酸敏感离子通道阻断剂amiloride对佐剂性关节炎大鼠关节软骨损伤的影响

目的探讨酸敏感离子通道(ASICs)阻断剂amiloride对佐剂性关节炎(AA)大鼠关节软骨损伤的影响。方法将大鼠随机分成正常组,AA模型组,amiloride50、100、150mg·kg-1组,地塞米松(0.2mg·kg-1)对照组。弗氏完全佐剂(CFA)致炎后第10天起,AA大鼠出现继发性炎症,此时腹腔注射amiloride、地塞米松,正常组与模型组灌胃等容量的无菌注射用水,连续7d。实验结束后,体外用激光共聚焦显微镜检测细胞Ca2+浓度,分析胞外低pH值和ASICs阻断剂amiloride对关节软骨细胞内钙离子浓度([Ca2+]i)的影响;用足容积法测量继发侧足肿胀度,用免疫组织化学方法分析大鼠关节软骨Ⅱ型胶原的表达,用alcian蓝染色检测大鼠关节软骨蛋白多糖的变化。结果细胞外pH(pH6.5)可使关节软骨细胞内Ca2+水平短暂性升高,amiloride能明显抑制pH6.5诱导的关节软骨胞内Ca2+水平;各用药组能明显升高AA大鼠关节软骨细胞基质成分Ⅱ型胶原和蛋白多糖表达量。结论amiloride可能通过阻断酸敏感离子通道减轻AA大鼠关节软骨破坏,发挥关节保护作用。

Therapeutic effect of amiloride on articular cartilage injury in rats with adjuvant arthritis

Aim To investigate the effects of ASICs blocker on articular cartilage injury in rats with adjuvant arthritis.Methods The rats were randomly divided into normal group,AA model group,amiloride(50,100,150 mg·kg-1)group and dexamethasone(0.2 mg·kg-1)control group. Adiuvant arthritis rat model was induced by a single intradermal injection of 100 μl of Freund′s complete adjuvant (CFA). The secondary inflammation of adjuvant-induced arthritis (AA) rats appeared on the d 10 after CFA injection.Amiloride (50,100,150 mg·kg-1) was given for 7 days starting after secondary inflammation appeared once daily intraperitoneally and dexamethasone was used as a positive control.Fura-3/AM probe and laser scanning microscope were used in vitro to detect intracellular Ca2+ concentration.Right hind paw volume was determined with volume meter. Immunohistochemistry was used to detect type Ⅱ collagen expression in rat articular cartilage. Alcian blue staining analyses were used to detect proteoglycan.Results Rapid extracellular pH (6.5) drop induced the marked increase of [Ca2+]i in the articular chondrocytes of AA rats. Amiloride almost completely blocked [Ca2+]i increase at low pH in AA rat articular chondrocytes. The type Ⅱ collagen (COⅡ) and proteoglycan (PG) of extracellular matrix of articular cartilage were increased significantly by amiloride.Conclusion ASICs blocker amiloride can have protective effects in articular cartilage in AA rats by inhibiting ASICs.