目的 本研究旨在探讨Renalase是否可以延缓肾脏纤维化.方法 体外培养人近端肾小管上皮细胞,给予TGF-β1和(或)不同浓度的Renalase与细胞共同孵育,观察细胞形态的变化,应用免疫荧光观察E-cadherin、α-SMA的分布和表达情况;应用Western blot(WB)法和RT-PCR检测细胞中E-cadherin、α-SMA的蛋白和mRNA表达,应用WB检测FN和Col-Ⅰ蛋白表达,并检测细胞内信号分子p-Smad-2/3、p-ERK1/2、p-p38水平的变化,探讨其可能的分子生物学机制.结果 给予TGF-31刺激后,E-cadherin表达下调、α-SMA、FN、Col-Ⅰ表达增加.应用不同浓度Renalase与TGF-β1共孵育细胞,上述现象改善,且呈剂量依赖性.同时,与单独TGF-β1刺激相比,添加Renalase共孵育后,细胞内p-smad 2/3和p-p38表达无明显变化,但p-ERK 1/2表达明显下调,差异有统计学意义.而应用质粒转染过表达ERK信号通路后,Renalase抑制TGF-β1诱导的EMT和纤维化的作用被抵消.结论 本研究首次证实Renalase可以减轻TGF-β1所致的肾小管上皮细胞间充质转分化和纤维化,其机制可能与抑制ERK 1/2信号通路的激活相关,为临床延缓CKD进展提供新的治疗靶点和理论依据.%Objective To explore whether Renalase can delay renal fibrosis.Methods Human proximal renal tubular epithelial cells were cultured in vitro and incubated with TGF-β1 with or without various concentrations of Renalase.The expression of E-cadherin,α-SMA,FN,Col-Ⅰ was detected by different methods to evaluate the level of EMT and fibtosis.Then,the expression of p-Smad 2/3,p-ERK 1/2 and p-p38 was detected by Western blot(WB) to explore the possible molecular biological mechanism.Results The expression of E-cadherin,α-SMA,FN and Col-Ⅰ increased after TGF-β1 stimulation.When incubated with different concentrations of Renalase,the appeal phenomenon was improved in a dose-dependent manner.The expression of p-smad 2/3 and p-p38 in the cells incubated with Renalase was almost the same with that of TGF-β1 alone,while the expression of p-ERK 1/2 was significantly down-regulated.When ERK signaling pathways was overexpressed,the inhibition of Renalase in TGF-β1-induced EMT and fibrosis role was offset.Conclusion Renalase can reduce TGF-β1-induced renal tubular epithelial-mesenchymal transition and fibrosis in a dose-dependent manner.This mechanism may be related to the inhibition of ERK 1/2 signaling pathway activation,which provides a new therapeutic target and theoretical basis for the clinical development of delayed CKD.
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