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。     

透明质酸钠对兔骨关节炎软骨诱导型NO合酶基因表达的影响

目的观察透明质酸钠(Na—HA)关节腔注射对骨关节炎(OA)模型关节软骨中诱导型NO合酶(iNOS) mRNA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％Na—HA 0.3ml，每周1次，连续5周，对照组则注射等量生理盐水。术后10周处死动物，观察两组股骨内髁关节软骨的大体形态学和组织学病理改变，采用反转录一聚合酶链反应(RT—PCR)方法检测软骨iNOS mRNA的表达。结果实验组软骨退变程度较对照组明显减轻，实验组软骨中iNOS mRNA的表达水平与对照组比较差异无统计学意义。结论Na—HA能有效地减轻早期OA关节软骨的退变程度，Na—HA对早期OA软骨中的iNOS的表达没有下调作用。     

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。     

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.     

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。     

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。     

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。     

去卵巢对兔膝关节软骨及基质金属蛋白酶-13表达的影响

目的探讨去卵巢对兔膝关节软骨及基质金属蛋白酶-13(MMP-13)表达的影响。方法将20只新西兰大白兔随机分为去势组和正常对照组,每组10只。去势组新西兰大白兔行双侧卵巢切除,正常对照组新西兰大白兔不作任何处理。去除卵巢10 w后,取血检测血清雌二醇水平;取左股骨髁关节软骨切片观察软骨形态,进行Mankin评分,免疫组化检测MMP-13蛋白的表达;取右股骨髁关节软骨进行RT-PCR检测MMP-13mRNA的表达。结果去势组大白兔血清雌二醇水平较正常对照组显著下降(P<0.01);去势组大白兔关节软骨出现明显的退变;去势组Mankin评分比正常对照组显著增高(P<0.01);去势组的MMP-13蛋白表达比正常对照组显著增高(P<0.01),去势组MMP-13 mRNA表达比正常对照组增高。结论去卵巢兔血清雌二醇水平明显降低,膝关节软骨退变,关节软骨中MMP-13表达增高。     

基质金属蛋白酶-1/13与信号通路激酶ERK1/2 在兔骨关节炎软骨中的表达

目的利用家兔膝关节前交叉韧带切断（ACLT）及内侧半月板前1／3切除的方法制作骨关节炎（OA）模型，测定关节炎发病的不同时期，关节软骨中基质金属蛋白酶-1（MMP-1），基质金属蛋白酶-13（MMP-13）以及细胞外信号调节激酶1／2（ERK1／2）蛋白表达水平的变化情况，探索它们之间的变化规律及关系。方法实验组家兔30只行ACLT及内侧半月板前1／3切除术造模，分别于术后4W、8W、12W处死10只，假手术对照组家兔10只于术后12W处死。进行股骨髁关节软骨退变的大体评分；取退变的软骨组织，用Western blot印记杂交方法测定软骨组织中MMP-1，MMP-13和ERK1／2的蛋白表达水平。结果ACLT及内侧半月板前1／3切除术后4W即可见软骨退变，8W和12W时退变进一步加重，对照组元明显软骨退变，不同组动物关节软骨评分差异有统计学意义。MMP-1和ERK1／2的蛋白表达在正常对照组水平均较低，造模4W开始升高，到第8W及第12W持续升高；而MMP-13在造模4w时表达开始升高，造模第8W时持续升高，但在造模第12W时MMP-13的蛋白表达下降。结论家兔膝关节ACLT及内侧半月板前1／3切除制作OA模型的方法简便可行，MMP-1和MMP-13在骨关节炎的发病过程中有不同程度的升高．可以作为OA研究的评价指标。本实验结果提示，在OA发展的过程中，MMP-1和MMP-13的表达不平行．两者上游信号调控通路可能存在差异。蛋白激酶ERK1／2的表达水平从造模第4w开始持续升高，说明ERK1／2信号转导通路在骨关节炎发病过程中持续发挥作用。     

兔前交叉韧带切断骨关节炎模型中MMP-1 MMP-13及TIMP-1的mRNA表达研究

目的；研究兔膝关节前交叉韧带切断（ACLT）骨关节炎模型关节软骨及滑膜中基质金属蛋白酶（MMP）－1，MMP－13及组织源性基质金属蛋白酶抑制剂（TIMP）－1在不同造模时期 的表达情况，探讨上述因子在骨关节炎（OA）发病过程中的作用。为该模型作OA防治研究提供理论依据。方法：实验组ACLT模型兔20史，分别于造模4周各处死10只，假手术对照组大白兔10只于术后8周处死。解剖显微镜下行股骨髁关节软骨退变的大体评分，取股骨内髁内侧退变软骨及邻近滑膜，用反转录－多聚酶链反应（RT－PCR）的方法检测MP－1，MMP－13及TIMP－1的mRNA表达。结果：ACLT术后4周即可见软骨退变，8周时退变进一步加重（P＜0．02），对照组无明显软骨退变。对照组软骨及滑膜中MMP－1及TIMP－1的检出率较低，造模4周时检出率明显增高（P＜0．05），部分阳性标本表达量有一定增高，造模8周时MMP－1仍有很高的检出率，表达量持续增高，而TIMP－1检出率较4周时下降；MMP－13在对照组滑膜中没有检测出，造模4周时有一定的检出率，8周时检出率明显增加（P＜0．002），软骨 中对照组MPP－13的表达及表达量都很低，造模4周时表达率明显增高（P＜0．05），8周时表达率却明显下降。结论：MMP－1、MMP－13及TIMP－1在骨关节炎软骨退变过程中起重要作用。其中MMP－1在造模4周和8周都有很高的检出率，且从阳性标本的电泳情况看，其表达量逐渐增高，适合作为OA防治研究中的评价指标。     

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.     

Phonoarthrography,musculoskeletal ultrasonography,and biochemical biomarkers for the evaluation of knee cartilage in osteoarthritis

The aim of this study was to examine the relationship among three different parameters used to assess cartilage in osteoarthritis (OA) of the knee. These parameters are phonoarthrography (Phono-A), musculoskeletal ultrasonography (MSUS) from the 4 condyles, and biochemical markers; notably, matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of proteinase (TIMP-1). A total of 100 knees with chronic idiopathic OA diagnosed according to the American College of Rheumatology (ACR) criteria were studied, together with 50 normal knees. The knee sounds were recorded by Phono-A and the cartilage thickness was measured by MSUS. All patients and controls had MMP-3 and TIMP-1 measured in a blood sample, using an enzyme-linked immunosorbent assay (ELISA). Conventional knee X-rays were obtained for diagnosis and for Kellgren–Lawrence (K-L) grading purposes. The results showed that Phono-A values were inversely correlated with cartilage thickness, both of these being sensitive parameters for cartilage degeneration. Phono-A values were higher in patients than in controls, denoting more degeneration of cartilage, and the cartilage thickness of all 4 condyles showed significant reductions in patients compared with normal controls. Most of the patients were categorized as grade 2 (36%) and grade 3 (30%) of the K-L classification. Mean levels of MMP-3 and TIMP-1 were significantly elevated in both groups but they were not correlated with each other. MMP-3 continued to rise with increasing radiological grades until grade 4, where it fell unexpectedly. In conclusion, Phono-A and cartilage thickness measured by MSUS seem to support each other. They can be used as parameters for following up cartilage in OA of the knees. The first deals with the roughness of the cartilage surface and the second with its thickness, complementing each other. MMP-3 continues to rise in early and middle grades of OA, denoting cartilage destruction.     

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.