Lysine specific demethylase 1 inhibits sodium arsenite activation of HSCs by regulating SESN2/AMPK/ULK1 signaling pathway activity

Lysine specific demethylase 1 (LSD1) is a histone demethylase that specifically catalyzes the demethylation of histone H3K4 (H3K4me1/2) and regulates gene expression. In addition, it can mediate the process of autophagy through its demethylase activity. Sestrin2 (SESN2) is a stress‐induced protein and a positive regulator of autophagy. In NaAsO2‐induced mouse fibrotic livers and activated hepatic stellate cells (HSCs), LSD1 expression is decreased, SESN2 expression is increased, and autophagy levels are also increased. Overexpression of LSD1 and silencing of SESN2 decreased the level of autophagy and attenuated the activation of HSCs induced by NaAsO2. LSD1 promoted SESN2 gene transcription by increasing H3K4me1/2 in the SESN2 promoter region. 3‐methyladenine (3‐MA) and chloroquine were used to inhibit autophagy of HSCs, and the degree of activation was also alleviated. Taken together, LSD1 positively regulates SESN2 by increasing H3K4me1/2 enrichment in the SESN2 promoter region, which in turn increases the level of autophagy and promotes the activation of HSCs. Our results may provide new evidence for the importance of LSD1 in the process of autophagy and activation of HSCs induced by arsenic poisoning. Increasing the expression and activity of LSD1 is expected to be an effective way to reverse the autophagy and activation of HSCs induced by arsenic poisoning.

as early as possible is the key to reverse the occurrence and development of hepatic fibrosis in clinical practice. 5Therefore, there is an urgent need to find the key targets of HSCs activation to reverse hepatic fibrosis.
Autophagy is a self-engulfing process that degrades damaged proteins, cellular metabolites and diseased organelles to maintain normal physiological functions of cells. 6With the deepening of the research on autophagy, autophagy plays an irreplaceable role in the occurrence, development and outcome of fibrotic diseases, and the increase of autophagy level can promote the occurrence of fibrotic diseases in various organs. 7,8Autophagy is initiated mainly through the Unc.1ike kinase 1 (ULKl) complex (ULK1-Atg13-FIP200-Atg101), and both ULKl complex and autophagy are inhibited under the condition of energy abundance.Some scholars have found that the activation process of HSCs is completed by autophagy, and inhibition of autophagy can effectively prevent its activation.In addition, autophagy of HSCs can promote the metabolism of intracellular lipid droplets, thereby providing energy to activate HSCs. 9,10 recent years, epigenetics has gradually become a new research hotspot.2][13] Epigenetics refers to the fact that the sequence information of DNA is not changed, but the gene expression is changed inheritable. 14Lysine specific demethylase 1 (LSD1), also known as KDM1A, is a member of the amine dependent oxidase superfamily (FAD), 15,16 which specifically demethylates H3K4(me1, me2).However, it cannot demethylation H3K4me3. 17In addition, LSD1 mediates the expression of certain proteins through epigenetic modification to affect autophagy. 18LSD1 can also participate in signal transduction and transcription of downstream effector molecules in the process of autophagy, and autophagy can be induced by knocking down its expression or inhibiting its demethylase activity. 19Some scholars have reported that the level of histone H3K4me3 and autophagy in LX-2 cells increased after hypoxia induced activation.Treatment with autophagy inhibitors inhibited the increase of H3K4me3 and autophagy levels.However, autophagy and activation of HSCs induced by hypoxia were inhibited by H3K4me3 methylation inhibitor. 20This suggests that histone H3K4 methylation is involved in the regulation of HSCs autophagy, but the specific mechanism is still unclear.Therefore, regulating the expression of LSD1 further specifically regulates the level of histone H3K4 methylation, and its regulatory role in the process of HSCs autophagy can be explored.
The Sestrins (SESNs) protein family is a group of evolutionarily conserved stress-induced proteins.Sestrin2 (SESN2) is one of the SESNs family members, which is upregulated when stress occurs.
Once activated, it can maintain cell homeostasis and promote their survival through autophagy, apoptosis, endoplasmic reticulum stress, DNA damage, and other ways. 213][24][25] Therefore, LSD1 and SESN2 have a strong correlation with autophagy and activation of HSCs.However, it is not clear whether LSD1 mediates SESN2 signaling pathway to regulate autophagy and activation of HSCs induced by arsenic poisoning.
In this study, NaAsO 2 was used to induce hepatic fibrosis in mice and activation of human HSCs LX-2, and the level of histone H3K4 methylation modification, SESN2 signaling pathway status and autophagy level were evaluated.To clarify the role of LSD1, we transfected LX-2 cells with LSD1 overexpression plasmid and observed the changes in the above evaluated indexes.In addition, small interfering RNA (siRNA) was used to knock down the expression of SESN2 in LX-2 cells, and the level and activation of autophagy in LX-2 cells were evaluated.Chromatin immunoprecipitation (ChIP) was used to detect the enrichment level of H3K4me1/2 in SESN2 promoter region.Finally, autophagy inhibitors 3-MA and CQ were used to inhibit the autophagy of HSCs, and the activation state was observed to further clarify the relationship between autophagy and activation of HSCs.Our study reveals that LSD1 is a new target in NaAsO 2induced autophagy and activation of HSCs, which may provide a new idea for reversing NaAsO 2 -induced hepatic fibrosis.

| Reagents and antibodies
Sodium arsenite (Shandong West Asia Chemical Industry Co., LTD., China), OG-L002 (a specific inhibitor of LSD1) and 3-methyladenine T A B L E 1 Primer sequences of target genes used to perform qRT-PCR.

Gene
Forward, 5 0 -3 0 Reverse, 5 0 -3 The mice were intraperitoneally injected with NaAsO 2 at a dose of 5 mg/kg for 8 weeks, and the control animals were treated with the same volume of phosphate buffer solution (PBS) for the same time.

| H&E staining and sirius red staining
Freshly removed mouse liver tissues were fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin, and then cut into 3 μm thick sections.The sections were stained with H&E staining and sirius red staining kit to evaluate the degree of liver injury and hepatic fibrosis.

| Immunohistochemistry
Paraffin-embedded liver sections (3 μm) were deparaffinized, and the tissue sections were antigen repaired using citrate buffer.The sections were then immersed in 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity, blocked with 5% FBS for 1 h, and next, the relevant indicator antibodies were incubated overnight in a 4 C refrigerator.The corresponding secondary antibodies were incubated for 1 h at room temperature.Finally, the color reagent was prepared according to the DAB color development kit.After the color reaction was stopped, the nucleus was counterstained with hematoxylin, and the tissue sections were dehydrated and sealed.The changes of the corresponding indexes were observed after the sealed tablets were dried.

| Immunofluorescence
The procedure for tissue sections was partially the same as that for immunohistochemical staining, except that tissue sections were permeabilized with 0.5% Triton X-100 before antigen retrieval.The secondary antibodies used were fluorescently labeled secondary antibodies.For cell experiments, the corresponding number of cell slides were placed in 12-well plates, and LX-2 cells were seeded in the plates at a density of 1 Â 10 4 cells/well.The LX-2 cells were cultured in the incubator (37 C, 5% CO 2 ) for 24 h and the corresponding drugs were added to continue the culture.The cells were permeabilized with 0.5% Triton X-100 for 30 min at room temperature and blocked in 5% F I G U R E 2 Legend on next page.

| Western blot analysis
The treated cells were washed twice with precooled PBS, then 200 μL cell lysate mixture (cell lysate: protease inhibitor = 99:1) was added and incubated on ice for 5 min.The cell suspension was collected and transferred to a 1.5 mL EP tube.Cells were lysed by sonication, centrifuged (12 000 rpm, 25 min), the precipitate was discarded, the supernatant was collected, quantified by BCA method, protein loading buffer was added, and proteins were denatured by metal bath (100 C, 10 min).For SDS-PAGE gel electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% skim milk for 1.5 h, membranes were washed 3 times /5 min with Tris Buffered Saline with Tween-20 (TBST), and incubated with primary antibodies overnight at 4 C.The next day, the PVDF membrane was washed by TBST and incubated with secondary antibodies for 1 h at room temperature.The developed images were analyzed by Image J for gray value.

| Quantitative real-time polymerase chain reaction
Total RNA was extracted from HSCs using Trizol reagent, the RNA was reverse transcribed into cDNA according to the reverse transcription kit, and the amplification reaction was performed according to the qRT-PCR kit operating instructions, with GAPDH as the reference gene.The target genes primer sequences used to perform the qRT-PCR are shown in Table 1.The protein expression levels of SESN2, P-AMPK, P-ULK1, and LC3II

| Transfection and silencing
were significantly increased in the model group compared with the control group (Figure 1G,H), and the immunofluorescence results also showed increased LC3 expression (Figure 1I).Our results showed that NaAsO 2 induced liver fibrosis in mice, and we hypothesized that NaAsO 2 -induced liver fibrosis and activation of HSCs may be related to the increased H3K4me1/2 modification level and autophagy level caused by LSD1 knockdown.2A).We chose NaAsO 2 at a final concentration of 8 μmol/L for the subsequent experiments, and the action time was 24 h.In the model group, the cell bodies of HSCs became larger, the aggregation was enhanced, the connections between cells were tighter, most of the cells extended pseudopodia, and most of the cells changed into long spindle shapes (Figure 2B).The protein expression of α-SMA and collagen 1 was increased, the mRNA and protein expression of LSD1 was decreased, and the histone H3K4me1/2 modification was increased (Figure 2C).In addition, SESN2 signaling pathway in HSCs was detected.The expression levels of SESN2 mRNA and protein in the model group were significantly increased, and the protein levels of P-AMPK, P-ULK1, and LC3II were also significantly increased (Figure 2D).Immunofluorescence results showed increased LC3 expression (Figure 2E) Our results showed that 8 μmol/L NaAsO 2 activated HSCs and increased the level of autophagy in HSCs in vitro.Combined with the in vivo results, we further confirmed that the down-regulation of LSD1 in the presence of NaAsO 2 was strongly correlated with the increase of liver fibrosis and activation and autophagy levels of HSCs in mice.

| Overexpression of LSD1 inhibited NaAsO2induced activation of HSCs
To determine the role of LSD1 in the development of NaAsO 2induced liver fibrosis, LX-2 cells were transfected with LSD1 overexpression plasmid.Green fluorescence was observed by fluorescence microscopy after 6-8 h, indicating that the LSD1 overexpression plasmid was successfully transferred into the cells, and the expression level of LSD1 protein in the LSD1 group was significantly higher than that in the NC group.This indicated that LSD1 was successfully overexpressed in HSCs (Figure 3A).The expression level of LSD1 protein was significantly lower in the OG-L002 group than in the LSD1 group, indicating that the expression of LSD1 was specifically inhibited by OG-L002 (Figure 3A).At 24 h after the cells were transfected with LSD1 overexpression plasmid, compared with the NC group and the OG-L002 group, the HSCs in the LSD1 group had round cell bodies, pseudopodia retraction, loose connections between cells, and poor cell aggregation (Figure 3B).However, the protein expression of collagen-1, α-SMA and H3K4me1/2 in HSCs in LSD1 group was significantly lower than that in NC group and OG-L002 group (Figure 3C,D).These results indicate that upregulation of LSD1 in HSCs can effectively inhibit NaAsO 2 -induced activation of HSCs.

| Overexpression of LSD1 inhibited SESN2 signaling pathway and autophagy in HSCs
To investigate the relationship between LSD1 and SESN2 during the development of NaAsO 2 -induced liver fibrosis, we examined SESN2 signaling pathway and autophagy marker LC3 after LSD1 overexpression.Interestingly, the protein expression levels of SESN2, P-AMPK, P-ULK1, and LC3II in the LSD1 group were significantly lower than those in the NC group and the OG-L002 group (Figure 4A,B).Immunofluorescence results also showed that the LC3 expression level was decreased after upregulation of LSD1 (Figure 4C).F I G U R E 6 Legend on next page.

| SESN2 knockdown inhibited SESN2 signaling pathway and autophagy level in HSCs, and inhibited the activation of HSCs
To further confirm the regulatory role of SESN2 in HSCs autophagy and activation, SESN2 siRNA was used to knock down the gene in HSCs.We found that most of the HSCs in the siSESN2 group changed from long spindle shape to round or oval shape, cell pseudopodia retracted, and cell body shrank (Figure 5A).Compared with the siNC group, the protein expression levels of collagen1, α-SMA, SESN2, P-AMPK, P-ULK1, and LC3II in the siSESN2 group were significantly decreased (Figure 5B-D), and the expression level of LC3 in HSCs was also decreased by immunofluorescence (Figure 5E).These results confirmed that knockdown of SESN2 could effectively inhibit the activity of SESN2/AMPK/ULK1 signaling pathway and reduce the level of autophagy in HSCs, and attenuate the activation of HSCs by NaAsO 2 .

| Inhibition of autophagy can effectively inhibit HSCs activation
In order to confirm the relationship between autophagy and activation of HSCs, we used the autophagy inhibitors 3-MA and CQ to inhibit NaAsO 2 -induced autophagy of HSCs.We observed that the degree of activation of HSCs was weakened after inhibition of the level of autophagy, which was manifested as: some HSCs changed from long spindle shape to round or oval shape, cell pseudopodia retracted, cell body shrank, and cell aggregation was weakened (Figure 6A).The protein expression levels of collagen I and α-SMA in 3-MA group and CQ group were significantly lower than those in the model group, while the expression level of LC3II in 3-MA group was significantly lower than that in the model group, and the expression level of LC3II in CQ group was significantly higher than that in the model group and 3-MA group (Figure 6B,C).The same changes in LC3II protein expression were also observed by immunofluorescence (Figure 6D).These results of our study indicated that inhibition of autophagy attenuated the activation of HSCs by NaAsO 2 .

| DISCUSSION
Arsenic poisoning is still a global health problem of great concern.
Long-term exposure to arsenic can cause various damage to the body.
As the liver is the main target organ of arsenic metabolism, the structure and function of the liver will be affected to varying degrees during arsenic poisoning.7][28] Activation of HSCs is the central link in the occurrence and development of hepatic fibrosis.
When the liver is affected by various damage and stimulation factors, HSCs change from a resting state to an activated state and transform into myofibroblasts, and various fibrin secreted by HSCS produces a large number of extracellular matrix (ECM).Moreover, the dynamic balance between matrix metalloproteinases (MMPs) and tissue inhibitors metalloproteinases (TIMPs) is broken, and the degradation of ECM is unbalanced.The level of degradation decreases, and a large amount of ECM accumulates in the liver to promote the occurrence of hepatic fibrosis.Therefore, inhibiting the activation of HSCs is the key to reverse hepatic fibrosis.
Autophagy is a process of self-phagocytosis that maintains the normal physiological function of cells and tissues during nutrient deficiency and various stresses.showed that NaAsO 2 induced hepatic fibrosis in mice.In vitro results showed that 8 μmol/L NaAsO 2 can transform LX-2 cells from quiescent to activated, while the activation of HSCs is a dynamic process and highly dependent on autophagy.In the absence of energy, mTOR signaling pathway is inhibited, and autophagy is initiated to produce ATP to complete the transformation process of HSCs into myofibroblasts. 31SESN2 is a stress-induced protein, which can initiate anti-stress mechanisms by affecting multiple signaling pathways after induction. 32Under oxidative stress conditions, SESN2 plays an antioxidative stress role by activating AMPK and thus inhibiting mTORC1. 33This suggests that AMPK/mTORC1 signaling is critical for of autophagy levels upon inhibition of LSD1 expression, which is consistent with our own findings. 37,38Conversely, we observed an elevation in LSD1 expression accompanied by a reduction in cellular autophagy.In our study, upregulation of LSD1 expression ameliorated sodium arsenite-induced liver fibrosis.0][41][42] These findings underscore the significance of targeting LSD1 for the treatment of diverse diseases.In summary, our study highlights the pivotal role of LSD1 as a key mediator in the pathogenesis and progression of various diseases, including arsenicinduced liver fibrosis.
SESN2 is a positive regulator of autophagy, and we hypothesized that its upregulation plays a key promoting role in NaAsO 2 -induced HSCs activation and autophagy, in order to confirm the relationship between SESN2 and autophagy induction after LSD1 downregulation by NaAsO 2 .We used siRNA technology to knock down SESN2, and our results showed that knockdown of SESN2 expression attenuated HSCs autophagy by inhibiting SESN2 signaling pathway, and also inhibited HSCs activation, to a certain extent reversing HSCs activation.In our study, autophagy and activation of HSCs changed in the same direction, and our study further confirmed that SESN2-regulated autophagy is essential for the activation of HSCs induced by NaAsO 2 .Therefore, we hypothesized that autophagy may be a key link in the transformation of HSCs from quiescent to activated.To confirm this speculation, we treated HSCs with the early autophagy inhibitor 3-MA in combination with the late autophagy inhibitor CQ. 3-MA inhibited the formation of autophagosome at the early stage, as indicated by the decreased expression of LC3II protein.
However, CQ can increase the pH value of lysosomes, block the fusion of autophagosomes and lysosomes and the degradation of lysosomal proteins, thereby promoting the accumulation of autophagosomes. 43In this study, our results after CQ intervention were consistent with the study results of Liu et al., showing that the expression of LC3II protein increased. 44The activation process of HSCs can be reversed and liver fibrosis can be alleviated through the inhibition of autophagy. 45This is consistent with our study results.Interestingly, we found that the activation of HSCs was inhibited after inhibition of autophagy activity by early autophagy inhibitors and late autophagy inhibitors.This suggests that NaAsO 2 promotes the activation of HSCs by inducing autophagy in our study.
In conclusion, our study showed that NaAsO 2 regulated autop- fetal bovine serum for 1 h.Next, the corresponding antibodies (200 μL/well) were added and incubated overnight at 4 C.The next day, the primary antibody was recovered and the fluorescent secondary antibody was incubated for 1 h at room temperature in the dark.Finally, the slides were taken out and inverted onto the slide, which had been added with about 10 μL anti-fluorescence quench agent (containing DAPI).The slides were observed and photographed by laser confocal microscope.F I G U R E 1 Intraperitoneal injection of NaAsO 2 (5 mg/kg) for 8 weeks induced liver fibrosis in mice, inhibited the expression of lysine specific demethylase 1 (LSD1), upregulated the modification level of histone H3K4me1/2 and increased the level of autophagy in the liver.(A) The degree of liver injury and liver fibrosis was assessed by H&E staining and sirius red staining.(B, C) Immunohistochemical staining and immunofluorescence detection of mouse liver LSD1 and α-SMA expression.(D) Effect of NaAsO 2 on α-SMA, collagen I, LSD1, and H3K4me1/2 protein expression levels by Western blot.(E) Detection of mouse Sestrin2 (SESN2) expression by immunohistochemical staining.(F) Colocalization of SESN2 and AMPK in mice was assessed by immunofluorescence.(G and H) Effect of NaAsO 2 on SESN2, P-AMPK, P-ULK1, and LC3 protein expression levels by Western blot.(I) LC3 expression measured by immunofluorescence.GAPDH or H3 was used as protein expression standard reference or LC3II/LC3I was used for statistical analysis.Values are expressed as mean ± SD (n = 3), **p < .01,***p < .001,verus control group.

3 | RESULTS 3 . 1 |
NaAsO 2 induced liver fibrosis in mice in vivo, downregulated LSD1 and upregulated autophagy level HE staining and sirius red staining showed that the structure of liver lobules in the control group was normal, and the hepatocytes were arranged radially and regularly around the central portal.The liver tissue of mice in the model group showed disordered arrangement of hepatocytes, swelling and degeneration, fibrous scar formation, and collagen deposition in the central portal area (Figure1A).Compared with the control group, the expression of α-SMA and collagen 1 was increased, the expression of LSD1 was decreased, and the level of H3K4me1/2 was significantly increased in the model group (Figure1B-D).The results of immunohistochemical staining showed increased SESN2 expression (Figure1E), and immunofluorescence showed increased colocalization of SESN2 with AMPK (Figure1F).F I G U R E 2 In vitro, 8 μmol/L NaAsO 2 activated hepatic stellate cells (HSCs), down-regulated lysine specific demethylase 1 (LSD1) expression, upregulated histone H3K4me1/2 modification, and increased autophagy level of HSCs.(A) LX-2 cells were treated with different concentrations of NaAsO 2 (4, 8, 12, 16, 20, and 24 μmol/L), and the proliferation of HSCs was monitored by RTCA in real time.(B) Cell morphological changes were observed under inverted microscope.(C) Effect of NaAsO 2 on α-SMA, collagen I, LSD1, and H3K4me1/2 protein expression levels by Western blot and LSD1 mRNA expression level by qRT-PCR.(D) Effect of NaAsO 2 on Sestrin2 (SESN2), P-AMPK, P-ULK1, and LC3 protein expression levels by Western blot and changes in SESN2 mRNA expression level by qRT-PCR.(E) LC3 expression detected by immunofluorescence.GAPDH or H3 was used as protein expression standard reference or LC3II/LC3I was used for statistical analysis.Values are expressed as mean ± SD (n = 3), **p < .01,***p < .001,versus control group.F I G U R E 3 Upregulation of Lysine specific demethylase 1 (LSD1) expression reduced the modification level of histone H3K4me1/2 and inhibited the activation of hepatic stellate cells (HSCs).(A) After overexpression of LSD1 for 24 h, fluorescence microscopy was used to observe whether LSD1 was transferred into cells, and then Western blot was used to detect the effect of LSD1 overexpression in HSCs and the effect of OG-L002 inhibition of LSD1 expression.(B) The morphological changes of LX-2 cells in each group were observed by inverted microscope.(C, D) protein expression levels of α-SMA, collagen I, and H3K4me1/2 were determined by Western blot.GAPDH was used as the standard reference for α-SMA and collagen I protein expression.H3 was used as the standard reference for H3K4me1/2 protein expression.Values are expressed as mean ± SD (n = 3), *p < .05,**p < .01,***p< .001,versus LSD1 group.F I G U R E 4 Overexpression of lysine specific demethylase 1 (LSD1) inhibited Sestrin2 (SESN2) pathway and autophagy in hepatic stellate cells (HSCs), while OG-L002 inhibited LSD1 expression and activated SESN2 pathway and induced autophagy in HSCS.8 μmol/L NaAsO 2 increased the level of histone H3K4 me1/2 modification in SESN2 gene promoter region in HSCs.(A, B) Changes in SESN2, P-AMPK, P-ULK1, and LC3 protein expression levels after overexpression of LSD1 or inhibition of LSD1 expression with OG-L002 were analyzed by Western blot.(C) Immunofluorescence analysis of LC3 expression.(D) Agarose gel electrophoresis results showed that most of the chromatin fragments of HSCs were in the range of 100-1000 bp, which met the conditions for subsequent chromatin immunoprecipitation (ChIP) experiments.(E) ChIP-qPCR statistics showing an increased level of histone H3K4 me1/2 modification in the SESN2 gene promoter region, and changes in enrichment levels are shown to be normalized on IgG controls.GAPDH was used as the protein expression standard reference or LC3II/LC3I was used for statistical analysis.Values are expressed as mean ± SD (n = 3), *p < .05,**p < .01,***p < .001,versus LSD1 group.## p < .01,versus control group.F I G U R E 5 Legend on next page.
The transcriptional activation of genes is usually related to the level of histone H3K4 methylation.Therefore, in order to determine whether H3K4 methylation is involved in the regulation of SESN2 gene transcription in the process of autophagy and activation of HSCs induced by arsenic poisoning, ChIP experiments were performed.The results of agarose gel electrophoresis showed that most of the chromatin fragments of HSCs were in the range of 100--1000 bp, which met the conditions of subsequent ChIP experiments (Figure4D).Our results showed that 8 μmol/L NaAsO2 significantly increased H3K4me1/2 modification level in SESN2 gene promoter region in HSCs (Figure4E).Our results showed that overexpression of LSD1 inhibited SESN2 signaling activity and downregulated autophagy in HSCs, indicating that LSD1 was involved in the regulation of SESN2/AMPK/ULK1 signaling pathway and autophagy.Mechanologically, NaAsO2 promoted SESN2 transcription in HSCs by increasing H3K4me1/2 modification in the SESN2 promoter region.F I G U R E 5 Silencing Sestrin2 (SESN2) inhibits the SESN2 pathway in hepatic stellate cells (HSCs), as well as their autophagy and activation.(A) After SESN2 was silenced by small interfering RNA technology, LX-2 cells were treated with 8 μmol/L NaAsO 2 for 24 h, and the morphological changes of cells were observed under inverted microscope.(B-D) protein expression levels of SESN2, P-AMPK, P-ULK1, and LC3 were determined by Western blot.(E) Immunofluorescence shows LC3 expression levels.GAPDH was used as the protein expression standard reference or LC3II/LC3I was used for statistical analysis.Values are expressed as mean ± SD (n = 3), *p < .05,**p < .01,***p < .001,versus siSESN2 group.

SESN2 to maintain cell
metabolism and survival under stress conditions.In this study, we found that NaAsO 2 activated SESN2-AMPK-ULK1 signaling pathway and induced autophagy.Our results suggest that activation of HSCs is inseparable from increased autophagic activity.Therefore, inhibition of HSCs autophagy may be a potential protective measure to weaken the activation of HSCs and reduce ECM production induced by arsenic poisoning, and thus become the key to reverse hepatic fibrosis induced by arsenic poisoning.F I G U R E 6 3-MA and chloroquine (CQ) inhibited autophagy and attenuated the activation of hepatic stellate cells (HSCs).(A) LX-2 cells were pretreated with 3-MA and CQ for 1 h and then treated with 8 μmol/L NaAsO 2 for 24 h.Morphological changes were observed under inverted microscope.(B, C) protein expression levels of collagen I, α-SMA, and LC3 were determined by Western blot.(D) LC3 expression was detected by immunofluorescence.GAPDH was used as the protein expression standard reference or LC3II/LC3I was used for statistical analysis.Values are expressed as mean ± SD (n = 3), *p < .05,**p < .01,***p < .001,versus model group.### p < .001versus 3-MA group.LSD1 is a member of FAD family.As a demethylase, LSD1 can specifically demethylate histone H3K4me1/2, and plays an irreplaceable role in maintaining histone methylation status and regulating the signal transduction and transcription of downstream effector molecules during autophagy.LSD1 not only interacts with the androgen receptor (AR) in a ligand-dependent manner to demethylate H3K9me1 and H3K9me2,34 It also catalyzes the methylation modification of nonhistone substrates such as tumor suppressor P53, transcription factor E2F1, DNA methyltransferase 1 (Dnmt1), and activated transcription factor 3 (STAT3).35The latest research findings have demonstrated that the inhibition of LSD1 expression leads to an elevation in the level of H3K4me2 on the SESN2 gene promoter, thereby facilitating the transcription process and ultimately enhancing autophagy.36However, the mechanism of LSD1 in NaAsO 2 -induced HSCs activation and autophagy is still unclear.Therefore, we detected LSD1 and histone H3K4me1/2 modification in HSCs in arsenic poisoning model.Since the transcriptional activation of certain genes is usually associated with histone H3K4 methylation, HSCs were transiently transfected with LSD1 overexpression plasmid.Interestingly, upregulation of LSD1 inhibited the level of H3K4me1/2 modification, while SESN2 signaling pathway, and autophagy and activation of HSCs were inhibited.To further clarify the role of LSD1, we used the LSD1 specific inhibitor OG-L002 as an intervention, and the results of intervention were consistent with those of NaAsO 2 .These results suggest that upregulation of LSD1 expression may inhibit SESN2 signaling pathway and attenuate autophagy of HSCs by down-regulating H3K4 methylation in HSCs, at least in part.CHIP assay was then performed and mechanically we found that LSD1 promoted SESN2 transcription in HSCs by regulating H3K4me1/2 in the SESN2 promoter region.Recent studies have demonstrated a significant upregulation hagy and activation of HSCs by regulating LSD1 expression to regulate histone H3K4me1/2 modification, which in turn affected the activity of SESN2 signaling pathway, thereby regulating autophagy and activation of HSCs.Our results enrich the study on the mechanism of NaAsO 2 -induced transformation of HSCs from quiescent to activated form from the perspective of epigenetic regulation, and also lay a certain foundation for reversing NaAsO 2À induced hepatic fibrosis.5| CONCLUSIONNaAsO 2 increases the level of autophagy in HSCs by downregulating the expression of LSD1 and increasing the level of H3K4me1/2, thereby increasing the transcription of SESN2 gene to promote the activation of HSCs.Conversely, upregulation of LSD1 expression inhibited autophagy and reversed NaAsO 2 -induced HSCs activation, which was achieved by regulating the SESN2 signaling pathway.AUTHOR CONTRIBUTIONS Date curation, validaion, and writing-original draft: Yingwan Zhang.Date curation and validaion: Tian Tian.Formal analysis, investigation, and software: Cai Liang, Junli Wang, Jiayuan Zhang, and Shanshan Tian.Project administration, resources, supervision, and visualization: Rujia Xie and Ting Yang.Conceptualization, project administration, supervision, and writing-review & editing: Bing Han.
NaAsO 2 ).HSCs after cell adherence were treated with NaAsO 2 for 24 h, and ChIP assay was performed using the ChIP kit.Human LX-2 cells were cross-linked using 1% formaldehyde according to the ChIP kit instructions, and crosslinking was terminated with 1Â glycine.Cells were lysed by adding 2 μL Human LX-2 cells were transiently transfected with LSD1 overexpression plasmid and SESN2 siRNA for 24 h and 48 h with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and treated with 8 μmol/L NaASO 2 for 24 h.The expression levels of corresponding indicators were detected.The sequences of the overexpressed LSD1 gene and the siRNA SESN2 gene are shown in Table2.2.12 | Statistical analysisGraphpad Prism 8.0 was used uniformly for statistical analysis, and data are presented as mean ± standard deviation.Combined one-way analysis of variance and unpaired t test were used to calculate and analyze the experimental data.p value < .05indicates a statistically significant difference in the data.