槲皮素通过抑制NF-κB减弱类风湿关节炎大鼠软骨细胞基质降解和细胞凋亡

目的探讨槲皮素(quercetin,Quer)对NF-κB活性的影响及对类风湿关节炎大鼠软骨细胞基质降解和细胞凋亡的作用。方法建立Ⅱ型胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠模型,设立CIA模型组、Quer治疗组并以正常大鼠作为对照组,比较各组大鼠AI指数和足趾容积;ELISA检测关节腔液中IL-1β、MMP-13含量;Western blot检测关节软骨组织中p-p65、MMP-13蛋白的表达。体外培养大鼠软骨组织细胞,分为control组、IL-1β处理组,检测软骨组织培养液中MMP-13、Hyp和蛋白多糖含量。将体外培养的软骨细胞分为3组:control组、IL-1β组、IL-1β+Quer组,检测软骨细胞内p-p65、MMP-13、COL2A1蛋白表达,流式检测细胞凋亡。结果与control组相比,CIA组大鼠AI指数和足趾容积明显增加,关节腔液中IL-1β、MMP-13含量以及软骨组织中p-p65、MMP-13蛋白表达明显增加;给予Quer治疗可显著降低大鼠的AI指数和足趾容积,使软骨组织内p-p65、MMP-13蛋白表达量明显减少,关节腔液中IL-1β、MMP-13含量明显降低。IL-1β处理可明显升高软骨组织培养基中MMP-13、Hyp含量,使蛋白多糖含量明显减少。IL-1β处理明显上调软骨细胞内p-p65、MMP-13蛋白表达,使COL2A1蛋白表达明显减少,软骨细胞凋亡明显增加;给予Quer处理则明显抑制软骨细胞内p-p65、MMP-13蛋白表达,使COL2A1表达量增加,细胞凋亡明显减少。结论 Quer可通过抑制NF-κB激活,减弱炎症环境下软骨细胞内MMP-13产生、基质降解和细胞凋亡,起到保护软骨的作用。     

Lunasin减轻大鼠运动性关节软骨损伤

目的探讨多肽Lunasin对运动性关节软骨损伤的治疗作用及机制。方法将大鼠随机分为对照组和模型组,在注射给予木瓜蛋白酶造模后,标准饲养28 d,然后将模型大鼠分为单纯模型组、0.01、0.05和0.1 mmol/L Lunasin治疗组(每组10只,治疗时间1个月)。治疗结束后分别用ELISA和RT-PCR检测血清及组织中IL-1β、IL-6、TNF-α、MMP-6及MMP-8 mRNA的表达;用试剂盒检测SOD活性及i NOS,用Western blot检测NRF2、Keap1、LC-3Ⅱ、Bax、Beclin1、p-AMPK和AMPK蛋白的表达。结果模型组中IL-1β、IL-6、TNF-α、MMP-6以及MMP-8在血清及组织中含量较对照组均显著升高(P0.05),Lunasin治疗后上述因子均显著回降(P0.05);并且Lunasin治疗能显著提高SOD的活性(P0.05),上调NRF2的表达并降低i NOS的产生(P0.05);另外,Lunasin能够上调受损关节软骨组织中自噬蛋白Beclin1以及LC-3Ⅱ的表达,抑制软骨细胞的凋亡蛋白Bax的表达。结论 Lunasin能够通过降低炎性介质的产生、激活氧化应激系统以及自噬通路,促进受损关节的修复。     

丹参酮ⅡA对COPD大鼠炎性反应及肺组织MMP-9与MCP-1表达的影响

目的 探讨丹参酮ⅡA对COPD大鼠炎性反应及肺组织MMP-9与MCP-1表达的影响.方法 36只SD雄性大鼠随机分为对照组、模型组与丹参酮Ⅱ A组,各12只.选用熏烟和气管内注入脂多糖(LPS)法建立大鼠COPD模型.收集支气管肺泡灌洗液(BALF)进行细胞计数,采用酶联免疫吸附(ELISA)方法测定BALF中TNF-α、IL-1β与IL-6的含量;分别用Western blot方法与逆转录-聚合酶链反应(RT-PCR)方法测定肺组织中MMP-9与MCP-1蛋白和mRNA的表达.结果 与对照组相比,COPD组大鼠BALF中巨噬细胞(AMC)、中性粒细胞(NEU)和淋巴细胞(LYM)的细胞总数显著升高(P＜0.01),炎性因子TNF-α、IL-1 β与IL-6的含量显著升高(PP＜0.01),肺组织中MMP-9与MCP-1的蛋白与mRNA表达均显著升高(P＜0.01).给予丹参酮Ⅱ A预处理后,COPD组大鼠BALF中AMC、NEU和LYM的细胞总数显著降低(P＜0.01),炎性因子TNF-α、IL-1β与IL-6的含量显著降低(P＜0.01),肺组织中MMP-9与MCP-1的蛋白与mRNA表达均显著降低(P＜0.01).结论 丹参酮ⅡA能够降低COPD大鼠炎性反应,下调肺组织MMP-9与MCP-1的表达.     

NF-κB抑制剂减低LPS致兔腰椎间盘髓核细胞锌指蛋白A20表达升高

目的观察脂多糖(LPS)刺激退变兔椎间盘髓核细胞前后,锌指蛋白A20(A20)、NF-κB及相关炎性因子的表达及其相互关系。方法获取正常和退变兔腰椎间盘髓核细胞,分为正常组、退变组、LPS刺激组和NF-κB抑制剂组,HE染色观察椎间盘髓核及纤维环的形态,免疫组化检测椎间盘A20、NF-κB p65蛋白及Ⅱ型胶原(COL-Ⅱ)的表达。Real-time PCR分别检测各组A20、IL-1β、TNF-α、NF-κB和COL-ⅡmRNA的表达。Western blot观察A20、p65和COL-Ⅱ的表达。ELISA检测细胞培养上清液中IL-1β、TNF-α的表达。结果退变组髓核细胞数量明显减少,髓核细胞聚集成团,COL-Ⅱ表达下降,A20和p65蛋白表达升高;与正常组相比,退变组A20、TNF-α、IL-1β和p65表达在mRNA及蛋白水平均明显升高,COL-Ⅱ表达降低(P0.05);LPS刺激组中A20、TNF-α、IL-1β和p65表达较退变组更显著,COL-Ⅱ降低明显(P0.05);NF-κB抑制剂组较LPS刺激组A20、TNF-α、IL-1β和p65表达回降,COL-Ⅱ表达回升(P0.05)。结论髓核细胞炎性反应与兔椎间盘退变的发生和发展密切相关,其中A20可能发挥了重要作用。     

肺气肿模型大鼠肾组织水通道蛋白2的表达及机制研究

目的: 观察肺气肿模型大鼠肾组织AQP2表达以及血中ET、IL-1β、TNF-α水平的变化,探讨在体液代谢过程中肺与肾脏之间关系。方法: 用气管内注入脂多糖加熏香烟方法复制肺气肿大鼠模型,应用免疫组化、Western blotting、RT-PCR方法测定肾组织AQP2蛋白及mRNA表达的变化,应用放免法测定血中、肺组织匀浆中ET、IL-1β及TNF-α含量。结果: 与对照组比较,肾组织AQP2蛋白及mRNA表达增强(P<0.01);同时模型组血、肺组织ET、IL-1β及TNF-α含量明显升高(P<0.01)。结论: 肺气肿模型大鼠肾组织AQP2表达上调;其机制可能与血中ET、IL-1β、TNF-α含量升高有关。     

基于Hedgehog通路探讨补肾活血方对实验性膝骨性关节炎兔退变软骨的保护作用

目的:探究补肾活血方(BSHX)对实验性膝骨性关节炎兔退变软骨的保护作用及其可能的机制。方法:将新西兰兔分为对照(control)组、模型(model)组、BSHX(1.86 g/kg)组、Hedgehog通路激活剂SAG(20 mg/kg)组和BSHX+SAG组,每组6只。测定兔膝关节宽度,处死兔后观察膝关节软骨大体情况,对软骨退变情况进行评定;HE染色和番红O染色观察膝关节软骨组织病理形态学变化;ELISA法检测软骨组织中肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)水平;免疫组化法检测软骨组织中基质金属蛋白酶13(MMP-13)和II型胶原(Col-II)蛋白表达;Western blot法检测软骨组织中Shh、Ptch1、Smo和Gli1蛋白表达。结果:与control组相比,model组膝关节宽度增大,软骨退变评分、软骨分级和Mankin评分升高,软骨组织中TNF-α和IL-1β水平升高,MMP-13阳性表达率升高,Col-II阳性表达率降低,Shh、Ptch1、Smo和Gli1蛋白表达升高(P<0.05)。与model组相比,BSHX组膝关节宽度减小,软骨退变评分、软骨分级和Mankin评分降低,软骨组织中TNF-α和IL-1β水平降低,MMP-13阳性表达降低,Col-II阳性表达升高,Shh、Ptch1、Smo和Gli1蛋白表达降低;SAG组膝关节宽度增大,软骨退变评分、软骨分级和Mankin评分升高,软骨组织中TNF-α和IL-1β水平升高,MMP-13阳性率表达升高,Col-II阳性表达率降低,Shh、Ptch1、Smo和Gli1蛋白表达升高(P<0.05)。BSHX+SAG组膝关节宽度,软骨退变评分,软骨分级,Mankin评分,软骨组织中TNF-α和IL-1β水平,MMP-13阳性表达率,以及Shh、Ptch1、Smo和Gli1蛋白表达均低于SAG组,Col-II阳性表达率高于SAG组(P<0.05)。结论:补肾活血方对实验性膝骨性关节炎兔退变软骨具有保护作用,其机制可能与抑制Hedgehog通路有关。     

脉冲电磁场通过Wntβ-catenin信号通路改善膝骨关节炎大鼠炎症反应的机制

目的:探讨脉冲电磁场通过Wntβ-catenin信号转导机制改善膝骨关节炎大鼠炎症反应的机制。方法:选取90只SD大鼠,均分为正常组、模型组和脉冲电磁场组,除正常组,其余各组均构建膝骨关节炎模型。测定各组大鼠局部皮温、膝关节周径和Lequesne MG评分;采取甲苯胺蓝染色进行Mankins评分;Western Blot检测软骨细胞凋亡调控蛋白Caspase-3和Caspase-8水平;ELISA法测定滑膜IL-1β、MMP-13、TNF-α表达;并检测Wnt和β-catenin mRNA和蛋白表达差异;检测大鼠线粒体膜电位。结果:与正常组相比,模型组大鼠膝关节局部皮温、膝关节周径、Lequesne MG评分、Mankins评分、Wntβ-catenin表达以及滑膜IL-1β、MMP-13、TNF-α水平均上调（P<0.05）;与模型组比较,脉冲电磁场组膝关节周径、Lequesne MG评分,Mankins评分、Wntβ-catenin表达以及滑膜IL-1β、MMP-13、TNF-α水平均下调（P<0.05）。相较于正常组,其余2组的关节软骨线粒体膜电位均有所下调。与模型组相比,脉冲电磁场组线粒体膜电位有所上调,脉冲电磁场组优于模型组。结论:脉冲电磁场能抑制膝骨关节炎模型大鼠炎症反应,有效修复软骨损伤,降低炎症因子表达,抑制Wntβ-catenin信号转导。     

丹参注射液治疗对膝骨性关节炎模型兔关节软骨中TNF-α、IL-6、MMP-3表达的影响

目的 研究丹参注射液治疗膝骨性关节炎(knee osteoarthritis,KOA)模型兔关节软骨中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-6、基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)的表达情况,探讨丹参注射液治疗膝骨性关节炎的分子机制。方法 选用40只健康白兔,随机分成四组,即假手术组、KOA模型(KOA)组、KOA+透明质酸钠(sodium hyaluronate,SH)组、KOA+丹参组。假手术组只作切口,不切断半月板,其余组均采用切断兔右膝关节半月板的方式,建立一个兔关节失稳的实验动物模型。KOA组给予0.9%生理盐水(0.7 mL/d),KOA+SH组给予兔关节内注射SH(0.4 mL/d),KOA+丹参组给予丹参注射液(0.7 mL/d),均连续注射5周。采用矿场实验验证兔失稳模型建立是否对兔的运动能力产生影响,并采用聚合酶链反应(polymerase chain reaction,PCR)和Western blot法分别检测兔膝关节软骨中TNF-α、IL-6、MMP-3的mRNA含量和蛋白表达量。酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)检测兔膝关节液中TNF-α、IL-6、MMP-3的含量。结果 在KOA模型建立之前,各实验组动物均无明显的运动能力差异,模型建立之后,KOA组兔子运动的距离明显低于假手术组[(150±5)比(580±9),t=60.610,P<0.05],而KOA+丹参组与KOA组相比,家兔移动的距离明显增加,差异具有统计学意义[(438±27)比(150±5),t=16.730,P<0.05]。KOA组兔关节液中TNF-α、IL-6和MMP-3含量明显高于假手术组[(22.62±2.18)mg/mL比(11.74±2.09)mg/mL,(4.01±0.14)mg/mL比(1.76±0.11)mg/mL,(0.57±0.05)mg/mL比(0.27±0.03)mg/mL,t值分别为4.413、17.620、6.495,P值均<0.05];KOA+丹参组兔关节液中TNF-α、IL-6、MMP-3含量较KOA组明显降低,差异具有统计学意义[(15.01±2.37)mg/mL比(22.62±2.18)mg/mL,(2.47±0.19)mg/mL比(4.01±0.14)mg/mL,(0.40±0.02)mg/mL比(0.57±0.05)mg/mL,t值分别为3.725、8.803、9.185,P值均<0.05]。在兔膝关节软骨的mRNA的检测中,KOA组TNF-α、IL-6、MMP-3的含量较假手术组明显升高[(1.90±0.02)比(1.00±0.15),(1.70±0.02)比(1.00±0.07),(1.60±0.20)比(1.00±0.13),t值分别为11.050、24.250、14.850,P值均<0.05];KOA+丹参组中TNF-α、IL-6、MMP-3的含量较KOA组相比明显降低,差异具有统计学意义[(1.09±0.05)比(1.90±0.02),(1.13±0.09)比(1.70±0.02),(1.04±0.08)比(1.60±0.20),t值分别为32.190、8.822、5.868,P值均<0.05)。在兔膝软骨的蛋白检测中,KOA组TNF-α、IL-6、MMP-3的蛋白表达量较假手术组明显升高,差异具有统计学意义(t值分别为65.280、12.320、22.280,P值均<0.05);KOA+丹参组中TNF-α、IL-6、MMP-3的蛋白含量较KOA组明显降低,差异具有统计学意义(t值分别为26.150、9.053、24.670,P值均<0.05)。结论 丹参注射液可以明显改善KOA组兔的运动能力,同时也降低了TNF-α、IL-6、MMP-3的水平,因此丹参注射液治疗膝骨性关节炎可能是抑制了骨基质蛋白酶的释放,抑制关节软骨的降解,对关节软骨的退行性病变起到控制作用,最终改善膝骨性关节炎的症状。     

火针对膝骨关节炎大鼠关节软骨MMP-3、TGF-β1、TNF-α的影响

目的:探讨火针对膝骨关节炎大鼠关节软骨MMP-3、TGF-β1、TNF-α的影响。方法:20只健康SD雄性大鼠,剃去后肢髋关节至脚趾毛,踝关节背屈70～90度,伸展膝关节至160～180度,纱布固定,将石膏从腹股沟至脚趾缠绕大鼠后肢,脚趾露出,绷带固定,建立KOA大鼠模型,另外10只不作任何处理作为正常组。六周后模型制作成功。20只模型大鼠随机分为2组,模型组和火针组。火针组选取膝前穴、阿是穴和阳陵泉穴,进行火针治疗,每3 d行一次针,共治疗6次。治疗前后对各组大鼠进行Lequesne MG评分评估,全自动生物化学分析仪测定各组大鼠血清MMP-3、TGF-β1、TNF-α的表达水平,HE染色后观察大鼠膝关节软骨病理形态变化,采用放射免疫分析法检测大鼠软骨组织中MMP-3、TGF-β1、TNF-α含量。结果:(1)与对照组比较,模型组大鼠血清MMP-3、TGF-β1和TNF-α表达水平显著增高,差异有统计学意义(P0. 05)。(2)模型组大鼠膝骨关节软骨表面粗糙,软骨细胞肥大,表层细胞坏死,巢状增生,潮线消失,呈明显退行性病变。(3)与对照组比较,模型组大鼠膝关节软骨组织中MMP-3、TGF-β1和TNF-α含量均显著升高(P0. 05)。(4)与模型组比较,火针组大鼠血清MMP-3、TGF-β1和TNF-α表达水平均明显降低,差异有统计学意义(P0. 05)。(5)火针组大鼠膝骨关节软骨表面较光滑,细胞排列规则,表层细胞轻度增生,潮线较完整。(6)与模型组比较,火针组大鼠膝关节软骨组织MMP-3、TGF-β1和TNF-α含量均明显降低,差异有统计学意义(P0. 05)。结论:火针治疗可有效减轻大鼠膝关节软骨损伤,降低血清及膝关节软骨组织中MMP-3、TGF-β1和TNF-α水平,进而发挥抗炎作用,可能是火针治疗膝骨关节炎的一个作用机制。     

HSYA对骨关节炎大鼠膝关节软骨损伤的修复作用研究

目的探讨羟基红花黄色素A(HSYA)对骨关节炎(OA)大鼠膝关节及MMP-3、TGF-β1、Notch1的影响。方法将25只大鼠随机分为正常组(假手术)、OA组(模型大鼠)、低HSYA组(模型大鼠尾部注射2.5 mg/kg HSYA)、中HSYA组(模型大鼠尾部注射5.0 mg/kg HSYA)、高HSYA组(模型大鼠尾部注射10.0 mg/kg HSYA)。采用ELISA检测血清指标IL-1β、TNF-α,Pelletier评分评估膝关节软骨损伤情况,HE染色观察组织病理形态并以Mankin评分进行评估,免疫组化检测MMP-3阳性表达,Western blot检测TGF-β1、Notch1蛋白水平。结果OA组血清中IL-1β、TNF-α水平高于其余各组(P<0.05),高HSYA组IL-1β、TNF-α水平低于低HSYA组和中HSYA组(P<0.05)。各组大鼠Pelletier评分组间比较差异有统计学意义(P<0.05)。OA组软骨和滑膜组织Mankin评分高于其余各组(P<0.05),高HSYA组Mankin评分低于低HSYA组、中HSYA组(P<0.05)。各组大鼠关节组织MMP-3阳性细胞率组间比较差异有统计学意义(P<0.05)。各组大鼠关节组织TGF-β1、Notch1蛋白水平组间比较差异有统计学意义(P<0.05)。结论HSYA能够降低OA大鼠血清炎性因子IL-1β、TNF-α水平,缓解滑膜组织损伤,且存在浓度依赖性,其机制与抑制MMP-3和Notch1表达、提高TGF-β1水平相关。     

Chrysin Attenuates IL-1β-Induced Expression of Inflammatory Mediators by Suppressing NF-κB in Human Osteoarthritis Chondrocytes

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. Chrysin, a natural flavonoid extracted from honey and propolis, has been reported to have anti-inflammatory effects. However, the anti-inflammatory effects of chrysin on OA have not been reported. This study aimed to assess the effects of chrysin on human OA chondrocytes. Human OA chondrocytes were pretreated with chrysin (1, 5, 10 μM) for 2 h and subsequently stimulated with IL-1β for 24 h. Production of NO, PGE2, MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 was evaluated by the Griess reaction and ELISAs. The messenger RNA (mRNA) expression of COX-2, iNOS, MMP-1, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, aggrecan, and collagen-II was measured by real-time PCR. The protein expression of COX-2, iNOS, p65, p-p65, IκB-α, and p-IκB-α was detected by Western blot. The protein expression of collagen-II and p65 nuclear translocation was evaluated by immunofluorescence. We found that chrysin significantly inhibited the IL-1β-induced production of NO and PGE2; expression of COX-2, iNOS, MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5; and degradation of aggrecan and collagen-II. Furthermore, chrysin dramatically blocked IL-1β-stimulated IκB-α degradation and NF-κB activation. Taken together, these results suggest that chrysin may be a potential agent in the treatment of OA.     

红姜提取物保护早期膝骨关节炎模型大鼠的关节软骨

背景:研究报道,联合使用姜提取物降低血清促炎细胞因子白细胞介素1β、白细胞介素6、肿瘤坏死因子α等水平与膝骨关节炎中软骨损伤的减轻有关。目的:观察红姜提取物灌胃对早期膝骨关节炎大鼠关节软骨保护情况及血清白细胞介素1β、白细胞介素6、肿瘤坏死因子α和软骨Col2α1 mRNA水平表达的影响,探讨红姜提取物对早期膝骨关节炎大鼠关节软骨保护作用及可能机制。方法:将50只SPF级SD大鼠随机分为空白组、模型组、红姜低剂量组、红姜高剂量组、阳性对照组,每组各10只。除空白组外,其余40只大鼠膝关节腔注射4%木瓜蛋白酶0.2 mL+0.03 mol/L的L-半胱氨酸混合溶液,建造膝骨关节炎模型。空白组与模型组常规饲养;红姜低剂量组、红姜高剂量组、阳性对照组分别予50 mg/kg的红姜提取物水溶液、100 mg/kg红姜提取物水溶液、18 mg/kg的塞来昔布胶囊水溶液灌胃,所有干预每日1次,共持续4周。治疗4周后取大鼠膝关节软骨进行番红O-固绿染色,并对关节软骨行Mankin评分,检测血清中白细胞介素1β、白细胞介素6、肿瘤坏死因子α及软骨中Col2α1 mRNA表达水平。实验方案经广州中医药大学动物实验伦理委员会批准,批准号:20190917002。结果与结论:①膝关节软骨的病理切片显示,模型组及各治疗组均有软骨基质流失,各治疗组Mankin评分均比空白组评分高(P<0.05),比模型组评分低(P<0.05),其中红姜高剂量组与阳性对照组评分差异无显著性意义(P>0.05),均显著低于红姜低剂量组(P<0.05);②血清白细胞介素1β、白细胞介素6、肿瘤坏死因子α结果显示,阳性对照组、红姜高剂量组、红姜低剂量组均比空白组表达上调(P<0.05),均比模型组表达下调(P<0.05),且各治疗组间水平阳性对照组<红姜高剂量组<红姜低剂量组(P<0.05);③软骨中Col2α1 mRNA结果显示,空白组与红姜高剂量组、阳性对照组的Col2α1 mRNA表达差异无显著性意义(P>0.05),模型组和红姜低剂量组Col2α1 mRNA表达相较其他3组均显著上调(P<0.05);④结果说明,红姜提取物可能主要通过抑制白细胞介素1β、白细胞介素6、肿瘤坏死因子α等炎症因子的表达,达到了保护膝骨关节炎关节软骨作用,从而延缓膝骨关节炎的发展进程;且相对于低剂量组,红姜提取物高剂量组的抗炎效果更好。     

Expression and correlation of matrix metalloproteinase-7 and interleukin-15 in human osteoarthritis

Objectives: To investigate the expression and correlation of matrix metalloproteinase (MMP)-7 and interleukin (IL)-15 in human osteoarthritis (OA). Methods: From October 2013 to December 2014, 30 patients with OA were enrolled. In addition, anther 30 patients with simple meniscus injury were collected as a control group. There were no significant differences in age and gender between the two groups. Articular cartilage tissue was obtained from both OA patients and control group patients. Protein, mRNA, and serum expression levels of MMP-7 and IL-15 in the both two groups were determined by immunohistochemical (IHC), in situ hybridization, and enzyme-linked immunosorbent assay (ELISA) assay, respectively. Additionally, correlation between MMP-7 and IL-15 expression level in cartilage tissue and serum was assessed using Pearson correlation analysis. Results: Protein, mRNA, and serum expression levels of MMP-7 and IL-15 in patients with OA were all significantly increased in OA patients compared with the control group. Besides, there were strong positive relationships between articular MMP-7 level and serum MMP-7 level (R² = 0.573, P = 0.018), between articular IL-15 level and serum IL-15 level (R² = 0.861, P = 0.023), and between serum IL-15 level and serum MMP-7 level (R² = 0.602, P = 0.012). Conclusion: These results suggest that MMP-7 and IL-15 might play important roles in the pathogenesis of OA, and IL-15 and MMP-7 has positive correlation in OA.     

Gene expression and localization of high-mobility group box chromosomal protein-1 (HMGB-1)in human osteoarthritic cartilage

We investigated the expression and localization of high-mobility group box chromosomal protein-1 (HMGB-1) in human osteoarthritic (OA) cartilage in relation to the histopathological grade of cartilage destruction, and examined the role of HMGB-1 in the regulation of proinflammatory cytokine expression in chondrocytes. An immunohistochemical study demonstrated that total HMGB-1-positive cell ratios increase as the Osteoarthritis Research Society International (OARSI) histological grade increased. The population of cytoplasmic HMGB-1-positive chondrocytes was especially increased in the deep layers of higher-grade cartilage. The ratios and localization of receptors for advanced glycation end products (RAGE) expression by chondrocytes in Grade 2, 3, and 4 were significantly higher than those in Grade 1. In vitro stimulation with IL-1β, but not TNFα, significantly upregulated the expression of HMGB-1 mRNA by human OA chondrocytes. Both IL-1β and TNFα promoted the translocation of HMGB-1 from nuclei to cytoplasm. IL-1β and TNFα secretions were stimulated at higher levels of HMGB-1. The results of our study suggest the involvement of HMGB-1 in the pathogenesis of cartilage destruction in OA.     

Expression of matrix metalloproteinases in normal and damaged articular cartilage from human knee and ankle joints

The objectives of this study were the following: (a) describe the appearance of histopathologic changes observed in human articular cartilage from the knee and ankle joints of organ donors with no symptomatic joint disease; (b) compare by in situ hybridization mRNA expression of six matrix metalloproteinases (MMP) in these cartilages; (c) compare MMP mRNA expression with the histology of the cartilage; and (d) test whether the effect of interleukin-1beta (IL-1beta) on the MMP mRNA expression could be detected with in situ hybridization. Human articular cartilages from the knee (tibiofemoral) and ankle (talocrural) joints of 41 different donors (aged 18 to 84 years) were obtained through the Regional Organ Bank of Illinois. The microscopic appearance of the cartilages was graded on a histopathologic scale from 0 to 13 with the highest grade representing severely damaged cartilage. In situ hybridization was performed using oligonucleotide probes to three collagenases (MMP-1, MMP-8, MMP-13), gelatinase A (MMP-2), stromelysin (MMP-3), and matrix type-1 metalloproteinase (MMP-14). Cartilages from some donors were cultured with IL-1beta and then analyzed for MMP expression using in situ hybridization. The histopathology grades of the cartilages from the asymptomatic donors covered the entire scale even in the ankle. Based on their grades, the cartilages were described as either normal (grades 0 to 5) or damaged (grades 6 to 13). The cartilages contained message for all six MMP tested with no detectable differences in expression of MMP-1, -2, -13, and -14 between the normal and damaged cartilages. However the expression of MMP-3 and MMP-8 was elevated in the damaged cartilages. In normal knee cartilage, mRNA expression of MMP-3 and MMP-8 was low, whereas in normal ankle cartilage, MMP-8 expression was below the detection limit. MMP-3 and MMP-8 message was up-regulated in the damaged cartilage from both joints, or if the tissue was cultured in the presence of IL-1beta. From this study we conclude the following: (a) similar histopathologic changes occur in both knee and ankle cartilages; (b) MMP-1, -2, -13, and -14 are constitutively expressed in adult human cartilage; and (c) only up-regulation of mRNA expression of MMP-3 and MMP-8 could be detected with naturally occurring cartilage damage and IL-1beta induction.     

SGTB Promotes the Caspase-Dependent Apoptosis in Chondrocytes of Osteoarthritis

The purpose of this study is to investigate the expression of small glutamine-rich tetratricopeptide repeat (TPR)-containing β (SGTB) in articular cartilage of osteoarthritis (OA) and analyze the relationship between SGTB and chondrocyte apoptosis. We established an OA rat model by the meniscal/ligamentous injury (MLI) modeling method and observed the expression of SGTB in articular cartilage by immunohistochemistry and RT-PCR. Human SW1353 chondrosarcoma cells were treated with interleukin-1β (IL-1β) to mimic the OA-like chondrocyte injury in vitro, and Western blot was employed to examine the IL-1β-induced expression of SGTB and active caspase-3. The co-localization of SGTB and active caspase-3 was confirmed by immunofluorescence. We knocked down SGTB expression by RNA interference (RNAi) and overexpressed SGTB by plasmid transfection. Western blot was carried out to detect the knockdown/overexpressing efficiency of SGTB and evaluate its effects on IL-1β-stimulated expression of active caspase-3 in SW1353 cells. Annexin V/propidium iodide staining was used to detect chondrocyte apoptosis. Then, Western blot was carried out to examine the IL-1β-induced expression of Hsp70 and evaluate SGTB effects on IL-1β-stimulated expression of Hsp70 in SW1353 cells. SGTB expression was significantly up-regulated in articular cartilage of OA rat model. IL-1β stimulation increased the expression of SGTB and active caspase-3 in SW1353 cells. SGTB co-localized with active caspase-3 in IL-1β-treated SW1353 cells. SGTB inhibition significantly reduced IL-1β-stimulated expression of active caspase-3 in SW1353 cells. In line with this, overexpressing SGTB via Myc-SGTB transfection increased the active caspase-3 level in IL-1β-stimulated SW1353 cells. Moreover, flow cytometry assay demonstrated that SGTB knockdown alleviated IL-1β-induced apoptosis, but it was increased in SW1353 cells that overexpressed SGTB. Overexpressing SGTB via Myc-SGTB transfection decreased the Hsp70 level in IL-1β-stimulated SW1353 cells. Our results suggested that SGTB positively regulate the activation of caspase-3 by negatively regulating the activity of Hsp70 and might promote chondrocyte apoptosis in OA. This study may provide a novel insight into the pathophysiology of OA and a potential therapeutic target for its treatment.