背景:可溶性鸡Ⅱ型胶原分散组合物(soluble dispersal mixture of chicken collagen type Ⅱ,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,SmCC Ⅱ(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20 mg/L与80 mg/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200 g/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,1 mL/d;SmCCⅡ低剂量组(n=24)与高剂量组(n=24):分别用20 mg/L与80 mg/L SmCCⅡ1 mL灌胃;赋形剂组(n=24):2.5 g/L甘露醇灌胃,1 mL/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,1 mL/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、Cath K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-1 mRNA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、Cath K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-1 mRNA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.
揹景:可溶性鷄Ⅱ型膠原分散組閤物(soluble dispersal mixture of chicken collagen type Ⅱ,SmCC Ⅱ)治療類風濕性關節炎安全有效,骨關節炎與類風濕性關節炎在免疫髮病機製及軟骨退變方麵有許多類似之處,其是否有延緩骨關節炎軟骨退變的作用有待研究.目的:觀察國產SmCCⅡ對大鼠骨關節炎的防治效果併分析關節軟骨中基質金屬蛋白酶與組織蛋白酶等水平的變化.設計:隨機對照觀察.單位:上海交通大學附屬第六人民醫院臨床藥學研究室.材料:選用258隻6週齡Wistar大鼠,清潔級,雌雄各半,SmCC Ⅱ(白色粉末)由上海本草生物醫學工程研究所任庚伕教授惠贈,使用前配成CCII有效成分分彆為20 mg/L與80 mg/L溶液,批號:00031004.賦形劑:甘露醇(江囌天元醫藥股份有限公司),灌胃前用無菌生理鹽水將200 g/L甘露醇溶液稀釋成0.25%的溶液.方法:實驗于2003-03/2006-02在上海交通大學附屬第六人民醫院整體動物實驗室及實驗中心完成,①預防性實驗:取132隻大鼠摸毬法隨機分為5組:模型組(n=36):無菌生理鹽水灌胃,1 mL/d;SmCCⅡ低劑量組(n=24)與高劑量組(n=24):分彆用20 mg/L與80 mg/L SmCCⅡ1 mL灌胃;賦形劑組(n=24):2.5 g/L甘露醇灌胃,1 mL/d;以上4組均採用Hulth氏手術法稍做改良誘導大鼠骨關節炎模型.正常組(n=24):大鼠不造模,但給予無菌生理鹽水灌胃,1 mL/d,各組均在造模噹天開始給藥.②治療性實驗:取126隻大鼠分為5組,除模型組動物數目為30隻外,其餘各組分組方式、組內大鼠數目和榦預措施同預防性實驗.但藥物榦預時間在術後6週.藥物榦預持續時間均為8週,所有大鼠在完成治療1週後取下大鼠右後肢膝關節,將標本沿冠狀麵切開備用.③採用囌木精-伊紅染色觀察關節軟骨形態學變化併計算Mankin積分評估關節炎嚴重程度;採用免疫組化染色法(ABC法)原位測定關節軟骨中基質金屬蛋白酶-13,基質金屬蛋白酶-9及組織蛋白酶K暘性軟骨細胞百分比;用半定量RT-PCR法測定大鼠關節軟骨組織中基質金屬蛋白酶-13、基質金屬蛋白酶-9、Cath K及基質基質金屬蛋白酶組織抑製劑-1的mRNA水平.主要觀察指標:①兩實驗中各組大鼠關節軟骨形態學改變.②各組大鼠關節軟骨基質金屬蛋白酶-13,基質金屬蛋白酶-9及組織蛋白酶K水平及相應mRNA水平.結果:納入大鼠258隻均進入結果分析.①預防性實驗:模型組大鼠關節軟骨齣現明顯退行性變,Mankin積分高于SmCCⅡ高、低劑量組[(6.44±0.81),(2.75±0.85),(2.70±0.81)分,P＜0.05],關節軟骨中基質金屬蛋白酶-13、基質金屬蛋白酶-9、組織蛋白酶K暘性軟骨細胞百分比及mRNA其均高于SmCCⅡ高、低劑量組(P＜0.05～0.01),蛋白酶組織抑製劑-1 mRNA與SmCCⅡ高、低劑量組差異不顯著(P＞0.05).賦形劑組大鼠關節軟骨基質金屬蛋白酶-13、基質金屬蛋白酶-9、Cath K水平與模型組差異不顯著(P＞0.05).①治療性實驗:模型組大鼠Mankin積分高于SmCCⅡ高、低劑量組[(6.96±1.02),(3.08±0.45),(4.00±0.94)分,P＜0.05～0.01],關節軟骨中基質金屬蛋白酶-13、基質金屬蛋白酶-9、組織蛋白酶K暘性軟骨細胞百分比及其mRNA均高于SmCCⅡ高、低劑量組mRNA(P＜0.05～0.01),蛋白酶組織抑製劑-1 mRNA與SmCCⅡ高、低劑量組差異不顯著(P＞0.05).結論:SmCCⅡ能緩解大鼠骨關節炎關節軟骨的退行性變,對骨關節炎防治有效,其作用機製可能與其在轉錄水平降低基質金屬蛋白酶-13、基質金屬蛋白酶-9及組織蛋白酶K的閤成有關.
배경:가용성계Ⅱ형효원분산조합물(soluble dispersal mixture of chicken collagen type Ⅱ,SmCC Ⅱ)치료류풍습성관절염안전유효,골관절염여류풍습성관절염재면역발병궤제급연골퇴변방면유허다유사지처,기시부유연완골관절염연골퇴변적작용유대연구.목적:관찰국산SmCCⅡ대대서골관절염적방치효과병분석관절연골중기질금속단백매여조직단백매등수평적변화.설계:수궤대조관찰.단위:상해교통대학부속제륙인민의원림상약학연구실.재료:선용258지6주령Wistar대서,청길급,자웅각반,SmCC Ⅱ(백색분말)유상해본초생물의학공정연구소임경부교수혜증,사용전배성CCII유효성분분별위20 mg/L여80 mg/L용액,비호:00031004.부형제:감로순(강소천원의약고빈유한공사),관위전용무균생리염수장200 g/L감로순용액희석성0.25%적용액.방법:실험우2003-03/2006-02재상해교통대학부속제륙인민의원정체동물실험실급실험중심완성,①예방성실험:취132지대서모구법수궤분위5조:모형조(n=36):무균생리염수관위,1 mL/d;SmCCⅡ저제량조(n=24)여고제량조(n=24):분별용20 mg/L여80 mg/L SmCCⅡ1 mL관위;부형제조(n=24):2.5 g/L감로순관위,1 mL/d;이상4조균채용Hulth씨수술법초주개량유도대서골관절염모형.정상조(n=24):대서불조모,단급여무균생리염수관위,1 mL/d,각조균재조모당천개시급약.②치료성실험:취126지대서분위5조,제모형조동물수목위30지외,기여각조분조방식、조내대서수목화간예조시동예방성실험.단약물간예시간재술후6주.약물간예지속시간균위8주,소유대서재완성치료1주후취하대서우후지슬관절,장표본연관상면절개비용.③채용소목정-이홍염색관찰관절연골형태학변화병계산Mankin적분평고관절염엄중정도;채용면역조화염색법(ABC법)원위측정관절연골중기질금속단백매-13,기질금속단백매-9급조직단백매K양성연골세포백분비;용반정량RT-PCR법측정대서관절연골조직중기질금속단백매-13、기질금속단백매-9、Cath K급기질기질금속단백매조직억제제-1적mRNA수평.주요관찰지표:①량실험중각조대서관절연골형태학개변.②각조대서관절연골기질금속단백매-13,기질금속단백매-9급조직단백매K수평급상응mRNA수평.결과:납입대서258지균진입결과분석.①예방성실험:모형조대서관절연골출현명현퇴행성변,Mankin적분고우SmCCⅡ고、저제량조[(6.44±0.81),(2.75±0.85),(2.70±0.81)분,P＜0.05],관절연골중기질금속단백매-13、기질금속단백매-9、조직단백매K양성연골세포백분비급mRNA기균고우SmCCⅡ고、저제량조(P＜0.05～0.01),단백매조직억제제-1 mRNA여SmCCⅡ고、저제량조차이불현저(P＞0.05).부형제조대서관절연골기질금속단백매-13、기질금속단백매-9、Cath K수평여모형조차이불현저(P＞0.05).①치료성실험:모형조대서Mankin적분고우SmCCⅡ고、저제량조[(6.96±1.02),(3.08±0.45),(4.00±0.94)분,P＜0.05～0.01],관절연골중기질금속단백매-13、기질금속단백매-9、조직단백매K양성연골세포백분비급기mRNA균고우SmCCⅡ고、저제량조mRNA(P＜0.05～0.01),단백매조직억제제-1 mRNA여SmCCⅡ고、저제량조차이불현저(P＞0.05).결론:SmCCⅡ능완해대서골관절염관절연골적퇴행성변,대골관절염방치유효,기작용궤제가능여기재전록수평강저기질금속단백매-13、기질금속단백매-9급조직단백매K적합성유관.
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.
