敲低CPEB4通过抑制Wnt/β–catenin通路改善糖尿病肾病大鼠肾间质纤维化

李玮; 任艳杰; 史雅静; 续婷婷; 路鹏雁

敲低CPEB4通过抑制Wnt/β–catenin通路改善糖尿病肾病大鼠肾间质纤维化

Study on the knockdown of CPEB4 improving renal interstitial fibrosis in rats with diabetic nephropathy by inhibiting the Wnt/β-catenin pathway

摘要

摘要:
目的： 旨在探讨细胞质多聚腺苷酸化元件结合蛋白（CPEB4）在糖尿病肾病（DKD）中的作用，并基于Wnt/β–连环蛋白（β–catenin）信号通路探讨其作用机制。
方法： 采用高脂高糖饮食 + 腹腔注射链脲佐菌素（STZ）制备DKD大鼠模型。将造模成功的SD大鼠分为DKD组、二甲双胍组（100 mg/kg，灌胃）、shRNA–NC组（300 μL shRNA–NC，尾静脉注射）、shRNA–CPEB4组（300 μL shRNA–CPEB4，尾静脉注射）和LiCl组（Wnt/β–catenin通路激活剂，300 μL shRNA–CPEB4 + 15 mg/kg LiCl，尾静脉注射），每组15只。另选15只大鼠作为对照（Con）组。采用RT–qPCR和Western Blot方法评估CPEB4在DKD大鼠肾组织中的表达水平；采用全自动生化分析仪检测尿素氮（BUN）、肌酐（Scr）和血尿酸（UA）水平；采用HE、Masson染色进行肾脏病理组织学分析；采用Western Blot法检测肾间质纤维化（RIF）相关蛋白I型胶原蛋白（Col I）、纤连蛋白（FN）、α–平滑肌肌动蛋白（α–SMA）和Wnt/β–catenin通路相关蛋白表达水平。
结果： 相较于Con组，DKD组大鼠肾小管萎缩和间质增宽，伴有严重炎性细胞浸润和胶原沉积，CPEB4 mRNA及蛋白水平、BUN、SCr、UA、Col I、FN、α–SMA、Wnt1、p–GSK–3β、p–β–catenin水平均升高（P ＜ 0.05）；相较于DKD组，二甲双胍组、shRNA–CPEB4组和LiCl组大鼠肾小球病变程度减轻，肾间质损伤减轻，少量系膜增生和炎性细胞浸润，胶原纤维沉积相对减少，CPEB4 mRNA及蛋白水平、BUN、SCr、UA、Col I、FN、α–SMA、Wnt1、p–GSK–3β、p–β–catenin水平均降低（P ＜ 0.05）；相较于shRNA–NC组，二甲双胍组、shRNA–CPEB4组和LiCl组大鼠肾小球萎缩、间质增宽和炎性浸润现象逐渐减轻，肾组织间有较多胶原纤维，CPEB4 mRNA及蛋白水平、BUN、SCr、UA、Col I、FN、α–SMA、Wnt1、p–GSK–3β、p–β–catenin水平均降低（P ＜ 0.05）；相较于二甲双胍组，shRNA–CPEB4组和LiCl组大鼠肾小球结构较为规则，细胞排列有序，无系膜增生，有少量炎性细胞浸润，未见明显纤维化；CPEB4 mRNA及蛋白水平、BUN、SCr、UA、Col I、FN、α–SMA、Wnt1、p–GSK–3β、p–β–catenin水平均降低（P ＜ 0.05）；相较于LiCl组，shRNA–CPEB4组大鼠肾组织恢复程度接近Con组，CPEB4 mRNA及蛋白水平、BUN、SCr、UA、Col I、FN、α–SMA、Wnt1、p–GSK–3β、p–β–catenin水平均降低（P ＜ 0.05）。
结论： 敲低CPEB4能够缓解肾组织病理损伤和胶原沉积，减轻肾功能损伤，抑制DKD大鼠RIF，其机制可能与抑制Wnt/β–catenin信号通路有关。

Abstract:
Objective To explore the role of cytoplasmic polyadenylation element-binding protein (CPEB4) in diabetic nephropathy (DKD), and its action mechanism based on the Wnt/β -catenin signaling pathway.
Methods The DKD rat model was established by a high-fat and high-sugar diet combined with intraperitoneal injection of streptozotocin (STZ). The SD rats model were divided into the DKD group, metformin group (100 mg/kg, gavage), shRNA-NC group (300 μL shRNA-NC, tail vein injection), and shRNA-CPEB4 group (300 μL shRNA-CPEB4, tail vein injection) and LiCl group (Wnt/β-catenin pathway activator, 300 μL shRNA-CPEB4 + 15 mg/kg LiCl, tail vein injection), 15 rats in each group. Another 15 rats were selected as the control (Con) group. The expression levels of CPEB4 in the renal tissues of DKD rats were evaluated by RT-qPCR and Western Blot methods. The levels of urea nitrogen (BUN), creatinine (Scr) and blood uric acid (UA) were detected by an automatic biochemical analyzer. Renal histopathological analysis was performed using HE and Masson staining. The expression levels of renal interstitial fibrosis (RIF)-related proteins type I collagen (Col I), fibronectin (FN), α-smooth muscle actin (α-SMA) and Wnt/β-catenin pathway-related proteins were detected by Western Blot method.
Results Compared with the Con group, the rats in the DKD group had atrophy of renal tubules and widening of interstitium, accompanied by severe inflammatory cell infiltration and collagen deposition. The levels of CPEB4 mRNA and protein, BUN, SCr, UA, Col I, FN, α-SMA, Wnt1, p-GSK-3β and p-β-catenin all increased (P ＜ 0.05); Compared with the DKD group, the degree of glomerular lesions in the metformin group, shRNA-CPEB4 group and LiCl group was alleviated, the renal interstitial injury was reduced, there was a small amount of mesangial hyperplasia and inflammatory cell infiltration, and the deposition of collagen fibers was relatively reduced. The levels of CPEB4 mRNA and protein, BUN, SCr, UA, Col I, FN, α-SMA, Wnt1, p-GSK-3β and p-β-catenin were all decreased (P ＜ 0.05). Compared with the shRNA-NC group, the phenomena of glomerular atrophy, interstitial widening and inflammatory infiltration in the metformin group, shRNA-CPEB4 group and LiCl group were gradually alleviated, and there were more collagen fibers between the renal tissues. The levels of CPEB4 mRNA and protein, BUN, SCr, UA, Col I, FN, α-SMA, Wnt1, p-GSK-3β and p-β-catenin were all decreased (P ＜ 0.05). Compared with the LiCl group, the recovery degree of renal tissue in the shRNA-CPEB4 group was close to that in the Con group, and the levels of CPEB4 mRNA and protein, BUN, SCr, UA, Col I, FN, α-SMA, Wnt1, p-GSK-3β and p-β-catenin were all decreased (P ＜ 0.05).
Conclusions Knockdown of CPEB4 can alleviate the pathological damage and collagen deposition in renal tissue, reduce renal function impairment, and inhibit RIF in DKD rats. The mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.

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