Angiotensin II Facilitates Fibrogenic Effect of TGF-β1 through Enhancing the Down-Regulation of BAMBI Caused by LPS: A New Pro-Fibrotic Mechanism of Angiotensin II

Angiotensin II has progressively been considered to play an important role in the development of liver fibrosis, although the mechanism isn't fully understood. The aim of this study was to investigate a possible pro-fibrotic mechanism, by which angiotensin II would enhance the pro-fibrotic effect of transforming growth factor beta 1 (TGF-β1) through up-regulation of toll-like receptor 4 (TLR4) and enhancing down-regulation of TGF-β1 inhibitory pseudo-receptor—BAMBI caused by LPS in hepatic stellate cells (HSCs). Firstly, the synergistic effects of angiotensin II, TGF-β1 and LPS on collagen 1α production were confirmed in vitro by ELISA, in which angiotensin II, LPS and TGF-β1 were treated sequentially, and in vivo by immunofluorescence, in the experiments single or multiple intra-peritoneally implanted osmotic mini-pumps administrating angiotensin II or LPS combined with intra-peritoneal injections of TGF-β1 were used. We also found that only LPS and TGF-β1 weren't enough to induce obvious fibrogenesis without angiotensin II. Secondly, to identify the reason of why angiotensin II is so important, the minute level of TLR4 in activated HSCs - T6 and primary quiescent HSCs of rat, up-regulation of TLR4 by angiotensin II and blockage by different angiotensin II receptor type 1 (AT1) blockers in HSCs were assayed by western blotting in vitro and immunofluorescence in vivo. Finally, BAMBI expression level, which is regulated by LPS-TLR4 pathway, was detected by qRT-PCR and results showed angiotensin II enhanced the down-regulation of BAMBI mRNA caused by LPS in vitro and in vivo, and TLR4 neutralization antibody blocked this interactive effect. These data demonstrated that angiotensin II enhances LPS-TLR4 pathway signaling and further down-regulates expression of BAMBI through up-regulation of TLR4, which results in facilitation of pro-fibrotic activity of TGF-β1. Angiotensin II, LPS and TGF-β1 act synergistically during hepatic fibrogenesis, showing crosstalks between angiotensin II-AT1, LPS-TLR4 and TGF-β1-BAMBI signal pathways in rat HSCs.

尽管具体机制尚未完全阐明，但血管紧张素II（Angiotensin II）已被逐步证实与肝纤维化的发生发展密切相关。本研究旨在探讨一种潜在的促肝纤维化机制：即血管紧张素II可通过上调Toll样受体4（TLR4）的表达，并增强脂多糖（LPS）诱导肝星状细胞（HSCs）对转化生长因子β1（TGF-β1）抑制性假受体BAMBI的下调作用，从而增强TGF-β1的促纤维化效应。首先，本研究通过体外酶联免疫吸附试验（ELISA）验证了血管紧张素II、TGF-β1与LPS对Ⅰ型胶原1α生成的协同促进作用：体外实验采用依次给药血管紧张素II、LPS与TGF-β1的处理方式；体内实验则通过单次或多次腹腔植入渗透微型泵给药血管紧张素II或LPS，联合腹腔注射TGF-β1的造模方案，结合免疫荧光染色完成验证。同时本研究发现，若无血管紧张素II参与，仅LPS与TGF-β1不足以诱导显著的肝纤维化进程。其次，为明确血管紧张素II发挥关键作用的分子机制，本研究分别针对活化肝星状细胞T6与大鼠原代静息肝星状细胞内TLR4的低表达水平、血管紧张素II对TLR4的上调作用，以及血管紧张素Ⅱ1型受体（AT1）拮抗剂对该过程的阻断效应，通过体外蛋白质印迹法（western blotting）与体内免疫荧光染色进行了检测。最后，本研究通过实时荧光定量聚合酶链反应（qRT-PCR）检测了受LPS-TLR4通路调控的BAMBI表达水平，结果显示：无论在体内还是体外，血管紧张素II均可增强LPS诱导的BAMBI mRNA下调效应，而TLR4中和抗体可阻断这一相互作用。上述实验数据表明，血管紧张素II可通过上调TLR4的表达增强LPS-TLR4通路的信号转导，并进一步下调BAMBI的表达，从而促进TGF-β1的促纤维化活性。在大鼠肝星状细胞中，血管紧张素II、LPS与TGF-β1在肝纤维化进程中发挥协同作用，揭示了血管紧张素II-AT1、LPS-TLR4与TGF-β1-BAMBI三条信号通路之间的交叉调控关系。