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目的:观察下瘀血汤药物血清对脂多糖(LPS)诱导活化后RAW 264.7细胞表达基质金属蛋白酶(MMP-2,9)及基质金属蛋白酶组织抑制剂(TIMP-1,2)的影响,以探讨其抗肝纤维化的作用机制。方法:100 g/L LPS刺激RAW264.7细胞,ELISA法分别测定0.5 h,1 h,2 h,4 h,6 h,12 h细胞培养上清中肿瘤坏死因子-α(TNF-α)含量,选择模型时间点;生化法测定RAW264.7细胞培养上清中乳酸脱氢酶(LDH)含量,分析10%和20%的正常大鼠血清和下瘀血汤药物血清的细胞毒性;Western blot法检测RAW 264.7细胞MMP-2、MMP-9、TIMP-1、TIMP-2的蛋白表达。结果:与10%浓度的正常大鼠血清相比,10%下瘀血汤药物血清对RAW264.7细胞的无毒性作用。LPS刺激细胞培养1 h时,细胞培养上清中TNF-α含量开始增加;4 h时达到最高;6 h时开始下降。与相应时间点正常组相比,4 h、6 h模型组RAW 264.7细胞MMP-2、MMP-9、TIMP-1和TIMP-2蛋白表达均显著升高(P<0.01);与相应时间点模型组相比,4 h、6 h下瘀血汤药物血清组上述指标均降低,其中6 h组RAW 264.7细胞MMP-2蛋白表达、4 h与6 h细胞MMP-9、TIMP-1和TIMP-2蛋白表达显著降低(P<0.01)。结论:下瘀血汤可能通过抑制枯否细胞(KCs)活化,抑制其MMP2,9/TIMP1,2的表达而发挥抗肝纤维化的作用。
OBJECTIVE: To observe the effect of serum from Xiaoyu Xue Tang on expression of matrix metalloproteinase (MMP-2, MMP-9) and matrix metalloproteinase inhibitor (TIMP-1,2) in RAW 264.7 cells induced by lipopolysaccharide To explore its mechanism of anti-liver fibrosis. Methods: RAW264.7 cells were stimulated with 100 g / L LPS, and the levels of tumor necrosis factor-α (TNF-α) in the culture supernatants of 0.5 h, 1 h, 2 h, 4 h, 6 h, , And the time point of the model was selected. The content of lactate dehydrogenase (LDH) in the culture supernatant of RAW264.7 cells was determined by biochemical method. The cytotoxicity of 10% and 20% Method to detect the protein expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in RAW 264.7 cells. Results: Compared with 10% normal rat serum, the drug serum of 10% Xiaoyuyu Decoction had no toxic effect on RAW264.7 cells. At 1 h after LPS stimulation, the TNF-α level in the cell culture supernatant began to increase, reaching the highest level at 4 h and then decreasing at 6 h. The mRNA expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in RAW 264.7 cells in 4 h and 6 h groups were significantly increased compared with the normal group at the corresponding time point (P <0.01) Compared with model group, the indexes of serum, serum hs-CRP and serum MMP-2, MMP-2, TIMP-1 and TIMP in RAW 264.7 cells at 6 h and 4 h, -2 protein expression was significantly lower (P <0.01). Conclusion: Xiaoyu Xuexiao decoction could inhibit hepatic fibrosis by inhibiting the activation of KCs and inhibiting the expression of MMP2,9 / TIMP1,2.