目的 探討二甲雙胍對高糖誘導(dǎo)的腎小管上皮HK-2細(xì)胞上皮間質(zhì)轉(zhuǎn)化（EMT）的影響及其機(jī)制。方法 以0、7.5、15.0、30.0、60.0、120.0 mmol/L二甲雙胍作用48 h后，采用噻唑藍(lán)（MTT）法檢測HK-2細(xì)胞活力以篩選合適的二甲雙胍作用濃度；將體外培養(yǎng)的HK-2細(xì)胞分為對照組（5.5 mmol/L D-葡萄糖）、高滲組（24.5 mmol/L甘露醇和5.5 mmol/L D-葡萄糖）、高糖組（30 mmol/L D-葡萄糖）、高糖+7.5 mmol/L二甲雙胍組（30 mmol/L D-葡萄糖+7.5 mmol/L二甲雙胍）和高糖+15 mmol/L二甲雙胍組（30 mmol/L D-葡萄糖+15 mmol/L二甲雙胍），倒置顯微鏡觀察各組HK-2細(xì)胞形態(tài)，免疫印跡法（Western blotting）檢測各組HK-2細(xì)胞中α-平滑肌肌動蛋白（α-SMA）、E-鈣黏附蛋白（E-cadherin）、轉(zhuǎn)化生長因子-β（TGF-β）、細(xì)胞外信號調(diào)節(jié)激酶（ERK）、磷酸化（p）-ERK、基質(zhì)金屬蛋白酶9（MMP-9）蛋白表達(dá)水平，實時熒光定量PCR（qRT-PCR）檢測α-SMA、E-cadherin、TGF-β、ERK、MMP-9 mRNA表達(dá)水平。結(jié)果 與對照組相比，30.0、60.0 mmol/L二甲雙胍作用后HK-2細(xì)胞活力明顯升高，120.0 mmol/L二甲雙胍作用后HK-2細(xì)胞活力明顯降低（P<0.05），而7.5、15.0 mmol/L二甲雙胍作用后HK-2細(xì)胞活力差異無統(tǒng)計學(xué)意義（P>0.05）；與對照組比較，高滲組細(xì)胞形態(tài)無明顯改變，且細(xì)胞中α-SMA、E-cadherin、TGF-β、MMP-9蛋白和mRNA表達(dá)水平以及p-ERK/ERK蛋白、ERK mRNA表達(dá)水平差異均無統(tǒng)計學(xué)意義（P>0.05），但高糖組細(xì)胞失去原有形態(tài)變?yōu)殚L梭形，且細(xì)胞中α-SMA、TGF-β蛋白和mRNA表達(dá)水平以及p-ERK/ERK蛋白、ERK mRNA表達(dá)水平明顯升高，而MMP-9、E-cadherin蛋白和mRNA表達(dá)水平明顯降低（P<0.05）；與高糖組比較，高糖+7.5 mmol/L二甲雙胍組、高糖+15.0 mmol/L二甲雙胍組細(xì)胞形態(tài)由長梭形逐漸變成圓形或橢圓形，且α-SMA、TGF-β蛋白和mRNA表達(dá)水平以及p-ERK/ERK蛋白、ERK mRNA表達(dá)水平明顯降低，而MMP-9、E-cadherin蛋白和mRNA表達(dá)水平明顯升高（P<0.05），且高糖+15.0 mmol/L二甲雙胍組細(xì)胞上述指標(biāo)變化幅度大于高糖+7.5 mmol/L二甲雙胍組。結(jié)論 二甲雙胍可抑制高糖誘導(dǎo)的腎小管上皮HK-2細(xì)胞EMT，其作用機(jī)制可能與抑制TGF-β/ERK/MMP-9通路活化有關(guān)。

Objective To investigate the effect of metformin on high glucose-induced epithelial mesenchymal transition (EMT) in HK-2 cells in renal tubular epithelium. Methods After treated with 0, 7.5, 15.0, 30.0, 60.0 and 120.0 mmol/L metformin for 48 h, the viability of HK-2 cells was detected by MTT method to select the appropriate concentration of metformin. HK-2 cells cultured in vitro were divided into control group (5.5 mmol/L D-glucose), hypertonic group (24.5 mmol/L mannitol and 5.5 mmol/L D-glucose), high glucose group (30 mmol/L D-glucose), high glucose + 7.5 mmol/L metformin group (30 mmol/L D-glucose + 7.5 mmol/L metformin) and high glucose + 15.0 mmol/L metformin group (30 mmol/L D-glucose + 15.0 mmol/L metformin). The morphology of HK-2 cells was observed under inverted microscope. Western blotting method was used to detect the protein expression levels of α-smooth muscle actin (α-SMA), E-cadherin, transforming growth factor-β (TGF-β), extracellular signal regulated kinase (ERK), phosphorylation (p)-ERK and matrix metalloproteinase-9 (MMP-9) in HK-2 cells, and real time fluorescent quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of α-SMA, E-cadherin, TGF-β, ERK and MMP-9. Results Compared with 0 mmol/L, 30 and 60 mmol/L metformin increased the viability of HK-2 cells significantly. After treated with 120.0 mmol/L metformin, the viability of HK-2 cells decreased significantly (P<0.05). Compared with those in the control group, there was no significant difference in HK-2 cell viability between the treat of 7.5 mmol/L and 15.0 mmol/L metformin (P>0.05). Compared with those in the control group, there was no significant change in cell morphology in the hypertonic group, the expression levels of α-SMA, E-cadherin, TGF-β and MMP-9 protein and mRNA, and p-ERK/ERK protein and ERK mRNA were not significantly different (P>0.05). However, the cells in high glucose group lost their original shape and became long spindle shape, the expression levels of α-SMA and TGF-β protein and mRNA, p-ERK/ERK protein and ERK mRNA were significantly higher, and the expression levels of MMP-9 and E-cadherin protein and mRNA were significantly lower (P<0.05). Compared with those in high glucose group, the morphology of cells in high glucose + 7.5 mmol/L metformin group and high glucose + 15.0 mmol/L metformin group gradually changed from long spindle shape to round or oval shape, the expression levels of α-SMA and TGF-β protein and mRNA, p-ERK/ERK protein and ERK mRNA were significantly lower, the expression levels of MMP-9 and E-cadherin protein and mRNA were significantly higher (P<0.05). The changes of the above indexes in high glucose + 15.0 mmol/L metformin group were greater than those in high glucose + 7.5 mmol/L metformin group. Conclusion Metformin can inhibit high glucose-induced EMT in HK-2 cells in renal tubular epithelium, which may be related to the inhibition of TGF-β/ERK/MMP-9 pathway activation.

R966

河南省醫(yī)學(xué)科技攻關(guān)計劃項目（2018020300）