可溶性鸡Ⅱ型胶原分散组合物延缓骨关节炎大鼠关节软骨形态学的变化:不同口服剂量及赋形剂比较(英文)

Soluble dispersive mixture of chicken collagen type Ⅱ delays the morphological changes of articular cartilage in rats with osteoarthritis: Comparison among different oral doses and excipients

背景:可溶性鸡Ⅱ型胶原分散组合物(soluble dispersal mixture of chicken collagen typeⅡ,SmCC Ⅱ)治疗类风湿性关节炎安全有效,骨关节炎与类风湿性关节炎在免疫发病机制及软骨退变方面有许多类似之处,其是否有延缓骨关节炎软骨退变的作用有待研究。目的:观察国产SmCCⅡ对大鼠骨关节炎的防治效果并分析关节软骨中基质金属蛋白酶与组织蛋白酶等水平的变化。设计:随机对照观察。单位:上海交通大学附属第六人民医院临床药学研究室。材料:选用258只6周龄Wistar大鼠,清洁级,雌雄各半,SmCCⅡ(白色粉末)由上海本草生物医学工程研究所任庚夫教授惠赠,使用前配成CCII有效成分分别为20mg/L与80mg/L溶液,批号:00031004。赋形剂:甘露醇(江苏天元医药股份有限公司),灌胃前用无菌生理盐水将200g/L甘露醇溶液稀释成0.25%的溶液。方法:实验于2003-03/2006-02在上海交通大学附属第六人民医院整体动物实验室及实验中心完成,①预防性实验:取132只大鼠摸球法随机分为5组:模型组(n=36):无菌生理盐水灌胃,1mL/d;SmCCⅡ低剂量组(n=24)与高剂量组(n=24):分别用20mg/L与80mg/LSmCCⅡ1mL灌胃;赋形剂组(n=24):2.5g/L甘露醇灌胃,1mL/d;以上4组均采用Hulth氏手术法稍做改良诱导大鼠骨关节炎模型。正常组(n=24):大鼠不造模,但给予无菌生理盐水灌胃,1mL/d,各组均在造模当天开始给药。②治疗性实验:取126只大鼠分为5组,除模型组动物数目为30只外,其余各组分组方式、组内大鼠数目和干预措施同预防性实验。但药物干预时间在术后6周。药物干预持续时间均为8周,所有大鼠在完成治疗1周后取下大鼠右后肢膝关节,将标本沿冠状面切开备用。③采用苏木精-伊红染色观察关节软骨形态学变化并计算Mankin积分评估关节炎严重程度;采用免疫组化染色法(ABC法)原位测定关节软骨中基质金属蛋白酶-13,基质金属蛋白酶-9及组织蛋白酶K阳性软骨细胞百分比;用半定量RT-PCR法测定大鼠关节软骨组织中基质金属蛋白酶-13、基质金属蛋白酶-9、CathK及基质基质金属蛋白酶组织抑制剂-1的mRNA水平。主要观察指标:①两实验中各组大鼠关节软骨形态学改变。②各组大鼠关节软骨基质金属蛋白酶-13,基质金属蛋白酶-9及组织蛋白酶K水平及相应mRNA水平。结果:纳入大鼠258只均进入结果分析。①预防性实验:模型组大鼠关节软骨出现明显退行性变,Mankin积分高于SmCCⅡ高、低剂量组[(6.44±0.81),(2.75±0.85),(2.70±0.81)分,P<0.05],关节软骨中基质金属蛋白酶-13、基质金属蛋白酶-9、组织蛋白酶K阳性软骨细胞百分比及mRNA其均高于SmCCⅡ高、低剂量组(P<0.05～0.01),蛋白酶组织抑制剂-1mRNA与SmCCⅡ高、低剂量组差异不显著(P>0.05)。赋形剂组大鼠关节软骨基质金属蛋白酶-13、基质金属蛋白酶-9、CathK水平与模型组差异不显著(P>0.05)。①治疗性实验:模型组大鼠Mankin积分高于SmCCⅡ高、低剂量组[(6.96±1.02),(3.08±0.45),(4.00±0.94)分,P<0.05～0.01],关节软骨中基质金属蛋白酶-13、基质金属蛋白酶-9、组织蛋白酶K阳性软骨细胞百分比及其mRNA均高于SmCCⅡ高、低剂量组mRNA(P<0.05～0.01),蛋白酶组织抑制剂-1mRNA与SmCCⅡ高、低剂量组差异不显著(P>0.05)。结论:SmCCⅡ能缓解大鼠骨关节炎关节软骨的退行性变,对骨关节炎防治有效,其作用机制可能与其在转录水平降低基质金属蛋白酶-13、基质金属蛋白酶-9及组织蛋白酶K的合成有关。

BACKGROUND： Soluble dispersive mixture of domestic chicken collagen type Ⅱ （SmCC Ⅱ ） has been proven to be safe and effective for rheumatoid arthritis （RA） treatment while there are some similar articular cartilage degradation and autoimmune pathogenesis between osteoarthritis （OA） and RA, so it is worth studying whether SmCC Ⅱ is effective for the precaution or treatment of OA.OBJECTIVE： To observe the prophylactic and therapeutic effects of SmCC Ⅱ on rat OA and analyze the concomitant matrix metalloproteinase （MMPs） and tissue protease changes in osteoarthritic rats.DESIGN： Randomized and controlled observation.SETTING： Department of Clinical Pharmacy, Sixth People＇s Hospital, Shanghai Jiao Tong University.MATERIALS： Totally 258 rats of clean grade and either gender, aged 6 weeks were involved in this experiment. SmCC Ⅱ （white powder, Batch No.00031004） was provided by Professor Ren Geng-Fu from Shanghai Engineering Institute of Herbal Biomedicine. The 20 mg/L and 80 mg/L solution of SmCC Ⅱ effective component were prepared before use.While 0.25% excipient （mannitol, product of Jiangsu Tianyuan Medical Co., Ltd） solution was prepared by 200 g/L mannitol dissolved in sterile saline solution.METHODS： The study was carried out in the Whole Animal Laboratory and Experimental Center, Sixth People＇s Hospital,Shanghai Jiao Tong University between March 2003 and February 2006. ①Prophylactic study： Totally 132 rats were randomized into 5 groups： OA group （n =36）： treated with sterile saline solution orally by a 16G syringe, 1mL/d; Low- and high-dose SmCC Ⅱ groups （n =24, respectively）： treated with 20 mg/L and 80 mg/L SmCC Ⅱ solution orally, 1 mL/d;Excipient group （n =24）： treated with 2.5g/L mannitol, 1 mL/d. OA models were surgically induced in these 4 groups by Hilth＇s method; Normal group （n =24）： Rats without operation were treated with sterile saline solution orally, 1 mL/d. All the rats were administrated on the day of modeling. ②Therapeutic study： Totally 126 rats were randomized into 5 groups, grouping, administration and rat number in different groups were the same as those in prophylactic study,respectively, except for the n =30 in OA group. In addition, all the rats were adminstrated for 8 weeks since 6 weeks after operation. The knee joints of right hind limb of all the rats were taken off after 1-week treatment. And sample was cut open along coronal incision for use. ③Morphological study of articular cartilage was conducted by HE staining and Mankin score was calculated to evaluate the severity of arthritis, immunohistochemical studies of matrix metalloproteinase （MMP）-13, MMP-9 and cathepsin K （Cath K） were carried out by ABC method in situ and the percentage of positive-stained chondrocytes were calculated while the mRNA level for MMP-13, MMP-9, Cath K and the tissue inhibitor of metalloproteinase 1 （TIMP1） were determined by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） method.MAIN OUTCOME MEASURES： ①The morphological changes of articular cartilage in different groups in prophylactic or therapeutic study.②The level of MMP-13, MMP-9, Cath K and their corresponding mRNA levels.RESULTS： All the 258 rats were involved in the final analysis. ①Prophylactic study： Apparent degeneration appeared in the rats of OA group and the mankin score in OA group was higher than that in high- or low- SmCC Ⅱ groups [（6.44±0.81）, （2.75±0.85）, （2.70±0.81） points respectively, P 〈 0.05], the mRNA and positive-stained chondrocyte percentage of MMP-13, MMP-9 and Cath K in OA group was higher than that in high- or low-dose SmCC Ⅱ group, respectively （P 〈0.05-0.01） while the TIMP1mRNA level in OA group was not significantly higher than that in high or low SmCC Ⅱ group （P〉 0.05）. There were no significant changes on the level of MMP-13, MMP-9 and Cath K between excipient and OA group （P 〉 0.05）. ②Therapeutic study： The Mankin＇s score of OA group was higher than high or low SmCC Ⅱ group [（6.96±1.02）, （3.08±0.45）, （4.00±0.94） respectively, P 〈 0.05-0.01] while the mRNA and positive-stained chondrocyte percentage of MMP-13, MMP-9 and Cath K in OA group was higher than that in high- or low-dose SmCC Ⅱ group,respectively （P 〈 0.05-0.01） while the TIMP1mRNA level in OA group was not significantly higher than that in high- or low-dose SmCC Ⅱ group （P 〉 0.05）.CONCLUSION： SmCC Ⅱ can delay the degradation of articular cartilage of OA rats and maybe effective for OA prevention or treatment. The mechanism maybe related to SmCC Ⅱ reducing the synthesis of MMP-13, MMP-9 and Cath K at transcriptional level.