GSK3β/eEF2K信号通路对小鼠肺纤维化过程的影响

Effects of GSK3β/eEF2K signaling pathway on pulmonary fibrosis in mice

目的:探讨糖原合成酶激酶-3(GSK3β)/真核延伸因子激酶2(eEF2K)信号通路对肺纤维化进程的影响,为临床治疗肺纤维化寻找新的思路。方法:采用一次性气管注射法构建C57BL/6雄性小鼠博莱霉素肺纤维化模型,造模14 d后将动物分成模型组、阴性抑制组与抑制组(n=5),另设空白组不作处理。抑制组使用腹腔注射TDZD-8(4 mg/kg),阴性抑制组腹腔注射二甲基亚砜(DMSO)溶液,28 d后处死采集指标。采用苏木精-伊红染色法检测小鼠肺脏病变情况;试剂盒水解法检测肺组织中羟脯氨酸(Hyp)的含量;采用Western blot法检测肺脏中GSK3β、磷酸化GSK3β(p-GSK3β)、eEF2K、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、基质金属蛋白酶-2前体蛋白(pro-MMP-2)、基质金属蛋白酶-2(MMP-2)蛋白表达水平,使用免疫组织化学法检测肺脏中MMP-2、胶原蛋白I(Col I)、胶原蛋白Ⅲ(ColⅢ)、α-平滑肌蛋白(α-SMA)的表达。结果:与空白组相比,模型组中GSK3β、p-GSK3β、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、pro-MMP-2、MMP-2、Col I、ColⅢ、α-SMA蛋白表达水平升高,eEF2K蛋白表达水平降低(P<0.05);与模型组相比,抑制组GSK3β、p-GSK3β、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、pro-MMP-2、MMP-2、Col I、ColⅢ、α-SMA蛋白表达降低,eEF2K蛋白表达升高(P<0.05)。结论:GSK3β能通过Ser70、Ser392、Ser470这3个位点磷酸化激活eEF2K,增加纤维化指标含量,促进肺纤维化形成,加重肺组织病变。

Objective:To investigate the effects of glycogen synthase kinase-3β(GSK3β)/eukaryotic extension factor kinase 2(eEF2K)signaling pathway on the process of pulmonary fibrosis through in vivo experiments,and find new ideas for clinical treatment of pulmonary fibrosis.Methods:The pulmonary fibrosis model of C57BL/6 male mice was induced by bleomycin with intratracheal injection at the dose of 2 mg/kg.After 14 days of modeling,animals were divided into model group,negative inhibition group and inhibition group(n=5 for each group),and control group was not processed.The inhibition group was treated with TDZD-8(4 mg/kg)after modeling,the negative inhibition group was given DMSO solution after modeling,and the samples were collected after 28 days.Hematoxylin-eosin staining method was used to detect lung fibrosis in mice and scored according to Ashcroft scale.Expression levels of GSK3β,p-GSK3β,eEF2K,p-eEF2K(Ser70,Ser392,Ser470),precursor protein of matrix metalloproteinase-2(pro-MMP-2),matrix metalloproteinase-2(MMP-2),collagen I(Col I),collagenⅢ(ColⅢ)andα-smooth muscle actin(α-SMA)were detected by Western blot.Results:Compared with control group,the fibrosis score was up-regulated,the expression levels of GSK3β,p-GSK3β,p-eEF2K(Ser70,Ser392,Ser470),pro-MMP-2,MMP-2,Col I,ColⅢandα-SMA were increased,while that of eEF2K was decreased in model group(P<0.05).Compared with model group,the fibrosis score,expression levels of GSK3β,p-GSK3β,p-eEF2K(Ser70,Ser392,Ser470),pro-MMP-2,MMP-2,Col I,ColⅢandα-SMA were decreased,but the expression level of eEF2K was increased in inhibition group(P<0.05).Conclusion:GSK3βcan activate eEF2K by phosphorylation at the sites of Ser70,Ser392 and Ser470,increase the contents of fibrosis indicators,promote the formation of pulmonary fibrosis,and aggravate lung tissue lesions.