目的 研究山姜素對脂多糖誘導的結腸NCM460細胞損傷修復及對維生素D3受體(VDR)/核因子E2相關因子2(Nrf2)/血紅素加氧酶-1(HO-1)通路的調節(jié)作用。方法 使用脂多糖建立NCM460細胞損傷模型,分別設置對照組,山姜素低、中、高劑量組及白藜蘆醇組,山姜素低、中、高劑量組分別加入終濃度為25、50、100μmol/L的山姜素,白藜蘆醇組加入終濃度為120μmol/L的白藜蘆醇,繼續(xù)培養(yǎng)72 h后,CCK-8法測定NCM460細胞增殖率,酶聯(lián)免疫吸附法(ELISA)測定NCM460細胞上清液腫瘤壞死因子-α(TNF-α)、白細胞介素(IL)-17、IL-8、IL-6、IL-1β及NCM460細胞勻漿液超氧化物歧化酶(SOD)、一氧化氮(NO)、丙二醛(MDA)水平,流式細胞術測定NCM460細胞凋亡率,使用試劑盒測定NCM460細胞線粒體膜電位水平,實時定量聚合酶鏈式反應(RT-qPCR)測定NCM460細胞VDR、Nrf2及HO-1 mRNA水平,免疫印跡法(Western blotting)測定NCM460細胞VDR、Nrf2及HO-1蛋白水平。結果 與模型組比較,山姜素各劑量組及白藜蘆醇組NCM460細胞增殖率、SOD水平、線粒體膜電位、VDR、Nrf2及HO-1 mRNA及蛋白水平顯著升高(P<0.05),NCM460細胞上清液TNF-α、IL-17、IL-8、IL-6、IL-1β水平、細胞勻漿液NO及MDA水平、NCM460細胞凋亡率顯著降低(P<0.05)。結論 山姜素能夠顯著抑制脂多糖誘導的結腸NCM460細胞損傷后炎癥因子的釋放及細胞凋亡,恢復NCM460細胞氧化損傷的修復能力及線粒體膜電位改變,促進NCM460細胞增殖,其機制可能與調節(jié)VDR/Nrf2/HO-1通路有關。

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, 100 μmol/L of alpinetin, respectively, and resveratrol group was added with final concentration of 120 μmol/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-1β level,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 NCM460cells. The mechanism may be related to the regulation of VDR/Nrf2/HO-1 pathway.

R285

海南省衛(wèi)生健康行業(yè)科研項目(22A200144)