Objective: To investigate the effect of HBP-A on meniscal injuries and the expressions of genes associated with pathological hypertrophy and calcification of the meniscusinduced by abnormal loading.
Methods: Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 3 h and treated with 0.6 mg/mL of HBP-A. The cell viability in the meniscus explants after 72 hin culture was determined using live/dead staining and the expression levels of genes associated with pathological hypertrophy and calcification of the meniscus (ANKH, ENPP1, ALP, MMP13, and IL-1) were measured using real-time PCR and Western blotting. The conditioned medium was collected for testing sulfated glycosaminoglycan (GAG) release.
Results: The number of dead cells, loss of proteoglycan content, and the expressions of ANKH, ENPP1, ALP and MMP13, and IL-1 at both the mRNA and protein levels were all significantly lower in the meniscus explants treated with 0.6 mg/mL HBP-A than in the explants with only 25% abnormal pressure stimulation (n=3, P<0.05).
Conclusion: HBP-A can effectively alleviate meniscal injuries induced by abnormal loading and suppress the expressions of genes related with pathological hypertrophy and calcification of the meniscus, and can serve as a potential drug for treatment of knee osteoarthritis.
目的: 观察中药单体河蚌葡聚糖 (HBP-A) 对因异常力学刺激引起的半月板组织损伤及病理性过度肥大和钙化相关基因蛋白表达的影响。
方法: 将牛的半月板组织切割成统一大小的圆柱形，施加使组织发生可压缩25%的异常压力刺激3 h，将半月板随机分为对照组、力学压力刺激3 h组、药物组 (力学压力刺激3 h+0.6 mg/mL HBP-A培养72 h)。采用细胞活性检测方法观察各组半月板细胞的成活率，二甲基亚甲基蓝检测培养基中蛋白多糖的含量以此来定量各组半月板组织蛋白多糖的丢失程度。通过RT-qPCR和Western blot来检测HBP-A对病理性过度肥大和钙化相关因子ANKH、ENPP1、ALP和MMP13、IL-1在mRNA和蛋白水平上表达的影响。
结果: 在药物组，细胞死亡 (红色荧光) 数量虽然多于正常对照组，但仍明显低于力学压力刺激3 h组。在培养液中，力学压力刺激3 h组蛋白多糖的含量是正常对照组的1.4倍 (P < 0.05)，是药物组蛋白多糖含量的1.3倍 (P < 0.05)，说明该药物明显抑制了半月板蛋白多糖的丢失。同时，在药物组，半月板病理性过度肥大和钙化相关因子ANKH、ENPP1、ALP和MMP13、IL-1在mRNA和蛋白水平上的表达也明显低于力学压力刺激3 h组 (P < 0.05)。
结论: HBP-A能够明显地抑制因异常力学刺激引起的半月板组织损伤 (细胞死亡、多糖丢失) 及病理性过度肥大和钙化相关基因蛋白的表达，可能是治疗膝骨关节炎的潜在性药物。