姜黄素对脂多糖诱导肺细胞损伤的作用及其机制研究

Mechanism of curcumin in alleviating LPS-induced lung cell injury

摘要:

目的 探究姜黄素通过介导长链非编码RNA THRIL（lncRNA THRIL）表达对脂多糖（LPS）诱导的人正常肺上皮细胞（BEAS-2B）损伤的影响。方法 LPS诱导BEAS-2B细胞复制体外急性肺损伤细胞模型，并加入姜黄素处理。采用Lipofectamine^?2000转染试剂将lncRNA THRIL过表达/敲降载体或空载体转染到BEAS-2B细胞中。通过实时荧光定量聚合酶链反应和酶联免疫吸附试验检测lncRNA THRIL及炎症细胞因子［白细胞介素1β（IL-1β）、白细胞介素6（IL-6）、肿瘤坏死因子-α（TNF-α）］水平；CCK-8法和流式细胞术分别检测LPS、姜黄素及THRIL对细胞活性和细胞凋亡的影响。结果 姜黄素组与对照组lncRNA THRIL相对表达量比较，差异无统计学意义（P >0.05），LPS组较对照组升高（P <0.05），LPS+姜黄素2.5 μmol/L组、LPS+姜黄素5 μmol/L组、LPS+姜黄素7.5 μmol/L组较LPS组降低（P <0.05）。姜黄素组与对照组细胞凋亡率比较，差异无统计学意义（P >0.05），LPS组较对照组升高（P <0.05），LPS+姜黄素2.5 μmol/L组、LPS+姜黄素5 μmol/L组、LPS+姜黄素7.5 μmol/L组较LPS组降低（P <0.05）。姜黄素组与对照组IL-1β、IL-6和TNF-α相对表达量及浓度比较，差异无统计学意义（P >0.05），LPS组较对照组升高（P <0.05），LPS+姜黄素2.5 μmol/L组、LPS+姜黄素5 μmol/L组、LPS+姜黄素7.5 μmol/L组较LPS组降低（P <0.05）。姜黄素组与对照组细胞活性比较，差异无统计学意义（P >0.05），LPS组较对照组降低（P <0.05），LPS+姜黄素2.5 μmol/L组、LPS+姜黄素5 μmol/L组、LPS+姜黄素7.5 μmol/L组较LPS组升高（P <0.05）。敲降THRIL组lncRNA THRIL的相对表达量较敲降阴性对照组降低（P <0.05）。LPS+敲降阴性对照组与LPS组细胞凋亡率比较，差异无统计学意义（P >0.05），LPS组较对照组升高（P <0.05），LPS+敲降THRIL组较LPS+敲降阴性对照组降低（P <0.05）。LPS+敲降阴性对照组与LPS组IL-1β、IL-6和TNF-α的相对表达量及浓度比较，差异无统计学意义（P >0.05），LPS组较对照组升高（P <0.05），LPS+敲降THRIL组较LPS+敲降阴性对照组降低（P <0.05）。LPS+敲降阴性对照组与LPS组细胞活性比较，差异无统计学意义（P >0.05），LPS组较对照组降低（P <0.05），LPS+敲降THRIL组较LPS+敲降阴性对照组升高（P <0.05）。过表达THRIL组lncRNA THRIL相对表达量较过表达阴性对照组升高（P <0.05）。LPS+姜黄素+过表达阴性对照组与LPS+姜黄素组细胞活性比较，差异无统计学意义（P >0.05），LPS组较对照组降低（P <0.05），LPS+姜黄素组较LPS组升高（P <0.05），LPS+姜黄素+过表达THRIL组较LPS+姜黄素+过表达阴性对照组细胞活性降低（P <0.05）。LPS+姜黄素+过表达阴性对照组与LPS+姜黄素组细胞凋亡率比较，差异无统计学意义（P >0.05），LPS组较对照组升高（P <0.05），LPS+姜黄素组较LPS组降低（P <0.05），LPS+姜黄素+过表达THRIL组较LPS+姜黄素+过表达阴性对照组升高（P <0.05）。LPS+姜黄素+过表达阴性对照组与LPS+姜黄素组IL-1β、IL-6和TNF-α的相对表达量及浓度比较，差异无统计学意义（P >0.05），LPS组较对照组升高（P <0.05），LPS+姜黄素组较LPS组降低（P <0.05），LPS+姜黄素+过表达THRIL组较LPS+姜黄素+过表达阴性对照组升高（P <0.05）。结论 姜黄素通过抑制THRIL表达，改善LPS诱导的BEAS-2A细胞凋亡和炎症反应。

Abstract:

Objective To explore the effect of curcumin on lipopolysaccharide (LPS)-induced lung epithelial injury by mediating the expression of long non-coding RNA THRIL.Methods The BEAS-2B cells were subject to LPS to establish the acute lung epithelial injury cell models in vitro, and they were treated with curcumin. LncRNA THRIL overexpression/knockdown vectors or empty vectors were transfected into BEAS-2B cells via Lipofectamine 2000. The expression levels of lncRNA THRIL and inflammatory cytokines (IL-1β, IL-6, and TNF-α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or enzyme-linked immunosorbent assay (ELISA). The effects of LPS, curcumin and lncRNA THRIL on cell viability and apoptosis were measured by flow cytometry and CCK-8.Results There was no difference in the expression of lncRNA THRIL between the curcumin group and the control group (P > 0.05). However, the expression of lncRNA THRIL was higher in the LPS group than in the control group (P < 0.05), whereas it was lower in the LPS + 2.5 μmol/L curcumin group, LPS + 5 μmol/L curcumin group and LPS + 7.5 μmol/L curcumin group than in the LPS group (P < 0.05). There was no difference in the levels of IL-1β, IL-6, and TNF-αbetween the curcumin group and the control group (P > 0.05). In contrast, the levels of IL-1β, IL-6, and TNF-α were higher in the LPS group than in the control group (P < 0.05), while they were lower in the LPS + 2.5 μmol/L curcumin group, LPS + 5 μmol/L curcumin group and LPS + 7.5 μmol/L curcumin group than in the LPS group (P < 0.05). The expression of lncRNA THRIL was lower in the THRIL knockdown group than that in the knockdown negative control group (P < 0.05). There was no difference in the apoptosis rate between the LPS + knockdown negative control group and the LPS group (P > 0.05), while the apoptosis rate was higher in the LPS group compared with the control group (P < 0.05) and was lower in the LPS + THRIL knockdown group compared with the LPS + knockdown negative control group (P < 0.05). The levels of IL-1β, IL-6, and TNF-α were not different between the LPS + THRIL knockdown group and the LPS group (P > 0.05), while they were higher in the LPS group than those in the control group (P < 0.05) and were lower in the LPS + THRIL knockdown group than those in the LPS + knockdown negative control group (P < 0.05). The cell viability was not different between the LPS + knockdown negative control group and the LPS group (P > 0.05), but was lower in the LPS group than that in the control group (P < 0.05) and was higher in the LPS + THRIL knockdown group than that in the LPS + knockdown negative control group (P < 0.05). The expression of lncRNA THRIL was higher in the THRIL overexpression group than that in the overexpression negative control group (P < 0.05). There was no difference in the cell viability between the LPS + curcumin + overexpression negative control group and the LPS + curcumin group (PP > 0.05), while the cell viability was lower in the LPS group than that in the control group (P < 0.05) and was higher in the LPS + curcumin group than that in the LPS group (P < 0.05). Besides, the cell viability was lower in the LPS + curcumin + THRIL overexpression group than that in the LPS + curcumin + overexpression negative control group (P < 0.05). There was no difference in the apoptosis rate between the LPS + curcumin + overexpression negative control group and the LPS + curcumin group (P > 0.05), whereas the apoptosis rate was higher in the LPS group than that in the control group (P < 0.05) and was lower in the LPS + curcumin group than that in the LPS group (P < 0.05). Compared with the LPS + curcumin + overexpression negative control group, the apoptosis rate was higher in the LPS + curcumin + THRIL overexpression group (P < 0.05). The levels of IL-1β, IL-6, and TNF-α were not different between the LPS + curcumin + overexpression negative control group and the LPS + curcumin group (P > 0.05), while they were higher in the LPS group than those in the control group (P < 0.05), were lower in the LPS + curcumin group than those in the LPS group (P < 0.05), and were higher in the LPS + curcumin + THRIL overexpression group than those in the LPS + curcumin + overexpression negative control group (P < 0.05).Conclusions Curcumin ameliorates the apoptosis and inflammatory response induced by LPS in BEAS-2A cells by inhibiting the expression of lncRNA THRIL.