Attractively existing reports suggest that

Attractively, existing reports suggest that cellular ferroptosis also probably plays vital role in liver fibrosis [[62], [63], [64]]. Carlson BA et al. illuminated that the ferroptosis regulator GPX4 was important for hepatocyte survival and proper liver function [62]. Sun X et al. showed that Nrf2 was critical for preventing HCC Liquidambaric acid from ferroptosis [63]. Sui M et al. found that magnesium isoglycyrrhizinate ameliorated liver fibrosis and hepatic stellate cell activation by regulating ferroptosis signaling pathway [64]. More interestingly, artesunate, an artemisinin derivative with a double oxygen bridge structure, was identified as a potential ferroptosis inducer. Ooko E et al. verified that artemisinin derivatives induced iron-dependent cell death (ferroptosis) in tumor cells [65]. Lin R et al. displayed that artemisinin derivative dihydroartemisinin specifically caused head and neck cancer cell death through contributing ferroptosis and apoptosis [66]. Roh JL et al. demonstrated that inhibition of the Nrf2-ARE pathway increased the sensitivity of artesunate-induced ferroptosis in resistant HNC cells [67]. However, artesunate-induced HSC ferroptosis and underlying deep-going mechanism is unknown. Therefore, whether artesunate could induce HSC ferroptosis in turn alleviate fibrosis deserves to analyze and study. Hence we performed experiments in vivo and in vitro to test our conjecture. Consistent with previous literature, various biological methods were carried out and our data showed that artesunate treatment surely promoted activated HSC ferroptosis in fibrotic livers and HSC-LX2 cell line. The main manifestations are following: artesunate induced-ferroptosis markers GPX4 and Ptgs2 co-localization with fibrosis markers a-SMA, great Fe2+ release, lipid peroxidation excess, and antioxidant capacity loss. More importantly, depletion of ferroptosis by specific inhibitors DFO reversed artesunate-induced anti-fibrosis effect. These experimental results corroborated that artesunate-induced activated HSC ferroptosis was necessary for artesunate-induced anti-fibrosis effect. Ferritinophagy, as the process of degrading ferritin and releasing iron, is involved in iron metabolism related-pathophysiologic process [[68], [69], [70], [71]]. Tang M et al. reported that blocking ferritinophagy to control iron level would provide a novel therapeutic strategy to suppress urinary tract infections [68]. Furthermore, Sun Y et al. found that inhibition of ferritinophagy by NCOA4 or Atg5/Atg7 siRNA relieved pUL38-deficient HCMV infection-induced cell death [69]. Besides, Zacharski LR et al. demonstrated that phlebotomy-triggered ferritinophagy released redox-active iron and aggravated diabetes and cardiovascular disease [70]. In addition, Baksi S et al. attested that a-syn impaired ferritinophagy, resulting in the enrichment of ferritin in the outer retina in vivo and retinal-pigment-epithelial (RPE) cells in vitro [71]. Currently, accumulating documents identified ferritinophagy as the upstream pathway of ferroptosis [[40], [41], [42], [43], [44]]. Our further experimental data also testified that ferritinophagy was responsible for artesunate-induced ferroptosis in activated HSCs. Firstly, the expression of ferritinophagy markers LC3, FTH1, NCOA4, substrate p62 and Atgs (Atg3, Atg5, Atg6/Beclin1, and Atg12) were detected by immunoblotting assay or real-time PCR analysis. The results showed that artesunate triggered HSC ferritinophagy. GFP-LC3 transfection experiment and TCM analysis displayed that autophagosome generation and autophagic flux increased after artesunate treatment, which was vital for artesunate-induced anti-fibrosis effect. In contrast, interdiction of ferritinophagy by CQ completely destroyed the efficacy of artesunate-induced HSC ferroptosis and HSC activation inhibition. In summary, our results clearly demonstrated that artesunate targeted activating hepatic stellate cells ferroptosis, and its effect was associated with activation of ferritinophagy. These findings will provide further mechanisms for understanding the protective effect of artesunate against CCl4-induced liver fibrosis (Fig. 6).