过敏毒素 C3a、C5a 对体外肾小管上皮细胞-肌成纤维细胞转分化的影响。

Effect of anaphylatoxin C3a, C5a on the tubular epithelial-myofibroblast transdifferentiation in vitro.

机构信息

Division of Nephrology, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, China.

出版信息

Chin Med J (Engl). 2011 Dec;124(23):4039-45.

Abstract

BACKGROUND

Tubulointerstitial renal fibrosis is the common end point of progressive kidney diseases, and tubular epithelial-myofibroblast transdifferentiation (TEMT) plays a key role in the progress of tubulointerstitial renal fibrosis. Anaphylatoxin C3a and C5a are identified as novel profibrotic factors in renal disease and as potential new therapeutic targets. The aim of this study was to investigate whether C3a, C5a can regulate TEMT by transforming growth factor-β1 (TGF-β)/connective tissue growth factor (CTGF) signaling pathway and the effects of C3a and C5a receptor antagonists (C3aRA and C5aRA) on C3a- and C5a-induced TEMT.

METHODS

HK-2 cells were divided into C3a and C5a groups which were subdivided into four subgroups: control group, 10 ng/ml TGF-β1 group, 50 nmol/L C3a group, 50 nmol/L C3a plus 1 µmol/L C3aRA group; control group, 10 ng/ml TGF-β1 group, 50 nmol/L C5a group, 50 nmol/L C5a plus 2.5 µmol/L C5aRA group. TGF-β1 receptor antagonist (TGF-β1RA) 10 µg/ml was used to investigate the mechanism of C3a- and C5a-induced TEMT. Electron microscopy was used to observe the morphological changes. Immunocytochemistry staining, real-time PCR and Western blotting were used to detect the expressions of a smooth muscle actin (α-SMA), E-cadherin, Col-I, C3a receptor (C3aR), C5aR, CTGF and TGF-β1.

RESULTS

HK-2 cells cultured with C3a and C5a for 72 hours exhibited strong staining of α-SMA, lost the positive staining of E-cadherin, and showed a slightly spindle-like shape and loss of microvilli on the cell surface. The expressions of α-SMA, E-cadherin, Col-I, C3aR, C5aR, TGF-β1 and CTGF in C3a- and C5a-treated groups were higher than normal control group (P < 0.05). C3aRA and C5aRA inhibited the expressions of α-SMA, Col-I, C3aR, C5aR, and up-regulated the expression of E-cadherin (P < 0.05). TGF-β1 and CTGF mRNA expressions induced by C3a and C5a were partly blocked by TGF-β1RA (P < 0.05).

CONCLUSION

C3a and C5a can induce TEMT via the up-regulations of C3aR and C5aR mRNA and the activation of TGF-β1/CTGF signaling pathway in vitro.

摘要

背景

肾小管间质肾纤维化是进行性肾脏疾病的共同终点，而肾小管上皮-肌成纤维细胞转分化（TEMT）在肾小管间质肾纤维化的进展中起着关键作用。过敏毒素 C3a 和 C5a 被确定为肾脏疾病中的新型促纤维化因子，也是潜在的新治疗靶点。本研究旨在探讨 C3a、C5a 是否可以通过转化生长因子-β1（TGF-β）/结缔组织生长因子（CTGF）信号通路调节 TEMT，以及 C3a 和 C5a 受体拮抗剂（C3aRA 和 C5aRA）对 C3a 和 C5a 诱导的 TEMT 的影响。

方法

将 HK-2 细胞分为 C3a 和 C5a 组，再分为四个亚组：对照组、10ng/ml TGF-β1 组、50nmol/L C3a 组、50nmol/L C3a 加 1µmol/L C3aRA 组；对照组、10ng/ml TGF-β1 组、50nmol/L C5a 组、50nmol/L C5a 加 2.5µmol/L C5aRA 组。使用 TGF-β1 受体拮抗剂（TGF-β1RA）10µg/ml 来研究 C3a 和 C5a 诱导的 TEMT 的机制。电子显微镜用于观察形态变化。免疫细胞化学染色、实时 PCR 和 Western blot 用于检测平滑肌肌动蛋白（α-SMA）、E-钙黏蛋白、Col-I、C3a 受体（C3aR）、C5aR、CTGF 和 TGF-β1 的表达。

结果

培养 72 小时的 HK-2 细胞用 C3a 和 C5a 处理后，α-SMA 染色强，E-钙黏蛋白阳性染色丢失，细胞表面出现轻微的纺锤形和微绒毛丢失。C3a 和 C5a 处理组的α-SMA、E-钙黏蛋白、Col-I、C3aR、C5aR、TGF-β1 和 CTGF 表达均高于正常对照组（P<0.05）。C3aRA 和 C5aRA 抑制了α-SMA、Col-I、C3aR、C5aR 的表达，并上调了 E-钙黏蛋白的表达（P<0.05）。TGF-β1RA 部分阻断了 C3a 和 C5a 诱导的 TGF-β1 和 CTGF mRNA 表达（P<0.05）。