独活寄生汤含药血清抑制IL-1β诱导的软骨细胞炎症反应的机制研究

On the Mechanism of Serum Medicated with Duhuo Jisheng Tang(独活寄生汤) Inhibiting Chondrocyte Inflammation Reaction Induced by IL-1β

目的:探讨独活寄生汤含药血清抑制白细胞介素-1β(IL-1β)诱导的软骨细胞炎症反应的机制。方法:采用机械-Ⅱ型胶原酶消化法从4周龄SD大鼠获取膝关节软骨细胞,Ⅱ型胶原免疫组化方法鉴定软骨细胞。将10只8周龄SD大鼠随机分为独活寄生汤组与空白组,每组5只,独活寄生汤组按9.3 g·kg^(-1)·d^(-1)剂量灌胃独活寄生汤,空白组灌服等量生理盐水,连续灌胃1周,末次给药1 h后腹主动脉采血,收集血清备用。将第3代软骨细胞分为正常组(10%空白血清的DMEM培养液干预)、模型组(含15 ng·mL^(-1) IL-1β的10%空白血清的DMEM培养液干预)、独活寄生汤组(含15 ng·mL^(-1) IL-1β的10%独活寄生汤含药血清的DMEM培养液干预),干预12 h后,分别采用免疫荧光技术和Western blot技术检测软骨细胞中基质金属蛋白酶-13(MMP-13)表达变化。结果:Ⅱ型胶原免疫组化染色后软骨细胞胞浆呈棕黄色,为阳性。Western blot及免疫荧光染色结果显示,与正常组比较,模型组细胞中MMP-13表达明显提高,差异有统计学意义(P<0.05);与模型组比较,独活寄生汤组细胞中MMP-13表达明显下降,差异有统计学意义(P<0.05)。结论:独活寄生汤能抑制IL-1β诱导的软骨细胞炎症反应,延缓关节软骨的退变。

Objective：To investigate the mechanism of serum medicated with Duhuo Jisheng Tang （独活寄生汤 ） inhibiting chondrocyte inflammation reaction induced by IL-1β.Methods：The mechanical type Ⅱ collagenase digestion method was used to obtain chondrocytes of knee joint from 4-week-old SD rats and the type Ⅱ collagen immunohistochemical method was used to identify chondrocytes.Ten 8-week-old SD rats were randomly divided into a Duhuo Jisheng Tang group and a control group,5 rats in each.Rats in the Duhuo Jisheng Tang group were given intragastric administration of Duhuo Jisheng Tang （ 9.3 g · kg^-1 · d^-1 ） and those in the control group were orally given the same amount of normal saline for a month.One hour after the last medication,abdomen aortic blood was collected for serum.Cartilage cells of the third generation were divided into a normal group （ intervened with 10% of DMEM of blank serum ） ,a model group （ intervened with 10% of DMEM of blank serum containing 15 ng · mL^-1 of IL-11） ） ,and a Duhuo Jisheng Tang group （ intervened with 10% of DMEM of serum medicated with Duhuo Jisheng Tang containing 15 ng· mL^-1 of IL-1β ） .Alter 12 hours, Immunofluorescence and Western blot were respectively used to detect the changes of MMP-13 expression in chondrocytes.Results：After the type Ⅱ collagen staining,the cytoplasm of the chondrocytes was brownish yellow.The results of Western blot and immunofluorescence assay showed that the expression of MMP-13 in cells of the model group significantly increased compared with that in the normal group,and the difference was statistically significant （ P 〈 0.05 ） .Compared with the model group,the expression of MMP-13 in cells of the Duhuo Jisheng Tang group significantly decreased, and the difference was statistically significant （ P 〈 0.05 ） .Conclusion：Duhuo Jisheng Tang can inhibit the chondrocytes inflammation reaction induced by IL-1β and delay the degeneration of articular cartilage.