Sestrin2 Overexpression Ameliorates Endoplasmic Reticulum Stress-Induced Apoptosis via Inhibiting mTOR Pathway in HepG2 Cells

Sestrin2 is a highly conserved stress-inducible protein, acting as a crucial part in regulating homeostasis in response to various stress conditions in the cell. However, the role of Sestrin2 in regulating cell apoptosis related to endoplasmic reticulum (ER) has not been fully investigated. Our study presented here aims to reveal the effect of Sestrin2 in tunicamycin (TM)-induced cell apoptosis related to ER stress and its underlying molecular mechanisms. The results demonstrated that Sestrin2 expression was significantly upregulated correlated with ER stress responses in TM treated HepG2 cells. Sestrin2 overexpression obviously alleviated ER stress with the determination of ER stress-related proteins expression. In addition, Sestrin2 overexpression inhibited cell apoptosis with the examination of apoptosis-related proteins and TUNEL assay. However, Sestrin2 knockdown further promoted the ER stress-mediated cell apoptosis. The further mechanistic study revealed that Sestrin2 overexpression inhibited TM-induced mTOR pathway activation. Taken together, our current study indicated that Sestrin2 overexpression ameliorates ER stress-induced apoptosis via inhibiting mTOR pathway in HepG2 cells.


Introduction
Te liver is a highly complex organ with multiple metabolic functions including transamination, gluconeogenesis, lipogenesis, bile, and protein synthesis [1]. Te liver disease remains a major public health problem worldwide, with incidence increasing with each passing decade. In China, approximately 300 million people are aficted with major liver diseases, including viral hepatitis, alcoholic liver disease, and nonalcoholic fatty liver disease (NAFLD). Globally, more than 2 million people die of liver diseases each year, accounting for about 4% of all deaths [2,3]. Liver injury, induced by overdosed acetaminophen (APAP), alcohol ingestion, hepatitis virus, insulin resistance, and NAFLD, is the common pathological basis of various liver diseases [4][5][6][7]. Among those risk factors provoking liver injury, APAP overdosage may induce hepatocellular necrosis and cytolysis, leading to acute liver failure. Also, excessive alcohol ingestion causes hepatocyte injury through oxidative stress, which is linked to the production of ROS and inhibition of the expression of antioxidant enzymes by ethanol [8][9][10]. However, despite extensive exploration of the pathophysiology of liver diseases, there is still no efective targeted therapy regimen. Terefore, it is necessary to explore the underlying mechanism of liver injury and seek out potential therapeutic targets.
Hepatocytes are the main functional cells of the liver, accounting for about 70% of all cells in the normal liver, and play important roles in regulating homeostasis, regeneration, and injury recovery [11]. As the main region of protein synthesis, hepatocytes contains abundant endoplasmic reticulum (ER), an organelle which takes charge of protein assembly, protein glycosylation, disulfde conformation adjustment, and cellular calcium storage, allowing this cell type be sensitive to changes of intracellular homeostasis [6,12,13]. If alcohol triggers oxidative stress, misfolded proteins accumulate in the ER, or other damage factors exists, ER will initiate unfolding protein response (UPR) to facilitate repair of tissue damage and restore homeostasis. However, if hepatocytes fail to adapt to the stressor, then excessive or prolonged UPR activation, namely, ER stress [14], leads to the development of liver injury through hepatocyte apoptosis [15], steatosis [16], and fbrosis [17]. ER stress consists of a complex network of signal transduction pathways from ER to the nucleus and is initiated by three ER transmembrane proteins, namely, PERK, ATF6, and IRE1, which keeps inactive under physiological conditions. When ER stress occurs, these transmembrane proteins are activated sequentially. Activated PERK induces phosphorylation of eukaryotic initiation factor 2α (eIF2α) and then promotes the translation of transcription factor ATF4. By regulating the expression of protein synthesis (e.g., ER chaperones GRP78 and GRP94), degrading protein aggregates and initiates autophagy, and PERK/ATF4 pathway helps to restore ER homeostasis and promotes survival. But sustained ER stress promotes apoptosis through ATF4/CHOP pathway. Similarly, activated ATF6 and IRE1 help ER restore function by regulating the expression of another transcription factor XBP1 or otherwise result in ER stress associated apoptosis through IRE1/ JNK and ATF6/CHOP pathway [6,12,13]. Besides, ER stress also activates apoptosis via apoptotic-related genes (Bax and Bcl-2) and an apoptotic caspase cascade in which Caspase-12, Caspase-3, and Caspase-9 are involved [18,19]. Previous studies showed that ER stress plays an important role in liver injury through up-regulating expression of apoptotic protein such as Caspase-3, Caspase-12, JNK, and CHOP, along with the increase of GRP78, ATF6, and XBP1, which are hall markers of ER stress [12,13]. Moreover, numerous studies have shown that through these signal pathway, ER stress results in various liver diseases, including viral hepatitis, alcoholic liver disease (ALD), NAFLD, insulin resistance, and ischemia-reperfusion injury [6,20].
Sestrin2 is a member of the sestrin family, also known as a highly conserved stress-inducible protein, induced by kinds of stress conditions, including DNA damage, oxidative stress, ER stress, and metabolic stress [21,22]. Numerous research studies have demonstrated that Sestrin2 expression can be upregulated under ER stress conditions and exert cytoprotective functions [22,23]. Han et al. found that Sestrin2 expression is upregulated in cholestatic liver injury. Once induced, Sestrin2 in turn protects hepatocyte from cholestasis-induced injury by ameliorating hepatic ER stress [24]. In addition, a study conducted by Li et al. revealed that overexpression of sestrin2 limits ER stress, promoting neuronal survival and improving functional recovery after spinal cord injury [25]. All these studies indicated that Sestrin2 might serve as an important regulator that maintains regulating cellular homeostasis under excessive ER stress. Terefore, understanding how Sestrin2 is regulated with diferent stress conditions is very helpful for us to study Sestrin2.
Te mammalian target of rapamycin (mTOR) is one of the fve components which make up rapamycin complex 1 (mTORC1). As a master growth regulator and an autophagy inhibitor, mTOR plays a crucial part in controlling cell proliferation, metabolism, and autophagy through perception of change of amino acids, hormones, and growth factors. Amino acids, especially leucine, as one of critical environmental stimulator, are quite important in mTORC1 activation [26]. In addition, Sestrin2, playing the role of leucine sensor, regulates mTORC1 pathway by sensing the change of leucine through its afnity for leucine by structural change [27]. Recently, Gao et al. demonstrated that kinsenoside alleviates alcoholic liver injury by inhibiting ER stress via AMPK/mTOR pathway [28]. Accumulating studies have demonstrated that mTOR hyperactivity takes a pivotal role in regulating fatty acid synthesis, hepatic insulin resistance, and type 2 diabetes [29,30]. Several mTOR signaling pathways are involved in the development of liver injury, including the LKB1/AMPK/mTOR metabolic axis, NF-κB/ mTOR signaling, and TSC/mTOR signaling. Te team of Zhou et al. discovered that Puerarin inhibits hepatocellular apoptosis in rats with 2-AAF/PH-induced liver injury through inhibition of mTOR signal pathway by upregulating the expression of p-mTOR [31]. Gao et al. demonstrated that kinsenoside can reverse the ethanolinduced inhibition of the AMPK in mice model with ethanol/CCl4-induced alcoholic liver injury through LKB1/ AMPK/mTOR pathway and leads to the inhibiting of phosphorylation of mTOR, which thereby activates autophagy to remove damaged proteins [28]. Considering that Sestrin2 is an upstream regulator of mTOR and closely related to ER stress, in the present study, we try to investigate whether Sestrin2 is involved in the regulation of ER stressinduced hepatocyte apoptosis through mTOR pathway.
In this study, we found Sestrin2 overexpression ameliorates ER stress-induced apoptosis via inhibiting mTOR pathway in HepG2 cells. Tese results indicated the potential target of Sestrin2 for modulating the mTOR signaling pathway to alleviate apoptosis in response to cell stress in liver injury. Understanding the precise functions and the underlying mechanism of Sestrin2 in regulating cellular homeostasis will provide the evidence for future experimental studies and facilitate the development of novel therapeutic strategies for liver diseases.

Cell
Culture. HepG2 was cultured in DMEM medium, supplemented with 10% FBS and 1% penicillin/streptomycin. We applied varying concentration of TM (0, 0.5, 1, 2, 5, and 10 μg/mL, dissolved in DMSO) to induce ER stress in HepG2 cells. Te dose of TM was decided according to previous studies with minor modifcations [32]. Concentration of 5 μg/mL of TM was selected for future analysis.

Cell Viability Test. We performed cell viability tests with
Cell Counting Kit (CCK-8, TargetMol, USA). 96-well plate was incubated with 100 μL cell for 24 h, and the process followed the instruction of the suppliers. We added 10 μL CCK-8 directly in culture medium in each well after treatment with various concentration of TM for 24 h. Ten, the incubation process lasted for 1 h with the temperature of 37°C. We exerted absorbance test for multiple times with Synergy TM H1 multidetection microplate reader (Bio-tek Instruments Inc., USA) at 450 nm. Cell viability was tested by the results of absorbance. Te 100% viable parameter was obtained from the cells in control medium.

Transient Transfection.
For Sestrin2 overexpression and knockdown experiments, the plasmids of pcDNA3.1-Ses-trin2, and pLKO.1-shSestrin2 and corresponding control plasmids were synthesized from Tsingke Biotechnology (Guangzhou, China). Te sequences used for shSestrin2 were shown in Supplementary Table 1. Te transfection was performed with ViaFect reagent (Vigorous Biotechnology, Beijing, China) according to the manufacturer's instructions. Tissue culture plate with 6-well was used for cells culturing. After 24 h, the culture medium was replaced prior to transfection. Te dosages of vector and plasmid were 2 μL and 5 μg for one well. We incubated the cell for 12 h, and then treatment of TM for 24 h was applied. Te sample was ready for analysis such as mRNA and protein expression test.

Western
Blot. Te HepG2 cells were lysed with radioimmunoprecipitation assay (RIPA) bufer (Fd Bioscience, Hangzhou, China) supplemented with protease/phosphatase inhibitor cocktail (Fd Bioscience) on ice for 30 min and centrifuged at 12000 rmp at 4°C for 5 min. Ten, BCA protein assay kit (Beyotime, Shanghai, China) was used to detect the concentration of protein. Te equal amounts (30 μg/lane) of total protein were separated using a 12% SDS-PAGE gel and transferred to PVDF membranes. Te membranes were blocked in 1 × TBST with 5% (w/v) skim milk or 1 × TBST with 5% w/v bovine serum albumin (BSA) at room temperature. To ensure the specifcity of primary antibodies, antibodies specifcity and the molecular weight of target proteins were tested with HepG2 cells extracts frst of all (Supplementary Figure 1). Afterwards, the whole PVDF membranes were cut into several rectangular bands for incubation of corresponding primary antibodies according to the molecular weight of the target protein under the direction of the protein ladder, and then the membranes were fnally scanned using MP ChemiDoc imaging system (Bio-Rad, USA). Te relative expression of the proteins was normalized to that of β-Actin.

Quantitative Real-Time PCR (qRT-PCR).
We extracted RNAs from HepG2 cells with Trizol reagent (Invitrogen) following with the instructions of manufacturer. Te integrity and concentration of RNA were evaluated by NanoDrop One ultraviolet spectrophotometer (Termo Fisher Science, USA). We reversely transcribed RNA into cDNA with the reverse transcription reagent kit (Applied Biological Materials, Vancouver, Canada) following with the instructions of suppliers. SYBR Green Master Mix (Roche Life Science, Basel, Switzerland) was used in the Light-Cycleer 480 system (Roche) to perform the qPCR test. Conditions were set to initial 95°C for 5 min, then 45 cycles with the same pattern, that is 10 s on 95°C, 15 s on 60°C and 20 s on 72°C. We ran the melting routine immediately after the last cycle. To exclude primer dimers' infuence, the routine is held for 10 s at 4°C and read every 1.0°C. Tese experiments were tested in triplicate. We used the 2 −ΔΔCt method to standardize the relative abundance of mRNAs with β-Actin mRNA as the invariant control. Te following primers were used: Sestrin2-F: 5′-TCTTACCTGGTAGGC TCCCAC-3′, Sestrin2-R: 5′-AGCAACTTGTTGATCTCG CTG-3′,β-Actin-F: 5′-CGCCGCCAGCTCACCATGGAT-3′,β-Actin-R: 5′-CCACCATCACGCCCTGGTGC-3′.

TdT Mediated dUTP Nick End Labeling (TUNEL)
Staining. We applied the TUNEL test by TUNEL FITC Apoptosis Detection Kit (Vazyme Biotech, Shanghai, China). Concentration of 0.01% poly-L-Lysine-coated glass coverslips was used to plate HepG2 cells in 24-well tissue culture plates. PBS was used to rinse the slides after the treatment and then fxed the slides with cold 4% paraformaldehyde (PFA) in PBS for 25 min with temperature of 4°C. Subsequently, we rinsed the slides twice for 5 min with PBS. PBS was used to permeabilize the cells with 0.2% Triton X-100 for 5 min. Finally, we used equilibration bufer to incubate the cells for 20 min at 25°C. In a humidifed and dark environment, TNNEL reaction mixture was added at 37°C, and counterstaining with DAPI (Invitrogen). A fuorescence microscope (DMI 8, Leica) was used to evaluate the sections.

Statistical
Analysis. Data were presented as mean-± standard error of the mean (SEM). Statistical analyses were processed with the SPSS 23 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Unpaired Student's t test was performed to evaluate the signifcance of the diference between two groups. Comparisons of means for multiple treatment groups against control group were conducted using one-way ANOVA followed by Dunnett's 2sidedpost-hoc test. For the correlation analysis between the protein levels of Sestrin2 and apoptotic-related markers, the Spearman's rank-order method was used for all tests since

Sestrin2 Expression Was Signifcantly Up-Regulated Correlated with ER Stress Responses in TM Treated HepG2
Cells. TM, as a natural antibiotic, induces endoplasmic reticulum (ER) stress by inhibiting glycosylation of newly synthesized proteins in the ER leading to the disruption of their maturation [33][34][35]. In order to study the efect of ER stress induced by TM on Sestrin2 expression, HepG2 cells were treated with varying concentrations of TM from 0 to 10 μg/mL. qPCR and western blot analysis showed that Tm increased Sestrin2 levels in a concentration-dependent manner (Figures 1(a)-1(c)). In addition, the protein expression of ER stress markers, such as the GRP78, CHOP, and XBP1s/XBP1u was signifcantly upregulated (Figures 1(b), 1(d)-1(f )). Te data indicated that Sestrin2 expression correlates with ER stress responses in TM-treated HepG2 cells. To further eliminate the possibility of unwanted cytotoxicity efects caused by excessive concentration of TM, we performed a cell viability assay to assess its efcacy at the preferred doses. Te results indicated that with the concentration of TM increasing, the cell viability was decreased dramatically; as expected, higher TM treatment (10 μg/mL) resulted in signifcant destructive efects on cell viability compared with 5 μg/mL TM treated group (Figure 1(g)).
Taken together, considering the TM at a dose of 5 μg/mL yielded obviously ER stress induction with limited adverse efects, we propose the TM dose of 5 μg/mL, which was a preference choice when to investigate the efect of ER stress-induced HepG2 cell apoptosis.

Overexpression of Sestrin2 Ameliorates the TM-Induced ER-Stress.
Firstly, in order to study the efect of Sestrin2 overexpression on ER stress induced by TM, we transfected HepG2 with pcDNA3.1-Sestrin2 or control plasmid and followed with 5 μg/mL TM treatment. Te Sestrin2 expression and ER stress biomarkers, such as the GRP78, ATF6, CHOP, XBP1s, and XBP1u were examined with western blot analysis. With the comparison to the control group, Sestrin2 was obviously overexpressed in the pcDNA3.1-Sestrin2 group (Figure 2). What is more is that Sestrin2 overexpression ameliorates the TM-induced ER stress, as well as the ER stress-related proteins expression, for example, GRP78 and ATF6 ( Figure 2).

Overexpression of Sestrin2 Ameliorates Tunicamycin-Induced HepG2 Cells Apoptosis.
To examine the detail function of Sestrin2 on TM-induced HepG2 cells apoptosis, cell apoptosis-related proteins, such as BAX, BCL2, Cleaved-caspase3, Caspase3, Cleaved-caspase12, Caspase12, and CytoC were detected with western-blot assay after overexpression of Sestrin2. In addition, we performed a TUNEL assay to test HepG2 cells apoptosis. Te results demonstrated that the expression of BAX/BCL2, Cleaved-caspase3/ Caspase3, and CytoC was inhibited in the Sestrin2 overexpression group (Figures 3(a) and 3(b)). Furthermore, compared to the control group, TUNEL-positive cells were obviously decreased in the Sestrin2 overexpression group (Figure 3(c)). All these fndings indicated that overexpression of Sestrin2 signifcantly alleviates the apoptosis of HepG2 cells.

Te Expression Level of Sestrin2 was Negatively Correlated with Tat of Apoptotic-Related Markers in HepG2 Cells.
In order to check the reliability of the inhibiting potential of Sestrin2 in ER stress-induced apoptosis, the Sestrin2 gene expression was reprogrammed in HepG2 cells by transient transfection of either pcDNA3.1-Sestrin2 or shSestrin2 plasmids. Te protein abundance of Sestrin2 and apoptoticrelated markers was detected in a total of 48 individual samples of HepG2 cell culture from 5 independent experiments using western blot analysis. Te bands obtained were quantifed using densitometry analysis by ImageJ, and then Spearman's rank-order correlation analysis was used to compare the fold change of the normalized expression of indicated proteins. Intriguingly, we found strong negative correlations between the protein levels of Sestrin2 and apoptotic-related markers, including Bax/Bcl2 (Spearman's rho � −0.739, P < 0.001, Figure 5(a)), C-caspase12/Cas-pase12 (Spearman's rho � −0.453, P � 0.001, Figure 5(b)), C-caspase3/Caspase3 (Spearman's rho � −0.511, P < 0.001, Figure 5(c)), and CytoC (Spearman's rho � −0.725, P < 0.001 , Figure 5(d)). Tese results further corroborated our fnding that Sestrin2 may play an important role in regulating ER stress-induced apoptosis in HepG2 cells.

Sestrin2 Overexpression Inhibits TM-Induced mTOR
Pathway Activation. We then research the potential mechanism of the inhibition of Sestrin2 overexpression on ER stress induced by TM and cell apoptosis. Expression of mTOR, P70S6K, and 4E-BP1 and their phosphorylated morphology were tested for their roles as mTOR pathway related proteins. Te outcome proved that mTOR, 4E-BP1, and p-P70S6K/P70S6 expression was dramatically inhibited in the pcDNA3.1-Sestrin2 group ( Figure 6). Te data altogether demonstrated that Sestrin2 overexpression inhibits TM-induced mTOR pathway activation. We propose that Sestrin2 may inhibit TM-induced ER stress and cell apoptosis through mTOR pathway.

Discussion
ER stress is the main cause of liver injury [36], and the molecular mechanism of ER stress is complex. Sestrin2 exerts as an important part in the process of ER stress, but its action pathway is not fully elucidated. In our study, we observed that TM could induce ER stress in HepG2 cells, and Sestrin2 expression was elevated under ER stress. Sestrin2 overexpression obviously alleviated TM-induced ER stress and cell apoptosis. Further mechanistic study revealed that Sestrin2 overexpression inhibited TM-induced mTOR pathway activation. In conclusion, we propose that Sestrin2 overexpression ameliorates ER stress-induced apoptosis via inhibiting mTOR pathway in HepG2 cells. Tis study demonstrated the potential target of Sestrin2/mTOR pathway for the treatment of cell apoptosis associated with ER stress. Tunicamycin is a bacterial toxin which activates mammalian cells' UPR by blocking of N-linked glycosylation [37]. It causes ER stress in hepatocytes through the UPR [38]. By detecting the marker proteins of ER stress, we noticed the ER stress induce by TM treatment, and the expression of Ses-trin2 was signifcantly increased. Tis is consistent with previous research fndings [32]. Interestingly, GRP78 and CHOP expression increased frst and decreased gradually while the TM concentration elevated. According to previous reports, GRP78 and CHOP are the important biomarkers of ER stress [38,39], and Sestrin2 protects against bavachininduced ER stress [40]. So, we proposed that a high concentration of TM treatment leads to overexpression of Sestrin2, and overexpression of Sestrin2 reversely inhibited the expression of GRP78 and CHOP, which eventually suppressed the ER stress.
To test this hypothesis, Sestrin2 was overexpressed with transfected with pcDNA3.1-Sestrin2 plasmid in HepG2 cells. By detecting the marker proteins of ER stress, we found that the expression of ER stress-related proteins was signifcantly inhibited after overexpression of Sestrin2. Te outcomes altogether are in accordance with previous studies, in which Park et al. demonstrated that Sestrin2 has an important regulatory role in obesity-induced ER stress [23], and Yang et al. showed that Sestrin2 can alleviate bavachin-induced ER Stress [40].
Autophagy is modulated by ER stress, and this process induces cell survival or death. However, the interaction between them is still obscured [41]. One of the primary  International Journal of Endocrinology pathogenesis in the liver disease is apoptosis, and autophagic activity modulated the hepatic apoptosis. Hepatic apoptosis is regulated by autophagic activity. Park et al. found that elevated Sestrin2 attenuates obesity-induced liver apoptosis [23]. What is more is that Sesn2 inhibits APAP-induced hepatocyte death [42]. CytoC, BAX, and BCL2 are recognized as biomarkers of apoptosis [43,44]. In our studies, by detecting the expression of the proteins mentioned above, as well as the TUNEL positive cells, we proved that Sestrin2 overexpression can reduce TM-induced apoptosis in HepG2 cells. mTOR, as a serine/threonine kinase, modulates numerous basic processes in cell, such as protein synthesis, growth, metabolism, aging, regeneration, autophagy [45]. Yang et al. have found that Sestrin2 regulated the BVinduced ER stress through the signaling pathway of AMPK/mTORC1 [40]. Han et al. demonstrated that Sestrin2 defciency resulted in hepatic ER stress induced by cholestasis, but overexpression of Sestrin2 ameliorated ER stress induced by bile acid [24]. Terefore, to investigate the systematic process of the Sestrin2 inhibition on ER stress induced by TM and cell apoptosis, the main protein expression involved in the mTOR pathway was evaluated, and the results indicated that Sestrin2 overexpression inhibits TM-induced mTOR pathway activation. We propose that Sestrin2 may inhibit TM-induced ER stress and cell apoptosis through mTOR pathway.
However, further validation should be conducted in this study, such as the mTOR activators should be used when the Sestrin2 was overexpressed to test whether mTOR activation could suppress the protective efect of Sestrin2. Furthermore, in vivo experiments could be performed to validate the inhibition efect of Sestrin2 on liver injury.

Conclusions
Tese fndings indicate that Sestrin2 overexpression ameliorates ER stress-induced apoptosis via inhibiting mTOR pathway in HepG2 cells. Sestrin2 might be a therapeutic target with potential for modulating the signaling pathway of mTOR to alleviate apoptosis in response to cell stress in liver injury.

Data Availability
Te research data presented in the paper are available from the corresponding authors with reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.  International Journal of Endocrinology 9