br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion Studies have suggested that the Hh signaling pathway, which plays as an essential autocrine viability factor for MFBs [10], [11], is a potential therapeutic target for liver fibrosis. Previously reported works have proven the effectiveness of the Hh pathway inhibitors such as cyclopamine and GDC-0449 in anti-hepatic fibrosis [9], [27]. Although the underlying mechanisms involved in the anti-fibrotic effects of Hh signaling antagonists have not been completely characterized, the HSCs viability inhibition may play an important role [10], [11]. As the transcription factor Gli constitutes the final step in the canonical or Smo-independent Hh pathway, its inhibitor GANT61 may be a more specific and powerful Hh signaling antagonist [12], [28]. Therefore, we hypothesized that GANT61 could also induce MFBs apoptosis and might be a potential antifibrotic agent for liver fibrosis treatment. The human hepatic stellate cell line (LX-2 cell) used in our experiment were virtually identical to activated HSCs, and exhibited robust endogenous Hh pathway activity which was comparable with the activated primary HSCs [11], [29]. Our experimental results bear out our supposition that GANT61 caused extensive apoptosis in LX-2 cells (Fig. 1). Several studies suggested that Hh signaling could suppress autophagy in certain types of malignant cells, while in hippocampal neurons and vascular endothelial cells, it did the opposite [7], [16], [17], [19], [30], [31]. Our results showed that GANT61 suppressed Hh signaling and boosted autophagy in LX-2 cells, indicating that Hh signaling pathway inhibits autophagy in activated HSCs (Fig. 2). Previous reported works demonstrated that GANT61-induced autophagy was detrimental in several cancer SZL P1-41 [7], [16], [17]. However, it turned out to be a protective factor in activated HSCs in our study (Fig. 3). Cellular energy demands conferred by fibrogenesis and proliferation were sharply increased after HSC transdifferentiation, but it could be resolved by Hh- and autophagy-mediated metabolic reprogramming in HSCs [32], [33]. That explains why both Hh and autophagy play as viability factors for activated HSCs. A recent study demonstrated that PERK and phosphorylation-eIF2a were repressed during Hh signaling activation [19], indicating that PERK-induced signaling transduction could be activated when Hh pathway is blocked with GANT61. The elevation of BiP suggested that ER stress was activated in LX-2 cells (Fig. 4A and D). Indeed, our findings demonstrated that GANT61 activated the PERK-eIF2a-ATF4 pathway (Fig. 4B–D), which was the adaptation response to relieve the burden of ER by facilitating the folding/assembly of amassed proteins or attenuating protein translation [25]. We also found that CHOP, which is closely associated with ER stress related cell apoptosis [24], was upregulated after GANT61 incubation (Fig. 4C). Although emerging evidence reveals that ER stress plays a role in the regulation of apoptosis, whether it is salutary or detrimental is also context dependent [25]. Therefore, we used salubrinal, a selective phosphatase inhibitor of eIF2a [26], to decrease ER stress and clarify its role in GANT61-induced apoptosis. The results showed that salubrinal significantly increased GANT61-induced HSCs apoptosis (Fig. 4F), suggesting that ER stress protects cells against GANT61-caused cytotoxicity. Additionally, we observed that salubrinal suppressed GANT61-induced autophagy in LX-2 cells (Fig. 4E). Previous studies suggests that ER is an essential organelle for autophagy induction, since it provides calcium signaling for autophagy activation and is the potential membrane source of autophagosomes [34], [35]. Moreover, the autophagy system can be activated by ER stress and facilitate the degradation of accumulated proteins result from ER stress [36]. Consistent with these explanations, our results suggest that ER stress may be a trigger for GANT61-induced autophagy.