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目的探讨骨化三醇对晚期糖基化终末产物(advanced glycation end products,AGEs)诱导的肾小管上皮细胞间质转化(epithelial-mesenchymal transition,EMT)的作用及可能机制。方法先单独用骨化三醇刺激细胞,再采用AGEs处理NRK52E细胞,建立由AGEs诱导的细胞EMT模型。分为4组:对照组、模型组、低剂量骨化三醇(10nmol/L)预处理的模型组、高剂量骨化三醇(100nmol/L)预处理的模型组。利用Western blot、逆转录实时定量PCR等方法检测E-cadherin、α-SMA、AGEs特异性受体(receptor for advanced glycation end products,RAGE)及NF-κB的磷酸化等的表达。结果骨化三醇单独刺激时,大鼠肾小管上皮细胞上的E-cadherin和α-SMA的表达未发生明显变化;较对照组,AGEs培养后,NRK52E细胞E-cadherin表达显著下调,α-SMA表达明显上调,AGEs特异性受体显著表达增多,NF-κB途径活化;以上变化都能够被骨化三醇所逆转(P<0.05),抗RAGE中和抗体(AF1616)可抑制AGEs引起的E-cadherin下调和α-SMA上调,骨化三醇抑制AGEs诱导RAGE的上调能够被NF-κB磷酸化抑制剂(IMD0354)部分阻断。结论骨化三醇通过降低RAGE的表达和阻断NF-κB的磷酸化来抑制肾小管上皮细胞上皮间质转化。
Objective To investigate the effects and possible mechanisms of calcitriol on advanced glycation end products (AGEs) induced epithelial-mesenchymal transition (EMT). Methods The cells were stimulated with calcitriol alone and treated with AGEs for NRK52E cells to establish a cell EMT model induced by AGEs. The rats were divided into 4 groups: control group, model group, low-dose calcitriol (10nmol / L) pretreatment model group and high-dose calcitriol (100nmol / L) pretreatment model group. The expressions of E-cadherin, α-SMA, receptor-specific glycoprotein (AGEs) specific receptors (RAGE) and phosphorylation of NF-κB were detected by Western blot and RT-PCR. Results The expression of E-cadherin and α-SMA in rat renal tubular epithelial cells did not change significantly when calcitriol was stimulated alone. Compared with the control group, the expression of E-cadherin in NRK52E cells was significantly down-regulated after AGEs culture, The expression of AGEs was significantly upregulated, AGEs specific receptor was significantly increased and NF-κB pathway was activated. All the above changes were reversed by calcitriol (P <0.05). Anti-RAGE neutralizing antibody (AF1616) E-cadherin down-regulation and α-SMA upregulation, and calcinetriol inhibition of AGEs-induced RAGE upregulation can be partially blocked by NF-κB phosphorylation inhibitor (IMD0354). Conclusion Calcitriol can inhibit epithelial-mesenchymal transition of renal tubular epithelial cells by decreasing the expression of RAGE and blocking the phosphorylation of NF-κB.