葛根素抑制软脂酸诱导的小鼠3T3-L1细胞炎性因子的表达及其机制

Inhibition effect of puerarin on the expression of inflammatory factors in 3T3-L1 cells induced by palmitic acid and its mechanism in mice

目的探讨葛根素对小鼠3T3-L1细胞炎性因子的表达及其机制。方法用200μmol·L^(-1)软脂酸(PA)诱导小鼠3T3-L1成熟脂肪细胞成Toll样受体4(TLR4)介导的炎性细胞模型。将细胞分为4组:对照组(未经PA诱导的3T3-L1脂肪细胞)、模型组和低、高2个剂量实验组(葛根素5,10μmol·L^(-1))。用RT-PCR技术检测各组TLR4 mRNA表达,用酶联免疫吸附法检测各组细胞培养液中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平,以免疫印迹法检测核因子κB激酶抑制剂β(IKKβ)、核因子κB抑制蛋白(IκB)及核因子κB-p65(NF-κB-p65)蛋白表达。结果对照组与模型组的TLR4 mRNA分别为1.20±0.20,6.23±0.80;这2组的TNF-α水平分别为(20.40±2.0),(60.20±8.0)pg·m L^(-1);这2组的IL-6表达水平分别为(4.20±0.2),(14.40±0.4)pg·m L^(-1),模型组与正常组的这3个指标比较,差异均有统计学意义(均P<0.01)。低、高2个剂量实验组的这3个指标分别为4.80±0.60和(38.80±6.0),(35.60±5.0)pg·m L^(-1);3.56±0.50和(8.80±0.5),(6.56±0.4)pg·m L^(-1),低、高2个剂量实验组与模型组比较,差异均有统计学意义(P<0.05,P<0.01)。模型组的IKKβ、IκB、NF-κB-p65蛋白相对表达水平分别为2.10±0.4,4.20±0.5,5.50±0.2;高剂量实验组的这3个指标分别为1.40±0.3,2.10±0.4,3.60±0.6,高剂量实验组与模型组相比,差异均有统计学意义(均P<0.01)。结论高剂量葛根素可抑制PA诱导的3T3-L1细胞炎性因子的表达,其机制可能是通过减少3T3-L1细胞TLR4基因表达、下调IKKβ、IκB、NF-κB-p65活化并降低TNF-α、IL-6炎性因子表达来实现的。

Objective To explore the effect and mechanism of puerarin on inflammatory response in murine 3 T3-L1 adipocytes. Methods Tolllike receptor 4（ TLR4） mediated inflammatory cell models were based on palmitic acid（ PA） 200 μmol·L^-1 stimulated 3 T3-L1 adipocytes cells. And then,these cells were divided into 4 groups： control group（ without PA induction in 3 T3-L1 cells）,model group,low and high dose experimental groups（ 5,10 μmol · L^-1 puerarin）. The mRNA expression of TLR4 was detected by Real-time PCR. The tumor necrosis factor α（ TNF-α） and interleukin-6（ IL-6） were performed using enzyme-linked immuno sorbent assay. The protein expressions of inhibitor of nuclear factor kappa-B kinase β（ IKKβ）,nuclear factor kappa Binhibitory protein（ IκB）,nuclear factor kappa B-p65（ NF-κB p65） in 3 T3-L1 adipocytes cells were assessed by Western blot. Results The mRNA expression of TLR4 in control group and model group were 1. 20 ± 0. 20,6. 23 ± 0. 80; the level of TNF-α in the two groups were（ 20. 40 ± 2. 0）,（ 60. 20 ± 8. 0） pg·m L^-1; the level of IL-6 in the two groups were（ 4. 20 ± 0. 2）,（ 14. 40 ± 0. 4） pg·m L^-1. Comparison between control group and model group,the differnce on the three factors had significantly（ all P〈0. 01）. Meanwhile,after administration of puerarin,the protein expressions of IKKβ,IκB and NF-κ-p65 in model group were 2. 10 ± 0. 4,4. 20 ± 0. 5,5. 50 ± 0. 2; that in experimental-H group were 1. 40 ± 0. 3,2. 10 ± 0. 4,3. 60 ± 0. 6. Comparison between experimental-H group and model group,the differnce on the three factors had significantly（ all P〈0. 01）. Conclusion Experimental-H group attenuates palmitate-induced inflammatory response in 3 T3-L1 adipocytes,its underlying mechanism include inhibiting expression of TLR4 and downregulating activation of IKKβ,IκB,NF-κB-p65 and decreased expression of TNF-α,IL-6 levels.