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该研究旨在探讨Hedgehog-Gli1(HH)信号在肾小管上皮细胞表型转化(epithelial-mesenchymal transition,EMT)和胶原累积中的作用及与TGF-β1信号的对话机制。该实验通过体外培养大鼠肾小管上皮细胞NRK-52E,以溶剂作为对照组,以1~50 ng/m L重组蛋白sonic hedgehog(Shh)或5 ng/m L TGF-β1作为诱导组,以加入或不加入HH信号特异性阻断剂环靶明(cyclopamine,Cyp)5μmol/L为干预组。细胞培养24 h,采用ELISA、q RT-PCR、免疫细胞荧光染色和Western blot等方法检测HH信号相关分子(Ptch1、Smo和Gli1)、TGF-β1、EMT相关分子(Rac1蛋白、肌成纤维细胞标志物α-SMA和上皮细胞标志物E-cadherin)、III型胶原m RNA或蛋白的表达。结果发现,外源性Shh上调Smo和Gli1表达,抑制Ptch1表达,继而激活HH信号;HH信号活化抑制肾小管上皮细胞E-cadherin的表达,上调α-SMA、III型胶原和TGF-β1的表达。环靶明干预后,Smo表达下调,进而抑制HH信号、EMT和胶原累积,并下调TGF-β1的表达。应用TGF-β1诱导小管上皮细胞EMT,同时也上调HH信号分子Smo和Gli1的表达,下调Ptch1的表达,提示TGF-β1可诱导HH信号活化。综上所述,HH信号和TGF-β1均参与了肾小管上皮细胞EMT和胶原累积过程。HH信号活化可促进TGF-β1的表达,同时TGF-β1能激活HH信号,推测TGF-β1与HH信号可能存在交叉对话以调控EMT和胶原累积。
This study aimed to investigate the role of Hedgehog-Gli1 (HH) signaling in the epithelial-mesenchymal transition (EMT) and collagen accumulation and the mechanism of the interaction with TGF-β1 signaling. In this experiment, rat renal tubular epithelial cells NRK-52E were cultured in vitro and the control group was treated with 1 ~ 50 ng / mL recombinant protein sonic hedgehog (Shh) or 5 ng / mL TGF-β1 Cyclopamine (Cyp) (5μmol / L), a specific inhibitor of HH signaling, was used as the intervention group. The cells were cultured for 24 h. The expression of Hch signaling related molecules (Ptch1, Smo and Gli1), TGF-β1, EMT related molecules (Rac1, myofibroblasts Marker alpha-SMA and epithelial cell marker E-cadherin), type III collagen m RNA or protein. The results showed that exogenous Shh up-regulated the expressions of Smo and Gli1 and down-regulated the expression of Ptch1, and then activated the HH signaling. HH signaling inhibited the expression of E-cadherin and up-regulated the expression of type III collagen and TGF-β1 in renal tubular epithelial cells . After the target was knocked down, Smo expression was down-regulated, which further inhibited the accumulation of HH signal, EMT and collagen and down-regulated the expression of TGF-β1. Application of TGF-β1 induces tubular epithelial cell EMT, up-regulates the expression of HH signaling molecules Smo and Gli1 and down-regulates the expression of Ptch1, suggesting that TGF-β1 can induce HH signaling. In summary, both HH signaling and TGF-β1 are involved in EMT and collagen accumulation in renal tubular epithelial cells. Activation of HH signal can promote the expression of TGF-β1, and TGF-β1 can activate HH signal. It is speculated that there may exist cross-talk between TGF-β1 and HH signal to regulate EMT and collagen accumulation.