独活寄生汤对脂多糖诱导的大鼠软骨细胞炎症模型IL-1β、TNF-α表达的影响

Effects of DuhuoJisheng decoction on the expression of IL-1β and TNF-α of inflammatory Chondrocytes

目的观察独活寄生汤水提物对体外培养的炎症模型大鼠软骨细胞IL-1β和TNF-α的影响。方法采用4周龄SD大鼠分离培养体外软骨细胞,Ⅱ型胶原免疫组织化学染色法对第二代软骨细胞进行鉴定。脂多糖诱导软骨细胞,建立软骨细胞的炎症模型,确定最佳干预浓度和时间;模型复制成功后,随机分为空白对照组(脂多糖浓度为0 ng/ml)、模型组(脂多糖浓度为10 ng/ml)和独活寄生汤组,分别干预软骨细胞,elisa法检测各组细胞上清液中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的表达变化。结果造模实验中,各组干预8 h后,脂多糖浓度为10 ng/ml、100 ng/ml、1 000 ng/ml、2 000 ng/ml时,细胞上清液中IL-1β和TNF-α表达高于空白对照组。脂多糖浓度为10 ng/ml时,IL-1β和TNF-α表达高于100 ng/ml、1 000 ng/ml、2 000 ng/ml,差异具有统计学意义(P<0.01);10 ng/ml脂多糖分别干预软骨细胞4 h,8 h,12 h和24 h,细胞上清液中IL-1β(t=-30.869,t=-8.422,t=-13.999,t=-19.532,P<0.05)和TNF-α(t=-9.441,t=-14.053,t=-4.238,t=-4.124,P<0.05)的表达均高于空白对照组,且干预8 h时细胞上清液中IL-1β(F=21.48,P<0.01)和TNF-α(F=104.15,P<0.01)的表达高于4 h,12 h和24 h。独活寄生汤组应用独活寄生汤干预8 h造模细胞后,独活寄生汤浓度为100μg/ml、200μg/ml、300μg/ml时,细胞上清液中IL-1β和TNF-α的表达均低于空白对照组,差异具有统计学意义(F=323.95,F=2789.80,P<0.01),浓度300μg/ml时IL-1β和TNF-α表达低于100μg/ml和200μg/ml时,差异具有统计学意义(F=34.46,F=4.373,P<0.01)。结论脂多糖浓度为10 ng/ml时,可成功建立体外软骨细胞炎症模型,独活寄生汤浓度为300μg/ml时可有效抑制软骨细胞炎症模型IL-1β、TNF-α的表达。

Objective To observe the efficacy of Duhuo Jisheng decoction (DHJSD) on the expression of IL- 1β and TNF- α in lipopolysaccharide (LPS)- treated Chondrocytes in vitro. Methods Chondrocytes was isolated from 4-week Sprague-Dawley rats and cells were cultured in vitro. The second generation of chondrocytes were identified with Type II collagen immunohistochemical staining.Chondrocytes were incubated with different concentrations of LPS in different time intervals to establish the optimal inflammatory model. Once it was duplicated successfully, three series groups were randomly divided into blank control group (0 ng/ml LPS), model group (10 ng/ml LPS) and DHJSD group to compare the expression levels of IL-1β and TNF-α using enzyme-linked immunosorbent assay (ELISA). Results In the modeling experiment, each group were initially intervened with different concentrations of LPS (100, 200, 300 μg/ml) for 8 h. Expression of IL-1 beta and TNF- alpha were detected and higher expression level were observed compare to the blank control group and chondrocytes intervened with 10 ng/ml LPS for 4 h, 8 h, 12 h and 24 h, the expression of supernatant IL-1 beta (P<0.01)and TNF- alpha (P<0.01) were all higher than the blank control group, the highest expression level occurred at 8 h after intervention (P<0.01). In the DHJSD group, 100 μg/ml, 200 μg/ml and 300 μg/ml DHJSD were used to intervene chondrocytes for 8 h,the expression of supernatant IL-1 beta (t=-30.869, t=-8.422, t=-13.999, t=-19.532, P<0.01) and TNFalpha(t=- 9.441, t=- 14.053, t=- 4.238, t=- 4.124, P<0.05) were lower than the blank control group,difference was statistically significant. The expression of supernatant IL-1 beta (F=21.48, P<0.01) and TNFalpha(F=104.15, P<0.01) were higher than 4 h, 12 h and 24 h compared to 8 h. And the expression of IL-1 beta (F=34.46, P<0.01) and TNF- alpha (F=4.373, P<0.05) at 300 μg/ml DHJSD was lower than 100 μg/ml and 200 μg/ml (P<0.01). Conclusion Chondrocyte inflammatory model can establish successfully with 10 ng/mL LPS, and DHJSD can effevtively reduce the expression of IL-1β and TNF-α at 300 μg/ml .