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目的研究熊果酸对瘦素诱导肝星状细胞(hepatic stellate cells,HSC)JAK2-STAT3信号通路活化、活性氧(ROS)产生及基质金属蛋白酶抑制因子-1(TIMP-1)、基质金属蛋白酶1(MMP-1)mRNA表达的影响。方法以重组大鼠瘦素作用于HSC-T6细胞,干预组在瘦素作用前分别加入熊果酸、N-乙酰-L半胱氨酸(NAC)、氯化二亚苯基碘嗡(DPI)及JAK抑制剂AG490干预,正常对照组不加任何药物。各组处理不同时间后,采用流式细胞检测细胞内ROS水平;采用免疫细胞化学检测磷酸化JAK2及STAT3蛋白表达;采用RT-PCR检测TIMP-1 mRNA、MMP-1 mRNA的表达。结果①免疫细胞化学检测显示,瘦素刺激HSC-T6细胞2 h后细胞质p-JAK2及细胞核内p-STAT3蛋白表达与正常对照组相比均明显增加(P<0.01),熊果酸干预后p-JAK2及p-STAT3蛋白表达明显低于瘦素刺激组[(85.50±3.78)vs(96.67±3.01),(26.50±3.66)vs(86.67±10.87),P<0.01]。NAC、DPI、AG490干预后p-JAK2及p-STAT3蛋白表达也低于瘦素刺激组(P<0.05或P<0.01)。②流式细胞术检测显示,熊果酸、NAC、DPI和AG490干预后的ROS水平均低于瘦素刺激组[(43.62±5.58)、(55.56±6.43)、(36.54±3.21)、(48.12±6.63)vs(90.86±3.86),P<0.01],而且熊果酸干预组的ROS水平低于NAC干预组(P<0.01)。③熊果酸干预12、24h后与瘦素刺激组相比,TIMP-1 mRNA表达明显下调[(0.28±0.03)vs(0.37±0.01),(0.29±0.04)vs(0.41±0.03),P<0.01],MMP-1 mRNA的表达明显升高[(0.33±0.02)vs(0.26±0.02),(0.37±0.04)vs(0.22±0.04),P<0.01]。结论熊果酸能阻断瘦素在肝星状细胞信号内信号转导,抑制TIMP-1、上调MMP-1的基因表达。
Objective To study the effects of ursolic acid on leptin-induced activation of JAK2-STAT3 signaling pathway, production of reactive oxygen species (ROS) and expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases (MMPs) in hepatic stellate cells (HSC) 1 (MMP-1) mRNA expression. Methods HSC-T6 cells were treated with recombinant leptin, and the intervention group was given ursolic acid, NAC, NADP, DPI ) And JAK inhibitor AG490 intervention, the normal control group without any drug. Flow cytometry was used to detect intracellular ROS levels after different time points. The expression of phosphorylated JAK2 and STAT3 proteins was detected by immunocytochemistry. The expressions of TIMP-1 mRNA and MMP-1 mRNA were detected by RT-PCR. Results ① Immunocytochemistry showed that leptin stimulated the expression of p-JAK2 and p-STAT3 protein in cytoplasm of HSC-T6 cells after 2 h compared with that of normal control group (P <0.01). After ursolic acid intervention The expression of p-JAK2 and p-STAT3 protein in leptin group was significantly lower than that in leptin group [(85.50 ± 3.78) vs (96.67 ± 3.01) vs (26.50 ± 3.66 vs 86.67 ± 10.87, P <0.01]. The expression of p-JAK2 and p-STAT3 protein after NAC, DPI and AG490 intervention were also lower than those of leptin stimulation group (P <0.05 or P <0.01). ② Flowcytometry showed that the levels of ROS in the ursolic acid, NAC, DPI and AG490 groups were lower than those in the leptin group [(43.62 ± 5.58), (55.56 ± 6.43), (36.54 ± 3.21), (48.12 ± 6.63) vs (90.86 ± 3.86), P <0.01], and the level of ROS in the ursolic acid intervention group was lower than that in the NAC intervention group (P <0.01). ③Compared with the leptin-stimulated group, the expression of TIMP-1 mRNA was significantly down-regulated after ursolic acid intervention for 12 and 24 hours [(0.28 ± 0.03) vs (0.37 ± 0.01), (0.29 ± 0.04) vs (0.41 ± 0.03), P <0.01], the expression of MMP-1 mRNA was significantly increased (0.33 ± 0.02 vs 0.26 ± 0.02, 0.37 ± 0.04 vs 0.22 ± 0.04, P <0.01). Conclusion UA can block the signal transduction of leptin in hepatic stellate cells, inhibit TIMP-1 and up-regulate the gene expression of MMP-1.