lncRNA A_30_P01029806减轻脓毒症急性肺损伤的机制研究

Mechanism of knocking down lncRNA A_30_P01029806 to reduce acute lung injury in sepsis

目的:探讨lncRNA A30P01029806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A30P01029806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A30P01029806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A30P01029806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A30P01029806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A30P01029806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A30P01029806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 A30P01029806 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 A30 P01029806 group(LPS+si-A30 P group)and transfection control group(LPS+si-NC group).Real-time fluorescence quantitative polymerase chain reaction(PCR)(q PCR)was used to detect the expression of A30P01029806 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-A30 P cells,and the same method was used to analyze the effects of BAY11-7082 on cell viability,apoptosis,and the expression of TNF-αand IL-6.24 male C57 BL/6 mice were randomly divided into sham operation group(Sham group),sepsis group(CLP group),knockdown A30P01029806 group(si-A30 P 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 A30P01029806,the apoptosis rate,the phosphorylation level of IκBαand p65 in the cells were significantly increased,and the content of TNF-αand 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-A30 P 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-αand 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-αand IL-6 content of the si-A30 P 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 A30P01029806 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.