2023 年5 期 第31 卷
论著![HTML下载 HTML下载](/Areas/201911120101/Content/img/icon_html.png)
![PDF下载 PDF下载](/Areas/201911120101/Content/img/icon_pdf.png)
二甲双胍对急性肺损伤细胞凋亡、迁移和上皮-间质转化的影响及机制研究
Effect and Mechanism of Metformin on Apoptosis, Cell Migration and Epithelial-Mesenchymal Transition in Acute Lung Injury
作者:欧阳运萍,陈涛,李鹏,赵博,杨小军
- 单位:
- 1.河北省唐山市协和医院急诊科2.河北省唐山市协和医院消化内科3.河北省唐山市协和医院重症医学科
- Units:
- 1.Department of Emergency, Tangshan Union Hospital, Tangshan 063000, China 2.Department of Gastroenterology, Tangshan Union Hospital, Tangshan 063000, China 3.Department of Critical Care Medicine, Tangshan Union Hospital, Tangshan 063000, China
- 关键词:
- 急性肺损伤; 肺泡上皮细胞; 二甲双胍; 细胞凋亡; 细胞迁移; 上皮-间质转化; p38丝裂原活化蛋白激酶类;
- Keywords:
- Acute lung injury; Alveolar epithelial cells; Metformin; Apoptosis; Cell migration; Epithelialmesenchymal transition; p38 mitogen-activated protein kinases
- CLC:
- DOI:
- 10.12114/j.issn.1008-5971.2023.00.118
- Funds:
- 2022年度河北省医学科学研究课题项目(20221833);
摘要:
目的 探讨二甲双胍对急性肺损伤细胞凋亡、迁移和上皮-间质转化(EMT)的影响及机制。方法本实验时间为2022年6—11月。将人肺泡上皮细胞(A549细胞)分为对照组、过氧化氢(H2O2)组、2.5 mmol/L二甲双胍组、5.0 mmol/L二甲双胍组、10.0 mmol/L二甲双胍组、SB203580组,对照组不做任何干预;H2O2组加入400μmol/L H2O2作用24 h,以构建急性肺损伤细胞模型;2.5 mmol/L二甲双胍组、5.0 mmol/L二甲双胍组、10.0 mmol/L二甲双胍组在H2O2组基础上分别加入2.5、5.0、10.0 mmol/L二甲双胍进行干预;SB203580组在H2O2组基础上加入10.0 mmol/L p38丝裂原活化蛋白激酶(p38 MAPK)信号通路抑制剂——SB203580进行干预,选取细胞活力最佳时的药物浓度进行后续实验。将A549细胞分为对照组、H2O2组、10.0 mmol/L二甲双胍组、SB203580组、抑制剂组和激活剂组,对照组、H2O2组、10.0 mmol/L二甲双胍组、SB203580组干预方法同前,抑制剂组和激活剂组在10.0 mmol/L二甲双胍组基础上分别加入10μmol/L SB203580和10μmol/L p38 MAPK信号通路激活剂——C16-PAF。采用CCK-8法测定对照组、H2O2组、2.5mmol/L二甲双胍组、5.0 mmol/L二甲双胍组、10.0 mmol/L二甲双胍组、SB203580组细胞活力。采用Hoechst 33258染色法测定对照组、H2O2组、10.0 mmol/L二甲双胍组、SB203580组、抑制剂组、激活剂组细胞凋亡率,划痕试验测定细胞迁移率,RT-qPCR法测定EMT相关因子[E-钙黏蛋白、N-钙黏蛋白、波形蛋白、纤维连接蛋白(FN)]mRNA相对表达量,Western blot法测定EMT相关因子蛋白及p38 MAPK信号通路相关蛋白(p38 MAPK、磷酸化p38 MAPK)相对表达量。结果 H2O2组细胞活力低于对照组,5.0 mmol/L二甲双胍组、10.0 mmol/L二甲双胍组、SB203580组细胞活力高于H2O2组,10.0 mmol/L二甲双胍组细胞活力高于5.0 mmol/L二甲双胍组(P<0.05),选取10.0 mmol/L二甲双胍进行后续实验。H2O2组细胞凋亡率、细胞迁移率,N-钙黏蛋白、波形蛋白、FN mRNA和蛋白相对表达量,磷酸化p38 MAPK相对表达量高于对照组,E-钙黏蛋白mRNA和蛋白相对表达量低于对照组(P<0.05);10.0 mmol/L二甲双胍组、SB203580组细胞凋亡率、细胞迁移率,N-钙黏蛋白、波形蛋白、FN mRNA和蛋白相对表达量,磷酸化p38 MAPK相对表达量低于H2O2组,E-钙黏蛋白mRNA和蛋白相对表达量高于H2O2组(P<0.05);抑制剂组细胞凋亡率、细胞迁移率,N-钙黏蛋白、波形蛋白、FN mRNA和蛋白相对表达量,磷酸化p38 MAPK相对表达量低于10.0 mmol/L二甲双胍组和SB203580组,E-钙黏蛋白mRNA和蛋白相对表达量高于10.0 mmol/L二甲双胍组和SB203580组(P<0.05);激活剂组细胞凋亡率、细胞迁移率,N-钙黏蛋白、波形蛋白、FN mRNA和蛋白相对表达量,磷酸化p38 MAPK相对表达量高于10.0 mmol/L二甲双胍组和SB203580组,E-钙黏蛋白mRNA和蛋白相对表达量低于10.0 mmol/L二甲双胍组和SB203580组(P<0.05)。结论 二甲双胍可抑制H2O2诱导的急性肺损伤的细胞凋亡、迁移和EMT,其作用机制可能与其抑制p38 MAPK信号通路激活有关。
Abstract:
Objective To investigate the effect and mechanism of metformin on apoptosis, cell migration and epithelial-mesenchymal transition (EMT) in acute lung injury. Methods The experiment was conducted from June to November 2022. Human alveolar epithelial cells (A549 cells) were divided into control group, hydrogen peroxide (H2O2) group, 2.5 mmol/L metformin group, 5.0 mmol/L metformin group, 10.0 mmol/L metformin group, SB203580 group, the control group were not intervened; the H2O2 group was added with 400 μmol/L H2O2 for 24 hours to construct an in vitro model of acute lung injury; the 2.5 mmol/L metformin group, 5.0 mmol/L metformin group, and 10.0 mmol/L metformin group were added with 2.5, 5.0, and 10.0 mmol/L metformin on the basis of the H2O2 group, respctively; the SB203580 group was added with 10.0 mmol/L p38 mitogenactivated protein kinase (p38 MAPK) signaling pathway inhibitor SB203580 on the basis of the H2O2 group. The drug concentration with optimal cell viability was selected for subsequent experiments. A549 cells were divided into control group, H2O2 group, 10.0 mmol/L metformin group, SB203580 group, inhibitor group and activator group, the intervention method was same as before in control group, H2O2 group, 10.0 mmol/L metformin group, SB203580 group, the inhibitor group and the activator group were added with 10 μmol/L SB203580 and 10 μmol/L p38 MAPK signaling pathway activator C16-PAF on the basis of the 10.0 mmol/L metformin group, respectively. CCK-8 method was used to measure the cell viability in the control group, H2O2 group, 2.5 mmol/L metformin group, 5.0 mmol/L metformin group, 10.0 mmol/L metformin group and SB203580 group. Hoechst 33258 staining method was used to determine the apoptosis rate, and scratch test was used to determine the cell migration rate, RT-qPCR was used to measure the mRNA relative expression of EMT-related factors [E-cadherin, N-cadherin, vimentin, and fibronectin (FN) ] , Western blot was used to detect the protein relative expression of EMT-related factors and relative expression of p38 MAPK signaling pathway-related proteins (p38 MAPK, p-p38 MAPK) in the control group, H2O2 group, 10.0 mmol/L metformin group, SB203580 group, inhibitor group, activator group. Results The cell viability of H2O2 group was lower than that of control group, the cell viability of 5.0 mmol/L metformin group, 10.0 mmol/L metformin group and SB203580 group was higher than that of H2O2 group, the cell viability of 10.0 mmol/L metformin group was higher than that of 5.0 mmol/L metformin group (P < 0.05) , and 10.0 mmol/L metformin was selected for subsequent experiments. The apoptosis rate, cell migration rate, N-cadherin, vimentin, and FN mRNA and protein relative expression, and p-p38 MAPK relative expression in the H2O2 group were higher than those in the control group, and the E-cadherin mRNA and protein relative expression was lower than that in the control group (P < 0.05) . The apoptosis rate, cell migration rate, N-cadherin, vimentin, and FN mRNA and protein relative expression, and p-p38 MAPK protein relative expression in the 10.0 mmol/L metformin group and SB203580 group were lower than those in the control group, and the E-cadherin mRNA and protein relative expression was higher than that in the H2O2 group (P < 0.05) . The apoptosis rate, cell migration rate, N-cadherin, vimentin, and FN mRNA and protein relative expression, and p-p38 MAPK relative expression in the inhibitor group were lower than those in the 10.0 mmol/L metformin group and SB203580 group, and the E-cadherin mRNA and protein relative expression was higher than that in the 10.0 mmol/L metformin group and SB203580 group (P < 0.05) . The apoptosis rate, cell migration rate, N-cadherin, vimentin, and FN mRNA and protein relative expression, and p-p38 MAPK relative expression in the activator group were higher than those in the 10.0 mmol/L metformin group and SB203580 group, and the E-cadherin mRNA and protein relative expression was lower than that in the 10.0 mmol/L metformin group and SB203580 group (P < 0.05) . Conclusion Metformin can inhibit the apoptosis, cell migration and EMT of H2O2-induced acute lung injury, and its mechanism may be related to the inhibition of p38 MAPK signaling pathway.
ReferenceList: