage is already present has minimal effects in slowing further disease progression. We sought to determine the treatment window following PTOA initiation in which inhibiting remodeling can attenuate progression of joint damage. We hypothesized that the most effective treatment would be to inhibit remodeling immediately after PTOA initiation. We used an animal model in which a single bout of mechanical loading was applied to the left tibia of 26-week-old male C57Bl/6 mice at a peak load of 9 N to initiate load-induced PTOA development. Following loading, we inhibited bone remodeling using daily alendronate (ALN) treatment administered either immediately or with 1 or 2 weeks' delay up to 3 or 6 weeks post-loading. A vehicle (VEH) treatment group controlled for daily injections. Cartilage and subchondral bone morphology and osteophyte development were analyzed and compared among treatment groups. Inhibiting remodeling using ALN immediately after load-induced PTOA initiation reduced cartilage degeneration, slowed osteophyte formation, and preserved subchondral bone volume compared to VEH treatment. Delaying the inhibition of bone remodeling at 1 or 2 weeks similarly attenuated cartilage degeneration at 6 weeks, but did not slow the development of osteoarthritis (OA)-related changes in the subchondral bone, including osteophyte formation and subchondral bone erosions. Immediate inhibition of subchondral bone remodeling was most effective in slowing PTOA progression across the entire joint, indicating that abnormal bone remodeling within the first week following PTOA initiation played a critical role in subsequent cartilage damage, subchondral bone changes, and overall joint degeneration. These results highlight the potential of anti-resorptive drugs as preemptive therapies for limiting PTOA development after joint injury, rather than as disease-modifying therapies after joint damage is established. © 2021 American Society for Bone and Mineral Research (ASBMR).     

壳聚糖微球转染人IL-1Ra与TGF-β1基因治疗兔骨关节炎

目的 探讨壳聚糖微球转染人IL-1Ra与TGF-β1基因治疗兔膝关节早期骨关节炎的方法与效果.方法 制备分别包裹IL-1Ra质粒DNA和TGF-β1质粒DNA的壳聚糖微球缓释系统,分组向兔膝关节早期骨关节炎模型关节腔注射含IL-1Ra基因和(或)TGF-β1基因的壳聚糖微球、不含上述基因的壳聚糖溶液,定期处死,使用ELISA检测关节腔灌洗液的IL-1Ra、TGF-β1浓度,取关节标本Mankin评分、苏木精-伊红(HE)染色、番红O染色及免疫组化检测.结果 经过60 d观察,壳聚糖微球转染基因组的IL-1Ra与TGF-β1的基因可持续表达,双基因组的软骨标本从外观、染色观察,损伤程度轻于单基因组.双基因组的Mankin评分明显低于单基因组(P＜0.05),单基因组的Mankin评分明显低于空白组(P＜0.05).结论 关节腔注射壳聚糖微球转染IL-1Ra与TGF-β1基因能抑制软骨的退变和促进软骨的修复.

Abstract：

Objective To explore the method and effect of transinfection of rabbit early knee osteoarthritis models via chitosan microsphere with gene of recombined human IL-1Ra gene and TGF-β1 gene. Methods Chitosan microspheres with plasmids of IL-1Ra gene and TGF-β1 gene, and rabbit early knee osteoarthritis models were prepared. Rabbits in different groups had intra-articular injections of chitosan microsphere containing IL-1Ra gene and / or TGF-β1 gene, and chitosan solution as control group before being executed regularly and randomly. The joint specimens were evaluated by HE staining, lycopene red O staining and immunohistochemical analysis and Mankin's score. ELISA was used for detection of IL-IRa and TGF-β1 concentration of articular cavity fluid in each group. Results The control group was consistent with the pathological changes of early OA. In co-transinfection group, judging from the appearance and staining of cartilage,the OA damage of the specimens was less serious than other groups'. Its Mankin's score was significantly lower than single-gene transinfection group (P ＜ 0.05), and the latters Mankin's score were significantly lower than control group (P ＜ 0.05). Conclusion Intra-articular injection of chitosan microspheres containing both IL-1Ra gene and TGF-β1 gene could inhibit the degeneration of cartilage and promote cartilage repair.     

Comparison between two rabbit models of posttraumatic osteoarthritis: A longitudinal tear in the medial meniscus and anterior cruciate ligament transection

Animal osteoarthritis (OA) models have been developed to understand OA progression and evaluate new OA therapies. However, individual variations in joint lesions remain a critical problem in most current OA models. We established a novel rabbit model by creating a longitudinal tear in the medial meniscus body that was reproducible and similar to posttraumatic biomechanical disturbances in human OA. New Zealand rabbits underwent surgery and were assessed for 9 weeks. The rabbits were randomized into the sham control, medial meniscal tear (MMT), and anterior cruciate ligament transection (ACLT) groups. The animals were sacrificed at 4, 6, and 9 weeks posttreatment. The knee joints were harvested for histological and gene expression assessments. Both the MMT and ACLT procedures led to time-dependent degenerative changes in the femoral condyle cartilage. At each time point, the MMT group cartilage showed more severe degenerative changes than did the ACLT group cartilage. Consistently, inflammatory cytokine and catabolic gene expression were significantly higher, and anabolic gene expression was significantly lower in the MMT group than in the ACLT group. MMT treatment caused more severe structural damage to the cartilage and higher catabolic gene expression levels than the ACLT model at each time point. The MMT model may be highly beneficial in investigating posttraumatic OA (PTOA) development, especially PTOA from a meniscal injury. The MMT model replicated key features of human PTOA, including meniscal lesions, inflammatory responses, and the progression to osteoarthritic cartilage degeneration, thereby providing an exciting new avenue for translating promising treatments to clinical practice.