水蛭提取物对电离辐射诱导的肺泡上皮细胞TGF-β/Smad通路及上皮-间质转化的影响  

Effects of leech extract on TGF-β/Smad pathway and epithelial-mesenchymal transition in alveolar epithelial cells induced by ionizing radiation

作　　者：杨吉帆 张涛[1] 李春梅[2] YANG Jifan;ZHANG Tao;LI Chunmei(Department of Oncology,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院肿瘤科,重庆400016 [2]重庆市巴南区第二人民医院儿科,重庆400054

出　　处：《中国免疫学杂志》2022年第10期1201-1206,共6页Chinese Journal of Immunology

基　　金：2017年重庆市巴南区科学技术项目([2017]47号)。

摘　　要：目的:探讨水蛭提取物对电离辐射诱导的肺泡上皮细胞L2上皮-间质转化(EMT)影响及机制。方法:采用MTT法检测水蛭提取物对L2细胞活力的影响以筛选适合的作用浓度。将体外培养的L2细胞分为正常对照组、8 Gy(^(60)Coγ照射剂量)组和8 Gy+0.50 g/L水蛭提取物组和8 Gy+1.00 g/L水蛭提取物组,倒置显微镜观察L2细胞形态变化,免疫荧光法检测L2细胞中E-钙黏附蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)表达,RT-qPCR和Western blot检测E-cadherin、α-SMA、TGF-β、Smad2、Smad3 mRNA及蛋白表达,Transwell细胞迁移实验和划痕实验检测细胞迁移能力。结果:与0 g/L相比,2.00和4.00 g/L水蛭提取物作用48 h后L2细胞存活率显著降低(P<0.05),0.125、0.25、0.50和1.00 g/L水蛭提取物对L2细胞存活率无明显毒性作用(P>0.05),因此选取0.50和1.00 g/L水蛭提取物进行后续实验。8 Gy^(60)Coγ照射后L2细胞由鹅卵形变为长梭形,给予0.50和1.00 g/L水蛭提取物后细胞上述形态变化逐渐恢复。与正常对照组相比,8 Gy^(60)Coγ照射后L2细胞E-cadherin荧光强度减弱,α-SMA荧光强度增强,E-cadherin表达显著降低,α-SMA、TGF-β、Smad2、Smad3表达显著升高,细胞迁移数和划痕愈合率显著升高(P<0.05);0.50和1.00 g/L水蛭提取物作用后可明显逆转8 Gy^(60)Coγ照射所引起的上述变化(P<0.05)。结论:水蛭提取物可抑制电离辐射诱导的肺泡上皮L2细胞EMT,其作用机制可能与抑制TGF-β/Smad通路活化有关。Objective:To investigate effect of leech extract on epithelial-mesenchymal transition(EMT)of alveolar epithelial cells L2 induced by ionizing radiation and its mechanism.Methods:MTT assay was used to detect effect of leech extract on L2 cell to select appropriate concentration.L2 cells were cultured in vitro and divided into normal control group,8 Gy(^(60)Coγ radiation dose)group,8 Gy+0.50 g/L leech extract group and 8 Gy+1.00 g/L leech extract group,morphological changes of L2 cells were observed by inverted microscope,expressions of E-cadherin and α-smooth muscle actin(α-SMA)were detected by immunofluorescence,mRNA and protein expressions of E-cadherin,α-SMA,TGF-β,Smad2 and Smad3 were detected by RT-qPCR and Western blot,Transwell cell migration assay and scratch test were used to detect cell migration.Results:Compared with 0 g/L,survival rate of L2 cells was decreased significantly after treated with 2.00 g/L and 4.00 g/L hirudo extract for 48 h(P<0.05),0.125,0.25,0.50 and 1.00 g/L lecch extract had no significant toxic effects on L2 cell survival rate(P>0.05),therefore,0.50 and 1.00 g/L leech extract were set for subsequent experiments.After 8 Gy irradiation,L2 cells changed from original goose eggs to long spindle shape,and these morphological changes gradually recovered after treatment with 0.50 and 1.00 g/L leech extract.Compared with normal control group,fluorescence intensity of E-cadherin was decreased,fluorescence intensity of α-SMA was increased,expression of E-cadherin was decreased,expressions of α-SMA,TGF-β,Smad2 and Smad3 were significantly increased,migration number and scratch healing rate were significantly increased(P<0.05),and the above changes induced by 8 Gy^(60)Coγ irradiation were significantly reverse after 0.50 g/L and1.00 g/L leech extract treatment(P<0.05).Conclusion:Leech extract can inhibit EMT of alveolar epithelial L2 cells induced by ionizing radiation,which may be related to inhibition of TGF-β/Smad pathway activation.

关 键 词：电离辐射 水蛭提取物 肺泡上皮细胞 上皮-间质转化 TGF-β/Smad通路 

分 类 号：R285.5[医药卫生—中药学]