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Autophagy and cellular senescence are stress responses essential for homeostasis. Therefore, they may represent new pharmacologic targets for drug development to treat diseases. In this

study, we sought to evaluate the effect of dihydroartemisinin (DHA) on senescence of activated hepatic stellate cells (HSCs), and to further elucidate the underlying mechanisms. We found

that DHA treatment induced the accumulation of senescent activated HSCs in rat fibrotic liver, and promoted the expression of senescence markers p53, p16, p21 and Hmga1 in cell model.

Importantly, our study identified the transcription factor GATA6 as an upstream molecule in the facilitation of DHA-induced HSC senescence. GATA6 accumulation promoted DHA-induced p53 and

p16 upregulation, and contributed to HSC senescence. By contrast, siRNA-mediated knockdown of GATA6 dramatically abolished DHA-induced upregulation of p53 and p16, and in turn inhibited HSC

senescence. Interestingly, DHA also appeared to increase autophagosome generation and autophagic flux in activated HSCs, which was underlying mechanism for DHA-induced GATA6 accumulation.

Autophagy depletion impaired GATA6 accumulation, while autophagy induction showed a synergistic effect with DHA. Attractively, p62 was found to act as a negative regulator of GATA6

accumulation. Treatment of cultured HSCs with various autophagy inhibitors, led to an inhibition of DHA-induced p62 degradation, and in turn, prevented DHA-induced GATA6 accumulation and HSC

senescence. Overall, these results provide novel implications to reveal the molecular mechanism of DHA-induced senescence, by which points to the possibility of using DHA based

proautophagic drugs for the treatment of liver fibrosis.

Liver fibrosis is a reversible wound-healing response following liver injury, and its end-stage cirrhosis is responsible for high morbidity and mortality worldwide.1, 2, 3 Liver

transplantation is the only treatment available for patients with advanced stages of liver fibrosis.4, 5, 6 Therefore, new therapeutic agents and strategies are needed for the management of

this disease.7, 8 Dihydroartemisinin (DHA), a natural and safe anti-malarial agent, exhibits an ample array of pharmacological activities such as anti-tumor,9 anti-bacterial10 and

anti-schistosomiasis properties.11 We previously reported that DHA treatment improved the inflammatory microenvironment of liver fibrosis in vivo,12 and inhibited activation and contraction

of hepatic stellate cells (HSCs) in vitro.13, 14, 15, 16 In the current study, we aimed to evaluate the effect of DHA on HSC senescence and to further elucidate the underlying mechanisms.

Cellular senescence is a terminal arrest of proliferation triggered by various cellular stresses including dysfunctional telomeres,17 DNA damage18 and oncogenic mutations.19 Cellular

senescence not only prevents the proliferation of damaged cells, thereby preventing tumorigenesis, but also affects the microenvironment through the secretion of pro-inflammatory cytokines,

chemokines, and proteases, a feature termed the senescence-associated secretory phenotype (SASP).20 The mechanisms underlying induction and maintenance of cell senescence remain entirely

elusive.21, 22, 23 Previous studies21, 22 have reported that p53 can lead to cell cycle arrest, DNA repair and apoptosis predominantly when it becomes transcriptionally active in response to

DNA damage, oncogene activation and hypoxia. Retinoblastoma 1 (pRb) inactivation mediated by p16 is also known to ensure durable cell cycle arrest, but is unlikely to be regulated by a

canonical DNA damage response.23 Attractively, it is interesting to explore the mechanism underlying the induction and maintenance of cell senescence in liver fibrosis.

Interestingly, several lines of evidence indicate a genetic relationship between autophagy and senescence.24, 25 However, whether autophagy acts positively or negatively on senescence is

still subject to debate.25 Through a specialized compartment known as the TOR-autophagy spatial coupling compartment (TASCC), autophagy generates a high flux of recycled amino acids, which

are subsequently used by mTORC1 for supporting the massive synthesis of the SASP factors and facilitating senescence.25 In contrast, increased levels of reactive oxygen species upon

autophagy inhibition partially contribute to cellular senescence.25 We previously reported that DHA treatment stimulated autophagy activation via a ROS-JNK1/2-dependent mechanism in liver

fibrosis.12 Attractively, whether autophagy activation contributes to DHA-induced HSC senescence is worth to further study.

In the present study, we evaluated the effect of DHA on HSC senescence, and to further elucidate the underlying mechanisms. We found that DHA could induce senescence of activated HSCs to

alleviate liver fibrosis via autophagy-dependent GATA6 accumulation. The results of the present study provide important information concerning the molecular mechanisms that underlie the

antifibrotic activities of DHA, which is essential for investigating its potential for clinical application.

Our previous data12, 13, 14, 15, 16 and the present results (Supplementary Figures 1A–C) have sufficiently demonstrated that DHA protected the liver against CCl4-induced injury and

suppressed hepatic fibrogenesis in the rat model. To investigate the mechanisms underlying the protective effects of DHA, we proposed that DHA might induce senescence of activated HSCs to

limit liver fibrosis. To identify senescent cells in situ, we stained liver sections from DHA and vehicle-treated rat for a panel of senescence-associated markers, including SA-β-gal, p53

and p21. Results from immunofluorescence staining showed that cells staining positive for each senescence-associated markers accumulated in fibrotic livers, and were invariably located along

the fibrotic scar by treatment with DHA in a dose-dependent manner (Figures 1a and b; Supplementary Figure 1D). Interestingly, we also found that these cells typically expressed multiple

senescence markers and were not proliferating. As shown in Figures 1c and d, of the p16-positive cells identified in DHA-treated livers, more than 80% were positive for p53 staining, whereas

less than 9% co-expressed the proliferation-association marker Ki67. Although hepatocytes represent the most abundant cell type in the liver, the location of senescent cells along the

fibrotic scar in rat livers raised the possibility that these cells were derived from activated HSCs, which initially proliferate following liver damage and are responsible for much of the

extracellular matrix production in fibrosis.4, 5, 6 In order to verify this hypothesis, the cells in DHA- and vehicle-treated liver sections were not only stained positive for the

senescence-associated markers p53 and p16, but also were positive for the HSC marker desmin. As expected, cells expressing the senescence markers p53 and p21 co-localized with those

expressing desmin (Figures 1e and f). Overall, these results indicate that DHA induces the accumulation of senescent activated HSCs in rat fibrotic liver.

DHA induces the accumulation of senescent activated HSCs in rat fibrotic liver. Rats were grouped as follows: group 1, vehicle control (no CCl4, no treatment); group 2, model group (with

CCl4, no treatment); group 3, DHA (3.5 mg/kg) and CCl4-treated group; group 4, DHA (7 mg/kg) and CCl4-treated group; group 5, DHA (14mg/kg) and CCl4-treated group. (a and b) Liver sections

were stained with immunofluorescence by using antibodies against p53 and p16. White arrows indicated p53 and p16-positive cells. (c) Liver sections were co-stained with p16 and Ki67. White

arrows indicated p16-positive cells, while green arrows indicated Ki67-positive cells. (d) Liver sections were co-stained with p16 and p53. White arrows indicated p16-positive cells, while

green arrows indicated p53-positive cells. (e) Liver sections were co-stained with desmin and p53. White arrows indicated desmin positive cells, while green arrows indicated p53-positive

cells. (f) Liver sections were co-stained with desmin and p16. White arrows indicated desmin positive cells, while green arrows indicated p16-positive cells. For the statistics of each panel

in this figure, data are expressed as mean±S.D. (n=6). Scale bars are 50 μm. For the statistics of each panel in this figure, data are expressed as mean±S.D. (n=6); *P