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lncRNA A_30_P01029806减轻脓毒症急性肺损伤的机制研究
作者:林润  罗凌青  饶平  李乐辉  陈耿仟 
单位:海南医学院第一附属医院 急诊科, 海南 海口 570102
关键词:lncRNA A_30_P01029806 脓毒症 急性肺损伤 炎症反应 NF-κB信号通路 
分类号:R563.2
出版年·卷·期(页码):2021·49·第八期(897-903)
摘要:

目的:探讨lncRNA A_30_P01029806对脓毒症急性肺损伤的影响及作用机制。方法:采用内毒素(LPS)处理体外培养的小鼠肺上皮细胞(MLE-12),实验分为对照组(Ctrl组)、LPS处理组(LPS组)、敲低A_30_P01029806组(LPS+si-A30P组)和转染对照组(LPS+si-NC组)。实时荧光定量PCR (qPCR)检测各组细胞中A_30_P01029806的表达量,噻唑蓝(MTT)实验检测细胞活力,流式细胞术检测细胞凋亡情况,酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)含量,蛋白质印迹法(Western blot)检测NF-κB信号通路中关键蛋白表达量。在LPS+si-A30P组细胞中添加NF-κB信号通路特异性抑制剂BAY11-7082,采用同样的方法分析BAY11-7082对细胞活力、凋亡以及TNF-α和IL-6表达的影响。将24只雄性C57BL/6小鼠随机分为假手术组(Sham组)、脓毒症组(CLP组)、敲低A_30_P01029806组(si-A30P组)和siRNA对照组(si-NC组),各组于造模24 h后取肺组织,进行肺损伤半定量评分(IQA),计算肺湿/干重比(W/D),采用ELISA检测肺组织中TNF-α和IL-6的含量。结果:与Ctrl组比,LPS组细胞活力明显降低,细胞中A_30_P01029806的表达量、凋亡率、IκBα和p65磷酸化水平明显升高,TNF-α和IL-6含量明显增多,差异均有统计学意义(P<0.05);与LPS组比,LPS+si-A30P组上述各指标均得到明显逆转,差异均有统计学意义(P<0.05);LPS组与LPS+si-NC组上述各指标间差异无统计学意义(P>0.05)。BAY11-7082能够部分逆转敲低A_30_P01029806对细胞活力提高、凋亡以及炎症因子分泌的抑制作用。与Sham组比,CLP组小鼠肺损伤评分、W/D值及肺组织中TNF-α和IL-6的含量均明显升高,差异均有统计学意义(P<0.05);与CLP组比,si-A30P组肺损伤评分、W/D值、TNF-α和IL-6含量均明显降低,差异均有统计学意义(P<0.05);CLP组与si-NC组上述各指标间差异无统计学意义(P>0.05)。结论:敲低A_30_P01029806能够减轻脓毒症急性肺损伤,其作用机制可能与阻断NF-κB信号通路抑制炎症反应有关。

Objective: To explore the effect of lncRNA A_30_P01029806 on acute lung injury in sepsis and its mechanism. Methods: Endotoxin(LPS) was used to treat mouse lung epithelial cells(MLE-12) cultured in vitro. The experiment was divided into control group(Ctrl group), LPS treatment group(LPS group), and knockdown A_30_P01029806 group(LPS+si-A30P group) and transfection control group(LPS+si-NC group). Real-time fluorescence quantitative polymerase chain reaction(PCR)(qPCR) was used to detect the expression of A_30_P01029806 in each group of cells. Thiazole blue(MTT) test was used to detect cell viability. Flow cytometry was used to detect apoptosis. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). Western blot was used to detect the expression of key proteins in the NF-κB signaling pathway. The NF-κB signaling pathway specific inhibitor BAY11-7082 was added to the LPS+si-A30P cells, and the same method was used to analyze the effects of BAY11-7082 on cell viability, apoptosis, and the expression of TNF-α and IL-6.24 male C57BL/6 mice were randomly divided into sham operation group(Sham group), sepsis group(CLP group), knockdown A_30_P01029806 group(si-A30P group) and siRNA control group(si-NC Group), lung tissues were taken from each group 24 h after modeling, and lung injury semi-quantitative scoring(IQA) was performed to calculate lung wet/dry weight ratio(W/D) content. Results In vitro experiments:Compared with the control group, the cell viability of the LPS group was significantly reduced, the expression level of A_30_P01029806, the apoptosis rate, the phosphorylation level of IκBα and p65 in the cells were significantly increased, and the content of TNF-α and IL-6 was significantly increased, the differences were statistically significant(P<0.05). Compared with the LPS group, the above indicators in the LPS+si-A30P group were significantly reversed, and the differences were statistically significant(P<0.05). There was no statistically significant difference of the above indicators between LPS group and LPS+si-NC group(P>0.05). BAY11-7082 partially reversed the inhibitory effects of knocking down A_30_P01029806 on cell viability, apoptosis and secretion of inflammatory factors.Compared with the Sham group, the lung injury score, W/D ratio and the content of TNF-α and IL-6 in the lung tissue of the CLP group were significantly increased, and the differences were statistically significant(P<0.05). Compared with the CLP group, the lung injury score, W/D ratio, TNF-α and IL-6 content of the si-A30P group were significantly reduced, and the differences were statistically significant(P<0.05).There was no statistically significant difference of the indicators between the CLP group and the si-NC group(P>0.05). Conclusion: Knocking down A_30_P01029806 can reduce acute lung injury in sepsis, and its mechanism may be related to blocking the NF-κB signaling pathway and inhibiting the inflammatory response.

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