MAPK signaling pathway

Author’s Accepted Manuscript 20(S)−25-methoxyl-dammarane-3β,12β,20-triol attenuates endoplasmic reticulum stress via ERK/MAPK signaling pathway Hongshuang Qin, Wei Li, Ying Sun, Yongli Bao, Luguo Sun, Zhenbo Song, Lihua Zheng, Yuqing Zhao, Yuxin Li www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(18)30447-3 https://doi.org/10.1016/j.ejphar.2018.08.001 EJP71926

To appear in: European Journal of Pharmacology Received date: 30 March 2018 Revised date: 31 July 2018 Accepted date: 3 August 2018 Cite this article as: Hongshuang Qin, Wei Li, Ying Sun, Yongli Bao, Luguo Sun, Zhenbo Song, Lihua Zheng, Yuqing Zhao and Yuxin Li, 20(S)−25-methoxyldammarane-3β,12β,20-triol attenuates endoplasmic reticulum stress via ERK/MAPK signaling pathway, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

20(S)-25-methoxyl-dammarane-3β,12β,20-triol attenuates endoplasmic reticulum stress via ERK/MAPK signaling pathway Hongshuang Qin1,4, Wei Li2, Ying Sun1, Yongli Bao1, Luguo Sun1, Zhenbo Song1, Lihua Zheng1,3,*, Yuqing Zhao2,*, Yuxin Li1,3 1

National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University,

2555 Jingyue Street, Changchun 130117, China 2

Key Laboratory of Structure-based Drug Design & Discovery, Ministry of Education, Shenyang

Pharmaceutical University, Shenyang 110016, China 3

Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal

University, 5268 Renmin Street, Changchun 130024, China 4

Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun

Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China *

Corresponding authors. Lihua Zheng, National Engineering Laboratory for Druggable Gene and Protein

Screening, Northeast Normal University, 2555 Jingyue Street, Changchun 130117, China. Tel.: +86-0431-8916-5950; Fax: +86-0431-8518-4723. E-mail addresses: [email protected] Prof. Yuqing Zhao, Key Laboratory of Structure-based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China. E-mail addresses: [email protected] Acknowledgements This research was supported by grants from the Fundamental Research Funds for the Central Universities (Grant no. 130028701-5), the Research Foundation of Jilin Provincial Science & Technology Development (Grant no.20150101188JC), the National Natural Science Foundation of China (Grant no. 81502284) and the Fundame ntal Research Funds for the Central Universities（Grant no. 2412017FZ020）.

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Abstract Endoplasmic reticulum (ER) stress, together with unfolded protein response (UPR), can remove unfolded proteins and promote survival. However, severe and prolonged ER stress leads to cell death, tissue injury, and many serious diseases. Therefore, it is essential to identify drugs that can attenuate ER stress for ER-related disease treatment. A great deal of research shows that selenoprotein S (SelS) is a sensitive and ideal marker of ER stress. Here, we used a firefly luciferase reporter driven by SelS gene promoter to screen natural compounds that can attenuate ER stress. Then we identified compound 20(S)-25-methoxyl-dammarane-3β,12β,20-triol (25-OCH3-PPD) could inhibit the promoter activity of SelS, further results showed that 25-OCH3-PPD effectively inhibited tunicamycin (TM) induced up-regulation of SelS expression in both mRNA and protein levels. Moreover, 25-OCH3-PPD significantly inhibited glucose-regulated protein 78 (GRP78; the major ER stress marker) expression in TM-induced ER stress in HepG2 and HEK293T cells, suggesting that 25-OCH3-PPD could attenuate ER stress in these cells. Mechanism studies showed that 25-OCH3-PPD significantly activated ERK/MAPK signaling pathway, and the inhibition of ERK/MAPK by U0126 dramatically abolished the inhibitory effect of 25-OCH3-PPD on ER stress, suggesting that 25-OCH3-PPD attenuated ER stress at least partially through activation of ERK/MAPK signaling pathway. Taken together, our studies indicate that 25-OCH3-PPD is a novel small molecular compound reducing ER stress, and a potential drug for treating diseases associated with ER stress. Key words: 20(S)-25-methoxyl-dammarane-3β,12β,20-triol; selenoprotein S; glucose-regulated protein 78; endoplasmic reticulum stress; ERK/MAPK signaling pathway

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1.

Introduction

Endoplasmic reticulum (ER) is an organelle with important roles in the modification, folding, and maturation of newly synthesized proteins (Healy et al., 2009; Wlodkowic et al., 2009). During cellular perturbations, such as hypoxia, nutrient deprivation, acidosis and a reduction in luminal Ca2+ concentration, the protein load in the ER exceeds its folding capacity, resulting in the retention of misfolded proteins within the ER, and consequently, ER stress (Matsuo et al., 2013). Strong and prolonged ER stress triggers cell death and organ damage, and participates in the occurrence and progression of many severe diseases such as Parkinson's disease, diabetes, and cardiovascular diseases (Tsujii et al., 2015; Kim et al., 2013; Sozen et al., 2015). Thus, screening and identifying drugs that can attenuate ER stress could be beneficial to some diseases. Various studies have demonstrated that selenoprotein S (SelS; also known as SEPS1 and Tanis) play an important role in ER stress(Gao et al., 2004). As a central component of retro-translocation channel in ERAD, SelS can remove misfolded proteins and reduce ER stress(Ye et al., 2004; Gao et al., 2007). The ER stress inducers tunicamycin (TM), thapsigargin, dithiothreitol (DTT), cycloheximide, staurosporine, β-mercaptoethanol and sodium selenite all can markedly increase SelS expression (Du et al., 2010; Kim et al., 2007). In contrast, SelS expression recovers to the normal level after ER stress is reversed(Du et al., 2010). Therefore, SelS has been used as a sensitive marker of ER stress to screen compounds capable of attenuating ER stress (Walder et al.,2002; Speckmann et al., 2014). Cellular behavior in response to extracellular stimuli is mediated by intracellular signaling pathways such as mitogen-activated protein kinase (MAPK) signaling pathways (Zhang et al., 2010). The MAPKs are central components of signal transduction pathways in the regulation of cell proliferation, differentiation, apoptosis, and responses to stress (Du et al., 2015). There are three well defined subgroups of MAPKs: the extracellular signal regulated kinases (ERKs), the c-jun N-terminal kinases (JNKs), and p38 MAPKs (Farrukh et al., 2015). All the

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three subgroups of MAPKs (ERK, JNK, and p38) are known to be activated in response to ER stress (Darling et al., 2014). Generally, the activation of ERK/MAPK signaling pathway is considered to inhibit ER stress and promote cell survival (Croft et al., 2014). For example, ERK1/2 activation reduced ER stress and protected against ER stress-induced cell death through up-regulation of MCL-1 (a pro-survival BCL2 protein) in human melanoma cells (Jiang et al., 2008); Inhibition of ERK1/2 signaling with U0126 promoted ER stress-induced apoptosis in human hepatocellular carcinoma cells (Dai et al., 2009). In contrast, JNK and p38 pathways act to aggravate ER stress and promote ER stress-induced cell death (Moton et al., 2003; Malumbres et al., 2005). For instance, inhibition of JNK signaling reduced UPR activation in human embryonic neuroepithelial cells and pancreatic epithelial cells, indicating that JNK signaling could increase ER stress (Verma et al., 2010; Li et al., 2012). Tunicamycin-induced activation of p38 could lead to cell cycle arrest and apoptosis in mouse fibroblast cells (NIH3T3 cells) (Faust et al., 2012; Wang et al., 1996). Overall, the MAPK signaling pathways are shown to play an important role in cell fate decision during ER stress. Here, we established a firefly luciferase reporter screening system driven by SelS promoter, and screened out 43 purified natural compounds. We found that 25-OCH3-PPD can inhibit TM-induced up-regulation of SelS expression. Mechanism studies showed that 25-OCH3-PPD attenuated ER stress at least partially through activation of ERK/MAPK signaling pathway. Collectively, our studies suggest that 25-OCH3-PPD is a novel small molecule reducing ER stress, and a potential drug for treating diseases associated with ER stress. 2. Materials and methods 2.1. Cell lines and cell culture HepG2 (human hepatocellular carcinoma cell) and HEK293T (human embryonic kidney cell) cells were purchased from the Chinese Academy of Sciences Shanghai Institute for Biological Sciences Cell Resource Center (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, CA,

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USA), which was supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 units penicillin and 100 μg/ml streptomycin (Ameresco, OH, USA) at 37 ºC with 5% CO2. 2.2. Antibodies and reagents The mouse monoclonal antibodies against SelS, JNK, p38 and the rabbit polyclonal antibody against GRP78 were obtained from Santa Cruz Biotechnology (CA, USA). The rabbit polyclonal antibody against ERK1/2 was purchased from Cell Signaling Technology (CST, Beverly, MA, USA). Phospho-specific antibodies against ERK1/2, JNK, and p38 were purchased from Cell Signaling Technology. The mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Kangcheng Biotech (Shanghai, China). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Beyotime (Shanghai, China). Dithiothreitol (DTT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), U0126, and tunicamycin (TM) were obtained from Sigma-Aldrich (MO, USA). 25-OCH3-PPD was isolated from the leaves of Panax notoginseng as previously described, and the purity of 25-OCH3-PPD was above 99.0% (Bi et al., 2009). Other natural compounds used in the study were extracted from plants and animals in our laboratory and the purity was over 95%. All compounds were dissolved in DMSO as a 10 mg/ml stock solution. 2.3. Screening of potential SelS expression inhibitors pSelS-luc reporter plasmid was constructed as described previously (Gao et al., 2004). HEK293T cells were plated at a concentration of 1×105 cells/well in a 24-well plate. After 24 h, cells were transfected with 1 μg of pSelS-luc plasmids or 1 μg of pGL3-basic vector plasmids per well plus 0.1 μg of pCMV-β-galactosidase plasmids using Calcium Phosphate Cell Transfection kit (Beyotime) according to the instructions of manufacturer. The cells were incubated for 24 h and then cells were treated with different compounds at a final concentration of 5 μg/ml in DMEM containing 3% FBS (v/v) (to reduce the complex

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interference caused by the composition of serum) for 24 h. DMSO was used as a negative control. Finally, luciferase activity was measured as described previously and normalized to the β-galactosidase activity using a FLUOstar OPTIMA system (BMG Labtech, Offenburg, Baden-Wuerttemberg, Germany) (Zhang et al., 2010). 2.4. RNA extraction and RT-PCR HepG2 cells were plated at a concentration of 5×105 cells/well in a 6-well plate. After culture for 24 h, cells were treated with TM (5 μg/ml) and 25-OCH3-PPD (1, 2, and 3 μg/ml) in 2 ml DMEM containing 3% FBS (v/v) for 12 h. Total RNA was prepared from the treated cells using Trizol reagent (Invitrogen) following the manufacturer’s instructions. RNA was quantified by measuring absorbance at 260/280 nm. 1 μg of the total RNA was reverse transcribed by oligo (dT) primers using the Reverse Transcription System (TAKARA, Dalian, Liaoning, China). RT-PCR was performed by RT-PCR kit (TransGen Biotech, Beijing, China) following the manufacturer’s instructions. The primers of SelS were 5′-GTTGCGTTGAATGATGTCTTCCT-3′ (sense) and 5′-AGAAACAAACCCCATCAACTGT-3′ (antisense). The primers of β-actin (the internal control) were 5′-TCGTGCGTGACATTAAGGAG-3′ (sense) and 5′-ATGCCAGGGTACAT GGTGGT-3′ (antisense). PCR was performed for 25 cycles (each cycle consisting of 94 ºC for 30 s, 55 ºC for 40 s, and 72 ºC for 30 s). The PCR products were examined by electrophoresis on 1% agarose gel (Sigma-Aldrich) stained with cyanine (KeyGEN, Nanjing, Jiangsu, China) and visualized under UV light (DNR Bio-imaging Systems, Jerusalem, Israel). 2.5. Protein preparation and western blot analysis To determine the expression of associated proteins, the whole-cell lysates were isolated and western blot assay was performed. Briefly, cells were harvested and resuspended in cell lysis buffer (0.015 M NaCl, 10 mM Tris-HCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 10 μg/ml pepstain A and 10 μg/ml leupeptin). Then the cell suspension was incubated on ice for 30 min. The cell lysates were centrifuged at 12,000×g for 8 min at 4 ºC.

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The supernatants were collected and mixed with one quarter volume of 4×SDS sample buffer, boiled for 10 min at 100 ºC, and then separated by 12% SDS-PAGE gel. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Then the PVDF membranes were blocked with 5% nonfat dry milk in TBST buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.6 and 0.05% Tween 20) for 2 h at room temperature. Then the membranes were probed with primary antibodies in TBST buffer overnight at 4 ºC. The membranes were then washed three times with TBST buffer, and incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature, and washed extensively with TBST buffer. Finally, protein bands were identified using enhanced chemiluminescence detection (ECL-Plus kit, Beyotime) and were digitally captured (MicroChemi, DNR Bio-imaging Systems, Jerusalem, Israel). Differences in protein loading were controlled by blotting for GAPDH. Immunoreactive bands were quantified by densitometry of unsaturated images with background density subtracted (Image J, NIH, Bethesda, USA). 2.6. MTT assay HepG2 or HEK293T cells were seeded into 96-well plates at a concentration of 1×104 cells/well. After 24 h, the cells were treated with 25-OCH3-PPD, TM or U0126 in the presence of 3% FBS (v/v) culture medium for 44 h at 37 ºC. Then MTT solution (20 μl, 5 mg/ml in PBS) was added to each well and incubated for 4 h at 37 ºC. The culture medium was removed. Then 100 μl DMSO was added to each well and shaken for 10 min at room temperature. Cell viability was analyzed by measuring the absorbance at 570 nm by plate reader (Bio-Rad, Hercules, CA, USA). 2.7. Statistical analysis All experiments were repeated at least three times. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used to calculate the statistical significance of the experimental results. The one-way analysis of variance was used to compare the results from each group. The significance level was set as *P < 0.05 and **P < 0.01. Error

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bars denote the standard deviation (S.D.). 3. Results 3.1. Screening of SelS expression inhibitors In order to easily detect SelS expression level, we established a firefly luciferase reporter screening system driven by SelS gene promoter (pSelS-luc). Then we screened out 43 nature compounds in HEK239T cells. The fold induction and P value of each compound were showed in Fig. 1A. 20(S)-25-methoxyl-dammarane-3β,12β, 20- triol (25-OCH3-PPD; see structure in Fig. 1B) with fold induction of 0.37 (P < 0.05) exhibited the most significantly inhibitory effect on the activity of SelS promoter, so the further study was focused on 25-OCH3-PPD. 3.2. The effect of 25-OCH3-PPD on cell viability In order to investigate the effect of 25-OCH3-PPD on ER stress at conditions free from cell viability influences, we tested the cytotoxicity of 25-OCH3-PPD by MTT assay in HepG2 and HEK293T cells (Fig. 2). 25-OCH3-PPD with the concentrations of 1, 2, and 3 μg/ml had no toxicity effect on HepG2 and HEK293T cells. 3.3. 25-OCH3-PPD inhibits TM-induced up-regulation of SelS and attenuates ER stress in HepG2 cells Then, we examined whether 25-OCH3-PPD could inhibit SelS expression in mRNA and protein levels in HepG2 cells. Tunicamycin (TM), a specific inhibitor of protein glycosylation, has been widely used to induce ER stress (Garcia-Marques et al., 2015). TM with concentrations less than 7 μg/ml had no cytotoxicity to HepG2 cells, and 5 μg/ml of TM and 3 μg/ml of 25-OCH3-PPD treated simultaneously had no cytotoxicity to HepG2 cells (Fig. 3A and 6C). RT-PCR results showed that TM increased SelS expression, and 25-OCH3-PPD significantly inhibited the up-regulation of SelS induced by TM at different concentrations (1, 2, and 3 μg/ml) in mRNA level (Fig. 3B-D). Western blot results also showed that 25-OCH3-PPD dramatically inhibited the up-regulation of

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SelS at different concentrations (1, 2, and 3 μg/ml) in protein level (Fig. 3E-G). Taken together, these studies confirmed that 25-OCH3-PPD could inhibit TM-induced up-regulation of SelS expression. SelS is a marker of ER stress (Darling et al., 2014). Since 25-OCH3-PPD could inhibit SelS expression, we next investigated whether 25-OCH3-PPD could attenuate ER stress. Glucose-regulated protein 78 (GRP78; also known as BiP/HSPA5), an ER molecular chaperone, plays a central role in protein folding and assembly in the ER and regulation of ER stress initiation mediators, such as IRE1α, ATF6, and PERK (Bertolotti et al., 2000; Inageda et al., 2010). GRP78 is the most critical UPR regulator and the major marker of ER stress (Chambers et al., 2012). As shown in Fig. 4A-C, TM induced ER stress representing by up-regulation of GRP78, after treatment with 25-OCH3-PPD (1, 2, and 3 μg/ml), the expression of GRP78 was significantly decreased. These results suggested that 25-OCH3-PPD could attenuate TM induced ER stress in HepG2 cells. 3.4 25-OCH3-PPD inhibits SelS expression in ER stress and attenuates ER stress in HEK293T cells Then we investigated the effects of 25-OCH3-PPD on SelS expression and ER stress in HEK293T cells. As shown in Fig. 5A-B, TM with concentrations less than 6 μg/ml had no cytotoxicity to HEK293T cells, and 5 μg/ml of TM and 3 μg/ml of 25-OCH3-PPD treated simultaneously had no cytotoxicity to HEK293T cells. In agreement with the observation in HepG2 cells, 25-OCH3-PPD dramatically reversed TM-induced up-regulation of SelS and GRP78 at different concentrations (1, 2, and 3 μg/ml) in HEK293T cells (Fig. 5C-E), suggesting that 25-OCH3-PPD could reverse TM-induced ER stress in HEK293T cells. Moreover, 25-OCH3-PPD could inhibit dithiothreitol (DTT; another ER stress inducer) induced up-regulation of SelS and GRP78 (Fig. 5F), suggesting that 25-OCH3-PPD could reverse DTT-induced ER stress. 3.5. 25-OCH3-PPD attenuates ER stress via ERK/MAPK signaling pathway A large body of evidence has shown that MAPK (ERK, JNK and p38) signaling pathways play important roles in response to ER stress (Hung et al., 2004; Li et al., 2004). To examine the underlying mechanisms by which

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25-OCH3-PPD attenuates ER stress, we investigated the effects of 25-OCH3-PPD on the MAPK signaling pathways in HepG2 cells. As shown in Fig. 6A, 25-OCH3-PPD could increase the phosphorylation of ERK1/2 (p-ERK1/2) at 15 min and 30 min, and the activation effect reached a significant level (P < 0.05) at 1 h, suggesting that 25-OCH3-PPD could activate ERK/MAPK signaling pathway. However, 25-OCH3-PPD had no significant effects on the phosphorylation of JNK and p38 (p-JNK and p-p38). We have demonstrated that 25-OCH3-PPD could attenuate ER stress and activate ERK/MAPK signaling pathway, so we hypothesized that 25-OCH3-PPD attenuated ER stress through activating ERK/MAPK signaling pathway. To test this hypothesis, we intended to assess the effect of 25-OCH3-PPD on ER stress after blocking ERK/MAPK signaling pathway in HepG2 cells. U0126, a small molecule inhibitor, specifically inhibits the phosphorylation and activation of ERK1/2 without affecting p38 or JNK pathways (DeSilva et al., 1998). U0126 with concentrations less than 11.415 μg/ml had no cytotoxicity to HepG2 cells, and 5 μg/ml of TM, 3 μg/ml of 25-OCH3-PPD and 7.61 μg/ml of U0126 treated simultaneously had no cytotoxicity to HepG2 cells (Fig. 6B-C). As shown in Fig. 6D, U0126 could effectively inhibit the phosphorylation of ERK1/2. Then we tested the effect of 25-OCH3-PPD on ER stress with inhibition of ERK/MAPK signaling by U0126, as shown in Fig. 6E, 25-OCH3-PPD inhibited TM-induced up-regulation of GRP78, and U0126 significantly reversed the inhibitory effect, suggesting that 25-OCH3-PPD attenuated ER stress, at least partially, through activation of ERK/MAPK signaling pathway. 4. Discussion ER stress involves in the pathogenesis of many diseases such as cancer, ophthalmology disorders, metabolic diseases, and Alzheimer's disease (Li et al., 2011; Kang et al., 2009; Cnop et al., 2012; Torres et al., 2015). Discovery of drugs that can reverse ER stress would be beneficial to the treatments of these diseases. SelS is a marker of ER stress (Walder et al., 2002; Speckmann et al., 2014). Therefore, SelS was used as a target to screen

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compounds capable of attenuating ER stress. Liver and kidney are the most important organs in the body, and perform many functions that are vital to life. ER stress in liver and kidney cells can lead to many severe diseases, such as hepatocellular carcinoma, acute hepatic failure, glomerular injury, and renal tubule interstitial injury (Kandel-Kfir et al., 2015; Taniguchi et al., 2015). Attenuating ER stress in hepatocytes and nephrocytes is essential to the body health, so we tested the effect of 25-OCH3-PPD on ER stress in liver and kidney cells (HepG2 and HEK293T cells). Our results showed that 25-OCH3-PPD could reverse TM-induced ER stress (Fig. 4, 5C-E), and further results showed that 25-OCH3-PPD also could attenuate DTT-induced ER stress (Fig. 5F). These results indicate that the inhibitory effect of 25-OCH3-PPD on ER stress is not specific to TM-induced ER stress, but is universal. We demonstrated that 25-OCH3-PPD with concentrations less than 3 μg/ml could reverse ER stress in HepG2 and HEK293T cells (Fig. 4, 5C-F), and these concentrations of 25-OCH3-PPD had no cytotoxicity as shown in Fig. 2A-B. These results suggested that 25-OCH3-PPD could reduce ER stress at sub-toxic concentrations in HepG2 and HEK293T cells. However, whether 25-OCH3-PPD can reverse ER stress at toxic concentrations in these cells needs further investigation. MAPK (ERK, JNK and p38) signaling pathways are involved in both cell growth and cell death, and the tight regulation of these signaling pathways is essential to determine the cell fate (Farrukh et al., 2015; Ravingerova et al., 2003). In ER stress, MAPK pathways are activated and form part of the UPR. Many studies have demonstrated that ERK/MAPK pathway could promote adaptation, survival and resistance to ER stress (Sale et al., 2013; Balmanno et al., 2009). Our results showed that 25-OCH3-PPD could activate ERK/MAPK signaling pathway (Fig. 6A). After inhibiting ERK/MAPK pathway, the inhibitory effect of 25-OCH3-PPD on ER stress was reversed, suggesting that 25-OCH3-PPD attenuated ER stress through activation of ERK/MAPK pathway (Fig. 6E). These results confirm the previous conclusion that ERK1/2 activation protects cells against ER stress.

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However, it is possible that other signals participate in 25-OCH3-PPD-induced resistance to ER stress, and the ERK/MAPK signaling may play a dominant role. Moreover, how ERK1/2 signaling interacts with the core components of UPR resulting in reducing ER stress is unknown, this still needs further study. In conclusion, our data highlight the role of 25-OCH3-PPD in reducing ER stress. Furthermore, we found that 25-OCH3-PPD attenuated ER stress via ERK/MAPK signaling pathway. Overall, our study provides a novel small molecule for attenuating ER stress.

Conflict of interest: The authors declare that they have no conflict of interest. Informed consent: Informed consent was obtained from all individual participants included in the study. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

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Figure legends Fig. 1 Screening of SelS expression inhibitors. a The effects of different compounds on the promoter activity of SelS. HEK293T cells were plated at a concentration of 1×105 cells/well in a 24-well plate. After 24 h, cells were co-transfected with pSelS-luc reporter plasmid or pGL3-basic vector and pCMV-β-galactosidase plasmid for 24 h, and were treated with different compounds (5 μg/ml) or DMSO for 24 h. Then luciferase activity was measured. Results are expressed as the fold induction (over the activity of the negative control) and P value (compared with negative control). The black data point represents 25-OCH3-PPD. b The structure of 25-OCH3-PPD Fig. 2 The cytotoxicity of 25-OCH3-PPD. a The cytotoxicity of 25-OCH3-PPD to HepG2 cells. HepG2 cells were plated in 96-well plates at a concentration of 1×104 cells/well. After 24 h, the cells were treated with different concentrations (1, 2, 3, 4, 5, 6, 7, and 8 μg/ml) of 25-OCH3-PPD for 44 h. MTT assays were conducted, as described in the materials and methods section. The results are expressed as relative cell viability compared to the untreated control and represent the means ± S.D. of three independent experiments. *P < 0.05 and **P < 0.01 compared with the control group. b The cytotoxicity of 25-OCH3-PPD to HEK293T cells. HEK293T cells were plated in 96-well plates at a concentration of 1×104 cells/well. After 24 h, the cells were treated with different concentrations (1, 2, 3, 4, 5, 6, 7, and 8 μg/ml) of 25-OCH3-PPD for 44 h. Then MTT assays were conducted. The results are expressed as cell viability and represent the means ± S.D. of three independent experiments. **P < 0.01 compared with the control group Fig. 3 25-OCH3-PPD inhibits TM-induced up-regulation of SelS in HepG2 cells. a The cytotoxicity of TM to HepG2 cells. HepG2 cells were plated in 96-well plates at a concentration of 1×104 cells/well. After 24 h, the cells were treated with different concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg/ml) of TM for 44 h. MTT assays were conducted, as described in the materials and methods section. The results are expressed as relative

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cell viability compared to the untreated control and represent the means ± S.D. of three independent experiments. **P < 0.01 compared with the control group. b-d 25-OCH3-PPD inhibits TM-induced up-regulation of SelS in mRNA levels. HepG2 cells were plated in 6-well plates at a concentration of 5×105 cells/well. The cells were simultaneously treated with TM (5 μg/ml) and different concentrations (1, 2, and 3 μg/ml) of 25-OCH3-PPD for 12 h. The control cells were treated with DMSO, 25-OCH3-PPD (1, 2, or 3 μg/ml), or TM (5 μg/ml). SelS mRNA levels were analyzed by RT-PCR. β-actin was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. e-g 25-OCH3-PPD inhibits TM-induced up-regulation of SelS in protein level. HepG2 cells were plated in 6-well plates at a concentration of 5×105 cells/well. The cells were simultaneously treated with TM (5 μg/ml) and different concentrations (1, 2, and 3 μg/ml) of 25-OCH3-PPD for 12 h. The control cells were treated with DMSO, 25-OCH3-PPD (1, 2, or 3 μg/ml), or TM (5 μg/ml). Then the expression of SelS was analyzed by western blot. Quantitative analysis was performed by Image J software and normalized by the anti-GAPDH signal. The quantitative results are shown as the means ± S.D. of three separate experiments. **P < 0.01 compared with the TM-induced group Fig. 4 25-OCH3-PPD attenuates TM-induced ER stress in HepG2 cells. a-c 25-OCH3-PPD inhibits TM-induced up-regulation of GRP78 in HepG2 cells. HepG2 cells were plated in 6-well plates at a concentration of 5×105 cells/well. The cells were simultaneously treated with TM (5 μg/ml) and different concentrations (1, 2, and 3 μg/ml) of 25-OCH3-PPD for 12 h. The control cells were treated with DMSO, 25-OCH3-PPD (1, 2, or 3 μg/ml), or TM (5 μg/ml). Then the expression of GRP78 was analyzed by western blot. Quantitative analysis was performed by Image J software and normalized by the anti-GAPDH signal. The quantitative results are shown as the means ± S.D. of three separate experiments. **P < 0.01 compared with the TM-induced group Fig. 5 25-OCH3-PPD inhibits SelS expression in ER stress and attenuates ER stress in HEK293T cells. a The cytotoxicity of TM to HEK293T cells. HEK293T cells were plated in 96-well plates at a concentration of 1×104

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cells/well. After 24 h, the cells were treated with different concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg/ml) of TM for 44 h. Then MTT assays were conducted. The results are expressed as relative cell viability compared to the untreated control and represent the means ± S.D. of three independent experiments. *P < 0.05 and **P < 0.01 compared with the control group. b The cytotoxicity of 25-OCH3-PPD and TM to HEK293T cells. HEK293T cells were plated in 96-well plates at a concentration of 1×104 cells/well. After 24 h, the cells were treated with 25-OCH3-PPD (3 μg/ml), TM (5 μg/ml), or simultaneously treated with 25-OCH3-PPD (3 μg/ml) and TM (5 μg/ml) for 44 h. Then MTT assays were conducted. The results are expressed as cell viability and represent the means ± S.D. of three independent experiments. c-e 25-OCH3-PPD inhibits TM-induced up-regulation of SelS and GRP78. HEK293T cells were plated in 6-well plates at a concentration of 6×105 cells/well. The cells were simultaneously treated with TM (5 μg/ml) and different concentrations (1, 2, and 3 μg/ml) of 25-OCH3-PPD for 12 h. The control cells were treated with DMSO, 25-OCH3-PPD (1, 2, or 3 μg/ml), or TM (5 μg/ml). Then the expression of SelS and GRP78 was analyzed by western blot. Quantitative analysis was performed by Image J software and normalized by the anti-GAPDH signal. The quantitative results are shown as the means ± S.D. of three separate experiments. *P < 0.05 and **P < 0.01 compared with the TM-induced group. f 25-OCH3-PPD inhibits DTT-induced up-regulation of SelS and GRP78. HEK293T cells were plated in 6-well plates at a concentration of 6×105 cells/well. The cells were simultaneously treated with DTT (0.09258 μg/ml) and 25-OCH3-PPD (3 μg/ml) for 12 h. The control cells were treated with DMSO, 25-OCH3-PPD (3 μg/ml), or DTT (0.09258 μg/ml). Then the cells were lysed and the expression of SelS and GRP78 was analyzed by western blot. Quantitative analysis was performed by Image J software and normalized by the anti-GAPDH signal. The quantitative results are shown as the means ± S.D. of three separate experiments. **P < 0.01 compared with the DTT-induced group Fig. 6 25-OCH3-PPD attenuates ER stress via ERK/MAPK signaling pathway. a The effects of 25-OCH3-PPD

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on the phosphorylation of ERK1/2, JNK and p38. HepG2 cells were plated in 6-well plates at a concentration of 5×105 cells/well. The cells were simultaneously treated with TM (5 μg/ml) and 25-OCH3-PPD (3 μg/ml) for 15 min, 30 min, and 1 h. The control cells were treated with DMSO, 25-OCH3-PPD (3 μg/ml), or TM (5 μg/ml). Then the cells were lysed and the phosphorylation levels of ERK1/2, JNK and p38 was analyzed by western blot. Quantitative analysis was performed by Image J software. Equal protein loading was evaluated by total ERK1/2, JNK and p38. The quantitative results are shown as the means ± S.D. of three separate experiments. **P < 0.01 compared with the TM-induced group. b The cytotoxicity of U0126 to HepG2 cells. HepG2 cells were plated in 96-well plates at a concentration of 1×104 cells/well. After 24 h, the cells were treated with different concentrations (1.9025, 3.805, 7.61, 11.415, 15.22, 19.025, 22.83, 26.635, and 30.44 μg/ml) of U0126 for 44 h. Then MTT assays were conducted. The results are expressed as relative cell viability compared to the untreated control and represent the means ± S.D. of three independent experiments. *P < 0.05 and **P < 0.01 compared with the control group. c The cytotoxicity of 25-OCH3-PPD, TM and U0126 to HepG2 cells. HepG2 cells were plated in 96-well plates at a concentration of 1×104 cells/well. After 24 h, the cells were treated with 25-OCH3-PPD (3 μg/ml), TM (5 μg/ml), U0126 (7.61 μg/ml), 25-OCH3-PPD (3 μg/ml) and TM (5 μg/ml), or simultaneously treated with 25-OCH3-PPD (3 μg/ml), TM (5 μg/ml) and U0126 (7.61 μg/ml) for 44 h. Then MTT assays were conducted. The results are expressed as cell viability and represent the means ± S.D. of three independent experiments. d U0126 inhibits the phosphorylation of ERK1/2. HepG2 cells were plated in 6-well plates at a concentration of 5×105 cells/well. The cells were treated with U0126 (7.61 μg/ml) for 30 min and 1 h. The control cells were treated with DMSO. Then the cells were lysed and the phosphorylation level of ERK1/2 was analyzed by western blot. Equal protein loading was evaluated by total ERK1/2. e U0126 reverses the inhibitory effect of 25-OCH3-PPD on ER stress. HepG2 cells were plated in 6-well plates at a concentration of 5×105 cells/well. The cells were simultaneously treated with TM (5 μg/ml), 25-OCH3-PPD (3 μg/ml) and U0126

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(7.61 μg/ml) for 12 h. The control cells were treated with DMSO, U0126 (7.61 μg/ml), TM (5 μg/ml), or simultaneously treated with TM (5 μg/ml) and 25-OCH3-PPD (3 μg/ml). Then the cells were lysed and the expression of GRP78 was analyzed by western blot. Quantitative analysis was performed by Image J software and normalized by the anti-GAPDH signal. The quantitative results are shown as the means ± S.D. of three separate experiments. **P < 0.01 compared with the control group

Fig. 1 Screening of SelS expression inhibitors

Fig. 2 The cytotoxicity of 25-OCH3-PPD

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Fig. 3 25-OCH3-PPD inhibits TM-induced up-regulation of SelS in HepG2 cells

Fig. 4 25-OCH3-PPD attenuates TM-induced ER stress in HepG2 cells

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Fig. 5 25-OCH3-PPD inhibits SelS expression in ER stress and attenuates ER stress in HEK293T cells.

Fig. 6 25-OCH3-PPD attenuates ER stress via ERK/MAPK signaling pathway.

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