Coombes Jason D, Choi Steve S, Swiderska-Syn Marzena, Manka Paul, Reid Danielle T, Palma Elena, Briones-Orta Marco A, Xie Guanhua, Younis Rasha, Kitamura Naoto, Della Peruta Marco, Bitencourt Shanna, Dollé Laurent, Oo Ye Htun, Mi Zhiyong, Kuo Paul C, Williams Roger, Chokshi Shilpa, Canbay Ali, Claridge Lee C, Eksteen Bertus, Diehl Anna Mae, Syn Wing-Kin
Regeneration and Repair Group, The Institute of Hepatology, Foundation for Liver Research, London, UK; Division of Transplantation Immunology and Mucosal Biology, King's College London, UK.
Division of Gastroenterology, Department of Medicine, Duke University, NC, USA; Section of Gastroenterology, Department of Medicine, Durham Veteran Affairs Medical Center, Durham, NC, USA.
Biochim Biophys Acta. 2016 Jan;1862(1):135-44. doi: 10.1016/j.bbadis.2015.10.028. Epub 2015 Oct 31.
Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH.
Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis.
MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFβ mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice.
OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.
当肝星状细胞（HSC）被激活成为产生胶原蛋白的肌成纤维细胞时，肝纤维化就会发生。在非酒精性脂肪性肝炎（NASH）中，脂肪因子瘦素上调，并通过刺猬信号通路直接激活HSC来促进肝纤维化。我们报道过刺猬信号调节的骨桥蛋白（OPN）在促进肝纤维化中起关键作用。在此，我们评估了OPN是否介导NASH中瘦素的促纤维化作用。
给瘦素缺陷型（ob/ob）和野生型（WT）小鼠喂食对照或蛋氨酸-胆碱缺乏（MCD）饮食。通过天狼星红、OPN和αSMA免疫组化以及对纤维化相关基因进行定量逆转录聚合酶链反应（qRT-PCR）来评估肝组织。在体外，用重组（r）瘦素和OPN中和或对照适配体处理稳定敲低OPN（或对照）的HSC。通过伤口愈合试验评估HSC对OPN缺失的反应。还将OPN适配体添加到精密肝切片（PCLS）中，并给予喂食MCD的WT（瘦素正常）小鼠，以确定OPN中和是否能消除纤维化。
喂食MCD的WT小鼠发生了NASH纤维化，OPN上调，且αSMA+细胞积聚。相反，喂食MCD的ob/ob小鼠发生的纤维化较少，积聚的αSMA+和OPN+细胞也较少。在体外，瘦素处理的HSC使OPN、αSMA、胶原蛋白1α1和转化生长因子β（TGFβ）mRNA上调近3倍，但这种作用因OPN缺失而减弱。抑制磷脂酰肌醇-3激酶（PI3K）和转导显性负性Akt可消除瘦素介导的OPN诱导，而组成型活性Akt则上调OPN。最后，OPN中和减少了PCLS和喂食MCD小鼠中瘦素介导的纤维化。
NASH中OPN的过表达通过PI3K/Akt增强了瘦素介导的纤维化。OPN中和可显著降低NASH纤维化，这强化了靶向OPN在晚期NASH患者治疗中的潜在效用。
