徐长卿丹皮酚对兔膝骨性关节炎软骨基质金属蛋白酶及其抑制剂的影响
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摘要
目的骨性关节炎是骨科领域最常见的关节病变，随着我国老龄化进程的发展，OA的发病率会不断升高，严重影响了中老年人的生活质量。骨性关节炎的主要病理学变化为关节软骨不同程度退行性变性或消失，以及软骨下骨质破坏。病理研究证实，关节软骨软骨变性是骨性关节炎特征性病理改变，也是OA最根基的病理改变。有关软骨退变机制及其细胞和分子基础成为近年来研究的热点。本课题利用徐长卿丹皮酚对基质金属蛋白酶的影响作为切入点，从细胞形态、电镜下超微病理结构的改变、分子生物学三个层面探讨徐长卿丹皮酚治疗骨性关节炎的作用机理，为临床推广徐长卿丹皮酚治疗骨性关节炎提供理论和实验依据。
方法40只体重2kg(±100g)健康新西兰成年大耳白兔，雌雄不限，随机分为正常组、对照组、生理盐水组、曲安奈德组、徐长卿丹皮酚组，每组8只。随机选取8只作为正常组，其余32只采用兔膝关节内侧半月板前1／3切除及前交叉韧带切断(ACLT)制备骨关节炎(OA)模型方法[4]进行造模，4周后造模成功。正常组：不作任何治疗。对照组：仅采用兔膝关节内侧半月板前1／3切除及前交叉韧带切断(ACLT)制备骨关节炎(OA)模型方法进行造模，不予其它处理。生理盐水组、曲安奈德组、徐长卿丹皮酚组：造模术后第1周开始各组分别予以右膝关节内注射0．9％生理盐水、曲安奈德、徐长卿丹皮酚各0.2ml，2次∕周，共10次，给药5周。各组动物分别于完成5周用药疗程后，耳缘静脉缓慢注入适量空气处死，暴露膝关节腔。①关节软骨肉眼观察表面光滑度、色泽、表面糜烂程度、是否有纤维性增生及软骨下骨暴露等形态学改变，拍照并按大体评分标准评分；取制备好的股骨内侧髁关节软骨标本，透射式电子显微镜下进行超微结构观察并拍照。②取右侧股骨内侧髁关节面约2mm厚关节软骨标本制作成石蜡切片后，两步法免疫组化染色，置光镜下对其组织结构进行详细的观察，并参照Mankin关节软骨病理评分标准进行积分统计Mankin评分，包括关节软骨结构，细胞数的变化，潮线的完整性及易染性四个方面；统计学分析关节软骨基质金属蛋白酶（MMP-1）和其抑制剂（TIMP-1）表达的阳性指数。③采用RNA原位杂交方法利用MMP-1和TIMP-1寡核苷酸探针，对兔关节软骨MMP-1和TIMP-1mRNA作定位和定量分析,检测兔膝关节骨性关节炎软骨组织MMP-1和TIMP-1mRNA表达。
结果①大体观察示正常组关节面光滑完整。对照组软骨改变集中在股骨内侧髁关节面，关节软骨有虫蚀样改变和软骨剥脱缺损。生理盐水组软骨表面粗糙，肥厚。徐长卿丹皮酚组关节面略粗糙，软骨轻度糜烂。曲安奈德组软骨糜烂程度较徐长卿组重，有的可见软骨下骨暴露。电镜观察示正常组细胞形态正常，胞核完整，胞浆密度均匀，可见大量细胞器，细胞表面的绒毛和胶原清晰可见。对照组大多数软骨细胞形态不规则，胞质内细胞器溶解，胶原排列紊乱。生理盐水组大部分软骨细胞肿胀明显。徐长卿组：细胞形态尚存，胶原轻度紊乱，胞质内粗面内质网丰富。曲安奈德组：呈颗粒状外观，细胞质内部分细胞器溶解，胶原纤维含量较少。②组织学观察示正常组Mankin评分0-1分；徐长卿丹皮酚组以轻度至中度损伤为主，Mankin评分4-6分；曲安奈德组以中等软骨损伤为主，Mankin评分8-10分；造模组以重度软骨损伤为主，Mankin评分11-12分；生理盐水组与造模组相似。徐长卿丹皮酚组软骨组织MMP-1表达的阳性指数较正常组增高，较曲安奈德组和造模组低，组间比较有统计学意义（P＜0.05）；徐长卿丹皮酚组软骨组织TIMP-1表达水平较曲安奈德组增高，两组间比较差异有统计学意义（P＜0.05），曲安奈德组TIMP-1表达水平与造模组、生理盐水组组间比较无统计学差异（P＞0.05）。③正常组关节软骨MMP-1和TIMP-1mRNA表达均低下或无表达；徐长卿丹皮酚组软骨组织MMP-1和TIMP-1mRNA表达均较低；曲安奈德组软骨组织MMP-1mRNA表达明显增强；造模组关节软骨MMP-1mRNA表达最强，TIMP-1mRNA全层无表达或低下；徐长卿丹皮酚组软骨组织MMP-1mRNA表达水平较正常组增高，较曲安奈德组和造模组低，组间比较有统计学意义（P＜0.05）；徐长卿丹皮酚组软骨组织TIMP-1mRNA表达水平较曲安奈德组增高，两组间比较差异有统计学意义（P＜0.05）。
结论实验结果提示关节损伤程度与MMP-1的表达成正相关，徐长卿丹皮酚可能具有抑制MMP-1的表达，消除MMP-1对软骨细胞外基质过度降解的作用。与此同时，MMP-1的特异性抑制物TIMP-1虽然随着MMP-1分泌的升高也有所升高,但与MMP-1的升高不同步，两者的分泌水平相差较大，可能使MMP-1对关节软骨的降解作用过度发挥。关节损伤程度与TIMP-1的表达成负相关，徐长卿丹皮酚可能具有促进TIMP-1的表达，从而达到抑制MMP-1表达的作用。徐长卿丹皮酚对骨性关节炎的治疗作用是通过抑制损伤软骨中MMP-1mRNA的表达和促进TIMP-1mRNA的表达这一双向调节机制来实现的。
Objective Osteoarthritis is the most common joint diseasedepartment of orthopedics field, with the rapid development ofChina's aging process, the incidence of OA will continue to rise,seriously affecting the quality of life in the elderly. The mainpathological changes of osteoarthritis of articular cartilage indifferent degree of degeneration or disappearance, and subchondralbone destruction. Pathological study confirmed, joint cartilagedegeneration of osteoarthritis is the characteristic pathologicalchanges, pathological changes of OA is the most basic. The cartilagedegeneration mechanism and the cellular and molecular basis ofbecome a research hotspot in recent years. The subject of the useof Cynanchum paniculatum paeonol effect on matrixmetalloproteinase as the breakthrough point, to explore themechanism of Radix Cynanchi paniculati Danpi treatment ofosteoarthritis from three aspects of cell morphology,ultrastructure changes under the electron microscope, molecularbiology, and provide theoretical and experimental basis for theclinical application of Radix Cynanchi paniculati paeonol for osteoarthritis of the knee.
Methods40weight2kg (±100g) healthy adult New Zealand whiterabbits, irrespective of gender, were randomly divided into normalgroup, control group, saline group, triamcinolone acetonide group,Radix Cynanchi paniculati paeonol group, with8rats in each group.Randomly selected8rats as normal group, the remaining32rabbitmedial meniscus of knee joint before1/3hepatectomy andtransection of the anterior cruciate ligament (ACLT) preparationof osteoarthritis (OA) model method of [4] modeling,4weeks Houzaomodel. Normal group: without any treatment. Control group: only therabbit knee medial meniscus of1/3hepatectomy and transectionof the anterior cruciate ligament (ACLT) preparation ofosteoarthritis (OA) model method of molding, no other treatment.The physiological saline group, Maria Nade group, Radix Cynanchipaniculati paeonol groups respectively: group right kneeintra-articular injection of0.9%saline, Maria Nade, Xu ChangqingDanpi0.2ml began modeling after first weeks,2times/week, a totalof10times, for5weeks. All animal were completed5weeks aftertreatment, the ear vein injection amount were exposed to air, kneejoint cavity. The naked eye observation of articular cartilagesurface smoothness, luster, surface erosion, whether there is afibrous hyperplasia and subchondral bone exposure morphologicalchanges, take pictures and score according to the general scorestandard; and the prepared the medial femoral condyle cartilagespecimens, transmission electron microscope ultrastructure wereobserved and photographed.②from the right medial femoralcondyle articular surface of about2mm thickness of articularcartilage were made into paraffin sections, two step immunohistochemical staining under light microscope, theorganizational structure of the detailed observation, and integralstatistics Mankin score according to Mankin articular cartilagepathology score standard, package structure includes jointcartilage, changes in cell number, tidal line of complete and easydyeing four aspects; statistical analysis of articular cartilagematrix metalloproteinases (MMP-1) and its inhibitor (TIMP-1)positive index expression. The use of MMP-1and TIMP-1oligonucleotide probe by RNA in situ hybridization method, as thelocalization and quantification of rabbit articular cartilage ofMMP-1and TIMP-1mRNA, detected the expression of rabbit kneeosteoarthritis cartilage tissue MMP-1and TIMP-1mRNA。
Results①gross observation showed the normal articularsurface was smooth and intact. The control group cartilage changeis concentrated in the medial femoral condyle, articular cartilagehas eroded and stripping of cartilage defect. Saline groupcartilage surface rough, hypertrophy. Cynanchum paniculatumpaeonol articular surface was slightly rough, mild cartilageerosion. Triamcinolone acetonide group cartilage erosion degreethan Cynanchum paniculatum weight group, some of the subchondralbone exposure. Electron microscope showed that the normal cells ofnormal morphology, nuclear, cytoplasmic density uniform, note theamount of organelles, cell surface villous and collagen clearlyvisible. The control group most cartilage cells are irregular inshape, cell organs in cytoplasm dissolved, collagen arranged indisorder. Saline group most cartilage cell swelling. Cynanchumpaniculatum group: cell morphology remained mild disorder,collagen, rough endoplasmic reticulum rich. Triamcinolone acetonide group: granular appearance, cytoplasmic organellesdissolved, less collagen fiber content.The examination of normalgroup Mankin score0-1points in the organization; Cynanchumpaniculatum paeonol group with mild to moderate injury, Mankinscore of4-6; triamcinolone acetonide group with medium cartilageinjury, Mankin score of8-10; model group with severe cartilageinjury, Mankin score11-12points; saline group was similar to modelgroup. Cynanchum paniculatum paeonol group cartilage tissue MMP-1expression positive index higher than the normal control group, theMaria Nade group and model group, there was significant differencebetween groups (P <0.05); the expression of Cynanchum paniculatumpaeonol group cartilage tissue TIMP-1level is Maria Nade groupincreased, compared between the two groups had significantdifference (P <0.05), the Maria Nade group the expression levelof TIMP-1and model group, saline group comparisons between groupsshowed no significant difference (P>0.05). The normal articularcartilage of MMP-1and TIMP-1mRNA expression was low or noexpression; expression of MMP-1and TIMP-1mRNA Cynanchumpaniculatum paeonol group cartilage tissue were lower; increase theexpression of triamcinolone acetonide group cartilage MMP-1mRNA;the model of articular cartilage strongest MMP-1mRNA expression,TIMP-1mRNA layer no expression or low expression of Cynanchumpaniculatum; paeonol group cartilage tissue MMP-1mRNA levelshigher than the normal control group, compared with triamcinoloneacetonide group low, with a significant difference between groups(P <0.05); Xu Qing expression of cartilage tissue of paeonol groupTIMP-1mRNA levels were triameinolone de group, there wasstatistically significant difference between two groups (P <0.05).
Conclusions these results suggest that expression of jointdamage and MMP-1positive correlation, Cynanchum paniculatum Danpicould inhibit the expression of MMP-1, the elimination of MMP-1incartilage of excessive extracellular matrix degradation. At thesame time, the MMP-1specific inhibitor TIMP-1with MMP-1secretionincreases also increased, but not in sync with the increase of MMP-1,the secretion level of their difference, may make the MMP-1excessive play on the degradation of articular cartilage.Expression of joint damage and TIMP-1are negatively correlated,Cynanchum paniculatum paeonol could promote the expression ofTIMP-1, and thus inhibit the expression of MMP-1function.Therapeutic effect of Radix Cynanchi paniculati Danpi onosteoarthritis is through inhibition of MMP-1mRNA expression incartilage injury and promote the expression of TIMP-1mRNA of thebidirectional regulation mechanism to achieve.
方法40只体重2kg(±100g)健康新西兰成年大耳白兔，雌雄不限，随机分为正常组、对照组、生理盐水组、曲安奈德组、徐长卿丹皮酚组，每组8只。随机选取8只作为正常组，其余32只采用兔膝关节内侧半月板前1／3切除及前交叉韧带切断(ACLT)制备骨关节炎(OA)模型方法[4]进行造模，4周后造模成功。正常组：不作任何治疗。对照组：仅采用兔膝关节内侧半月板前1／3切除及前交叉韧带切断(ACLT)制备骨关节炎(OA)模型方法进行造模，不予其它处理。生理盐水组、曲安奈德组、徐长卿丹皮酚组：造模术后第1周开始各组分别予以右膝关节内注射0．9％生理盐水、曲安奈德、徐长卿丹皮酚各0.2ml，2次∕周，共10次，给药5周。各组动物分别于完成5周用药疗程后，耳缘静脉缓慢注入适量空气处死，暴露膝关节腔。①关节软骨肉眼观察表面光滑度、色泽、表面糜烂程度、是否有纤维性增生及软骨下骨暴露等形态学改变，拍照并按大体评分标准评分；取制备好的股骨内侧髁关节软骨标本，透射式电子显微镜下进行超微结构观察并拍照。②取右侧股骨内侧髁关节面约2mm厚关节软骨标本制作成石蜡切片后，两步法免疫组化染色，置光镜下对其组织结构进行详细的观察，并参照Mankin关节软骨病理评分标准进行积分统计Mankin评分，包括关节软骨结构，细胞数的变化，潮线的完整性及易染性四个方面；统计学分析关节软骨基质金属蛋白酶（MMP-1）和其抑制剂（TIMP-1）表达的阳性指数。③采用RNA原位杂交方法利用MMP-1和TIMP-1寡核苷酸探针，对兔关节软骨MMP-1和TIMP-1mRNA作定位和定量分析,检测兔膝关节骨性关节炎软骨组织MMP-1和TIMP-1mRNA表达。
结果①大体观察示正常组关节面光滑完整。对照组软骨改变集中在股骨内侧髁关节面，关节软骨有虫蚀样改变和软骨剥脱缺损。生理盐水组软骨表面粗糙，肥厚。徐长卿丹皮酚组关节面略粗糙，软骨轻度糜烂。曲安奈德组软骨糜烂程度较徐长卿组重，有的可见软骨下骨暴露。电镜观察示正常组细胞形态正常，胞核完整，胞浆密度均匀，可见大量细胞器，细胞表面的绒毛和胶原清晰可见。对照组大多数软骨细胞形态不规则，胞质内细胞器溶解，胶原排列紊乱。生理盐水组大部分软骨细胞肿胀明显。徐长卿组：细胞形态尚存，胶原轻度紊乱，胞质内粗面内质网丰富。曲安奈德组：呈颗粒状外观，细胞质内部分细胞器溶解，胶原纤维含量较少。②组织学观察示正常组Mankin评分0-1分；徐长卿丹皮酚组以轻度至中度损伤为主，Mankin评分4-6分；曲安奈德组以中等软骨损伤为主，Mankin评分8-10分；造模组以重度软骨损伤为主，Mankin评分11-12分；生理盐水组与造模组相似。徐长卿丹皮酚组软骨组织MMP-1表达的阳性指数较正常组增高，较曲安奈德组和造模组低，组间比较有统计学意义（P＜0.05）；徐长卿丹皮酚组软骨组织TIMP-1表达水平较曲安奈德组增高，两组间比较差异有统计学意义（P＜0.05），曲安奈德组TIMP-1表达水平与造模组、生理盐水组组间比较无统计学差异（P＞0.05）。③正常组关节软骨MMP-1和TIMP-1mRNA表达均低下或无表达；徐长卿丹皮酚组软骨组织MMP-1和TIMP-1mRNA表达均较低；曲安奈德组软骨组织MMP-1mRNA表达明显增强；造模组关节软骨MMP-1mRNA表达最强，TIMP-1mRNA全层无表达或低下；徐长卿丹皮酚组软骨组织MMP-1mRNA表达水平较正常组增高，较曲安奈德组和造模组低，组间比较有统计学意义（P＜0.05）；徐长卿丹皮酚组软骨组织TIMP-1mRNA表达水平较曲安奈德组增高，两组间比较差异有统计学意义（P＜0.05）。
结论实验结果提示关节损伤程度与MMP-1的表达成正相关，徐长卿丹皮酚可能具有抑制MMP-1的表达，消除MMP-1对软骨细胞外基质过度降解的作用。与此同时，MMP-1的特异性抑制物TIMP-1虽然随着MMP-1分泌的升高也有所升高,但与MMP-1的升高不同步，两者的分泌水平相差较大，可能使MMP-1对关节软骨的降解作用过度发挥。关节损伤程度与TIMP-1的表达成负相关，徐长卿丹皮酚可能具有促进TIMP-1的表达，从而达到抑制MMP-1表达的作用。徐长卿丹皮酚对骨性关节炎的治疗作用是通过抑制损伤软骨中MMP-1mRNA的表达和促进TIMP-1mRNA的表达这一双向调节机制来实现的。
Objective Osteoarthritis is the most common joint diseasedepartment of orthopedics field, with the rapid development ofChina's aging process, the incidence of OA will continue to rise,seriously affecting the quality of life in the elderly. The mainpathological changes of osteoarthritis of articular cartilage indifferent degree of degeneration or disappearance, and subchondralbone destruction. Pathological study confirmed, joint cartilagedegeneration of osteoarthritis is the characteristic pathologicalchanges, pathological changes of OA is the most basic. The cartilagedegeneration mechanism and the cellular and molecular basis ofbecome a research hotspot in recent years. The subject of the useof Cynanchum paniculatum paeonol effect on matrixmetalloproteinase as the breakthrough point, to explore themechanism of Radix Cynanchi paniculati Danpi treatment ofosteoarthritis from three aspects of cell morphology,ultrastructure changes under the electron microscope, molecularbiology, and provide theoretical and experimental basis for theclinical application of Radix Cynanchi paniculati paeonol for osteoarthritis of the knee.
Methods40weight2kg (±100g) healthy adult New Zealand whiterabbits, irrespective of gender, were randomly divided into normalgroup, control group, saline group, triamcinolone acetonide group,Radix Cynanchi paniculati paeonol group, with8rats in each group.Randomly selected8rats as normal group, the remaining32rabbitmedial meniscus of knee joint before1/3hepatectomy andtransection of the anterior cruciate ligament (ACLT) preparationof osteoarthritis (OA) model method of [4] modeling,4weeks Houzaomodel. Normal group: without any treatment. Control group: only therabbit knee medial meniscus of1/3hepatectomy and transectionof the anterior cruciate ligament (ACLT) preparation ofosteoarthritis (OA) model method of molding, no other treatment.The physiological saline group, Maria Nade group, Radix Cynanchipaniculati paeonol groups respectively: group right kneeintra-articular injection of0.9%saline, Maria Nade, Xu ChangqingDanpi0.2ml began modeling after first weeks,2times/week, a totalof10times, for5weeks. All animal were completed5weeks aftertreatment, the ear vein injection amount were exposed to air, kneejoint cavity. The naked eye observation of articular cartilagesurface smoothness, luster, surface erosion, whether there is afibrous hyperplasia and subchondral bone exposure morphologicalchanges, take pictures and score according to the general scorestandard; and the prepared the medial femoral condyle cartilagespecimens, transmission electron microscope ultrastructure wereobserved and photographed.②from the right medial femoralcondyle articular surface of about2mm thickness of articularcartilage were made into paraffin sections, two step immunohistochemical staining under light microscope, theorganizational structure of the detailed observation, and integralstatistics Mankin score according to Mankin articular cartilagepathology score standard, package structure includes jointcartilage, changes in cell number, tidal line of complete and easydyeing four aspects; statistical analysis of articular cartilagematrix metalloproteinases (MMP-1) and its inhibitor (TIMP-1)positive index expression. The use of MMP-1and TIMP-1oligonucleotide probe by RNA in situ hybridization method, as thelocalization and quantification of rabbit articular cartilage ofMMP-1and TIMP-1mRNA, detected the expression of rabbit kneeosteoarthritis cartilage tissue MMP-1and TIMP-1mRNA。
Results①gross observation showed the normal articularsurface was smooth and intact. The control group cartilage changeis concentrated in the medial femoral condyle, articular cartilagehas eroded and stripping of cartilage defect. Saline groupcartilage surface rough, hypertrophy. Cynanchum paniculatumpaeonol articular surface was slightly rough, mild cartilageerosion. Triamcinolone acetonide group cartilage erosion degreethan Cynanchum paniculatum weight group, some of the subchondralbone exposure. Electron microscope showed that the normal cells ofnormal morphology, nuclear, cytoplasmic density uniform, note theamount of organelles, cell surface villous and collagen clearlyvisible. The control group most cartilage cells are irregular inshape, cell organs in cytoplasm dissolved, collagen arranged indisorder. Saline group most cartilage cell swelling. Cynanchumpaniculatum group: cell morphology remained mild disorder,collagen, rough endoplasmic reticulum rich. Triamcinolone acetonide group: granular appearance, cytoplasmic organellesdissolved, less collagen fiber content.The examination of normalgroup Mankin score0-1points in the organization; Cynanchumpaniculatum paeonol group with mild to moderate injury, Mankinscore of4-6; triamcinolone acetonide group with medium cartilageinjury, Mankin score of8-10; model group with severe cartilageinjury, Mankin score11-12points; saline group was similar to modelgroup. Cynanchum paniculatum paeonol group cartilage tissue MMP-1expression positive index higher than the normal control group, theMaria Nade group and model group, there was significant differencebetween groups (P <0.05); the expression of Cynanchum paniculatumpaeonol group cartilage tissue TIMP-1level is Maria Nade groupincreased, compared between the two groups had significantdifference (P <0.05), the Maria Nade group the expression levelof TIMP-1and model group, saline group comparisons between groupsshowed no significant difference (P>0.05). The normal articularcartilage of MMP-1and TIMP-1mRNA expression was low or noexpression; expression of MMP-1and TIMP-1mRNA Cynanchumpaniculatum paeonol group cartilage tissue were lower; increase theexpression of triamcinolone acetonide group cartilage MMP-1mRNA;the model of articular cartilage strongest MMP-1mRNA expression,TIMP-1mRNA layer no expression or low expression of Cynanchumpaniculatum; paeonol group cartilage tissue MMP-1mRNA levelshigher than the normal control group, compared with triamcinoloneacetonide group low, with a significant difference between groups(P <0.05); Xu Qing expression of cartilage tissue of paeonol groupTIMP-1mRNA levels were triameinolone de group, there wasstatistically significant difference between two groups (P <0.05).
Conclusions these results suggest that expression of jointdamage and MMP-1positive correlation, Cynanchum paniculatum Danpicould inhibit the expression of MMP-1, the elimination of MMP-1incartilage of excessive extracellular matrix degradation. At thesame time, the MMP-1specific inhibitor TIMP-1with MMP-1secretionincreases also increased, but not in sync with the increase of MMP-1,the secretion level of their difference, may make the MMP-1excessive play on the degradation of articular cartilage.Expression of joint damage and TIMP-1are negatively correlated,Cynanchum paniculatum paeonol could promote the expression ofTIMP-1, and thus inhibit the expression of MMP-1function.Therapeutic effect of Radix Cynanchi paniculati Danpi onosteoarthritis is through inhibition of MMP-1mRNA expression incartilage injury and promote the expression of TIMP-1mRNA of thebidirectional regulation mechanism to achieve.
引文
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