miR-153靶向抑制活化蛋白C(APC)加重脂多糖诱导的脓毒症大鼠肺损伤及机制

miR-153 aggravates lung injury induced by lipopolysaccharide via inhibiting activated protein C(APC)in rats with sepsis and its mechanism

目的探讨miR-153靶向活化蛋白C(APC)调控脂多糖(LPS)诱导的大鼠脓毒症肺损伤的机制。方法雌性SD大鼠分为对照组、LPS组、LPS联合miR-153抑制物(miR-153 inhibitor)组、LPS联合miR-153抑制物阴性对照(inhibitor NC)组、LPS和miR-153 inhibitor联合APC小干扰RNA(si-APC)组、LPS和miR-153 inhibitor联合APC小干扰RNA阴性对照(si-NC)组。除对照组外,其余各组通过给予相应处理后,再给予LPS诱导建立大鼠脓毒症损伤模型。通过实时定量PCR检测miR-153的表达,Western blot法检测APC、B淋巴细胞瘤因子2(Bcl2)和裂解型胱天蛋白酶3(c-caspase-3)的蛋白表达;分离并培养大鼠肺泡上皮细胞,CCK-8法检测细胞活力;流式细胞术检测细胞凋亡。ELISA检测大鼠血清中超氧化物歧化酶(SOD)、丙二醛(MDA)、白细胞介素6(IL-6)、IL-1β和肿瘤坏死因子α(TNF-α)的表达水平;双荧光素报告实验检测miR-153和APC的关系。结果与对照组相比,脓毒症肺损伤大鼠模型中,miR-153表达明显增加,APC明显降低。LPS组的细胞活力、Bcl2蛋白表达和SOD活性明显降低,细胞凋亡率、c-caspase-3蛋白表达、MDA、IL-6、IL-1β、TNF-α含量显著增加;与LPS联合inhibitor NC组相比,LPS联合miR-153 inhibitor组的细胞活力、Bcl2蛋白表达和SOD活性明显增加,细胞凋亡率、c-caspase-3蛋白表达、MDA、IL-6、IL-1β、TNF-α表达显著降低。miR-153可以靶向调控APC的表达,与LPS和miR-153 inhibitor联合si-NC组相比,LPS和miR-153 inhibitor联合si-APC组的细胞活力、Bcl2蛋白表达和SOD活性明显降低,细胞凋亡率、c-caspase-3蛋白表达、MDA、IL-6、IL-1β、TNF-α表达显著增加。结论LPS诱导肺组织miR-153表达增强,靶向抑制APC,促进脓毒症大鼠肺组织的细胞凋亡、氧化应激和炎症反应,加重损伤。

Objective To investigate the mechanism of miR-153 targeting activated protein C(APC)to regulate lipopolysaccharide(LPS)-induced lung injury in rats with sepsis.Methods Female SD rats were divided into control group,LPS group,LPS combined with miR-153 inhibitor(miR-153 inhibitor)group,LPS combined with miR-153 inhibitor negative control(inhibitor NC)group,LPS and miR-153 inhibitor combined with APC small interfering RNA(si-APC)group,LPS and miR-153 inhibitor combined with APC small interfering RNA negative control(si-NC)group.Except for the control group,the other groups were given corresponding treatments,and then LPS were given to establish rat sepsis injury models.Real-time quantitative PCR was used to detect the expression of miR-153 and Western blot analysis to detect the protein expression of APC,B-cell lymphoma 2(Bcl2)and cleaved caspase-3(c-caspase-3).The rat alveolar epithelial cells were isolated andcultured,and their cell viability was detected by CCK-8 assay,along with cell apoptosis detected by flow cytometry.ELISA was performed to test the expression levels of superoxide dismutase(SOD),malondialdehyde(MDA),interleukin 6(IL-6),IL-1β and tumor necrosis factorα(TNF-α)in rat serum;and the dual fluorescein reporter experiment detects the relationship between miR-153 and APC.Results Compared with the control group,rat model of sepsis lung injury showed significantly increased expression of miR-153 and reduced APC.The cell viability,Bcl2 protein expression and SOD activity of the LPS group were significantly reduced,and the apoptosis rate,c-caspase-3 protein expression,MDA,IL-6,IL-1β,and TNF-α content significantly escalated.Compared with the LPS combined with inhibitor NC group,the cell viability,Bcl2 protein expression and SOD activity of the LPS combined miR-153 inhibitor group significantly increased,while the apoptosis rate,c-caspase-3 protein expression,the content of MDA,IL-6,IL-1β and TNF-α dropped considerably.miR-153 can target and regulate the expression of APC.Compared with the LPS and miR-153 inhibitor combined with si-NC group,the cell viability,Bcl2 protein expression and SOD activity of the LPS and miR-153 inhibitor combined with si-APC group significantly down-regulated,and the apoptosis rate,c-caspase-3 protein expression,MDA,IL-6,IL-1β,and TNF-α contents increased significantly.Conclusion LPS induces increased expression of miR-153 in lung tissue,inhibits APC expression,promotes apoptosis,oxidative stress and inflammatory response in lung tissue of septic rats,and aggravates damage.