山姜素对脂多糖诱导的结肠NCM460细胞损伤及对VDR/Nrf2/HO-1通路的作用研究

Effects of alpinetin on lipopolysaccharids-induced colon NCM460 cell damage and VDR/Nrf2/HO-1 pathway

目的研究山姜素对脂多糖诱导的结肠NCM460细胞损伤修复及对维生素D3受体(VDR)/核因子E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)通路的调节作用。方法使用脂多糖建立NCM460细胞损伤模型,分别设置对照组,山姜素低、中、高剂量组及白藜芦醇组,山姜素低、中、高剂量组分别加入终浓度为25、50、100μmol/L的山姜素,白藜芦醇组加入终浓度为120μmol/L的白藜芦醇,继续培养72 h后,CCK-8法测定NCM460细胞增殖率,酶联免疫吸附法(ELISA)测定NCM460细胞上清液肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-17、IL-8、IL-6、IL-1β及NCM460细胞匀浆液超氧化物歧化酶(SOD)、一氧化氮(NO)、丙二醛(MDA)水平,流式细胞术测定NCM460细胞凋亡率,使用试剂盒测定NCM460细胞线粒体膜电位水平,实时定量聚合酶链式反应(RT-qPCR)测定NCM460细胞VDR、Nrf2及HO-1 mRNA水平,免疫印迹法(Western blotting)测定NCM460细胞VDR、Nrf2及HO-1蛋白水平。结果与模型组比较,山姜素各剂量组及白藜芦醇组NCM460细胞增殖率、SOD水平、线粒体膜电位、VDR、Nrf2及HO-1 mRNA及蛋白水平显著升高(P<0.05),NCM460细胞上清液TNF-α、IL-17、IL-8、IL-6、IL-1β水平、细胞匀浆液NO及MDA水平、NCM460细胞凋亡率显著降低(P<0.05)。结论山姜素能够显著抑制脂多糖诱导的结肠NCM460细胞损伤后炎症因子的释放及细胞凋亡,恢复NCM460细胞氧化损伤的修复能力及线粒体膜电位改变,促进NCM460细胞增殖,其机制可能与调节VDR/Nrf2/HO-1通路有关。

Objective To study the effects of alpinetin on lipopolysaccharide induced colon NCM460 cell damageand regulation of VDR/Nrf2/HO-1 pathway.Method NCM460 cells were treated with lipopolysaccharide to establish damage model.Control group,low,medium and high dose groups of alpinetin group,and resveratrol group were set up,respectively.Low,medium and high dose groups of alpinetin were added with final concentration of 25,50,100μmol/L of alpinetin,respectively,and resveratrol group was added with final concentration of 120μmol/L.Continue to develop after 72 h,CCK-8 method was used to detect NCM460 cell proliferation rate and enzyme-linked immunosorbent(ELISA)was used to measure the levels of TNF-α,IL-17,IL-8,IL-6,IL-1β,SOD,NO,MDA of NCM460 cells.The apoptosis rate of NCM460 cells was determined by flow cytometry,mitochondrial membrane potential of NCM460 cells was determined by kits,and mRNA levels of VDR,Nrf2 and HO-1 of NCM460 cells were determined by real-time quantitative polymerase chain reaction(RT-qPCR).The protein levels of VDR,Nrf2,and HO-1 in NCM460 cells were determined by Western blotting.Results Compared with model group,the proliferation rate of NCM460 cells,SOD level,mitochondrial membrane potential,VDR,Nrf2 and HO-1 mRNA and protein levels of NCM460 cells in various dosage groups of alpinetin and resveratrol group were significantly increased(P<0.05),and NCM460 cells that TNF-α,IL-17,IL-8,IL-6,IL-1βlevel,NO and MDA level,NCM460 apoptosis rate was significantly lower(P<0.05).Conclusions Alpinetin can significantly inhibit the release of inflammatory factors and apoptosis of colon NCM460 cells after lipopolysaccharida-induced injury,restore the repair ability of oxidative damage and the change of mitochondrial membrane potential of NCM460 cells,and promote the proliferation of NCM460 cells.The mechanism may be related to the regulation of VDR/Nrf2/HO-1 pathway.