葡萄糖依赖性肠促胰岛素类似物DAIa2GIP对大鼠膝关节损伤软骨的保护效应

Protective effect of glucose-dependent incretin analogue DAIa2GIP on knee cartilage injury of rats

背景:已有研究证实,葡萄糖依赖性肠促胰岛素类似物DAla2GIP可直接调节成骨细胞的生长发育,但有关其在软骨细胞上的作用鲜有报道。目的:探讨葡萄糖依赖性肠促胰岛素类似物DAIa2GIP对SD大鼠骨性关节炎软骨损伤的保护效应及潜在机制。方法:将50只雄性SD大鼠(山西医科大学实验动物中心提供)随机分5组,每组10只:正常对照组不进行任何干预;模型对照组、模型治疗组切断双后膝关节前交叉韧带、内侧副韧带并摘除内侧半月板;假手术对照组、假手术治疗组只打开双侧膝关节囊。造模后第8周,模型治疗组、假手术治疗组腹腔注射25 nmol/kg的DAIa2GIP,模型对照组、假手术对照组腹腔注射等量生理盐水,2次/周,注射8周。造模16周后,取各组大鼠膝关节,进行苏木精-伊红染色、番红O-固绿染色、透射电镜观察及免疫组织化学染色。实验方案经山西医科大学伦理委员会批准(2017008)。结果与结论:①苏木精-伊红染色显示,模型对照组软骨4层结构不清;模型治疗组软骨可见4层结构,软骨层较模型对照组增厚;其余3组软骨表面光滑,4层结构清晰;②番红O-固绿染色显示,模型对照组蛋白多糖表达明显不均匀,软骨厚度变薄;模型治疗组蛋白多糖表达基本均匀,软骨厚度较模型对照组增加;其余3组蛋白多糖表达均匀,软骨厚度适中;③透射电镜显示,模型对照组软骨细胞胞质内可见脂滴,线粒体、粗面内质网等细胞器消失;模型治疗组可见数量不等线粒体、粗面内质网等细胞器,并且结构基本恢复正常;其余组细胞结构正常;④免疫组织化学染色显示,与正常对照组比较,模型对照组胶原酶13蛋白表达升高(P<0.05),Ⅱ型胶原蛋白表达降低(P<0.05);与模型对照组比较,模型治疗组胶原酶13蛋白表达降低(P<0.05),Ⅱ型胶原蛋白表达升高(P<0.05);⑤结果表明,DAIa2GIP可能通过抑制关节软骨中胶原酶13表达、减少Ⅱ型胶原的破坏,对SD大鼠骨性关节炎中关节软骨发挥保护作用。

BACKGROUND:Glucose-dependent incretin analogue DAIa2GIP has been shown to directly regulate the growth and development of osteoblasts,but its effect on chondrocytes is rarely reported.OBJECTIVE:To investigate the protective effect and potential mechanism of glucose-dependent incretin analogue DAIa2GIP on cartilage injury in Sprague-Dawley rats with osteoarthritis.METHODS:Fifty male Sprague-Dawley rats provided by Laboratory Animal Center of Shanxi Medical University were randomly divided into five groups(n=10/group):normal control group(no intervention),model control group(anterior cruciate ligament and medial collateral ligament resection,and medial meniscus removal at bilateral posterior knees+normal saline),model treatment group(anterior cruciate ligament and medial collateral ligament resection,and medial meniscus removal at bilateral posterior knees+25 nmol/kg DAIa2GIP)and sham control group(opening articular capsule of bilateral knees+normal saline),and sham treatment group(opening articular capsule of bilateral knees+25 nmol/kg DAIa2GIP).The intraperitoneal administration was given at 8 weeks after modeling,twice weekly for 8 consecutive weeks.The knee joint was removed at 16 weeks after modeling for hematoxylin-eosin staining,safranin O-fast green staining,transmission electron microscope and immunohistochemistry.The study was approved by the Ethics Committee of Shanxi Medical University,approval number:2017008.RESULTS AND CONCLUSION:(1)Hematoxylin-eosin staining:the four layers of the cartilage structure in the model control group were obscure.The four layers of the cartilage structure were visible in the model treatment group,and the cartilage lay was thicker than that in the model group.In the other three groups,the cartilage surface was smooth with clear four layers.(2)Safranin O-fast green staining:the expression of proteoglycan in the model control group was uneven,and cartilage thickness was thin.The expression of proteoglycan in the model treatment group was even,and the cartilage thickness was increased compared with the model control group.The expression of proteoglycan in the other three groups was even,with moderate cartilage thickness.(3)Transmission electron microscope:there were lipid droplets in the model control group,and mitochondria and rough endoplasmic reticulum disappeared.In the model treatment group,some mitochondria and rough endoplasmic reticulum were observed,and the structure returned to be normal.The other three groups showed the normal structure.(4)Immunohistochemistry:compared with the normal control group,the expression level of collagenase 13 was significantly increased(P<0.05),and the expression level of collagen typeⅡwas significantly decreased in the model control group(P<0.05).Compared with the model control group,the expression level of collagenase 13 was significantly decreased(P<0.05),and the expression level of collagen typeⅡwas significantly increased in the model treatment group(P<0.05).(5)To conclude,DAIa2GIP exerts protective effect on articular cartilage in Sprague-Dawley rats with osteoarthritis possibly by inhibiting the expression of collagenase 13 and reducing collagen typeⅡdestruction in articular cartilage.