目的 探讨桔梗总皂苷对膝骨关节炎（knee osteoarthritis，KOA）大鼠软骨损伤及Ras同源基因家族成员A（Ras homolog gene family member A，RhoA）/Rho相关的卷曲螺旋激酶（Rho-associated coiled-coil kinase，ROCK）信号通路的影响。方法 采用膝关节注射木瓜蛋白酶制备KOA模型，随机分为模型组、硫酸氨基葡萄糖（0.2 g/kg）组及桔梗总皂苷低、高剂量（100、200 mg/kg）组和桔梗总皂苷（200 mg/kg）＋RhoA激活剂LPA（1 mg/kg）组，另设置假手术组，每组15只。连续给药4周后，Micro CT扫描观察各组大鼠膝关节变化，采用苏木素-伊红（hematoxylin-eosin，HE）和番红O-固绿染色观察软骨组织病理学变化，ELISA法检测关节液中基质金属蛋白酶-13（matrix metalloproteinase-13，MMP-13）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和白细胞介素-6（interleukin-6，IL-6）水平，TUNEL染色观察软骨细胞凋亡，透射电镜观察软骨细胞微观结构，Western blotting检测软骨组织聚集蛋白聚糖（aggrecan）、Ⅱ型胶原蛋白（type II collagen，Col-Ⅱ）、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）、剪切型半胱氨酸天冬氨酸蛋白酶3（cleaved cystein-asparate protease-3，cleaved Caspase-3）、RhoA、ROCK1和ROCK2蛋白表达。结果 与假手术组比较，模型组大鼠软骨组织病理损伤严重，软骨细胞超微结构破坏，Mankin评分和国际骨关节炎研究协会（osteoarthritis research society international，OARSI）评分、软骨细胞凋亡率及关节液中TNF-α、MMP-13、IL-6水平和软骨组织RhoA、ROCK1、ROCK2、Bax、cleaved Caspase-3蛋白表达水平均显著升高（P＜0.05），骨体积分数（bone volume/total volume，BV/TV）、骨小梁厚度（trabecular thickness，Tb.Th）和软骨组织Bcl-2、aggrecan、Col-Ⅱ蛋白表达水平显著降低（P＜0.05）；与模型组比较，硫酸氨基葡萄糖组和桔梗总皂苷各剂量组软骨组织病理损伤减轻，软骨细胞超微结构较为正常，Mankin评分、OARSI评分、软骨细胞凋亡率及关节液中TNF-α、MMP-13、IL-6水平和软骨组织RhoA、ROCK1、ROCK2、Bax、cleaved Caspase-3蛋白表达水平显著降低（P＜0.05），BV/TV、Tb.Th和软骨组织Bcl-2、aggrecan、Col-Ⅱ蛋白表达水平显著升高（P＜0.05）；联用RhoA激活剂LPA后，桔梗总皂苷对以上指标的改善作用均被部分逆转（P＜0.05）。结论 桔梗总皂苷通过抑制RhoA/ROCK通路，改善KOA大鼠软骨损伤。

Objective To explore the effect of total saponins of Platycodon grandiflorum on cartilage injury and Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinase (ROCK) signaling pathway in knee osteoarthritis (KOA) rats.Methods Knee joint injection of papain was used to prepare a KOA model. The rats were randomly divided into model group, glucosamine sulfate (0.2 g/kg) group, total saponins of P. grandiflorum low- and high-dose (100, 200 mg/kg) groups and total saponins of P. grandiflorum (200 mg/kg) + RhoA activator LPA (1 mg/kg) group. A sham group was also included, with 15 rats in each group. After four weeks of continuous administration, micro CT scanning was used to observe changes in knee joints of rats in each group. Hematoxylin-eosin (HE) and Safranin O-fast green staining were used to observe the pathological changes of cartilage tissue. ELISA was used to detect the levels of matrix metalloproteinase-13 (MMP-13), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in joint fluid. TUNEL staining was used to observe chondrocyte apoptosis, and transmission electron microscopy was used to observe the microstructure of chondrocytes. Western blotting was used to detect expressions of aggrecan, type II collagen (Col II), B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved cystein-asparate protease-3 (cleaved Caspase-3), RhoA, ROCK1 and ROCK2 proteins in cartilage tissue. Results Compared with sham group, rats in model group showed severe pathological damage to the cartilage tissue, destruction of the ultrastructure of chondrocytes, Mankin score, osteoarthritis research society international (OARSI) score, chondrocyte apoptosis rate, levels of TNF-α, MMP-13, IL-6 in joint fluid, as well as protein expression levels of RhoA, ROCK1, ROCK2, Bax and cleaved Caspase-3 in cartilage tissue were significantly increased (P < 0.05), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th) and protein expression levels of Bcl-2, aggrecan, Col-II in cartilage tissue were significantly reduced (P < 0.05). Compared with model group, glucosamine sulfate group and total saponins of P. grandiflorum groups showed reduced pathological damage to cartilage tissue, normal ultrastructure of chondrocytes, Mankin score, OARSI score, chondrocyte apoptosis rate, levels of TNF-α, MMP-13, IL-6 in joint fluid, as well as protein expression levels of RhoA, ROCK1, ROCK2, Bax and cleaved Caspase-3 in cartilage tissue were significantly decreased (P < 0.05). BV/TV, Tb.Th and protein expression levels of Bcl-2, aggrecan, Col-II in cartilage tissue were significantly increased (P < 0.05). After the combined use of RhoA activator LPA, the improvement effect of total saponins of P. grandiflorum on the above indicators was partially reversed (P < 0.05). Conclusion Total saponins of P. grandiflorum can improve cartilage damage in KOA rats by inhibiting RhoA/ROCK pathway.

R285.5

上海市科技计划项目（23ZR1457600）