柴胡疏肝散对炎性损伤肋软骨细胞活性的改善作用及其机制

Improvement effect of Chaihu Shugan powder on activity of inflammatory damaged costal chondrocytes and its mechanism

目的观察柴胡疏肝散对炎性损伤肋软骨细胞活性的改善作用,并探讨其机制。方法取对数生长期肋软骨细胞分为对照组、IL-1β组、柴胡疏肝散低剂量组、柴胡疏肝散中剂量组、柴胡疏肝散高剂量组。对照组常规培养肋软骨细胞;IL-1β组在含有肋软骨细胞的培养基中加入40 ng/mL的IL-1β;柴胡疏肝散高剂量组、柴胡疏肝散中剂量组、柴胡疏肝散低剂量组在含有肋软骨细胞的培养基中加入40 ng/mL的IL-1β后,再分别加入80、40、20μg/mL的柴胡疏肝散溶液。各组细胞继续培养24 h,采用CCK-8法检测各组细胞活性(以OD值表示),采用蛋白免疫印迹法检测各组细胞NFκB/NLRP3-Caspase-1通路相关蛋白p-NFκB、NLRP3、ASC、Caspase-1。结果IL-1β组细胞OD值低于其余各组(P均<0.05),柴胡疏肝散高剂量组、柴胡疏肝散中剂量组细胞OD值均高于其余各组(P均<0.05)。与对照组相比,IL-1β组、柴胡疏肝散低剂量组细胞中p-NFκB、NLRP3、ASC、Caspase-1蛋白相对表达量均显著增高(P均<0.05),柴胡舒肝散中剂量组中p-NFκB蛋白相对表达量也显著增高(P<0.05);与IL-1β组相比,柴胡疏肝散高剂量组、柴胡疏肝散中剂量组的p-NFκB、NLRP3、ASC、Caspase-1蛋白相对表达量均显著降低(P均<0.05),柴胡疏肝散低剂量组中Caspase-1蛋白相对表达量也显著降低(P<0.05);与柴胡疏肝散低剂量组比较,柴胡疏肝散高剂量组、柴胡疏肝散中剂量组的p-NFκB蛋白相对表达量显著降低(P均<0.05),柴胡疏肝散高剂量组ASC蛋白相对表达量显著降低(P<0.05);与柴胡疏肝散中剂量组相比,柴胡疏肝散高剂量组p-NFκB蛋白相对表达量显著降低(P<0.05)。结论40μg/mL、80μg/mL的柴胡疏肝散均可提高炎性损伤肋软骨细胞的细胞活性。柴胡疏肝散对炎性损伤肋软骨细胞活性的改善作用机制可能与调控NFκB/NLRP3-Caspase-1通路相关蛋白的表达有关。

Objective To observe the improvement effect of Chaihu Shugan powder on the activity of inflammatory damaged costal chondrocytes,and to explore its mechanism.Methods The costal chondrocytes in the logarithmic growth phase were divided into the control group,IL-1βgroup,and low-dose,medium-dose,and high-dose Chaihu Shugan powder groups,respectively.Costal chondrocytes in the control group were cultured conventionally.In the IL-1βgroup,40 ng/mL of IL-1βwas added into the medium containing costal chondrocytes.Costal chondrocytes in the highdose group,medium-dose group and low-dose Chaihu Shugan powder groups were added with 40 ng/mL of IL-1βin the medium containing costal chondrocytes,and then 80,40,20μg/mL of Chaihu Shugan powder solution were added,respectively.The cells in each group were continued to be cultured for 24 h,then CCK-8 was used to observe the cell activity of each group(expressed by OD value),and Western blotting was used to detect the expression levels of NFκB/NLRP3 signaling pathway-related proteins p-NFκB,NLRP3,ASC,and Caspase-1.Results The OD value of the IL-1βgroup was significantly lower than those of the other groups(all P<0.05),and the OD values of the high-dose and medium-dose Chaihu Shugan powder groups were higher than those of the other groups(all P<0.05).Compared with the control group,the protein expression levels of p-NFκB,NLRP3,ASC and Caspase-1 in the IL-1βgroup and low-dose Chaihu Shugan powder group significantly increased(all P<0.05),and the protein expression of P-NFκB in the medium-dose Chaihu Shugan powder group also significantly increased(P<0.05).Compared with the IL-1βgroup,the protein expression levels of PNFκB,NLRP3,ASC and Caspase-1 in the high-dose and medium-dose Chaihu Shugan powder groups significantly decreased(all P<0.05),and Caspase-1 protein in the low-dose Chaihu Shugan powder group also significantly decreased(P<0.05).Compared with the low-dose Chaihu Shugan powder group,the protein expression levels of p-NFκB in the highdose and medium-dose Chaihu Shugan powder groups significantly decreased(all P<0.05),and the protein expression of ASC in the high-dose group significantly decreased(P<0.05).Compared with the medium-dose Chaihu Shugan powder group,the expression of p-NFκB in the high-dose Chaihu Shugan powder group significantly decreased(P<0.05).Con⁃clusions Chaihu Shugan powder of 40 and 80μg/mL can enhance the cellular activity of inflammatory damaged costal chondrocytes.The mechanism of Chaihu Shugan powder in improving the activity of inflammatory damaged costal chondrocytes may be related to regulating the expression of NFκB/NLRP3-Caspase-1 pathway-related proteins.