br Funding br Ethics br Conflict of

Funding
Ethics
Conflict of interest
Acknowledgements
Introduction Hepatic fibrosis occurs in response to different etiologies of chronic liver injury, which is mainly accompanied by pathological microcystin lr of excessive extracellular matrix (ECM) of the liver, as the common reaction of multiple chronic liver diseases to liver cirrhosis [1,2]. Without effective management, liver fiber nodules are produced soon and normal liver structure and function are disrupted [3]. Therefore, hepatic fibrosis has become a major public health issue, and currently, effective anti-fibrotic strategies are still lacking in clinical practice. Inflammation is involved in the pathophysiological process of liver fibrosis [[4], [5], [6]]. Fibrotic liver is in the inflammatory microenvironment and surrounded by different inflammatory factors [[7], [8], [9]]. In normal liver, pro- and anti-inflammatory factors are kept balanced, which is broken upon injury [10,11]. Hence, it is feasible to fight against liver fibrosis by suppressing inflammation. Zhang et al. reported that dihydroartemisinin(DHA) alleviated liver fibrosis by inhibiting the expressions of pro-inflammatory factors and promoting those of anti-inflammatory factors, and such process was dependent on activation of autophagy in vivo and in vitro [7]. Li et al. showed that quercetin mitigated liver fibrosis in mice through modulating of HMGB1-TLR2/4-NF-κB signaling pathways [12]. Autophagy mediates the regulation of inflammation and plays a key role in the pathophysiology of many human disorders including hepatic fibrosis [[13], [14], [15], [16]]. It directly suppresses proinflammatory complexes, and also indirectly allows efficient clearance of damaged organelles or intracellular pathogenic microorganisms that both constitute potent inflammatory stimuli [7,17]. Therefore, targeting autophagy may be able to combat liver fibrosis. Wang et al. found that 3-methyladenine, a selective type III phosphatidylinositol 3-kinase inhibitor, relieved liver fibrosis mainly through inhibiting the autophagy of hepatic stellate cells (HSCs) regulated by the NF-κB signaling pathways [13]. Besides, Zhang et al. showed that ROS-JNK1/2-dependent activation of autophagy was required to induce the anti-inflammatory effects of DHA on liver fibrosis [7]. In this study, we thus focused on the above two aspects. CCl4 is a commonly used carcinogen, not only anesthetizing effect on the central nervous system but also seriously damaging to the liver and kidney [18]. It has been widely used to induce liver injury or fibrosis in animals including rats [19,20]. Due to short experimental period and stability, the in vivo model of CCl4-induced liver fibrosis was employed in this study. The key role of HSCs in liver fibrosis is well-documented [[21], [22], [23]]. HSCs activation is a critical process in the pathogenesis of liver fibrosis, because ECM deposit mainly originates from activated HSCs [24]. Therefore, PDGF-BB-activated HSCs were used herein to perform in vitro experiments. Natural products have attracted worldwide attention and are now important sources for anti-fibrotic agents. Catalpol, an iridoid glycoside extracted from traditional Chinese medicine Rehmannia glutinosa, has anti-asthma [25], antioxidant, anti-inflammatory [26], antidiabetic [27,28], antitumor [29] and other remarkable pharmacological effects. However, its effects on liver fibrosis remain largely unknown. In this study, the in vitro and in vivo anti-fibrotic effects of catalpol were assessed for the first time. Meanwhile, the mechanisms were clarified by examining the expressions of autophagy associated proteins and inflammatory factors in vitro and in vivo. Finally, the influence of autophagy inhibition on the anti-inflammatory effects of catalpol on liver fibrosis was evaluated in vitro. A new mechanism by which catalpol resisted fibrosis was confirmed. By regulating autophagy-mediated inflammation, catalpol alleviated liver fibrosis in vivo and in vitro.