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新型磷酸二酯酶5抑制剂CPD1对单侧肾缺血再灌注损伤后肾间质纤维化的影响

刘傲璐¹, 李壮¹, 卢美芝², 邱浩恒¹, 谢中莲¹, 刘晓晴¹, 赵子建¹, 穆云萍^1,*, 李芳红¹

¹广东工业大学生物医药学院，广州 510006；²广州市花都区人民医院，广州 510800

摘要

为观察新型磷酸二酯酶5 (phosphodiesterase 5, PDE5)抑制剂CPD1对单侧肾脏缺血再灌注损伤(unilateral renal ischemia- reperfusion injury, UIRI)后肾间质纤维化的影响，本文采用夹闭(35 min) BALB/c小鼠肾蒂建立UIRI动物模型，造模2 h后每天给予CPD1 (5 mg/kg)灌胃治疗，术后第10天切除对侧肾脏，第11天取患侧肾脏。通过HE、Masson三色和天狼星红染色观察肾组织结构损伤和胶原沉积情况，利用免疫组织化学染色、蛋白质印迹技术分析肾脏组织、大鼠肾脏成纤维细胞(NRK-49F)、人肾小管上皮细胞(HK-2)中纤维化标志蛋白的表达。结果显示：与假手术组相比，UIRI小鼠肾脏中出现明显的肾小管上皮细胞损伤和间质纤维化，I型胶原(collagen I)、纤维连接蛋白(fibronectin)、纤溶酶原激活物抑制剂1型(plasminogen activator inhibitor-1, PAI-1)及α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)表达显著增加；CPD1治疗可明显减轻肾小管损伤和间质纤维化程度，抑制UIRI小鼠肾脏中纤维化标志性蛋白fibronectin、collagen I、PAI-1及α-SMA的表达；同时，在NRK-49F细胞和HK-2细胞中，CPD1呈剂量依赖性抑制转化生长因子-β1 (transforming growth factor β1, TGF-β1)诱导的fibronectin和PAI-1蛋白的表达。以上结果提示，新型PDE5抑制剂CPD1通过抑制TGF-β信号通路，调节PAI-1介导的细胞外基质合成和降解平衡，减轻缺血再灌注引起的肾组织结构损伤和间质纤维化。

关键词： 磷酸二酯酶5抑制剂; CPD1; 肾缺血再灌注损伤; 肾间质纤维化; TGF-β信号通路

Effect of a novel phosphodiesterase 5 inhibitor, CPD1, on renal interstitial fibrosis after unilateral renal ischemia-reperfusion injury

LIU Ao-Lu¹, LI Zhuang¹, LU Mei-Zhi², QIU Hao-Heng¹, XIE Zhong-Lian¹, LIU Xiao-Qing¹, ZHAO Allan Zi-Jian¹, MU Yun-Ping^1,*, LI Fang-Hong¹

¹The School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China；²Huadu District People’s Hospital of Guangzhou, Guangzhou 510800, China

Abstract

This study was designed to evaluate the protective effect of CPD1, a novel phosphodiesterase 5 inhibitor, on renal interstitial fibrosis after unilateral renal ischemia-reperfusion injury (UIRI). Male BALB/c mice were subjected to UIRI, and treated with CPD1 once daily (i.g, 5 mg/kg). Contralateral nephrectomy was performed on day 10 after UIRI, and the UIRI kidneys were harvested on day 11. Hematoxylin-eosin (HE), Masson trichrome and Sirius Red staining methods were used to observe the renal tissue structural lesions and fibrosis. Immunohistochemical staining and Western blot were used to detect the expression of proteins related to fibrosis. HE, Sirius Red and Masson trichrome staining showed that CPD1-treated UIRI mice had lower extent of tubular epithelial cell injury and deposition of extracellular matrix (ECM) in renal interstitium compared with those in the fibrotic mouse kidneys. The results from immunohistochemistry and Western blot assay indicated significantly decreased protein expressions of type I collagen, fibronectin, plasminogen activator inhibitor-1 (PAI-1) and α-smooth muscle actin (α-SMA) after CPD1 treatment. In addition, CPD1 dose- dependently inhibited the expression of ECM-related proteins induced by transforming growth factor β1 (TGF-β1) in normal rat kidney interstitial fibroblasts (NRK-49F) and human renal tubular epithelial cell line (HK-2). In summary, the novel PDE inhibitor, CPD1, displays strong protective effects against UIRI and fibrosis by suppressing TGF-β signaling pathway and regulating the balance between ECM synthesis and degradation through PAI-1.

Key words: phosphodiesterase 5 inhibitors; CPD1; renal ischemia-reperfusion injury; renal interstitial fibrosis; TGF-β signaling pathway

收稿日期：　　录用日期：

通讯作者：穆云萍　　E-mail:

DOI: 10.13294/j.aps.2022.0103

引用本文：

刘傲璐, 李壮, 卢美芝, 邱浩恒, 谢中莲, 刘晓晴, 赵子建, 穆云萍, 李芳红. 新型磷酸二酯酶5抑制剂CPD1对单侧肾缺血再灌注损伤后肾间质纤维化的影响[J]. 生理学报 2023; 75 (1): 1-9.

LIU Ao-Lu, LI Zhuang, LU Mei-Zhi, QIU Hao-Heng, XIE Zhong-Lian, LIU Xiao-Qing, ZHAO Allan Zi-Jian, MU Yun-Ping, LI Fang-Hong. Effect of a novel phosphodiesterase 5 inhibitor, CPD1, on renal interstitial fibrosis after unilateral renal ischemia-reperfusion injury. Acta Physiol Sin 2023; 75 (1): 1-9 (in Chinese with English abstract).