SMYD2介导高糖诱导大鼠肾成纤维细胞活化的机制研究

Mechanism underlying the role of SMYD2 in mediating high glucose-induced activation of rat renal fibroblasts

摘要:

目的 探讨组蛋白赖氨酸甲基转移酶2（SMYD2）介导体外高糖环境下大鼠肾成纤维细胞（NRK-49F）活化的可能机制。方法 复制糖尿病（DM）小鼠模型，经全自动生化分析仪检测各组小鼠血糖（BG）、血肌酐（Scr）水平，经苏木精-伊红、Masson染色观察小鼠肾组织病理改变，经Western blotting检测各组小鼠肾组织纤维连接蛋白（Fibronectin）、Ⅰ型胶原蛋白（CollagenⅠ）、α-平滑肌肌动蛋白（α-SMA）、组蛋白H3赖氨酸4三甲基化（H3K4me3）、SMYD2、核转录因子-κB p65（NF-κB p65）及磷酸化NF-κB p65（NF-κB p-p65）蛋白表达，经免疫荧光检测各组小鼠肾组织α-SMA及SMYD2表达。复制体外DM细胞模型，使用正常糖（含5.5 mmol/L葡萄糖）或高糖（含30.0 mmol/L葡萄糖）处理NRK-49F细胞，经Western blotting检测SMYD2、α-SMA及细胞外基质（ECM）成分蛋白表达；经CCK-8法检测SMYD2特异性抑制剂AZ505的细胞毒性；在AZ505处理/不处理的条件下，经Western blotting检测SMYD2、α-SMA、H3K4me3、Fibronectin、CollagenⅠ、NF-κB p65、NF-κB p-p65蛋白表达，经免疫荧光检测α-SMA、SMYD2及NF-κB p-p65在细胞中的表达及空间定位情况。结果 DM组血清BG、Scr水平高于NC组（P <0.05）。DM组肾组织α-SMA、Fibronectin、CollagenⅠ、SMYD2、H3K4me3、NF-κB p-p65蛋白相对表达量高于NC组（P <0.05），两组NF-κB p65蛋白相对表达量比较，差异无统计学意义（P >0.05）。HG刺激不同时间点NRK-49F细胞α-SMA、Collagen I、Fibronectin、SMYD2蛋白相对表达量比较，差异均有统计学意义（P <0.05）。40 μmol/L组、60 μmol/L组NRK-49F细胞存活率较0 μmol/L组低（P <0.05）。HG组NRK-49F细胞Fibronectin、CollagenⅠ、SMYD2蛋白相对表达量较NG组高（P <0.05），HG + AZ505（20 μmol/L）组较HG组低（P <0.05）。HG组NRK-49F细胞Fibronectin、CollagenⅠ、α-SMA、H3K4me3、SMYD2蛋白相对表达量较NG组高（P <0.05），HG+AZ505组较HG组低（P <0.05）。HG组NRK-49F细胞NF-κB p-p65蛋白相对表达量较NG组高（P <0.05），HG+AZ505组较HG组低（P <0.05），各组大鼠NRK-49F细胞NF-κB p65蛋白相对表达量比较，差异无统计学意义（P >0.05）。结论 SMYD2可介导高糖诱导的NRK-49F细胞活化，其机制可能与激活NF-κB细胞信号通路有关。

Abstract:

Objective To investigate the possible mechanism underlying the role of the histone lysine methyltransferase SET and MYND domain-containing protein 2 (SMYD2) in mediating the activation of rat renal fibroblasts (NRK-49F) in a high glucose environment in vitro.Methods The mouse model of diabetes mellitus (DM) was established, and levels of blood glucose (BG) and serum creatinine (Scr) were measured via the automatic biochemical analyzer. The renal histopathological changes in mice were observed by hematoxylin-eosin and Masson staining. The protein expression levels of fibronectin, type Ⅰ collagen (collagen Ⅰ), alpha-smooth muscle actin (α-SMA), histone H3 lysine 4 trimethylation (H3K4me3), SMYD2, nuclear factor kappa B p65 (NF-κB p65) and phosphorylated NF-κB p65 (NF-κB p-p65) in the kidney tissues of DM mice were detected via Western blotting, and the expressions of α-SMA and SMYD2 in the kidney tissues of DM mice were also detected via immunofluorescence. The cell models of DM were replicated in vitro, where NRK-49F cells were treated with glucose of normal concentration (containing 5.5 mmol/L of glucose) or high concentration (containing 30 mmol/L of glucose). Then protein expressions of SMYD2, α-SMA and components of extracellular matrix (ECM) were measured by Western blotting, and cell counting kit-8 was used to detect the cytotoxicity of SMYD2-specific inhibitor AZ505. When treated with or without AZ505, protein expressions of SMYD2, α-SMA, H3K4me3, fibronectin, collagen Ⅰ, NF-κB p65 and NF-κB p-p65 were detected via Western blotting, and the expressions and distribution of α-SMA, SMYD2 and NF-κB p-p65 in NRK-49F cells were detected via immunofluorescence.Results The levels of BG and Scr in the DM group were higher than those in the NC group (P < 0.05). The relative protein expressions of α-SMA, fibronectin, collagen Ⅰ, SMYD2, H3K4me3 and NF-κB p-p65 in the kidney tissues of the DM group were higher than those of the NC group (P < 0.05). There was no difference in the relative protein expression of NF-κB p65 between the two groups (P > 0.05). The relative protein expressions of α-SMA, collagen I, fibronectin and SMYD2 in NRK-49F cells were different among the distinct time points when mice were treated with HG (P < 0.05). The survival rate of NRK-49F cells in the 40 μmol/L group and the 60 μmol/L group was lower than that in the 0 μmol/L group (P < 0.05). The relative protein expressions of fibronectin, collagen Ⅰ and SMYD2 in NRK-49F cells of the HG group were higher than those of the NG group (P < 0.05), while those of the HG + AZ505 (20 μmol/L) group were lower than those of the HG group (P < 0.05). The relative protein expressions of fibronectin, collagen Ⅰ, α-SMA, H3K4me3 and SMYD2 in NRK-49F cells of the HG group were higher than those of the NG group (P < 0.05), while those of the HG + AZ505 (20 μmol/L) group were lower than those of the HG group (P <0.05). The relative protein expression of NF-κB p-p65 in NRK-49F cells of the HG group was higher than that of the NG group (P < 0.05), while that of the HG + AZ505 group was lower than that of the HG group (P < 0.05). There was no difference in the relative protein expression of NF-κB p65 in NRK-49F cells among the groups (P > 0.05).Conclusions SMYD2 mediates high glucose-induced activation of NRK-49F cells by a mechanism that may be related to the activation of the NF-κB signaling pathway.