Sorafenib attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis via HIF‐1α/SLC7A11 pathway

Abstract Objectives Evidences demonstrate that sorafenib alleviates liver fibrosis via inhibiting HSC activation and ECM accumulation. The underlying mechanism remains unclear. Ferroptosis, a novel programmed cell death, regulates diverse physiological/pathological processes. In this study, we aim to investigate the functional role of HSC ferroptosis in the anti‐fibrotic effect of sorafenib. Materials and Methods The effects of sorafenib on HSC ferroptosis and ECM expression were assessed in mouse model of liver fibrosis induced by CCl4. In vitro, Fer‐1 and DFO were used to block ferroptosis and then explored the anti‐fibrotic effect of sorafenib by detecting α‐SMA, COL1α1 and fibronectin proteins. Finally, HIF‐1α siRNA, plasmid and stabilizers were applied to assess related signalling pathway. Results Sorafenib attenuated liver injury and ECM accumulation in CCl4‐induced fibrotic livers, accompanied by reduction of SLC7A11 and GPX4 proteins. In sorafenib‐treated HSC‐T6 cells, ferroptotic events (depletion of SLC7A11, GPX4 and GSH; accumulation iron, ROS and MDA) were discovered. Intriguingly, these ferroptotic events were not appeared in hepatocytes or macrophages. Sorafenib‐elicited HSC ferroptosis and ECM reduction were abrogated by Fer‐1 and DFO. Additionally, both HIF‐1α and SLC7A11 proteins were reduced in sorafenib‐treated HSC‐T6 cells. SLC7A11 was positively regulated by HIF‐1α, inactivation of HIF‐1α/SLC7A11 pathway was required for sorafenib‐induced HSC ferroptosis, and elevation of HIF‐1α could inhibit ferroptosis, ultimately limited the anti‐fibrotic effect. Conclusions Sorafenib triggers HSC ferroptosis via HIF‐1α/SLC7A11 signalling, which in turn attenuates liver injury and fibrosis.

and scar formation. 3 In response to liver injury, quiescent HSCs undergo a complex activation process and eventually transdifferentiate into activated HSCs. Markers of activated HSCs, such as cytoskeletal protein α-smooth muscle actin (α-SMA), collagen 1α1 (COL1α1) and fibronectin, are upregulated in this process. 4 Studies from our own and others laboratories have shown that inhibition of HSC decreases ECM deposition and alleviates liver fibrosis. [5][6][7] Ferroptosis, a novel type of programmed cell death, is characterized by intracellular iron overload and accumulation of lipid reactive oxygen species (ROS). 8 The morphology and mechanism of ferroptosis are different from traditional programmed cell death, such as apoptosis, autophagy and necrosis. Morphologically, ferroptotic cells exhibit reduced mitochondrial volume, condensed mitochondrial membrane density, absent mitochondrial cristae and even ruptured outer mitochondrial membranes. Mechanically, ferroptosis is associated iron metabolism disorder, lipid peroxidation accumulation, glutathione (GSH) and solute carrier family 7 member 11 (SLC7A11) deficiency. 9 Ferroptotic events have been found in HSC cell lines treated with erastin, sorafenib and buthionine sulfoximine, and HSC growth and ECM accumulation were significantly decreased in this progess. 10,11 Moreover, some traditional Chinese medicines exhibit anti-fibrotic effects through inducing HSC ferroptosis. 12,13 Sorafenib, a multiple kinases inhibitor, is well known for its antitumour effect. In addition, sorafenib regulates HSC viability via inhibiting cell proliferation and promoting apoptosis, afterwards, exhibits anti-fibrosis effect in liver. 14,15 Recent studies proved sorafenib as a ferroptotic inducer, which triggers a series of ferroptotic events in different kinds of cells. 9,16,17 Ferroptosis has been observed in sorafenib-treated HSCs as well. 10,11 Nevertheless, the underling mechanism remains unclear.
During the progress of fibrosis, regions of hypoxia were observed in liver, accompanied by increased expression of hypoxiainducible factor 1α (HIF-1α). 18,19 In nonalcoholic fatty liver disease model, knockout HIF-1α specifically in hepatocyte could reduce liver fibrosis. 20 Meanwhile, sorafenib decreased HIF-1α protein level and caused apoptosis of hepatocellular carcinoma cell. 21 Interestingly, decrease in HIF-1α with inhibitors or specific siRNA enhanced cell ferroptosis. 22 As a transcriptional factor, HIF-1α regulates several gene level including SLC7A11. 23,24 Knockout of HIF-1α sharply decreased SLC7A11 protein in rat brain tissue. 25 So we wondered whether HIF-1α/SLC7A11 is involved in sorafenib-induced HSC ferroptosis.
In the study, we find that sorafenib induces ferroptotic events in HSCs via HIF-1α/SLC7A11 pathway, and HSC ferroptosis is involved with reduction of α-SMA and CoL1α1. On the contrary, ferroptotic inhibitors, ferrostatin-1(Fer-1) and deferoxamine (DFO), dramatically reduce the anti-fibrotic effect of sorafenib. Intriguingly, sorafenib triggers ferroptosis in HSCs with no effect on hepatocytes or macrophages. Our study may provide a potential therapeutic target for sorafenib in the treatment of liver fibrosis.

| ALT/AST/HYP assay
The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hydroxyproline (HYP) in serum were assessed using ALT Kit (#C009-2-1, Jiancheng, China), AST Kit (#C010-2-1, Jiancheng, China) and HYP Kit (#A030-2-1, Jiancheng, China) according to manufacturer's instructions. then, the area was calculated. Afterwards, the entire staining of the tissue was selected to calculate the total area. The quantitative analysis of collagen staining (also known as the volume fraction of collagen) was the ratio of the positive collagen area to the total tissue area.

| Cell culture and treatment
Rat HSC line (HSC-T6) was obtained from the Chinese Academy of Science. HSC-T6 were cultured in Dulbecco's modified Eagle medium (DMEM; #SH30022.01, HyClone, USA) with 10% foetal bovine serum (FBS; Biological Industries, Israel) and incubated at 37°C with 5% CO 2 . Cells were grown to 70% confluence and then

| Cell viability and cell cytotoxicity analyses
HSC-T6 cells were seeded into 96-well plates. When the cell density reached 70%, different concentrations of sorafenib were added, and equal amounts of DMSO without drug were exposed to the con-

| Observation of mitochondria morphology in cells
HSC-T6 cells were inoculated into the cell culture dish, collected with or without sorafenib treatment (the final number of cells was no less than 10 7 ) and fixed, dehydrated, macerated and embedded.
Embedded blocks were cut into 70 nm-thick sections and stained with lead citrate. The images were acquired using a transmission electron microscope (TEM; #JEM1400, JEOL, JPN).

| Semi-quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR)
qRT-PCR assays were performed as described. 6 Specifically, total RNA was extracted from HSC-T6 cells using TRIzol reagent

| HIF-1α RNA interference and HIF-1α Overexpression analyses
Small short interfering RNAs (siRNAs) targeting HIF-1α gene se- process was carried out as described previously. 26 Then, qRT-PCR was used to determine the efficiency of gene knockout and overexpression.

| Immunofluorescence analysis
Immunofluorescence assays were performed as described in our previous description. 6 Primary antibodies were as follows: HIF-1α

| Western blot analysis
The total proteins of liver tissues and cells were extracted by RIPA (#P0013C, Beyotime, China) with phosphatase inhibitor and PMSF (#P0012S, Beyotime, China). Immunoblotting was performed as previously described. 6 Primary antibodies were as follows: α-SMA

| Statistical analysis
All experiments were performed at least three times, and statistical analysis was performed by using GraphPad Prism 5. Data were presented as mean ± SD, and statistical significance was determined by ANOVA with the post hoc test. All significance levels were set at 0.05.

| Ferroptosis occurs in mouse fibrotic liver induced by sorafenib
It has been proved that sorafenib attenuates liver fibrosis via regulating ECM accumulation and inflammatory reaction. 14,27 In line with the previous studies, we found that hepatocyte degeneration, inflammatory cell infiltration, fibrous scarring and collagen deposition were all aggravated in the CCl 4 -treated group, while these changes were remarkably mitigated in sorafenib-treated groups ( Figure 1A). Besides, sorafenib decreased the elevated liver/weight ratio triggered by CCl 4 ( Figure 1B). Moreover, HYP level elevated in the CCl 4 -treated group, which was significantly reduced by sorafenib ( Figure 1B). Sorafenib also significantly reduced the serum levels of ALT and AST ( Figure 1C). Fibrotic markers, α-SMA, COL1α1 and fibronectin, were overexpressed in the CCl 4 -treated group, while were notably reduced in sorafenibtreated groups ( Figure 1D).
Above data confirm that sorafenib alleviates CCl 4 -induced liver injury and fibrosis. To further explore the underlying mechanism, ferroptotic markers, GPX4, SLC7A11 and PTGS2, were stained in the lobes of livers. As illustrated in Figure S1, these markers were barely expressed in the vehicle group. GPX4 and SLC7A11 immunostaining signals were obviously observed in HSCs around the fibrotic scars induced by CCl 4 , whereas sorafenib treatment reduced GPX4 and SLC7A11 expressions ( Figure S1). It is worth mentioning that GPX4 and SLC7A11 proteins were also significantly increased in liver parenchymal after CCl 4 induction ( Figure S1). Additionally, PTGS2 signals in HSCs were enhanced in sorafenib-treated liver compared to fibrotic mouse liver ( Figure S1). α-SMA immunostaining signals were abated in sorafenib-treated livers ( Figure S1). Overall, these data suggest that sorafenib attenuates mouse liver fibrosis, which is accompanied by the ferroptotic markers in HSCs.

F I G U R E 3
Sorafenib triggers HSC ferroptosis with no effect on hepatocytes or macrophages. AML-12, RAW 264.7 and HSC-T6 cells

| Sorafenib inhibits HSC activation by triggering ferroptosis in vitro
Given that HSCs as the main contributor of ECM production, HSC-  Figure 2B). ZVAD-FMK, an apoptotic inhibitor, also impeded sorafenib inhibition effect in some degree ( Figure 2B). However, necrosulfonamide, an inhibitor of necroptosis, had no effect on the cell viability ( Figure 2B). Both ferroptotic inhibitors and apoptotic inhibitor hampered the suppression of cell viability in sorafenibtreated HSC-T6 cells, Fer-1 and DFO exhibited a higher ability to reverse the inhibition of HSC viability than that of ZVAD-FMK ( Figure 2B). Additionally, mitochondrial condensation and rupture were observed in the sorafenib-treated HSC-T6 cells ( Figure 2C).
Ferroptotic events were also found in sorafenib-treated HSC-T6 cells, which were evidenced by decreased GSH content, increased iron level and lipid peroxidation products ( Figure 2D). Protein levels of GPX4 and SLC7A11 were markedly decreased in sorafenibtreated HSCs ( Figure 2E). Additionally, the levels of α-SMA, COL1α1 and fibronectin were down-regulated by sorafenib compared with the control group ( Figure 2F). In conclusion, above data suggest that ferroptosis is involved in sorafenib-induced HSC inhibition and ECM reduction.

| Sorafenib triggers HSC ferroptosis with no effect on hepatocytes or macrophages
It is well known that liver is composed of hepatocytes, HSCs, macrophages, etc. 28 Figure 3D) and ROS ( Figure 3E) were elevated by sorafenib in HSC-T6 cells, while GSH content ( Figure 3C) was declined. However, exposed of AML-12 and RAW 264.7 cells to sorafenib neither of them showed the ferroptotic events ( Figure 3A-E). Together, the data indicate that sorafenib causes ferroptosis in HSCs with little effect on hepatocytes and macrophages.

| Blockade of HSC ferroptosis abolishes sorafenib-induced anti-fibrotic effect
To further investigate the functional role of HSC ferroptosis in the anti-fibrotic effect of sorafenib, HSC-T6 cells were exposed to Fer-1 and DFO for 24 h. As illustrated in Figure 4A and D, both Fer-1 and DFO could completely abolish sorafenib-induced ferroptotic events. Meanwhile, reduced GPX4 protein level in sorafenibtreated HSC-T6 cells could be reversed by Fer-1 and DFO ( Figure 4B and E), whereas SLC7A11 protein was partially elevated by DFO, but not affected by Fer-1 ( Figure 4B and E). In addition, reduced ECM expression in sorafenib-treated HSC-T6 cells was observed, and this process was impaired by Fer-1 and DFO ( Figure 4C and F). In summary, inhibition of HSC ferroptosis impairs sorafenibinduced anti-fibrotic efficacy.

| HIF-1α expression is decreased during sorafenib-induced anti-fibrosis
It is interesting to note that DFO, a ferroptotic inhibitor, is also a HIF-1α prolylhydroxylase inhibitor, which protects HIF-1α protein F I G U R E 7 Sorafenib triggers HSC ferroptosis via HIF-1α/SLC7A11-dependent mechanism. Exposure HIF-1α plasmid to HSC-T6 cells for 48 h, with or without sorafenib (10 μM) treatment for 24 h. HIF-1α (A) mRNA and (B) protein levels were measured by qRT-PCR and Western blot. Data were presented as the mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01. (C) Western blot analyses of SLC7A11 and GPX4 proteins were performed. Data were presented as the mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01. (D) The contents of iron, MDA, and GSH in cell lysates were measured by kits. Intracellular ROS generation was detected with DCFH-DA probe. Data were presented as the mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01. N.S. not significant. Exposure HIF-1α siRNA to HSC-T6 cells for 48 h, with or without sorafenib (10 μM) treatment for 24 h. HIF-1α (E) mRNA and (F) protein levels were measured by qRT-PCR and Western blot. Data were presented as the mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01. (G) The expression of SLC7A11 was measured by immunofluorescence (Scale bar: 50 μm). Data were presented as the mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01. (H) Western blot analyses of SLC7A11 and GPX4 proteins were performed. Data were presented as the mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01. (I) The contents of iron, MDA and GSH in cell lysates were measured by Kits. Intracellular ROS generation was detected with DCFH-DA probe. Data were presented as the mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01 F I G U R E 8 HIF-1α is involved in the anti-fibrotic effect of sorafenib. Western blot analyses of α-SMA and COL1α1 proteins were performed in(A) HIF-1α-overexpressed and (B) HIF-1α-silenced HSC-T6 cells, with or without sorafenib (10 μM) treatment for 24 h. Data were presented as the mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01. N.S. not significant from degradation. 29 HIF-1α protein level was significantly elevated in fibrotic liver compared to vehicle-treated liver, which was reduced by sorafenib ( Figure 5A). Furthermore, when exposed HSC-T6 cells to sorafenib, fluorescence staining assay showed a decline of HIF-1α both in cytoplasm and in nucleus ( Figure 5B). Western blot analysis confirmed that HIF-1α was decreased in sorafenib-treated HSC-T6 cells ( Figure 5C). Considering that HIF-1α is a transcription factor and acts its role in the nucleus mostly, the level of HIF-1α protein in cytoplasm and nucleus of HSC-T6 cells was detected respectively. As shown in Figure 5C, sorafenib decreased HIF-1α expression both in the cytoplasm and in the nucleus.
To further explore the role of HIF-1α in sorafenib-induced antifibrosis effect, two HIF-1α prolylhydroxylase inhibitors, dimethyloxallyl glycine (DMOG) and DFO, were exposed to stabilize HIF-1α protein in HSC-T6 cells for 24 h. As illustrated in Figure 5D, HIF-1α was significantly increased in the nucleus when treated with DMOG and DFO. In the cytoplasm, HIF-1α level was increased in DFOtreated cells, but not affected by DMOG ( Figure 5D). Interestingly, DMOG and DFO also offset sorafenib anti-fibrotic effect, as evidenced by α-SMA, COL1α1 and fibronectin expressions ( Figure 5E).
Overall, these data demonstrate that sorafenib decreases HIF-1α both in HSC nucleus and in cytoplasm. On the contrary, stabilization of HIF-1α protein impedes anti-fibrotic effect of sorafenib.

| Sorafenib triggers HSC ferroptosis via HIF-1α/ SLC7A11-dependent mechanism
To gain insight into the potential signalling pathways in sorafenibtriggered HSC ferroptosis, DFO and DMOG were applied to stabilize HIF-1α protein. As shown in Figure 6A, sorafenib-induced ferroptotic markers were mostly hindered by DFO and DMOG in HSC-T6 cells.
Similarly, protection of HIF-1α from degradation could abrogated the depletion of SLC7A11 induced by sorafenib ( Figure 6B). The data obtained with the pharmacological blockers were strengthened by the application of specific plasmid HIF-1α. As shown in Figure 7A and B, HIF-1α mRNA and protein levels were significantly increased in HSC-T6 cells after 48-h transfection. SLC7A11 and GPX4 proteins were significantly upregulated in HIF-1α-overexpressed HSC-T6 cells ( Figure 7C). Besides, sorafenib-induced ferroptosis was curbed by HIF-1α overexpression ( Figure 7D). Taken together, overexpression of HIF-1α elevates SLC7A11 protein level, which impairs sorafenibinduced HSC ferroptosis.
To further establish the vital role of HIF-1α/SLC7A11 in HSC ferroptosis, siRNA specific for rat HIF-1α (HIF-1α siRNA) was used to knockdown the HIF-1α expression. As illustrated in Figure S2, Fluorescence intensity of SLC7A11 was notably decreased by HIF-1α siRNA with or without sorafenib treatment ( Figure 7G).
GPX4 and SLC7A11 protein levels significantly reduced in HIF-1αsilenced HSC-T6 cells ( Figure 7H). In addition, depletion of HIF-1α strengthened ferroptosis induced by sorafenib ( Figure 7I). Finally, ECM accumulation was also determined. Low expressions of α-SMA and Col1α1 in sorafenib-treated HSC-T6 cells were reinforced by silence of HIF-1α and were abated partly by overexpression of HIF-1α ( Figure 8A and B). Based on the above data, we prove that HIF-1α/ SLC7A11 is involved in sorafenib-induced HSC ferroptosis, and silencing HIF-1α can intensify the anti-fibrotic effect of sorafenib.
F I G U R E 9 Sorafenib ameliorates liver fibrosis by triggering ferroptosis in HSC via HIF-1α/SLC7A11 pathway. Treatment with sorafenib induces a decrease in HIF-1α, which in turn reduces SLC7A11 expression in HSCs, then leads to GPX4, GSH depletion and ROS excess, and ultimately induces HSC ferroptosis and ECM reduction

| DISCUSS ION
HSC acts a critical role in pathogenesis of liver fibrosis. In response to injury, HSCs differentiate into activated myofibroblasts, synthesize and deposit fibrillar collagen, which result in significant fibrosis.
Therefore, inhibition of HSC is considered as a key strategy for therapeutic intervention of liver fibrosis. [5][6][7] Ferroptosis, a new type of programmed cell death, has been identified as a therapeutic target in liver fibrotic diseases. 10,11 In this study, we provide evidence that sorafenib ameliorates CCl 4 -induced liver fibrosis by triggering ferroptosis in HSCs through HIF-1α/SLC7A11 pathway. Strikingly, inhibition of HSC ferroptosis hampers the anti-fibrotic effect of sorafenib ( Figure 9).
Sorafenib has been proved to be a potential drug for treating liver fibrosis. In hepatotoxic and cholestatic animal models, sorafenib remarkably alleviated liver injury and decrease ECM deposition. 30 Figure S1). Likewise, SLC7A11 and GPX4 were expressed in mouse primary HSCs. 13,37 More importantly, it has been found that inhibitions of SLC7A11 and GPX4 are involved in cell ferroptosis. 9,38 Here, we focused on SLC7A11 and GPX4 expressions in HSCs around fibrotic scars. In the CCl 4 -treated group, SLC7A11 and GPX4 were significantly expressed around the scar, while decreased with sorafenib treatment ( Figure S1). These suggest that the anti-fibrotic process of sorafenib is accompanied by ferroptosis in HSCs.
Inducing HSC ferroptosis provides a new way for the treatment of liver fibrosis, while ferroptotic inducers can also trigger some other cells resided in the liver and finally may cause some side effects. Wang et al. found that ferroptosis occurs in hepatocytes and macrophages, which is hazardous to the recovery of liver injury. 17 Therefore, drug design aims at induction of ferroptosis in HSCs rather than in hepatocytes and macrophages. Our data showed that sorafenib (10 μM) induces ferroptosis in HSCs, with less effect on hepatocytes and macrophages. Indeed, GSH level, a major cellular defence system against ROS, is higher in hepatocytes than in HSCs. 39 That may be why hepatocytes are more resistant to sorafenib-induced ferroptosis than HSCs. In addition, the main function of macrophages is to engulf harmful microorganisms and destroy them, and these processes are largely dependent on the production of ROS. 40 Thus, macrophages may be able to sense the invasion of sorafenib and achieve coordinated ROS production and clearance, ultimately resist ferroptosis.
In this study, we found sorafenib-induced ferroptosis in HSCs accompanied by reduced SLC7A11 and HIF-1α proteins. Moreover, silencing HIF-1α decreased SLC7A11 protein level, and overexpression of HIF-1α with plasmid or stabilizers increased SLC7A11 protein level. Decreased HIF-1α and SLC7A11 in HSC-T6 cells strengthened sorafenib-induced cell ferroptosis and ECM reduction. On the contrary, increased expressions of HIF-1α and SLC7A11 inhibited HSC ferroptosis and impaired the sorafenib anti-fibrotic effect. It may be conceivable that sorafenib induces HSC ferroptosis, at least in part, via HIF-1α/SLC7A11.
PTGS2, also known as cyclooxygenase 2 (COX2), is a potent enzyme that initiates inflammation and promotes prostaglandin synthesis following stimulation by various inflammatory factors. 41,42 Recently, gene networks analysed by bioinformatics have shown that PTGS2 is the hub gene in the biology of cell ferroptosis. 43 As well, PTGS2 expression is increased during ferroptosis in various cells, such as vascular smooth muscle cells, neuronal cells and HSCs. [44][45][46] Consistent with the previous studies, PTGS2 expression was markedly increased both in sorafenib-treated fibrotic livers and in HSC-T6 cells, which was accompanied by ferroptotic events.
However, PTGS2 expression w as not affected by HIF-1α with or without sorafenib treatment ( Figure S3A, B, C and D). In other words, sorafenib elevating PTGS2 may be not through HIF-1α/SLC7A11 pathway, and the underlying mechanism needs to be further explored.
In summary, our findings in the present study demonstrate that HSC ferroptosis acts a vital role in sorafenib anti-fibrosis. And sorafenib induces HSC ferroptosis, at least in part, via HIF-1α/ SLC7A11 pathway. Blockade of ferroptosis with inhibitors offsets the reduction of ECM in liver. This study clearly demonstrates that sorafenib induces ferroptosis in HSCs, and these findings will provide new targets for the treatment of liver fibrosis.