透明质酸钠对兔骨关节炎软骨和滑膜中MMP-1-3及其组织抑制物mRNA表达的影响

目的观察透明质酸钠(HA)关节腔注射对骨关节炎(OA)模型关节软骨及滑膜中的基质金属蛋白酶(MMP)-1、MMP-3及其组织抑制物(TIMP)-1mRNA表达水平的影响。方法16只大耳白兔行单侧前交叉韧带切断术,术后5周将动物随机分为实验组和对照组,实验组关节腔注射1%HA0.3ml,每周1次,连续5周,对照组则注射等量生理盐水。术后10周观察两组动物股骨内髁关节软骨光镜下的病理改变,采用反转录-聚合酶链反应(RT-PCR)方法检测关节软骨及滑膜中MMP-1、MMP-3及TIMP-1mRNA的表达。结果实验组软骨退变程度较对照组明显减轻,实验组滑膜中MMP-3的mRNA表达水平显著低于对照组(0.40±0.10vs0.62±0.13),而软骨中的MMP-3的表达较对照组差异无显著性,MMP-1和TIMP-1在实验组和对照组软骨及滑膜中的mRNA表达差异无显著性。结论HA能有效地减轻早期OA关节软骨的退变,其对早期OA的治疗作用的机制之一可能是抑制滑膜MMP-3的表达。     

高脱乙酰度羧甲基壳聚糖对兔膝骨关节炎软骨诱导型一氧化氮合酶表达的影响

目的观察高脱乙酰度羧甲基壳聚糖(HD-CMC)经关节腔注射后对兔膝骨关节炎(OA)软骨诱导型一氧化氮合酶(iNOS)的mRNA和蛋白表达的影响,探讨其用于OA防治的机制及可能性。方法36只日本大耳白兔,行右侧膝关节前交叉韧带切断术(ACLT),术后随机分为2组,实验组于术后当天开始关节腔内注射2%HD-CMC 0．15mg/kg,每2周给药1次,对照组同一时间点关节腔内注射0．9%生理盐水0．15mg/kg。实验组与对照组于第6周分别随机处死大白兔各9只,剩余大白兔于第12周处死,对比两组大白兔股骨髁关节软骨的大体改变,用反转录聚合酶链反应(RT-PCR)及免疫组织化学的方法检测iNOS在软骨中的mRNA和蛋白的表达。结果实验组退变软骨的大体评分轻于对照组,NOS的mRNA和蛋白表达水平均低于对照组。结论HD-CMC能够降低OA退变软骨iNOS的表达,并延缓软骨的退行性变,对OA退变软骨具有修复保护作用。     

壳聚糖膝关节腔内注射疗法对兔骨关节炎关节软骨的影响

目的观察关节内注射羧甲基壳聚糖(CMCTS)对兔骨关节炎(OA)关节软骨退变及软骨基质金属蛋白酶(MMP)-1,-3mRNA表达的影响。方法24只大耳白兔行单侧膝关节前交叉韧带切断术,术后5周将动物随机分为3组,A组关节内注射2%CMCTS0.3ml,每2周1次;B组关节内注射1%透明质酸钠(HA)0.3ml,每周1次;C组关节内不注射药物。术后11周处死动物,观察各组股骨内髁关节软骨的大体变化,采用反转录-聚合酶链反应(RT-PCR)方法检测股骨内髁退变软骨中MMP-1和MMP-3的mRNA表达。结果CMCTS和HA注射组软骨退变程度较不用药组明显减轻,CMCTS注射组软骨MMP-1、MMP-3的mRNA表达明显低于HA注射组和不用药组。HA注射组软骨MMP-1和MMP-3的mRNA表达与不用药组比较,差异没有显著性意义。结论CMCTS能够明显抑制OA软骨MMP-1和MMP-3的mRNA表达,明显减轻软骨退变的程度,CMCTS对早期OA软骨有修复保护作用。     

兔前交叉韧带切断骨关节炎模型软骨MMP-1 MMP-3及诱导型一氧化氮合酶表达的研究

目的观察兔前交叉韧带切断（ACLT）骨关节炎（OA）模型软骨中不同造模时期基质金属蛋白酶（MMP）-1、3及诱导型一氧化氮合酶（iNOS）的表达，为该模型用于OA防治提供理论依据。方法36只大白兔，随机选择27只，每只均行右侧膝关节腔切开前交叉韧带切断术（ACLT），另9只单纯行右侧膝关节腔切开术作为假手术对照组。术后4、8及12周随机处死实验组大白兔各9只及假手术对照组大白兔3只。对比各组大白兔股骨髁关节软骨的大体改变，用反转录-聚合酶链反应及免疫组织化学的方法检测MMP-1、3及iNOS在软骨中的mRNA和蛋白的表达结果。结果ACLT造模术后4周即开始出现软骨早期退变表现，造模8周软骨退变加重，造模12周出现OA晚期软骨退变表现，不同造模时期的大体评分差异有统计学意义，造模4周MMP-1、3及iNOS表达量明显高于对照组，随着造模时间延长MMP-1、3及iNOS表达量进一步升高，MMP-1、3及iNOS在退变软骨的不同阶段表达分布有各自的特点。结论ACLT模型能够表现OA软骨降解退变的全过程，适宜用于OA防治研究，MMP-1、3及iNOS的高表达在OA软骨退变的病理过程中起着重要的作用，随着造模时间延长、软骨退变的加重，表达量持续升高，适合作为OA防治研究中的评价指标。     

透明质酸钠关节腔内注射对兔软骨的保护作用及对过氧化物酶体增殖物激活受体γ mRNA表达的影响

目的 观察关节腔内注射透明质酸钠(Na-HA)对骨关节炎(OA)软骨的保护作用及对过氧化物酶体增殖物激活受体γ(PPAR-γ)mRNA表达的影响,探讨Na-HA治疗OA的机制.方法 48只大耳白兔随机分为A、B、C 3组,每组16只,A组为正常对照组,B、C两组行单膝前交叉韧带切断术,术后第5周开始,B组关节腔内注射0.3 ml生理盐水;C组注射等量的高分子量1%Na-HA,每周1次,连续5周.术后11周处死动物,比较各组股骨内髁关节软骨的大体变化,苏木素-伊红(HE)染色观察股骨内髁软骨的病理变化,番红O染色观察软骨基质的改变,采用实时定量聚合酶链反应(Real-Time PCR)方法检测软骨PPAR-γ mRNA的表达水平.结果 大体评分显示B组软骨退变的程度明显重于A组和C组(P＜0.05);HE染色与A、C两组比较,B组软骨明显溃疡形成;番红O染色平均灰度值B组软骨基质染色明显高于A、c两组(P＜0.05);B组软骨PPAR-γ mRNA表达量明显高于A、C两组(P＜0.05);A、C两组大体评分和PPAR-γ mRNA表达量差异无统计学意义.结论 关节腔注射Na-HA可以抑制软骨PPAR-γmRNA表达,减轻软骨退变的程度,可能是其治疗骨关节炎的机制之一.     

羧甲基化几丁质降低兔实验性骨关节炎软骨基质金属蛋白酶-1表达的实验研究

目的 观察羧甲基化几丁质(CMC)经关节内注射后对兔前交叉韧带切断(ACLT)骨关节炎模型中软骨退变及软骨基质金属蛋白酶-1(MMP-1)表达及分布的影响。方法 20只大白兔行单侧ACLT，随机选取10只作实验组，于术后第1、3、5周分别给予0.3ml 2% CMC关节内注射，另10只于同一时间点给予0．3m1生理盐水关节内注射作对照组。术后第6周处死大白兔，对比两组大白兔股骨髁关节软骨的大体改变和光镜下的病理改变，并用反转录-聚合酶链反应（RT-PCR)及免疫组织化学的方法检测MMP—1在软骨中的mRNA和蛋白表达。结果 实验组软骨退变的大体评分和光镜下Mankin‘s评分都显著低于对照组，RT—PCR亦显示MMP-1在实验组中的表达明显低于其在对照组中的表达。免疫组织化学显示MMP-1主要表达于软骨表层及中上层，实验组MMP-1表达量亦明显低于对照组．结论 CMC能明显降低骨关节炎(OA)软骨中MMP-1的mRNA和生白表达水平，并明显降低软骨退变的程度，有可能成为防治OA的良好药物。     

一氧化氮合酶抑制剂在实验性兔关节软骨修复中的作用

目的：探讨一氧化氮合酶（iNOS)抑制剂S－甲基异硫脲在实验性关节软骨修复中的作用。方法：20只新西兰兔随机分为对照组和实验组，在双侧股骨髁间滑车关节面作全层软骨缺损，对照组应用纤维蛋白凝胶BMP复合物充填缺损，实验组同样充填缺损后，并皮下注射iNOS抑制剂S－甲基异硫脲，术后16周处死动物，作修复组织质量评价，在狼猩红－苦味酸染色检测I型和Ⅱ型胶原分布；反转录－多聚酶链反应（RT－PCR）检测iNOS和MMP－9 mRNA基因表达，BrdU检测软骨细胞增生情况，并利用图像仪进行分析。结果：形态学观察证实，术后16周，实验组关节软骨缺损修复在评分方面优于对照组（P＜0．05），天狼猩红－苦味酸染色显示实验组I型胶原明显少于对照组，II型胶原多于对照组；术后16周RT－PCR在对照组检测到iNOS mRNA和MMP－9 mRNA表达，实验组仅检测到少量MMP－9 mRNA表达，实验组BrdU阳性细胞8．5个／m^2明显多于对照组3．2个／m^2，结论：iNOS抑制剂S－甲基异硫脲的合理应用可明显提高软骨修复组织的质量。     

羧甲基壳聚糖对兔骨关节炎软骨一氧化氮合酶表达的影响

目的探讨关节腔注射羧甲基壳聚糖(CMCTS)对骨关节炎(OA)软骨诱导型一氧化氮合酶(iNOS)表达的影响。方法大耳白兔32只,每只兔单侧膝关节行前交叉韧带切断术,术后5周将兔随机分为4组,每组8只,A组：关节腔注射2%高相对分子质量的CMCTS 0．3ml,每2周重复1次；B组：同等条件下注射2%低相对分子质量的CMCTS 0．3ml；C组：关节腔注射1%透明质酸钠(Na-HA)0．3ml,每周重复1次；D组：关节腔不注射任何药物。术后11周处死动物。采用免疫组织化学、反转录聚合酶链反应(RT-PCR)方法检测软骨iNOS的表达。结果免疫组织化学及RT-PCR显示CMCTS注射组软骨iNOS的表达水平显著低于Na-HA注射组和不用药组,不同相对分子质量CMCTS注射组之间、Na-HA注射组和不用药组之间比较,iNOS的表达差异无统计学意义。结论CMCTS能够明显抑制OA软骨i NOS的表达,Na-HA对OA软骨iNOS的表达没有显著下调作用。     

透明质酸钠对兔骨关节炎滑膜iNOS的表达及滑液一氧化氮含量的影响

目的观察透明质酸钠(SH)对兔骨关节炎模型滑膜诱导型NO合酶(iNOS)的表达及滑液中NO含量的影响。方法16只大耳白兔行单膝前交叉韧带切断术,术后5周将动物随机分为实验组和对照组,实验组关节腔注射1％SH 0．3 ml,每周1次,连续5周；对照组则注射等量生理盐水。术后10周处死动物,采用反转录-聚合酶链反应(RT-PCR)方法检测滑膜iNOS的mRNA表达；采用硝酸还原酶法测定滑液NO的含量。结果实验组滑膜iNOS mRNA的表达水平(0．47±0．09)较对照组(0．65±0．12)显著降低(P＜0．01)。实验组滑液中NO的含量[(134±12)→μmol/L]与对照组[(152±16)μmol/L]比较明显下降(P＜0．05)。结论SH对兔骨关节炎关节滑液中NO的含量有降低作用,这可能与SH抑制滑膜iNOS的表达有关。     

一氧化氮合酶抑制剂调控白细胞介素1β和脂多糖对关节软骨的损害

目的：评价兔膝关节内注射白细胞介素－1β（IL-1β）和脂多糖（混合液后，一氧化氮合酶（iNOS）抑制剂S－甲基异硫脲（SMT）调控关节软骨代谢的作用。方法：实验分为3组，正常组：不使用任何干预药物；对照组：皮下注射生理盐水后，膝关节内给予白细胞介素－1（IL－1）和LPS；实验组：SMT持续皮下注射72h后，关节内注射内浓度IL－1β和LPS。经8h后通过检测硝酸盐和亚硝酸盐的含量来观察膝关节滑液、滑膜和软骨NO的释放量以及iNOS的活性；用原位杂交检测软骨iNOS mRNA的表达。48h后，通过Na2^35SO4掺入法检测关节软骨蛋白糖的合成率。结果：IL－1β和LPS混合液可诱发iNOS mRNA显著表达，使兔膝关节滑液、滑膜和软骨释放大量NO和增加NOS活性。关节软骨是关节内NO的主要来源。SMT减少了NO释放，降低了NOS活性，抑制了iNOS mRNA表达，并且部分逆转了蛋白多糖的抑制作用。结论：iNOS抑制剂SMT对关节软骨的损伤具有部分保护作用；体内高浓度药物的维持是其发挥作用的保证。     

Trichostatin A inhibits expression of cathepsins in experimental osteoarthritis

The aim of this study was to investigate the effects of trichostatin A (TSA) on expression of cathepsins in cartilage in experimental osteoarthritis (OA). OA was induced in 18 rabbits by bilateral anterior cruciate ligament transection (ACLT). Four weeks after surgery, rabbits received intra-articular injection with TSA dissolved in the dimethylsulphoxide (DMSO) in the right knees and DMSO in the left knees once a week for 5 weeks. Rabbits were killed 7 days after the last injection. The knee joints were assessed by morphological and histological examination. Messenger RNA expression of cathepsins K, B, L, S and cystatin C was studied by real-time PCR. TSA inhibited the expression of cathepsins K, B, L, S and cystatin C accompanied with the less degradation in cartilage. The results suggest that TSA exhibits protective effects against cartilage degradation in rabbits with OA and the effects may be associated with the inhibition of cathepsins.     

Effect of dehydroepiandrosterone on cartilage and synovium of knee joints with osteoarthritis in rabbits

The aim of this study was to investigate the effects of intra-articular injection of dehydroepiandrosterone (DHEA) on cartilage and synovium of knee joints with osteoarthritis (OA) in rabbits and the underlying mechanism. Forty rabbits underwent unilateral anterior cruciate ligament transaction and were divided into two groups. Rabbits were injected with 100 μmol/l DHEA dissolved in the dimethylsulphoxide (DMSO) in the knee joints 5 weeks after transaction, once a week for 5 weeks. Rabbits injected with DMSO under the same condition were served as a control. All rabbits were killed 1 week after the last injection. The knee joints were evaluated by gross morphology, histology, and gene expression analysis. Gross morphologic inspection and histological evaluation showed that the DHEA group appeared less damage in cartilage and synovium as compared with the control. Gene expression analysis revealed that the mRNA expression of matrix metalloproteinase-3 (MMP-3) in cartilage and synovium decreased significantly in the DHEA group and that of tissue inhibitor of metalloproteinase-1 (TIMP-1) increased. No significant difference of interleukin-1 beta (IL-1β) mRNA expression was found in the cartilage between two groups while the mRNA expression of IL-1β in the synovium was largely suppressed in the DHEA group. The study suggests that DHEA plays a protective role against cartilage degradation and synovium inflammation in rabbits with OA. This role may be achieved through the regulation of the MMP-3, TIMP-1, and IL-1β gene expression in the cartilage and synovium.     

Astaxanthin ameliorates cartilage damage in experimental osteoarthritis

Abstract

Purpose. Astaxanthin is a red-pigment carotenoid found in certain marine animals and plants. Astaxanthin has been shown to inhibit matrix metalloproteinases (MMPs) expression in vitro. However, the effect of astaxanthin on cartilage is still unclear. The aim of this study was to investigate the effects of astaxanthin on cartilage in experimental osteoarthritis (OA).

Methods. New Zealand rabbits underwent anterior cruciate ligament transection to induce OA in right knee. Rabbits received intra-articular injection containing 0.3 ml of vehicle (dimethyl sulfoxide) or astaxanthin (50 μM). Injection was started on the day of operation, and the injection were performed once weekly for six consecutive weeks. Then, rabbits were sacrificed and the right knees were harvested for study.

Results. Cartilage degradation was reduced by astaxanthin, as assessed by morphological and histological examination. Astaxanthin inhibited the gene expression of MMP-1, MMP-3, and MMP-13 in cartilage as compared with the vehicle group.

Conclusions. The results suggest that astaxanthin may be considered as pharmaceutical agent in OA treatment.     

Expression of MMP-1 in cartilage and synovium of experimentally induced rabbit ACLT traumatic osteoarthritis: immunohistochemical study

To observe the level and the distribution of MMP-1 in cartilage and synovium over the progression of OA in rabbit ACLT model, and to explore the relationship between the amount of MMP-1 expression and the degree of OA cartilage degradation. OA was induced in 20 rabbits by anterior cruciate ligament transection (ACLT) and ten rabbits were killed at 4 and 8 weeks after the operation, respectively. A further ten rabbits received unilateral knee arthrotomy as controls. Cartilage degradation was observed under dissecting microscope, immunohistochemical, and morphometric analysis were adopted to record the tissue level and distribution of MMP-1 in cartilage and synovium. Cartilage degradation in both ACLT groups was more severe than that in control group, and the degradation in 8-week ACLT group was more severe than that in 4-week ACLT group. MMP-1 was expressed predominantly in the superficial and upper intermediate layers of cartilage and in the lining layer of synovium. Its expression was increased steadily over the progression of OA both in cartilage and in synovium, but there was a little difference in the increase pattern between them: increase of MMP-1 in synovium lagged behind that in cartilage in early stage of OA. Conclusion: MMP-1 was involved in OA development in rabbit ACLT model and the amount of its expression was related with the degree of cartilage degradation. The increase of MMP-1 expression in synovium lagged behind that in cartilage, suggesting OA pathology was originated from cartilage, but synovitis may also paticipate in cartilage degradation, especially in middle and late stage of OA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.