Sandoval-Rodriguez Ana, Monroy-Ramirez Hugo Christian, Meza-Rios Alejandra, Garcia-Bañuelos Jesus, Vera-Cruz Jose, Gutiérrez-Cuevas Jorge, Silva-Gomez Jorge, Staels Bart, Dominguez-Rosales Jose, Galicia-Moreno Marina, Vazquez-Del Mercado Monica, Navarro-Partida Jose, Santos-Garcia Arturo, Armendariz-Borunda Juan
Department of Molecular Biology and Genomics Institute for Molecular Biology in Medicine and Gene Therapy Health Sciences University Center University of Guadalajara Guadalajara México.
Tecnologico de Monterrey Campus Guadalajara Zapopan México.
Hepatol Commun. 2020 Jan 16;4(3):434-449. doi: 10.1002/hep4.1474. eCollection 2020 Mar.
Nonalcoholic steatohepatitis (NASH) is recognized by hepatic lipid accumulation, inflammation, and fibrosis. No studies have evaluated the prolonged-release pirfenidone (PR-PFD) properties on NASH features. The aim of this study is to evaluate how PR-PFD performs on metabolic functions, and provide insight on a mouse model of human NASH. Male mice were fed with either normo diet or high-fat/carbohydrate diet for 16 weeks and a subgroup also fed with PR-PFD (300 mg/kg/day). An insulin tolerance test was performed at the end of treatment. Histological analysis, determination of serum hormones, adipocytokines measurement, and evaluation of proteins by western blot was performed. Molecular docking, site-directed mutagenesis, and experiments using HepG2 cultured cells were performed to validate PR-PFD binding to peroxisome proliferator-activated receptor alpha (PPAR-α), activation of PPAR-α promoter, and sirtuin 1 (SIRT1) protein expression. Compared with the high-fat group, the PR-PFD-treated mice displayed less weight gain, cholesterol, very low density lipoprotein and triglycerides, and showed a significant reduction of hepatic macrosteatosis, inflammation, hepatocyte ballooning, fibrosis, epididymal fat, and total adiposity. PR-PFD restored levels of insulin, glucagon, adiponectin, and resistin along with improved insulin resistance. Noteworthy, SIRT1-liver kinase B1-phospho-5' adenosine monophosphate-activated protein kinase signaling and the PPAR-α/carnitine O-palmitoyltransferase 1/acyl-CoA oxidase 1 pathway were clearly induced in high fat + PR-PFD mice. In HepG2 cells incubated with palmitate, PR-PFD induced activation and nuclear translocation of both PPARα and SIRT1, which correlated with increased SIRT1 phosphorylated in serine 47, suggesting a positive feedback loop between the two proteins. These results were confirmed with both synthetic PPAR-α and SIRT1 activators and inhibitors. Finally, we found that PR-PFD is a true agonist/ligand for PPAR-α. PR-PFD provided an anti-steatogenic effect and protection for inflammation and fibrosis.
非酒精性脂肪性肝炎（NASH）的特征为肝脏脂质蓄积、炎症和纤维化。尚无研究评估缓释型吡非尼酮（PR-PFD）对NASH特征的影响。本研究旨在评估PR-PFD对代谢功能的作用，并深入了解人类NASH的小鼠模型。雄性小鼠分别给予正常饮食或高脂/高碳水化合物饮食16周，其中一个亚组同时给予PR-PFD（300毫克/千克/天）。在治疗结束时进行胰岛素耐量试验。进行了组织学分析、血清激素测定、脂肪细胞因子测量以及蛋白质的蛋白质印迹评估。进行了分子对接、定点诱变以及使用HepG2培养细胞的实验，以验证PR-PFD与过氧化物酶体增殖物激活受体α（PPAR-α）的结合、PPAR-α启动子的激活以及沉默调节蛋白1（SIRT1）蛋白表达。与高脂组相比，接受PR-PFD治疗的小鼠体重增加、胆固醇、极低密度脂蛋白和甘油三酯水平较低，肝脏大脂肪变性、炎症、肝细胞气球样变、纤维化、附睾脂肪和总体脂量均显著降低。PR-PFD恢复了胰岛素、胰高血糖素、脂联素和抵抗素水平，同时改善了胰岛素抵抗。值得注意的是，在高脂+PR-PFD小鼠中，SIRT1-肝脏激酶B1-磷酸化5'-腺苷单磷酸激活蛋白激酶信号通路以及PPAR-α/肉碱O-棕榈酰转移酶1/酰基辅酶A氧化酶1途径明显被诱导。在用棕榈酸孵育的HepG2细胞中，PR-PFD诱导PPARα和SIRT1的激活和核转位，这与丝氨酸47处磷酸化的SIRT1增加相关，表明这两种蛋白之间存在正反馈回路。这些结果通过合成的PPAR-α和SIRT1激活剂及抑制剂得到了证实。最后，我们发现PR-PFD是PPAR-α的真正激动剂/配体。PR-PFD具有抗脂肪生成作用，并对炎症和纤维化具有保护作用。
