4-PBA inhibits LPS-induced inflammation through regulating ER stress and autophagy in acute lung injury models
Authors: Meichun Zeng, Wenhua Sang, Sha Chen, Ran Chen, Hailin Zhang, Feng Xue, Zhengmao Li, Yu Liu, Yongsheng Gong, Hongyu Zhang, Xiaoxia Kong
PII: S0378-4274(17)30081-4
DOI: http://dx.doi.org/doi:10.1016/j.toxlet.2017.02.023
Reference: TOXLET 9711
To appear in: Toxicology Letters
Received date: 15-12-2016
Revised date: 18-2-2017
Accepted date: 24-2-2017
Please cite this article as: Zeng, Meichun, Sang, Wenhua, Chen, Sha, Chen, Ran, Zhang, Hailin, Xue, Feng, Li, Zhengmao, Liu, Yu, Gong, Yongsheng, Zhang, Hongyu, Kong, Xiaoxia, 4-PBA inhibits LPS-induced inflammation through regulating ER stress and autophagy in acute lung injury models.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2017.02.023
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 proof before it is published in its final 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.
4-PBA inhibits LPS-induced inflammation through regulating ER stress and autophagy in acute lung injury models
Meichun Zenga, 1, Wenhua Sanga, 1, Sha Chena, Ran Chena, Hailin Zhangb, Feng Xuea, Zhengmao Lic, Yu Liud, Yongsheng Gonga, Hongyu Zhangc, *, Xiaoxia Konga, *
aSchool of Basic Medical Sciences, Institute of Hypoxia Research, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China;
[email protected](M.Z.); [email protected](W.S.); [email protected](S.C.) [email protected](R.C.);[email protected](Y.G.); [email protected](X.K.)
bDepartment of Children’s Respiration, The Second Affiliated Hospital &Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, Zhejiang, China;
[email protected](H.Z.)
cSchool of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, China; [email protected](Z.L.); [email protected](H.Z.)
dDepartment of Chest Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China;[email protected](Y.L.)
*Corresponding author at: School of Basic Medical Sciences, Institute of Hypoxia Research, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China(X.Kong); School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, China(H.Zhang);
Tel.: +86 577 86699163; fax: +86 577 86689163.
E-mail addresses: [email protected](X.Kong), [email protected](H.Zhang)
1 These authors contributed equally to this work.
Abbreviations
ALI Acute lung injury
LPS Lipopolysaccharide
ER stress Endoplasmic-reticulum stress
NF-κB Nuclear factor κB
IκB Inhibitor of κB
HIGHLIGHTS
⦁ 4-PBA protects LPS-induced acute lung injury and inflammation in mouse model.
⦁ 4-PBA decreases the levels of ER stress and autophagy induced by LPS in vivo and in vitro.
⦁ Inhibition of autophagy by 3-MA aggravates cell injury induced by LPS, ER stress-associated autophagy may play a protective effect in LPS-induced lung injury.
Abstract
Acute lung injury (ALI) is a common clinical disorder that causes substantial health problems worldwide. An excessive inflammatory response is the central feature of ALI, but the mechanism is still unclear, especially the role of endoplasmic-reticulum (ER) stress and autophagy. To identify the cellular mechanism of lung inflammation during lipopolysaccharide (LPS)-induced mouse model of ALI, we investigated the influence of classic ER stress inhibitor 4-phenyl butyric acid (4-PBA) on ER stress and autophagy, which partially affect the activation of inflammation, both in LPS-induced ALI mouse model and human alveolar epithelial cell model. We demonstrated that 4-PBA, which further prevented the activation of
the NF-κB pathway, decreased the release of the pro-inflammatory mediators IL-1β, TNF-α and IL-6, significantly inhibited LPS-activated ER stress. Moreover, it was found that autophagy was also decreased by the treatment of 4-PBA, which may play a protective role in ALI models through the classical AKT/mTOR signalling pathway. Inhibition of autophagy by 3-MA exacerbates cytotoxicity induced by LPS in A549 alveolar epithelial cells. Taken together, our study indicated that ER stress is a key promoter in the induction of inflammation by LPS, the protective effect of 4-PBA is related to the inhibition of ER stress and autophagy in LPS-induced ALI models. Furthermore, the role of autophagy that contributes to cell survival may depend on the activation of ER stress.
Keywords: ALI, LPS, endoplasmic reticulum stress, autophagy, NF-κB
⦁ Introduction
Acute lung injury (ALI), along with the more severe condition acute respiratory distress syndrome (ARDS), is induced by a variety of insults, including endotoxins, acid aspiration, complement activation, and hyperoxia (Ather et al., 2010), and it is characterized by an excessive inflammatory response within the lungs and severely impaired gas exchange resulting from alveolar-capillary barrier disruption and pulmonary oedema (Matthay et al., 2012; Wheeler and Bernard, 2007). Inflammation is regarded as an important and ubiquitous feature of respiratory airway diseases (Sethi, 2010), it is widely accepted that dysregulation of inflammatory responses
results in various aggravated lung diseases, such as ALI/ARDS, vascular diseases and even sepsis (Chung et al., 2009). Lipopolysaccharide (LPS) is able to activate signaling cascades for inflammatory mediators expression, such as tumor necrosis factor (TNF-α) and interleukin (IL)-6 (Rossol et al., 2011). The risks of inflammation are starkly demonstrated in a transgenic mouse that expressed a mutant version of the inhibitory protein IκB in which the activation of NF-κB in lung epithelial cells was sufficient to cause neutrophil recruitment, pulmonary oedema, arterial hypoxemia, and death in the absence of any infection or exogenous stimuli (Poynter et al., 2003), but the specific mechanism of this transcription factor remains to be further tested in ALI.
Endoplasmic reticulum (ER) stress is defined as the accumulation of unfolded or misfolded proteins in the ER and subsequently triggers the unfolded protein response (UPR), which are mediated by three transmembrane ER signaling proteins: pancreatic endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) (Hosoi and Ozawa, 2010). Engagement of UPR sensors triggers changes in downstream signaling such as X-box binding protein 1 (XBP1), CHOP, eukaryotic translation initiation factor 2 subunit alpha (eIF2α), which leads to the up-regulation of various UPR target genes to restore ER homeostasis (Ron and Walter, 2007; Wang et al., 2012)). Recently, studies have demonstrated that ER stress is involved in LPS-induced lung inflammation in vivo and the expression of GRP78 and CHOP is up-regulated in LPS-stimulated airway epithelial cells in vitro (Kim et al., 2013). In LPS induced A549 alveolar epithelial
type II cells injury model, LPS treatment caused the accumulation of p-PERK, p-elF2α and nuclear ATF4, which triggered the UPR (Ye et al., 2015). However, the role of ER stress in LPS-induced lung inflammation is not fully understood.
Autophagy is a major catabolic process that delivers proteins, cytoplasmic components, and organelles to lysosomes for degradation and recycling (Gutierrez et al., 2004; Mizushima and Komatsu, 2011; Sir et al., 2010; Thurston et al., 2009; Wang et al., 2014). In general, autophagy could signify two possible functions. Autophagy represents an early adaptive mechanism of the tissue to the clearing of organelles or proteins for maintaining cell homeostasis. Moreover, excess autophagy results in autophagic cell death (Mizushima et al., 2008; Terman and Brunk, 2005). Evidence has been presented that indicates autophagy can promote cell survival or cell death depending on the cell type, the specific circumstances and different stimulus (Jin et al., 2012). Some studies have shown that increased autophagy reportedly plays an important role in ischaemia reperfusion-induced lung injury (Gao et al., 2013; Zhang et al., 2013). It is definitely confirmed that ER stress has emerged as a novel autophagy inducer and gained increased attention (Lee et al., 2012; Ullman et al., 2008; Yorimitsu and Klionsky, 2007; Zhang et al., 2016). It was demonstrated that autophagosomes could arise from other intracellular membrane structures, such as the ER, further evidences have suggested that the ER could contribute to autophagosome formation (Axe et al., 2008; Hayashi-Nishino et al., 2009; Yla-Anttila et al., 2009). Recently, LPS has been shown to activate ER stress and autophagy in A549 alveolar epithelial type II cells (Ye et al., 2015). However, there is no available
information on the relationship between ER stress and autophagy in lung inflammation.
The inhibitor of ER stress, 4-phenyl butyric acid (4-PBA), is a low molecular weight compound that stabilizes protein conformation, improves the folding capacity of the ER and facilitates trafficking of mutant proteins to suppress ER stress (Kim et al., 2013; Yam et al., 2007). In this study, 4-PBA was added to suppress the ER stress signaling pathways, to evaluate protective effect of 4-PBA on LPS-induced acute lung injury mouse. In addition, we also employed a nonspecific inhibitor of PI3-kinase, 3-MA to further evaluate the link between ER stress and autophagy in LPS-stimulated A549 alveolar epithelial type II cells. Our data demonstrated that 4-PBA inhibited ER stress sensor proteins obviously and also decreased the level of autophagy, further inhibited inflammation. Moreover, the effect of 3-MA suggested that ER stress-mediated autophagy is likely to be an adaptive protective role in LPS-induced lung injury, the supplemental strategy to activate autophagy may contributes to the rescue of ALI.
⦁ Materials and methods
⦁ Animals and experimental protocol
Male ICR mice, 7 to 8 weeks of age and free of murine specific pathogens, were obtained from the Animal Center of the Chinese Academy of Science (Shanghai, China). Mice were randomized into groups consisting of control group, LPS (Sigma-Aldrich, L3129, LPS from E. coli 0127:B8) group, 4-PBA (Sigma Aldrich,
USA) +LPS group and 4-PBA group. Mice were treated once by intratracheal instillation with 5mg/kg of LPS in saline (or with saline as a control) under anesthesia using chloral hydrate (Matrix, Orchard Park, NY, USA). 4-PBA was administered 16–18 hours (i.p., 10 mg/kg) before LPS treatment. Bronchoalveolar lavage fluid (BAL fluid) and lung tissue was performed at 6 hours after intratracheal instillation of LPS. At the time of lavage, the mice (6 mice in each group) were narcotized using chloral hydrate. The chest cavity was exposed to allow for expansion, after which the trachea was carefully intubated. Precooled phosphate-buffered saline (PBS, Gibco-Invitrogen, Carlasbad, CA, USA) (0.8 ml each time) solution was slowly instilled into the lung and withdrawn. The collected solutions were pooled and then kept at 4°C. A part of each pool was used for total cell counting. After centrifugation, the BAL fluid supernatants were stored at -80°C until use. Cell pellets were resuspended with BSA for cell differentials or immunofluorescence staining. Total cell numbers were counted with a Nucleocounter (Chemometec., Gydevang, Denmark).
⦁ Histological study
Lung tissue for histological study was fixed in fresh 4% formaldehyde solution for 24h and then dehydrated and embedded in paraffin, finally 5μm thick sections were cut and stained with hematoxylineosin (H&E). The sections were counterstained with hematoxylin. The tissue sections were observed under a light microscope for the lung histopathology.
⦁ Enzyme-linked immunosorbent assay (ELISA)
The cytokine production of IL-1β, TNF-α and IL-6 in BAL fluid supernatants was quantified using a murine ELISA development kit (PEPROTECH, Rocky Hill, NJ) according to the manufacturer’s recommendation.
⦁ Cell culture and drug treatment
The human type II lung epithelial A549 cell line was obtained from The Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were incubated in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO, USA) in a humidified atmosphere at 37 °C with 5% CO2. 3-MA (5 mM) or 4-PBA (5 mM) dissolved in PBS were used to pretreat the cells for 2h before the administration of LPS dissolved in PBS. Sampling was performed at 24 hours after the treatment of LPS.
⦁ MTT assay
Cells were seeded at a density of 1×104 cells in each well of the 96-well plates and allowed to attach overnight. A series of concentrations of reagent were added to the wells for 20 hours. Next，20 μl of MTT (5 mg/ml in PBS) was added to each well and
incubated for 4 h at 37°C with 5% CO2. DMSO was then added (200 µl/well) to each
well to dissolve any crystals and the plates were agitated for 10 min. Absorbance values at 490 nm was measured using an automatic multiwell spectrophotometer.
⦁ Monodansylcadaverine(MDC)staining
Monodansylcadaverine (MDC) has been proposed as a tracer for autophagic vacuoles (Biederbick et al., 1995). A549 cells in the logarithmic growth phase were plated in 12-well plates at a density of 1×105 cells. Following incubation with culture medium without serum for 12h, corresponding drugs were added to the four experimental groups. Then, A549 cells were incubated with 0.05mM MDC in PBS at 37°C for 30min. and then fixed with 4% paraformaldehyde for 15 min. After three washes with PBS, inverted fluorescence microscopy was used to observe the change in autophagic vacuoles and to capture images.
⦁ Western blotting
Proteins were extracted from the lung tissues samples and A549 cells using RadioImmunoprecipitation Assay (RIPA) buffer (Beyotime, P0013B) with PMSF lysis buffer. Proteins were separated on 10%-12% SDS-PAGE gels and were then transferred to PVDF membranes (Immobilon, IPVH00010). Membranes were blocked in 5% non-fat milk in Tris-buffered saline (TBS) for 1.5h and probed with antibodies LC3 (Santa Cruz Biotechnology, 1:1000, sc-28266), GRP78 (Cell Signal, 1:1000, 3177s), phosphorylated PERK (Cell Signal, 1:1000, 3179), XBP-1s (Cell Signal, 1:1000,12782), ATF6 (Abcam, 1:100, ab135707), CHOP (Cell Signal, 1:1000, 2895s),
Caspase-12 (Abcam, 1:1000, ab82832), IKK (Sigma, 210440), IκB (Cell Signal, 1:1000, 4814s), p-IκB (Cell Signal, 1:1000, 2859s), NF-κB p65 (Sigma, 1:500,
510038), IL-6 (Santa Cruz Biotechnology, 1:1000, sc-1266), AKT (Abcam, 1:500,ab8805), p-AKT (Abcam, 1:500, ab38449), mTOR (Cell Signal, 1:1000, 2893s),
p-mTOR (Cell Signal, 1:1000, 5536s) and GAPDH (Bioworld, 1:10000, AA56133) overnight at 4°C. After three washes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Beyotime Institute of Biotechnology) for 1h at room temperature. The relative intensities of bands were analyzed by using image analysis software program (Image J 1.44p, Wayne Rasband, National Institutes of Health, USA).
⦁ Immunofluorescence microscopy analysis
For lung tissue, after deparaffinization, serum blocking and antigen retrieval, sections underwent immunofluorescence staining with primary antibodies against IL-1ß (1:200), IL-6 (1:200), TNFα (1:200), PDI (1:100) and LC3 (1:200), at 4°C
overnight. Alexa Fluor 488- or 594-conjugated secondary antibodies (Molecular Probes, Carlsbad, CA) were added to sections for 1h at room temperature. Treated with Hoechst blue fluorescent dye (Hoechst) for 10min was used for nuclear counterstaining. Sections were mounted with a coverslip, sealed with nail polish and stored in the dark at 4°C.
A549 cells were seeded in 6-well plates containing coverslips overnight before indicated treatments. Then the cells were pretreated with or without the 4-PBA (5mM) or 3-MA for 1 hours followed by treatment with LPS (5μg/ml) for 6 hours. After the treatment, cells were washed with PBS and fixed with 4% PFA in PBS for 20 min at
room temperature, and then permeabilized with 0.5% Triton X-100 in PBS for 15 min and blocked with 5% BSA in PBS for 45 min at room temperature. After blocking, cells were incubated with rabbit monoclonal antibody to NF-κB p65 (1:200) or LC3 (1:200) overnight at 4°C. After further washed three times with PBS, cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:200). The nucleus was stained with Hoechst. All images were captured on Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan).
⦁ Statistical analysis
All data were expressed as the mean ± SEM. Student’s t test was used for statistical analysis for comparison of two groups. For more than two groups, statistical evaluation of the data was performed using a one-way analysis-of-variance (ANOVA) test, followed by Tukey’s post hoc test. Statistical significance was accepted at P< 0.05.
⦁ Results
⦁ Inhibitory effects of 4-PBA on structural damage and the production of inflammatory mediators induced by LPS in a lung injury mouse model
To investigate the effect of 4-PBA on lung injury induced by LPS, the histopathologic features of lungs from the different groups were evaluated by light microscopy, and results for the normal group mice, LPS-induced mice, LPS-induced
mice pre-treated with 4-PBA for 2h, and mice administered only 4-PBA are shown. Histological analyses showed marked infiltration of inflammatory cells into the alveolar space, peribronchial wall thickening, and vascular congestion in the lungs of LPS-induced mice. These histologic changes were dramatically reduced by administration of 4-PBA (Fig.1A). To further identify the anti-inflammatory properties of 4-PBA, an important procedure is determining the number of polymorphonuclear neutrophils (PMNs) that infiltrate the lungs. Recruitment of excess numbers of inflammatory cells is necessary for the pathogenesis of inflammation, and the effect of LPS on the inflammatory cell count in BAL fluid is shown in Fig.1B. Compared with vehicle alone, treatment with LPS significantly increased the number of total cells (P<0.01). The increased number of inflammatory cells in BAL fluid from LPS-instilled mice was reduced by 4-PBA administration (P<0.01). IL-1β, TNF-α and IL-6 are representative pro-inflammatory mediators in various infectious disorders; to further evaluate the anti-inflammatory properties of 4-PBA, these three inflammatory mediators in BAL fluid were measured in our study. The results indicated that compared with the control group, LPS significantly increased the concentrations of the inflammatory cytokines IL-1β (Fig.1C), TNF-α (Fig.1D) and IL-6 (Fig.1E) in BAL fluid (P<0.01). However, pre-treatment with 4-PBA largely reduced the levels of IL-1β (P<0.05), TNF-α and IL-6 in BAL fluid (P<0.01). Subsequently, we analysed expression of the proteins IL-1β (Fig.1F), TNF-α (Fig.1G) and IL-6 (Fig.1H) using immunofluorescence staining, and we found results that were consistent with those seen in BAL fluid. These data showed that
4-PBA administration reduced LPS-induced lung injury and inflammatory mediator production.
⦁ 4-PBA prevents the nuclear translocation of NF-κB in LPS-instilled mice
In this study, the effects of 4-PBA on IKK, IκB and NF-κB activity were measured in vivo. Western blotting results showed that IKK is rapidly activated in mice that received LPS for 6h. Lower levels of IKK were observed in mice that were pre-treated with 4-PBA before LPS was instilled (P<0.05) (Fig.2A). In addition, the cytosolic content of p-IκBα was increased after LPS administration. In contrast, in 4-PBA-pre-treated mice, the expression of p-IκBα was significantly lower, which suggested that 4-PBA inhibited p-IκBα degradation (P<0.01) (Fig.2B). Moreover, as shown in Fig.2C and D, LPS administration caused translocation of NF-κB p65 into nuclear in the injured lung of mice with ALI, which was inhibited by the treatment of 4-PBA significantly (P<0.05). Furthermore, we determined the effect of 4-PBA on the protein expression of the inflammatory cytokine IL-6 induced by LPS in mice. Western blotting results showed that the protein expression of IL-6 was increased in LPS-induced lung injury compared with the control group, while 4-PBA reduced IL-6 expression (Fig.2E). Subsequently, we analyzed the level of IL-6 protein using immunofluorescence staining. The intensities of IL-6 in lung tissues from LPS-instilled mice were increased compared with the control group, but treatment with 4-PBA clearly decreased LPS-induced IL-6 expression (P<0.01) (Fig.2F), which
was consistent with the Western blotting. These results indicated that 4-PBA protects the lungs from LPS-induced injury through inhibition of LPS-activated NF-κB signaling and reduction of inflammatory cytokines.
⦁ 4-PBA decreases the level of ER stress and inhibits the activation of autophagy in mice with LPS-induced lung injury
To assess whether ER stress is activated upon LPS-induced lung injury, we analyzed the protein levels of GRP78, CHOP and Caspase-12 by Western blotting. The LPS-induced increases in the expression of GRP78 (Fig.3A), CHOP (Fig.3B) and Caspase-12 (Fig.3C) in lung tissues of LPS-instilled mice were reduced by 4-PBA administration (P<0.05). In addition, we determined the expression levels of protein disulfideisomerase (PDI) in lung tissues by confocal microscopy to further evaluate whether ER stress is triggered in LPS-instilled mice. As shown in Fig.3D, red puncta revealed PDI-labelled protein, and an increased amount of PDI-labelled protein was found in the LPS-treated mice, compared with the control group (P<0.01). Compared with the LPS group, the amount of PDI-labelled protein was reduced and the fluorescence intensity was decreased in the 4-PBA pre-treatment group (P<0.01). No significant (P>0.05) red puncta appeared in 4-PBA group. These results indicated that 4-PBA protects the lungs from LPS-induced injury through inhibiting ER stress.
To elucidate whether autophagy participates in LPS-induced lung injury and the effect of 4-PBA on autophagy, we first examined the expression of the microtubule-associated protein LC3, which is a marker of autophagosomes. Western
blotting results indicated that the expression of LC3-II increased significantly in the LPS group (P<0.01). And compared to the LPS group, pre-treatment with 4-PBA significantly decreased the expression of the LC3-II protein (P<0.01) (Fig.4A). Based on LC3 immunofluorescence staining, we found that autophagy was activated by increasing the formation of LC3 puncta after treatment with LPS for 6h compared with the control group but that 4-PBA pre-treatment can markedly decrease the expression of LC3 induced by LPS (P<0.05) (Fig.4B). These results indicated that 4-PBA inhibited ER stress, which further affected the activation of autophagy in lung tissues, suggested the crosstalk of ER stress and autophagy probably influenced the pathological process of LPS-induced lung injury together.
.
⦁ 4-PBA protects the viability of A549 cells induced by LPS
In order to mimic LPS-induced lung injury, the effect of LPS on the viability of A549 cells was determined by MTT assay. Treatment of the cells with LPS at concentrations of 1, 5 and 10 µg/ml resulted in significant decreases in cell viability at 24 h, whereas treatment of the cells with LPS at 0.1, and 0.5 µg/ml did not affect cell viability (P>0.05) (Fig.5A). These results suggested that LPS suppressed the growth of A549 cells in a dose-dependent manner. Based on this result, we chose 5 µg/ml LPS as the concentration used in further experiments. At the same time, the MTT cell viability assay showed that the decrease of viability after stimulation with LPS for 24h was rescued in the group pre-treated with 4-PBA (P<0.05) (Fig.5B). 4-PBA had
no significant effect on cell viability when used independently. These data suggested that 4-PBA has a protective effect on A549 cell injury induced by LPS.
⦁ 4-PBA protects A549 cells by attenuating the nuclear translocation of NF-κB p65
In order to test that NF-κB activation is a key signaling mechanism of LPS induced A549 cell injury, we examined the effect of 4-PBA on the protein expression of active NF-κB p65 and IκBα in A549 cells. As shown in Fig.6A-C, The protein level of IKK and nuclear protein NF-κB p65 in A549 cells increased significantly after LPS administration, compared with the control group. LPS also decreased the protein levels of IκB by enhancing IκBα phosphorylation. However, pre-treatment with 4-PBA clearly reduced nuclear translocation of NF-κB p65 induced by LPS (P<0.05). Subsequently, we analyzed the nuclear translocation of NF-κB p65 using immunofluorescence staining, pre-treatment with 4-PBA markedly decreased nuclear translocation of NF-κB p65 induced by LPS (Fig.6D). Furthermore, we detected inflammatory cytokine IL-6 protein levels. As shown in Fig.6E, LPS exposure appeared to increase the cytokine IL-6 protein level, but treatment with 4-PBA markedly attenuated the expression level of IL-6 protein. These results indicated that 4-PBA reduced LPS-induced NF-κB p65 nuclear translocation and pro-inflammatory cytokine production in A549 cells.
⦁ 4-PBA inhibits ER stress and autophagy induced by LPS through
the Akt/mTOR pathway in A549 cells
Our next goal was to demonstrate whether ER stress is involved in the reduction of viability and activation of inflammation in A549 cells induced by LPS. The expressions of three ER stress markers, GRP78, CHOP and Caspase-12, were detected by Western blotting. As shown in Fig.7A-C, LPS significantly increased the expression of GRP78, CHOP and Caspase-12 proteins in A549 cells, and pre-treatment with 4-PBA markedly inhibited the up-regulation of the GRP78, CHOP and Caspase-12 proteins induced by LPS (P<0.05). To further demonstrate which UPR components are involved in the stress ER stress induced by LPS, three proteins ATF6, XBP1 and PERK, respond to the accumulation of unfolded proteins in the ER lumen, were detected by Western blotting. We found that LPS treatment resulted in the accumulation of phosphorylated PERK (p-PERK, Fig.7D). Furthermore, XBP1 and ATF6 were also decreased by treatment with 4-PBA (Fig.7E and F). Collectively, these results suggested that ER stress plays an important role in LPS-induced A549 cell injury.
In order to investigate crosstalk of autophagy and ER stress in LPS-induced acute lung injury. We used MDC and LC3 immunofluorescence staining in our studies, we found that autophagy was activated in A549 cells with an increase in autophagosomes and the formation of LC3 puncta after treatment with LPS for 24h compared with the control group. Compared with the LPS group, autophagic vacuoles were reduced and fluorescence intensity was decreased in the 4-PBA-treated and LPS-induced group (Fig.8A and B). No significant autophagic vacuoles appeared in
the 4-PBA only group, these results are consistent with the results in vivo. Subsequently, we analyzed expression levels of LC3 by Western blotting. As shown in Fig.8C, LPS treatment resulted in accumulation of LC3-II, and the ratios of the amounts of LC3-II to LC3-I were significantly higher than in the control group. 4-PBA markedly inhibited the increase of LC3-II protein expression induced by LPS. All the above data indicated that ER stress might mediate LPS-induced autophagy in A549 cells.
To further probe the molecular mechanism by which 4-PBA inhibited LPS-induced autophagy in A549 cells, we analyzed the classical AKT/mTOR pathway. When cells were treated with 5 µg/ml LPS alone, p-AKT and p-mTOR decreased sharply, but returned to normal levels in the 4-PBA-treated and LPS-induced group (Fig.8D and E). These results indicated that the classical AKT/mTOR pathway is responsible for 4-PBA-mediated inhibition of autophagy induced by LPS in human A549 cells.
⦁ Inhibition of autophagy exacerbates cytotoxicity induced by LPS in A549 cells
In order to probe the specific role of autophagy in vitro, 3-MA was added. Western blotting analysis showed that the amount of LC3-II protein was lower in A549 cells treated with 3-MA, compared with the control group (Fig.9A). Consistent with previous studies, we demonstrated that LPS was able to induce injury in a
dose-dependent manner in A549 cells. MTT assay demonstrated that LPS exposure reduced the cell viability of A549 cells, while co-treatment with 3-MA significantly decreased LPS-induced cell viability compared with the control group. The 3-MA only group showed no changes in the proliferation rate (Fig.9B). These data indicated that autophagy played a protective role in LPS-induced A549 cell injury. To determine whether the protective mechanism of autophagy is related to ER stress, we detected the ER stress-related proteins in A549 cells induced by LPS. We found that 3-MA inhibits autophagy and exacerbates the expression of GRP78 and CHOP proteins induced by LPS (Fig.9C and D). Taken together, we showed that LPS-induced ER stress and cell death could be aggravated by autophagy inhibitor 3-MA, suggested that autophagy probably plays a protective role in LPS-induced A549 cell injury.
⦁ Discussion
Currently, there are no pharmacological approaches available for the treatment of ALI/ARDS, which highlights the urgent need for the development of therapeutic strategies for ALI/ARDS. LPS has been considered one of the important factors leading to lung injury (Opal et al., 1999). LPS challenge leads to the leakage of serous fluids into lung tissues, resulting in the typical symptoms of acute inflammatory responses in the lung. However, the mechanism of inflammation in the development of ALI is unclear. It is well known that ER stress is an adaptive mechanism by which cells react to perturbations in ER homeostasis. However, if ER homeostasis cannot be restored, the prolonged ER stress response may induce injury (Oyadomari and Mori,
2004). In Endo M’s study, the ER stress pathway proteins ATF4 and CHOP were induced after LPS treatment, apoptosis induced by LPS treatment was suppressed in the lung of CHOP knockout mice, and overexpression of CHOP induced apoptosis in a lung cancer-derived cell line (Endo et al., 2005). In ischaemia-reperfusion (I/R) injury, gypenoside markedly improved the cardiac structure and function of I/R injured rats through the blockade of the CHOP pathway and activation of the PI3K/AKT pathway (Yu et al., 2016). In livers from obese mice, the administration of LPS or tunicamycin results in IRE1α and PERK activation, leading to the overexpression of CHOP, which initiating hepatocyte interleukin-1β secretion (Lebeaupin et al., 2015). These results suggested that the ER stress pathway is activated and plays a key role in the pathogenesis of inflammatory disease. The same results were shown in our study. LPS increased expressions of GRP78, CHOP and Caspase-12 in lung, which were decreased by treatment with 4-PBA, which also inhibited the activation of phosphorylation of ER stress sensor proteins ATF6, XBP1 and PERK obviously. Collectively, we found that ER stress is an important player in the development of LPS-induced acute lung injury, providing additional evidence for the pro-inflammatory role of ER stress.
Autophagy is a fundamental homeostatic process and plays a key role in the regulation of intracellular protein, organelle, and metabolic homeostasis (Mizushima et al., 2008). In present study, our results showed that LPS increased the level of autophagy. However, whether this increase of autophagy induced by LPS is related to ER stress is unclear. It has been reported that ER stress-induced autophagy is partly
attributed to the downregulation of AKT/TSC/mTOR pathway in murine embryo fibroblasts (MEFs), pretreatment with 4-PBA partly rescues PI3K/AKT signalling pathway and decreases autophagy (Qin et al., 2010). Moreover, the loss of autophagy-related protein 7 (ATG7) leads to the expected decrease in autophagic flux and also results in ER stress in pancreatic epithelial cells (Antonucci et al., 2015). These diverse studies illustrated that a link exists among autophagy activation and ER stress in various tissues. Herein, our findings showed that LPS triggered ER stress and activated autophagy obviously, both in vivo and in vitro. We hypothesized that ER stress could be the upstream mediator of autophagy. Our data showed that treatment with 4-PBA obvious activated the AKT/mTOR pathway, decreased the expression of LC3II protein, suggested ER stress was responsible for activation of autophagy in LPS-induced A549 cells.
Under ER stress, autophagy is generally considered as a cytoprotective response to the overload of unfolded or misfolded proteins to attenuate apoptosis, which contributes to cardioprotection against ischaemia-reperfusion injury (Petrovski et al., 2011). Another previous study found that inhibiting autophagy promoted ER stress and the pro-inflammatory response in addition to cell death in HepG2 cells treated with LPS (Yin et al., 2016). Moreover, autophagy has also been shown to play a protective role in hyperoxia-induced lung injury (Tanaka et al., 2012). Inhibition of autophagy by LC3 knockdown protects mouse lung epithelial cells from CSE-induced apoptosis (Chen et al., 2008). However, it was also reported that excessive autophagy could result in the programmed cell death (Ding et al., 2007). These findings show
that autophagy is either an adaptive or potentially deleterious process. In our study, we found that treatment with 3-MA significantly decreased LPS-induced expression of LC3II protein in A549 cells. Interestingly, 3-MA treatment decreased cell viability and increased ER stress-related protein expression. These results suggested that inhibition of autophagy using 3-MA aggravates ER stress and A549 cell injury induced by LPS, indicated autophagy also plays a key role in the pathogenesis of LPS-induced lung injury.
In conclusion, our results demonstrated 4-PBA protected LPS-induced lung injury by inhibiting ER stress and inflammation, ER stress-mediated autophagy has cytoprotective effects on LPS-induced lung injury. This study provides a new concept that autophagy could be the downstream process of ER stress, as a cellular defence mechanism against LPS-induced lung injury. These findings may enhance the understanding of lung pathophysiological alterations at the cellular level during ALI.
Conflicts of Interest: The authors declare no conflict of interest.
Author Contributions: Hongyu Zhang and Xiaoxia Kong conceived and designed the experiments; Meichun Zeng performed the experiments; Wenhua Sang, Sha Chen, Zhengmao Li, Ran Chen, Yu Liu analyzed the data; Xiaoxia Kong and Meichun Zeng wrote the paper. Meichun Zeng, FengXue and Hailin Zhang contributed to the preparation of the manuscript.
Funding: This study was partly supported by research grants from the Zhejiang Provincial Natural Science Funding (LY17H010005, LY17H010009, Y17H160193). National Natural Science Funding of China (81472165). Zhejiang Provincial Program of Medical and Health Science (2014KYA131).
References
Antonucci, L., Fagman, J.B., Kim, J.Y., Todoric, J., Gukovsky, I., Mackey, M., Ellisman, M.H., Karin, M., 2015. Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress. Proc Natl Acad Sci U S A 112(45), E6166-6174.
Ather, J.L., Alcorn, J.F., Brown, A.L., Guala, A.S., Suratt, B.T., Janssen-Heininger, Y.M., Poynter, M.E., 2010. Distinct functions of airway epithelial nuclear factor-kappaB activity regulate nitrogen dioxide-induced acute lung injury. Am J Respir Cell Mol Biol 43(4), 443-451.
Axe, E.L., Walker, S.A., Manifava, M., Chandra, P., Roderick, H.L., Habermann, A., Griffiths, G., Ktistakis, N.T., 2008. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 182(4), 685-701.
Biederbick, A., Kern, H.F., Elsasser, H.P., 1995. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66(1), 3-14.
Chen, Z.H., Kim, H.P., Sciurba, F.C., Lee, S.J., Feghali-Bostwick, C., Stolz, D.B., Dhir, R., Landreneau, R.J.,
Schuchert, M.J., Yousem, S.A., Nakahira, K., Pilewski, J.M., Lee, J.S., Zhang, Y., Ryter, S.W., Choi, A.M., 2008. Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease. PLoS One 3(10), e3316.
Chung, H.Y., Cesari, M., Anton, S., Marzetti, E., Giovannini, S., Seo, A.Y., Carter, C., Yu, B.P., Leeuwenburgh, C., 2009. Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev 8(1), 18-30.
Ding, W.X., Ni, H.M., Gao, W., Hou, Y.F., Melan, M.A., Chen, X., Stolz, D.B., Shao, Z.M., Yin, X.M., 2007.
Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J Biol Chem 282(7), 4702-4710.
Endo, M., Oyadomari, S., Suga, M., Mori, M., Gotoh, T., 2005. The ER stress pathway involving CHOP is activated in the lungs of LPS-treated mice. J Biochem 138(4), 501-507.
Gao, M., Liu, D., Du, Y., Sun, R., Zhao, L., 2013. Autophagy facilitates ventilator-induced lung injury partly through activation of NF-kappaB pathway. Med Sci Monit 19, 1173-1175.
Gutierrez, M.G., Master, S.S., Singh, S.B., Taylor, G.A., Colombo, M.I., Deretic, V., 2004. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119(6), 753-766.
Hayashi-Nishino, M., Fujita, N., Noda, T., Yamaguchi, A., Yoshimori, T., Yamamoto, A., 2009. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 11(12), 1433-1437.
Hosoi, T., Ozawa, K., 2010. Endoplasmic reticulum stress in disease: mechanisms and therapeutic
opportunities. Clin Sci (Lond) 118(1), 19-29.
Jin, Y., Tanaka, A., Choi, A.M., Ryter, S.W., 2012. Autophagic proteins: new facets of the oxygen paradox.
Autophagy 8(3), 426-428.
Kim, H.J., Jeong, J.S., Kim, S.R., Park, S.Y., Chae, H.J., Lee, Y.C., 2013. Inhibition of endoplasmic reticulum stress alleviates lipopolysaccharide-induced lung inflammation through modulation of NF-kappaB/HIF-1alpha signaling pathway. Sci Rep 3, 1142.
Lebeaupin, C., Proics, E., de Bieville, C.H., Rousseau, D., Bonnafous, S., Patouraux, S., Adam, G., Lavallard, V.J., Rovere, C., Le Thuc, O., Saint-Paul, M.C., Anty, R., Schneck, A.S., Iannelli, A., Gugenheim, J., Tran, A., Gual, P., Bailly-Maitre, B., 2015. ER stress induces NLRP3 inflammasome activation and hepatocyte death. Cell Death Dis 6, e1879.
Lee, H., Noh, J.Y., Oh, Y., Kim, Y., Chang, J.W., Chung, C.W., Lee, S.T., Kim, M., Ryu, H., Jung, Y.K., 2012.
IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux. Hum Mol Genet 21(1), 101-114.
Matthay, M.A., Ware, L.B., Zimmerman, G.A., 2012. The acute respiratory distress syndrome. J Clin Invest 122(8), 2731-2740.
Mizushima, N., Komatsu, M., 2011. Autophagy: renovation of cells and tissues. Cell 147(4), 728-741.
Mizushima, N., Levine, B., Cuervo, A.M., Klionsky, D.J., 2008. Autophagy fights disease through cellular self-digestion. Nature 451(7182), 1069-1075.
Opal, S.M., Scannon, P.J., Vincent, J.L., White, M., Carroll, S.F., Palardy, J.E., Parejo, N.A., Pribble, J.P., Lemke, J.H., 1999. Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock. J Infect Dis 180(5), 1584-1589.
Oyadomari, S., Mori, M., 2004. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11(4), 381-389.
Petrovski, G., Das, S., Juhasz, B., Kertesz, A., Tosaki, A., Das, D.K., 2011. Cardioprotection by endoplasmic reticulum stress-induced autophagy. Antioxid Redox Signal 14(11), 2191-2200.
Poynter, M.E., Irvin, C.G., Janssen-Heininger, Y.M., 2003. A prominent role for airway epithelial NF-kappa B activation in lipopolysaccharide-induced airway inflammation. J Immunol 170(12), 6257-6265.
Qin, L., Wang, Z., Tao, L., Wang, Y., 2010. ER stress negatively regulates AKT/TSC/mTOR pathway to enhance autophagy. Autophagy 6(2), 239-247.
Ron, D., Walter, P., 2007. Signal integration in the endoplasmic reticulum unfolded protein response.
Nature reviews Molecular cell biology 8(7), 519-529.
Rossol, M., Heine, H., Meusch, U., Quandt, D., Klein, C., Sweet, M.J., Hauschildt, S., 2011. LPS-induced cytokine production in human monocytes and macrophages. Crit Rev Immunol 31(5), 379-446.
Sethi, S., 2010. Infection as a comorbidity of COPD. Eur Respir J 35(6), 1209-1215.
Sir, D., Tian, Y., Chen, W.L., Ann, D.K., Yen, T.S., Ou, J.H., 2010. The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication. Proc Natl Acad Sci U S A 107(9), 4383-4388.
Tanaka, A., Jin, Y., Lee, S.J., Zhang, M., Kim, H.P., Stolz, D.B., Ryter, S.W., Choi, A.M., 2012. Hyperoxia-induced LC3B interacts with the Fas apoptotic pathway in epithelial cell death. Am J Respir Cell Mol Biol 46(4), 507-514.
Terman, A., Brunk, U.T., 2005. Autophagy in cardiac myocyte homeostasis, aging, and pathology.
Cardiovasc Res 68(3), 355-365.
Thurston, T.L., Ryzhakov, G., Bloor, S., von Muhlinen, N., Randow, F., 2009. The TBK1 adaptor and
autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria. Nat Immunol 10(11), 1215-1221.
Ullman, E., Fan, Y., Stawowczyk, M., Chen, H.M., Yue, Z., Zong, W.X., 2008. Autophagy promotes necrosis in apoptosis-deficient cells in response to ER stress. Cell Death Differ 15(2), 422-425.
Wang, Y., Zheng, W., Bian, X., Yuan, Y., Gu, J., Liu, X., Liu, Z., Bian, J., 2014. Zearalenone induces apoptosis and cytoprotective autophagy in primary Leydig cells. Toxicol Lett 226(2), 182-191.
Wang, Z., Zhang, H., Xu, X., Shi, H., Yu, X., Wang, X., Yan, Y., Fu, X., Hu, H., Li, X., Xiao, J., 2012. bFGF
inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways. Toxicol Lett 212(2), 137-146.
Wheeler, A.P., Bernard, G.R., 2007. Acute lung injury and the acute respiratory distress syndrome: a clinical review. Lancet 369(9572), 1553-1564.
Yam, G.H., Gaplovska-Kysela, K., Zuber, C., Roth, J., 2007. Sodium 4-phenylbutyrate acts as a chemical chaperone on misfolded myocilin to rescue cells from endoplasmic reticulum stress and apoptosis. Invest Ophthalmol Vis Sci 48(4), 1683-1690.
Ye, L., Yang, Y., Zhang, X., Cai, P., Li, R., Chen, D., Wei, X., Zhang, X., Xu, H., Xiao, J., Li, X., Lin, L., Zhang, H., 2015. The Role of bFGF in the Excessive Activation of Astrocytes Is Related to the Inhibition of TLR4/NFkappaB Signals. Int J Mol Sci 17(1).
Yin, J.J., Xie, G., Zhang, N., Li, Y., 2016. Inhibiting autophagy promotes endoplasmic reticulum stress and the ROSinduced nodlike receptor 3dependent proinflammatory response in HepG2 cells. Mol Med Rep 14(4), 3999-4007.
Yla-Anttila, P., Vihinen, H., Jokitalo, E., Eskelinen, E.L., 2009. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5(8), 1180-1185.
Yorimitsu, T., Klionsky, D.J., 2007. Endoplasmic reticulum stress: a new pathway to induce autophagy.
Autophagy 3(2), 160-162.
Yu, H., Zhang, H., Zhao, W., Guo, L., Li, X., Li, Y., Zhang, X., Sun, Y., 2016. Gypenoside Protects against Myocardial Ischemia-Reperfusion Injury by Inhibiting Cardiomyocytes Apoptosis via Inhibition of CHOP Pathway and Activation of PI3K/Akt Pathway In Vivo and In Vitro. Cell Physiol Biochem 39(1), 123-136.
Zhang, G., Liu, K., Ling, X., Wang, Z., Zou, P., Wang, X., Gao, J., Yin, L., Zhang, X., Liu, J., Ao, L., Cao, J., 2016. DBP-induced endoplasmic reticulum stress in male germ cells causes autophagy, which has a cytoprotective role against apoptosis in vitro and in vivo. Toxicol Lett 245, 86-98.
Zhang, J., Wang, J.S., Zheng, Z.K., Tang, J., Fan, K., Guo, H., Wang, J.J., 2013. Participation of autophagy in lung ischemia-reperfusion injury in vivo. J Surg Res 182(2), e79-87.
Fig.1. Effect of 4-PBA on structural damage and production of inflammatory mediators induced by LPS in mice. (A) Lung sections of the mice were stained with representative H&E for histological examination (magnification × 200). (B) BAL fluid was performed to differentially count total cells. IL-1β (C), TNF-α (D) and IL-6 (E) in BAL fluid were analyzed by ELISA. Representative confocal laser immunofluorescence photomicrographs showing location of IL-1β (F), TNF-α (G) and IL-6 (H) in BAL cells from different group. Data are reported as mean± S.E (n=5). ** P<0.05versus control group. # P<0.05, ## P<0.01 in comparison to LPS group.
Fig.2. Effect of 4-PBA on NF-κB signaling induced by LPS. Expression of protein IKK (A), IκB (B), NF-κB p65 (Cytoplasm) (C), NF-κB p65 (Nucleus) (D) and IL-6 (E) in lung tissues were detected by Western blotting, relative expression levels of proteins were normalized against GAPDH. (F) Immunostaining analysis of IL-6 (green) in lung tissues from different group
(magnification × 400). Bars represent mean ± SE (n=5). * P<0.05, ** P<0.01 versus control group.
# P<0.05, ## P<0.01 in comparison to LPS group.
Fig.3. Effect of 4-PBA on ER stress induced by LPS. The ER stress relative proteins GRP78 (A), CHOP (B) and Caspase-12 (C) was determined by Western blotting, GAPDH served as a loading control. (D) Immunostaining analysis of PDI (red) in lung tissue from different group
(magnification × 400). * P<0.05, ** P<0.01 versus control group. # P<0.05, ## P<0.01 in
comparison to LPS group.
Fig.4. Effect of 4-PBA on autophagy induced by LPS. (A) The protein levels of LC3 were examined using Western blotting analyses, LC3 expression was quantified by densitometric analysis and normalized to GAPDH expression. (B) Immunostaining analysis of LC3 (green) in lung tissue from different group (magnification × 400). * P<0.05, ** P<0.01 versus control group;
# P<0.05, ## P<0.01 versus the LPS group.
Fig.5. Effect of 4-PBA on A549 cells viability induced by LPS. (A) After exposure to (0.1, 0.5, 1, 5, 10) μg/ml LPS for 24 hours, A549 cell viability was measured by MTT assay. (B) Cell viability was determined in A549 cells treated with by 4-PBA and (or ) LPS for 24 hours. Data are
reported as mean ± SE (n=4), * P<0.05, ** P<0.01 versus control group, # P<0.05 versus LPS
group.
Fig.6. 4-PBA protects A549 cells injury by attenuating nuclear translocation of NF-κB p65. Representative Western blotting of the IKK (A), I-κB (B), NF-κB p65 (C) and IL-6 (E) was showed respectively. GAPDH served as a loading control. (D) Immunofluorescence microscopy analysis was performed for identification of NF-κB p65 translocation (magnification × 1000). The
data are presented as the mean ± SE of 3 independent experiments. * P<0.05, ** P<0.01 versus
control group. # P<0.05, ## P<0.01 versus LPS group.
.
Fig.7. 4-PBA inhibits ER stress induced by LPS in A549 cells. The protein expression of CHOP (A), GRP78 (B), Caspase-12 (C), phosphorylated PERK (D), XBP1 (E) and cleaved ATF6 (F) were examined by Western blotting. GAPDH served as a loading control. Data are presented as
the mean± S.E of 3 independent experiments. * P<0.05, ** P<0.01 versus control group, # P<0.05,
## P<0.01 versus LPS group.
Fig.8. 4-PBA inhibits autophagy induced by LPS by Akt/mTOR signaling in A549 cells. (A) MDC fluorescence staining of autophagic vacuoles in A549 cells treated with LPS and (or) 4-PBA (magnification × 400). (B) Expression of LC3 (green) of A549 cells was analyzed by immunostaining (magnification × 400). (C) Western blotting was performed to detect levels of
protein LC3, p-AKT (D) and p-mTOR (E) in A549 cells, GAPDH served as a loading control.
Data are reported as mean±S.E (n=3). * P<0.05, ** P<0.01versus control group. # P<0.05, ##
P<0.01versus LPS group.
Fig.9. Inhibition of autophagy exacerbates cytotoxicity induced by LPS in A549 cells. (A) The expression of LC3-II protein in A549 cells treated with 3-MA and (or) LPS for 24 hours was determined by Western blotting. The bar graph indicates the relative abundance of the LC3-II protein (normalized to that of GAPDH). (B) Cell viability was determined by MTT assay. ER stress related proteins GRP78(C) and CHOP (D) were analyzed by Western blotting. GAPDH
served as a loading control. Data are reported as mean±S.E (n=3). * P<0.05, ** P<0.01 versus control group, # P<0.05, ## P<0.01 versus LPS group.