Apelin-13对高糖诱导肾小管上皮细胞-间充质细胞转分化的影响

摘要

目的 观察Apelin-13对高糖诱导肾小管上皮细胞-间充质细胞转分化(Epithelial-mesenchymal transition,EMT)的影响并探讨其可能机制.方法 用Apelin-13(10-8、10-7和106 mol/L)预处理HK-2细胞30 min后,用含D-葡萄糖(30 mmol/L)的培养基培养HK-2细胞48 h.免疫荧光检测HK-2细胞中间充质细胞的标志物α-平滑肌肌动蛋白(α-SMA)的表达,透射电镜观察HK-2细胞的超微结构,Western-Blot检测上皮细胞的标志物E-钙粘蛋白(E-cadherin)、α-SMA及自噬相关蛋白表达.结果 与对照组比较,高糖组的细胞呈现长梭形,细胞间隙明显增宽,E-cadherin蛋白表达显著性下调(t=4.751,P=0.011),而α-SMA蛋白表达显著上调(t=3.846,P=0.016),自噬体数量明显增多,Beclin-1、LC3-Ⅱ蛋白表达和LC3-Ⅱ/LC3-Ⅰ的比值均显著增加(t1=5.271,t2=4.695,t3=6.753,P1=0.009,P2=0.012,P3=0.002),p62表达显著降低(t=4.552,P=0.012).与高糖组比较,Apelin-13(10-7和10-6mol/L)+高糖与高糖组比较,明显抑制了细胞形态的变化,细胞形态大多数呈椭圆型,E-cadherin蛋白表达显著上调(t1=3.495,t2=4.124,P1=0.019,P2=0.013),而α-SMA蛋白表达显著下调(t1=4.014,t2=4.283,P1=0.014,P2=0.012),自噬体数量明显增多,Beclin-1、LC3-Ⅱ蛋白表达和LC3-Ⅱ/LC3-Ⅰ的比值均显著增加(t1=3.487,t2=4.059,t3=5.342,t4=4.271,t5=5.360,t6=5.851,P1=0.018,P2=0.014,P3=0.009,P4=0.011,P5=0.005,P6=0.004),p62表达显著降低(t1=4.342,t2=3.557,P1=0.012,P2=0.019).结论 Apelin-13抑制了高糖诱导的肾小管EMT,其机制可能与Apelin-13诱导肾小管上皮细胞自噬有关.%Objective To explore the effect of Apelin-13 on epithelia-mesenchymal transition (EMT) of renal tubular epithelial cell line HK-2 cells induced by high glucose.Methods After pretreatment with Apelin-13 (108,10-7 and 10-6 mol/L) for 30 min,HK-2 cells were cultured in the medium containing 30 mmol/L D-glucose for 48 h.Expression of mesenchymal cell markers alpha smooth muscle actin (α-SMA) in the HK-2 cells were tested by immunofluorescence assay.Ultrastructural changes of HK-2 cells were observed by transmission electron microscopy.Expressions of the epithelial marker E-cadherin,α-SMA,and autophagy related proteins were measured by western blot.Results Compared with the control group,HK-2 cells in the high glucose group showed long spindle shape and widened intercellular spaces,with down-regulated E-cadherin expression (t=4.751,P=0.011),up-regulated αt-SMA expression (t=3.846,P=0.016),increased number of autophagy,increased expression of Beclin-1,LC3-Ⅱ and LC3-Ⅱ/LC3-Ⅰ ratio (t=5.271,P=0.009 for Bectlin-1;t=4.695,P=0.012 for LC3-Ⅱ;t=6.753,P=0.002 for LC3-Ⅱ/LC3-Ⅰ ratio),and decreased expression of p62 (t=4.562,P=0.012).Compared with the high glucose group,KH-2 cells in the Apelin-13 (10-7 and 10-6 mol/L) + high glucose group showed oval shape,with up-regulated E-cadherin expression (t=3.495,P=0.019 for 10-7 mol/L Apelin-13;t=4.124,P=0.013 for 10-6 mol/L Apelin-13),down-regulated α-SMA expression (t=4.014,P=0.014 for 10-7 mol/L Apelin-13;t=4.283,P=0.012 for 10-6 mol/L Apelin-13),increased number of autophagy,increased expression of Beclin-1,and LC3-Ⅱ and LC3-Ⅱ/LC3-Ⅰ ratio (t=3.487,P=0.018 for Beclin-1 and 10-7 mol/L Apelin-13;t=4.059,P=0.014 for Beclin-1 and l0-6 mol/L Apelin-13;t=5.342,P=0.009 for LC3-Ⅱ and 10 7 mol/L Apelin-13;t=4.271,P=0.011 for LC3-Ⅱ and 10-6 mol/L Apelin-13;t=5.360,P=0.005 for LC3-Ⅱ/LC3-Ⅰ ratio and 10-7 mol/L Apelin-13;t=5.851,P=0.004 for LC3-Ⅱ/LC3-Ⅰ ratio and 10-6 mol/L Apelin-13),and decreased expression of p62 (t=4.342,P=0.012 for 107 mol/L Apelin-13;t=3.557,P=0.019 for 10-6 mol/L Apelin-13).Conclusions Apelin-13 inhibits the EMT of renal tubular epithelial cell line HK-2 cells induced by high glucose.The increase of autophagy induced by Apelin-13 may relate to the inhibition of EMT in HK-2 cells.