Objective To investigate the inhibition of proliferation and regulation of heaptic fibrosis-related genes in HSC-T6 cells by prolyloligopeptidase(POP)inhibitor in vitro. Methods The HSC-T6 cells were cultured,and the POP activity was inhibited by increasing concentrations of POP inhibitor(S17092). The intracellular level of N-acetyl- seryl-aspartyl-lysyl-proline(Ac-SDKP)was determined by ELISA. The cell apoptosis and cell proliferation were detected by flow cytometry and CCK-8 assay,respectively. The mRNA levels of TGF-β1,α-SMA,monocyte chemoattractant protein 1(MCP-1)and collagen I(Col I)were detected by realtime-PCR,and the POP,TGF-β1,α-SMA,Smad7,p-Smad2/3 and PPAR-γ expression were detected by Western blot. Results The intracellular Ac-SDKP level decreased significantly after co-culture with S17092,the POP inhibitor (P<0.05)as compared to that in the control; the proliferation of HSC-T6 cells was significantly inhibited(P<0.05) and the cell apoptosis was not influenced obviously after S17092 intervention(P>0.05);the Smad7(P<0.05)and PPAR-γ(P<0.05)protein levels were significantly down-regulated,and the α-SMA protein level was significantly up-regulated(P<0.05)after S17092 intervention;the MCP-1(P<0.05)and α-SMA(P<0.05)mRNA level were significantly up-regulated. Conclusion POP plays a pivotal role in anti-inflammation and anti-fibrosis in HSC cells,which might be related to the regulation of Smad7 and PPAR-γ expression.%目的 体外探讨肝星状细胞(HSC)脯氨酰寡肽酶(POP)的生物学功能,以进一步阐述其在肝脏炎症和纤维化发生发展中的作用.方法 取HSC-T6细胞,经不同浓度的POP抑制剂(S17092)处理,采用ELISA法检测HSC-T6细胞N- 乙酰基- 丝氨酸- 天冬氨酸- 赖氨酸- 脯氨酸(Ac-SDKP)水平,使用流式细胞术和CCK8检测HSC-T6凋亡和增殖水平,采用实时定量-PCR法检测相关炎症和肝纤维化基因转化生长因子-β1(TGF-β1)、α- 平滑肌肌动蛋白(α-SMA)、单核细胞趋化蛋白-1(MCP-1)、Ⅰ型胶原(Col I)水平,采用Western blot法检测相关蛋白(POP、TGF-β1、α-SMA、Smad7、p-Smad2/3、PPAR-γ)表达.结果 与对照组比,POP抑制剂干预组细胞内Ac-SDKP水平显著下降(P<0.05);POP被抑制后对HSC-T6凋亡没有显著的影响(P>0.05),但可抑制其增殖(P<0.05);与对照组比,抑制剂组HSC内α-SMA蛋白表达显著升高(P<0.05),而Smad7和PPAR-γ蛋白表达显著下降(P均<0.05);抑制剂组HSC内MCP-1和α-SMA mRNA水平显著上调(P均<0.05).结论 POP在肝星状细胞内可能发挥重要的抗炎抗纤维化作用,其机制可能与其调节Ac-SDKP及Smad7和PPAR-γ的表达有关.
展开▼
