Oxyresveratrol Induces Autophagy via the ER Stress Signaling Pathway, and Oxyresveratrol-Induced Autophagy Stimulates MUC2 Synthesis in Human Goblet Cells

Background: Autophagy is a cell protection system invoked to eliminate the damaged organelles and misfolded proteins that induce various stresses, including endoplasmic reticulum (ER) stress. Autophagy can control mucin secretion in goblet cells. Oxyresveratrol (OXY), an antioxidant, stimulates expression of MUC2. Thus, we investigated the effect of OXY on autophagy and found that OXY-induced autophagy stimulates MUC2 expression in human intestinal goblet cells. Methods: Autophagy-related genes and proteins were examined by quantitative real-time PCR (qPCR) and Western blotting, respectively. Autophagy was assessed by immunocytochemistry (ICC). To analyze the protein expression profiles of OXY-treated LS 174T goblet cells, two-dimensional electrophoresis (2DE) and peptide mass fingerprinting (PMF) were performed. MUC2 expression in cells was evaluated by ICC. Results: OXY significantly increased the expression levels of genes related to autophagy induction, and activated phagosome elongation resulted in the formation of autophagosomes. OXY also activated the ER stress signaling pathway and promoted MUC2 synthesis, which was inhibited by treatment with an autophagy inhibitor. Conclusion: OXY induces autophagy via the ER stress signaling pathway, and OXY-induced autophagy increases MUC2 production in intestinal goblet cells.


Introduction
Autophagy is a self-digesting process that is characterized by the removal of damaged organelles, misfolded proteins, and old or nonfunctional proteins [1,2]. This system is a type of survival mechanism that is triggered in response to diverse stresses, such as starvation and hypoxia, to maintain intracellular homeostasis. The primary role of autophagy is protection against cell death, and the process has a role in stress management, immunity, and preventing various disorders such as cancer [3], diabetes, and obesity [4]. Autophagy deficiency promotes tumorigenesis [5,6] and causes neurodegenerative diseases [7] and chronic inflammation [8].
Autophagy is divided into three types according to the mechanisms by which cargo is transported to the lysosomes or vacuoles. Macroautophagy is a mechanism that delivers cargo to the lysosome through autophagosomes that are composed of double membrane-bound vesicles. In microautophagy, cargo is directly transported to the lysosome itself. Chaperone-mediated autophagy Table 1. Primers used for qPCR analysis.

Autophagy Detection
LS 174T cells were seeded at a density of 1.25 × 10 5 cells/mL on a coverslip with 0.1% gelatin in 24-well plates and incubated with various concentrations of OXY at 37 • C for 18 h. The cells were stained with CYTO-ID ® Green Detection Reagent (Enzo Life Sciences, Farmingdale, IL, USA) for Antioxidants 2020, 9, 214 5 of 19 30 min at 37 • C and then washed with assay buffer according to the manufacturer's instructions. Nuclei were counterstained with Hoechst 33342 (Enzo Life Science, Farmingdale, IL, USA). Autophagic vacuole accumulation and flux were both detected by fluorescence microscopy using a Nikon C1 plus confocal laser scanning microscope (Nikon, Tokyo, Japan).

Statistical Analysis
All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA) version 24.0. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. The statistical significance of differences between samples was determined with Student's t-test. Statistical differences among groups were determined using one-way analysis of variance (ANOVA) followed by post hoc Tukey's HSD (honestly significant difference) test. A p value of < 0.05 was considered statistically significant.

Cytotoxicity of OXY in LS 174T Goblet Cells
The cytotoxic effect of OXY on LS 174T goblet cells was evaluated after treatment with OXY for 24 h using the MTT assay. The relative viabilities of cells treated with 2.5, 5, 10, and 20 µg/mL OXY were 101.7 ± 6.7%, 100.1 ± 4.7%, 99.4 ± 5.1%, and 91.6 ± 6.1%, respectively, compared with the negative control ( Figure 1). As the viability of the cells treated with 20 µg/mL OXY was significantly reduced, we used 2.5, 5, and 10 µg/mL OXY for ensuing experiments in this study.
Antioxidants 2020, 9, 214 5 of 19 LS 174T cells were seeded at a density of 1.25 × 10 5 cells/mL on a coverslip with 0.1% gelatin in 24-well plates and incubated with various concentrations of OXY at 37 °C for 18 h. The cells were stained with CYTO-ID ® Green Detection Reagent (Enzo Life Sciences, Farmingdale, IL, USA) for 30 min at 37 °C and then washed with assay buffer according to the manufacturer's instructions. Nuclei were counterstained with Hoechst 33342 (Enzo Life Science, Farmingdale, IL, USA). Autophagic vacuole accumulation and flux were both detected by fluorescence microscopy using a Nikon C1 plus confocal laser scanning microscope (Nikon, Tokyo, Japan).

Statistical Analysis
All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA) version 24.0. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. The statistical significance of differences between samples was determined with Student's t-test. Statistical differences among groups were determined using one-way analysis of variance (ANOVA) followed by post hoc Tukey's HSD (honestly significant difference) test. A p value of < 0.05 was considered statistically significant.

Cytotoxicity of OXY in LS 174T Goblet Cells
The cytotoxic effect of OXY on LS 174T goblet cells was evaluated after treatment with OXY for 24 h using the MTT assay. The relative viabilities of cells treated with 2.5, 5, 10, and 20 µg/mL OXY were 101.7 ± 6.7%, 100.1 ± 4.7%, 99.4 ± 5.1%, and 91.6 ± 6.1%, respectively, compared with the negative control ( Figure 1). As the viability of the cells treated with 20 µg/mL OXY was significantly reduced, we used 2.5, 5, and 10 µg/mL OXY for ensuing experiments in this study.

Effect of OXY on Autophagy Development at Specific Times
Time is an important factor in autophagy induction; thus, observation of autophagy at a specific time point is susceptible to inaccurate interpretations [23,24]. To determine the appropriate time to induce autophagy by treatment with OXY, the expression levels of autophagy-related proteins were measured by Western blotting at various time points. OXY (10 µg/mL) significantly increased the protein expression of Atg7, Atg5, and LC3B at 18 and 24 h but Beclin-1 significantly increased only at 18 h ( Figure 2). Therefore, subsequent assays were performed at 18 h or 24 h.

Effect of OXY on Autophagy Development at Specific Times
Time is an important factor in autophagy induction; thus, observation of autophagy at a specific time point is susceptible to inaccurate interpretations [23,24]. To determine the appropriate time to induce autophagy by treatment with OXY, the expression levels of autophagy-related proteins were measured by Western blotting at various time points. OXY (10 µg/mL) significantly increased the protein expression of Atg7, Atg5, and LC3B at 18 and 24 h but Beclin-1 significantly increased only at 18 h ( Figure 2). Therefore, subsequent assays were performed at 18 h or 24 h.

OXY Increases Expression of Autophagy-Related Factors
Phagophore formation is initiated by activation of the ULK complex consisting of ULK1, ULK2, Atg13, and Atg101 as well as Beclin-1, which plays a critical role in the initial step in phagophore formation [25]. To investigate the effect of OXY on the early stage of phagophore formation, the expression levels of phagophore formation-related genes were measured by qPCR after 10 µg/mL OXY treatment for 18 h. Levels of ULK2, Atg13, Atg101, and Beclin-1 in 2.5, 5, and 10 µg/mL OXYtreated cells increased significantly in a dose-dependent manner compared with the negative control ( Figure 3A). Levels of ULK1 and VPS34 were also significantly increased in 5 µg/mL OXY-treated cells compared with negative control cells. These results suggest that OXY induces autophagy.
Autophagosome elongation results in the formation of a double-membrane vesicle, which is mainly driven by the Atg5-Atg12-Atg16L1 complex and LC3B conjugation system [1,2]. To investigate the effect of OXY on autophagosome elongation, the expression levels of genes that induce double-membrane vesicle formation were assessed. Compared with the negative control, expression levels of Atg7, Atg4, Atg5, Atg16L1, and LC3B were significantly elevated by OXY at 2.5, 5, and 10 µg/mL in a dose-dependent manner ( Figure 3B), as were the protein expression levels of Beclin-1, Atg7, Atg5, and LC3B-II, which is converted from LC3B-I ( Figure 3C,D). To further investigate the effect of OXY on autophagic vesicle formation, cells were treated with a green fluorescence dye that

OXY Increases Expression of Autophagy-Related Factors
Phagophore formation is initiated by activation of the ULK complex consisting of ULK1, ULK2, Atg13, and Atg101 as well as Beclin-1, which plays a critical role in the initial step in phagophore formation [25]. To investigate the effect of OXY on the early stage of phagophore formation, the expression levels of phagophore formation-related genes were measured by qPCR after 10 µg/mL OXY treatment for 18 h. Levels of ULK2, Atg13, Atg101, and Beclin-1 in 2.5, 5, and 10 µg/mL OXY-treated cells increased significantly in a dose-dependent manner compared with the negative control ( Figure 3A). Levels of ULK1 and VPS34 were also significantly increased in 5 µg/mL OXY-treated cells compared with negative control cells. These results suggest that OXY induces autophagy.
Autophagosome elongation results in the formation of a double-membrane vesicle, which is mainly driven by the Atg5-Atg12-Atg16L1 complex and LC3B conjugation system [1,2]. To investigate the effect of OXY on autophagosome elongation, the expression levels of genes that induce double-membrane vesicle formation were assessed. Compared with the negative control, expression levels of Atg7, Atg4, Atg5, Atg16L1, and LC3B were significantly elevated by OXY at 2.5, 5, and 10 µg/mL in a dose-dependent manner ( Figure 3B), as were the protein expression levels of Beclin-1, Atg7, Atg5, and LC3B-II, which is converted from LC3B-I ( Figure 3C,D). To further investigate the effect of OXY on autophagic vesicle formation, cells were treated with a green fluorescence dye that stains autophagic vacuoles in live cells. Significantly increased fluorescent signals were observed in cells treated with OXY in a dose-dependent manner, particularly with regard to increased punctate structure formation (arrows in Figure 3E), compared with the negative control ( Figure 3E,F). The formation of green puncta indicates that LC3 was recruited to form autophagosomes [23]. The number of puncta increased with 10 µg/mL OXY treatment, whereas no puncta were observed in the negative control. These results suggest that OXY effectively enhances the formation of autophagosomes followed by autophagy induction.
structure formation (arrows in Figure 3E), compared with the negative control ( Figure 3E,F). The formation of green puncta indicates that LC3 was recruited to form autophagosomes [23]. The number of puncta increased with 10 µg/mL OXY treatment, whereas no puncta were observed in the negative control. These results suggest that OXY effectively enhances the formation of autophagosomes followed by autophagy induction.
The ultimate objective of autophagy is degradation of internal material after fusion with the lysosome [26]. To investigate the effect of OXY on autolysosome formation, the expression levels of genes related to autolysosome formation were measured after OXY treatment for 24 h. Levels of Rab7, STX17, VAMP8, and LAMP2 significantly increased in cells treated with 2.5, 5, and 10 µg/mL OXY in a dose-dependent manner compared with the negative control ( Figure 3G). These results show that OXY induces autophagosome-lysosome fusion by stimulating the expression of genes related to autolysosome formation. Figure 3. Effect of OXY on the expression levels of autophagy-related genes and proteins. Expression levels of genes related to phagophore formation were quantified by qPCR (A). Expression levels of autophagosome elongation-related genes were quantified by qPCR (B). Expression levels of autophagosome formation-related proteins were measured by Western blotting (C) and quantified (D). From each lysate, equal amounts of protein were loaded on a separate gel, blotted for actin and this signal was used for determining the ratio of the protein of interest/actin as displayed in the figure. The cropped blots are representative of three independent experiments. The full-length blots are shown in Supplementary Figure S2. The effect of OXY on autophagic vesicle formation was measured by staining with a fluorescence dye using a CYTO-ID autophagy detection kit; white arrows indicate autophagosomes (magnification 600×) (E), which were quantified (F). Expression levels of autolysosome formation-related genes were quantified by qPCR (G). Each value indicates the mean ± SD of three independent experiments performed in triplicate. The ultimate objective of autophagy is degradation of internal material after fusion with the lysosome [26]. To investigate the effect of OXY on autolysosome formation, the expression levels of genes related to autolysosome formation were measured after OXY treatment for 24 h. Levels of Rab7, STX17, VAMP8, and LAMP2 significantly increased in cells treated with 2.5, 5, and 10 µg/mL OXY in a dose-dependent manner compared with the negative control ( Figure 3G). These results show that OXY induces autophagosome-lysosome fusion by stimulating the expression of genes related to autolysosome formation.

3-Methyladenine (3-MA) Decreases the Effect of OXY on Autophagy
To confirm the effect of OXY on autophagy, we used 3-MA, which inhibits autophagy by blocking the early stage of autophagosome formation, and found that expression levels of phagosome-related genes decreased significantly in cells cotreated with OXY and 3-MA compared with those treated with OXY alone ( Figure 4A). In addition, the level of autophagic vesicle formation in cells cotreated with OXY and 3-MA decreased by 56.3% compared with the cells treated with OXY alone (Figure 4B,C). Interestingly, the significant inhibitory effect of 3-MA on autophagy was not observed in cells treated with 3-MA alone compared with the negative control, which may be because autophagy is less active under basal conditions. Therefore, the results suggest that OXY is an inducer of autophagosome formation.
Antioxidants 2020, 9, 214 8 of 19 Figure 3. Effect of OXY on the expression levels of autophagy-related genes and proteins. Expression levels of genes related to phagophore formation were quantified by qPCR (A). Expression levels of autophagosome elongation-related genes were quantified by qPCR (B). Expression levels of autophagosome formation-related proteins were measured by Western blotting (C) and quantified (D). From each lysate, equal amounts of protein were loaded on a separate gel, blotted for actin and this signal was used for determining the ratio of the protein of interest/actin as displayed in the figure.
The cropped blots are representative of three independent experiments. The full-length blots are shown in Supplementary Figure S2. The effect of OXY on autophagic vesicle formation was measured by staining with a fluorescence dye using a CYTO-ID autophagy detection kit; white arrows indicate autophagosomes (magnification 600×) (E), which were quantified (F). Expression levels of autolysosome formation-related genes were quantified by qPCR (G). Each value indicates the mean ± SD of three independent experiments performed in triplicate.

3-Methyladenine (3-MA) Decreases the Effect of OXY on Autophagy
To confirm the effect of OXY on autophagy, we used 3-MA, which inhibits autophagy by blocking the early stage of autophagosome formation, and found that expression levels of phagosome-related genes decreased significantly in cells cotreated with OXY and 3-MA compared with those treated with OXY alone ( Figure 4A). In addition, the level of autophagic vesicle formation in cells cotreated with OXY and 3-MA decreased by 56.3% compared with the cells treated with OXY alone ( Figure 4B,C). Interestingly, the significant inhibitory effect of 3-MA on autophagy was not observed in cells treated with 3-MA alone compared with the negative control, which may be because autophagy is less active under basal conditions. Therefore, the results suggest that OXY is an inducer of autophagosome formation.

OXY Stimulates the Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway
To examine the mechanism by which OXY induces autophagy, we investigated whether OXY affects the MAPK pathway, a potential autophagy induction pathway. The levels of MEK, ERK1, ERK2, JNK1, JNK2, and p38 expression increased significantly in 2.5, 5, and 10 µg/mL OXY-treated cells ( Figure 5). Accordingly, the results demonstrate that OXY stimulates the MAPK signaling pathway.

OXY Stimulates the Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway
To examine the mechanism by which OXY induces autophagy, we investigated whether OXY affects the MAPK pathway, a potential autophagy induction pathway. The levels of MEK, ERK1, ERK2, JNK1, JNK2, and p38 expression increased significantly in 2.5, 5, and 10 µg/mL OXY-treated cells ( Figure 5). Accordingly, the results demonstrate that OXY stimulates the MAPK signaling pathway.

Effect of MAPK kinase Inhibitors on Expression Levels of Autophagy-Related Genes and Proteins
To confirm that the OXY-stimulated MAPK signaling pathway is involved in autophagy, inhibitors of MEK/ERK (U0126), JNK (SP600125), and p38 (SB203580) were applied. In cells cotreated with OXY and U0126, expression levels of Beclin-1, Atg5, and Atg7 decreased significantly compared with OXY-only-treated cells, whereas the level of LC3B increased in cells cotreated with OXY and U0126. In contrast, cotreatment with OXY and SP600125 or SB203580 did not decrease gene expression ( Figure 6A). Interestingly cotreatment with OXY and SP600125 significantly increased expression levels of Beclin-1 and Atg7, which might indicate that OXY and an JNK inhibitor show somehow a costimulatory effect on autophagosome formation. The lack of an inhibitory effect on Beclin-1, Atg5, and Atg7 after exposure to SB203580 indicates that OXY-enhanced p38 signaling did not have autophagic effects. Thus, we only used U0126 in subsequent analyses to investigate whether OXY increases autophagic effects through the MEK/ERK signaling pathway.
Based on our gene expression data, we measured the level of ERK protein expression and found significant increases in phosphorylated ERK in 2.5, 5, and 10 µg/mL OXY-treated cells compared with the negative control ( Figure 6B,C). We further investigated the expression levels of LC3B-I and LC3B-

Effect of MAPK kinase Inhibitors on Expression Levels of Autophagy-Related Genes and Proteins
To confirm that the OXY-stimulated MAPK signaling pathway is involved in autophagy, inhibitors of MEK/ERK (U0126), JNK (SP600125), and p38 (SB203580) were applied. In cells cotreated with OXY and U0126, expression levels of Beclin-1, Atg5, and Atg7 decreased significantly compared with OXY-only-treated cells, whereas the level of LC3B increased in cells cotreated with OXY and U0126. In contrast, cotreatment with OXY and SP600125 or SB203580 did not decrease gene expression ( Figure 6A). Interestingly cotreatment with OXY and SP600125 significantly increased expression levels of Beclin-1 and Atg7, which might indicate that OXY and an JNK inhibitor show somehow a costimulatory effect on autophagosome formation. The lack of an inhibitory effect on Beclin-1, Atg5, and Atg7 after exposure to SB203580 indicates that OXY-enhanced p38 signaling did not have autophagic effects. Thus, we only used U0126 in subsequent analyses to investigate whether OXY increases autophagic effects through the MEK/ERK signaling pathway.
Based on our gene expression data, we measured the level of ERK protein expression and found significant increases in phosphorylated ERK in 2.5, 5, and 10 µg/mL OXY-treated cells compared with the negative control ( Figure 6B,C). We further investigated the expression levels of LC3B-I and LC3B-II, which revealed that U0126 significantly decreased the levels of phosphorylated ERK and Atg5 proteins ( Figure 6D,E). Conversely, U0126 had no significant inhibitory effect on LC3B-II protein expression. The results suggest that Atg5, which forms a complex with other Atg proteins and participates in autophagosome membrane elongation, is stimulated through MEK/ERK signaling induced by OXY treatment but that LC3B, which accounts for the major complex of double-membraned autophagosomes and is most widely used as an autophagy marker, is not associated.
II, which revealed that U0126 significantly decreased the levels of phosphorylated ERK and Atg5 proteins ( Figure 6D,E). Conversely, U0126 had no significant inhibitory effect on LC3B-II protein expression. The results suggest that Atg5, which forms a complex with other Atg proteins and participates in autophagosome membrane elongation, is stimulated through MEK/ERK signaling induced by OXY treatment but that LC3B, which accounts for the major complex of doublemembraned autophagosomes and is most widely used as an autophagy marker, is not associated. Figure 6. Effect of MAPK kinase inhibitors on the expression of autophagy-related genes and proteins. Cells were treated with OXY (10 µg/mL) with or without MEK inhibitor (U0126), JNK inhibitor (SP600125), and p38 inhibitor (SB203580). Expression levels of Beclin-1, Atg7, Atg5, and LC3B were determined by qPCR (A). Expression levels of phosphorylated ERK protein were measured by Western blotting (B) and quantified (C). Expression levels of phosphorylated ERK, Atg5, and LC3B proteins were measured by Western blotting (D) and quantified (E). From each lysate, equal amounts of protein were loaded on a separate gel, blotted for actin and this signal was used for determining the ratio of the protein of interest/actin as displayed in the figure. The cropped blots are representative of three independent experiments. The full-length blots are shown in Supplementary Figures S3 and S4. Each value indicates the mean ± SD of three independent experiments performed in triplicate.

OXY Stimulates the ER Stress Signaling Pathway
To further elucidate the mechanism by which OXY stimulates autophagy, we performed 2DE by selecting protein spots showing significant expression variation that deviated by over two-fold compared with the negative control. In total, 13 proteins were identified by MALDI-TOF-MS ( Table 2). The expression level of AP-1 complex subunit beta-1 (AP1B1) was markedly increased compared with other proteins. AP1B1 is found in the trans-Golgi network and mediates recruitment of clathrin, which interacts with Atg16L1 and is involved in the formation of early autophagosome precursors [27]. Among the 13 proteins, 5 (PHB, GANAB, HSPA5, HSP90B1, and HSP90AA1) are associated with protein processing in the endoplasmic reticulum (ER) according to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and a network of the 5 protein interactions based on STRING, a meta-database program that generates a network of protein interactions from high-throughput experimental data, is shown in Figure 7A. The ER stress response is a protective process that is triggered by the accumulation of misfolded proteins in the ER. As ER stress is known to induce autophagy, we explored the effect of OXY on the expression of ER stress-related genes and proteins. OXY significantly increased IRE1α, XBP1, PERK, and ATF4 mRNA expression in a dose-dependent manner compared with the negative control ( Figure 7B). Furthermore, protein expression of IRE1α, which is the most conserved factor of the ER stress signaling branch, was significantly increased in OXY-treated cells in a dose-dependent manner compared with the negative control ( Figure 7C,D).

An ER Stress Inhibitor Decreases Expression Levels of Autophagy-Related Genes and Proteins
To investigate whether the effect of OXY on autophagy involves the ER stress signaling pathway, 4-PBA that is a well-known ER stress inhibitor was used. Although ATF4 levels did not decrease

An ER Stress Inhibitor Decreases Expression Levels of Autophagy-Related Genes and Proteins
To investigate whether the effect of OXY on autophagy involves the ER stress signaling pathway, 4-PBA that is a well-known ER stress inhibitor was used. Although ATF4 levels did not decrease significantly, levels of Beclin-1, Atg7, Atg5, IRE1α, PERK, ATE4, and XBP1 were significantly decreased in cells cotreated with OXY and 4-PBA compared with cells treated only with OXY; in contrast, 4-PBA did not inhibit expression of LC3B ( Figure 8A). However, unlike U0126, 4-PBA significantly suppressed the conversion of LC3B-I to LC3B-II ( Figure 8B,C). Cotreatment with OXY and 4-PBA also significantly decreased the levels of Beclin-1, Atg7, and Atg5 expression compared with the treatment with OXY alone. Moreover, the degree of autophagic vesicle formation decreased significantly by 42.3% in cells cotreated with OXY and 4-PBA compared with OXY-only-treated cells ( Figure 8D,E). These results suggest that the effect of OXY on autophagy might be associated with ER stress signaling.

OXY-Induced Autophagy Affects Expression of MUC2
Goblet cells are colon epithelial cells that specialize in mucin production, and OXY stimulates MUC2 expression [17]. To investigate whether OXY-induced autophagy stimulates MUC2 production, the expression level of MUC2 was measured in cells treated with OXY with or without the autophagy inhibitor 3-MA. OXY significantly increased the level of MUC2 expression, which was significantly decreased in cells cotreated with OXY and 3-MA ( Figure 9A). Although expression level of MUC2 increased in 3-MA only-treated cells, the increase was not statistically significant compared with the negative control. Similarly, the level of MUC2 protein expression decreased significantly by 49.3% in cells cotreated with OXY and 3-MA compared with OXY only-treated cells ( Figure 9B,C). These results show that cotreatment of OXY with 3-MA significantly decreased OXY-dependent MUC2 expression. Unlike gene expression, the expression level of the MUC2 protein did not rise in cells treated with 3-MA alone compared with the negative control. This low correlation between mRNA and protein expression patterns may be because 3-MA inhibits PI3K protein expression. Therefore, OXY stimulates MUC2 expression, which might be associated with autophagy induction by OXY. Figure 8. Effect of an ER stress inhibitor on expression of autophagy-related genes and proteins. Cells were treated with OXY (10 µg/mL) with or without the ER stress inhibitor 4-PBA. Expression levels of IRE1α, XBP1, PERK, ATF4, Beclin-1, Atg5, Atg7, and LC3B were determined by qPCR (A). Expression levels of Beclin-1, Atg7, Atg5, and LC3B proteins were measured by Western blotting (B) and quantified (C). From each lysate, equal amounts of protein were loaded on a separate gel, blotted for actin and this signal was used for determining the ratio of the protein of interest/actin as displayed in the figure. The cropped blots are representative of three independent experiments. The full-length blots are shown in Supplementary Figure S6. Autophagic vesicle formation was detected using the CYTO-ID autophagy green dye and observed by fluorescence microscopy; white arrows indicate autophagosomes (magnification 600×) (D), which were quantified (E). Each value indicates the mean ± SD of three independent experiments performed in triplicate.

OXY-Induced Autophagy Affects Expression of MUC2
Goblet cells are colon epithelial cells that specialize in mucin production, and OXY stimulates MUC2 expression [17]. To investigate whether OXY-induced autophagy stimulates MUC2 production, the expression level of MUC2 was measured in cells treated with OXY with or without the autophagy inhibitor 3-MA. OXY significantly increased the level of MUC2 expression, which was significantly decreased in cells cotreated with OXY and 3-MA ( Figure 9A). Although expression level of MUC2 increased in 3-MA only-treated cells, the increase was not statistically significant compared 49.3% in cells cotreated with OXY and 3-MA compared with OXY only-treated cells ( Figure 9B,C). These results show that cotreatment of OXY with 3-MA significantly decreased OXY-dependent MUC2 expression. Unlike gene expression, the expression level of the MUC2 protein did not rise in cells treated with 3-MA alone compared with the negative control. This low correlation between mRNA and protein expression patterns may be because 3-MA inhibits PI3K protein expression. Therefore, OXY stimulates MUC2 expression, which might be associated with autophagy induction by OXY.

Discussion
OXY shows higher antioxidant activity than does resveratrol, a well-known antioxidant [15]. Antioxidants such as resveratrol, quercetin, and vitamin C protect cells and tissues against free radicals that cause damage to cells, proteins, and DNA. Thus, antioxidant supplementation can significantly improve the immune system against homeostatic disturbances [28][29][30]. The immune system is a complicated and exquisite network that is able to maintain homeostasis under normal physiological conditions [31], and antioxidants play a role in immune homeostasis. One of the mechanisms for maintaining homeostasis is the autophagy system, which induces the degradation of malfunctioning cellular molecules via autophagosomes and lysosomes [32]. Resveratrol induces autophagy by directly inhibiting the mTOR signaling pathway in HeLa cells [33], and OXY activates autophagy via inhibition of PI3K/AKT/mTOR and activation of p38 MAPK signaling pathways in neuroblastoma cells [34]. Although mTOR induces antioxidant activity, both reports did not show any direct evidences on autophagy induction by antioxidant properties of resveratrol and OXY. In

Discussion
OXY shows higher antioxidant activity than does resveratrol, a well-known antioxidant [15]. Antioxidants such as resveratrol, quercetin, and vitamin C protect cells and tissues against free radicals that cause damage to cells, proteins, and DNA. Thus, antioxidant supplementation can significantly improve the immune system against homeostatic disturbances [28][29][30]. The immune system is a complicated and exquisite network that is able to maintain homeostasis under normal physiological conditions [31], and antioxidants play a role in immune homeostasis. One of the mechanisms for maintaining homeostasis is the autophagy system, which induces the degradation of malfunctioning cellular molecules via autophagosomes and lysosomes [32]. Resveratrol induces autophagy by directly inhibiting the mTOR signaling pathway in HeLa cells [33], and OXY activates autophagy via inhibition of PI3K/AKT/mTOR and activation of p38 MAPK signaling pathways in neuroblastoma cells [34]. Although mTOR induces antioxidant activity, both reports did not show any direct evidences on autophagy induction by antioxidant properties of resveratrol and OXY. In general, as ROS increase, ER stress is induced. OXY treatment did not induce ROS production in neuroblastoma cells, which means that OXY-induced autophagy might be independent of antioxidant property of OXY. Therefore, further studies are needed to verify whether antioxidant property of OXY induces autophagy or OXY-induced autophagy leads to reduction of ROS production. In this study, we found that OXY induces autophagy in intestinal goblet cells through the ER stress signaling pathway, which might enhance mucin production.
In the autophagy process, a flat membrane (phagophore) is formed in response to various stimuli by two protein complexes; one complex is the ULK complex consisting of Atg13, Atg101, ULK1/2, and FIP200, and the other complex is the PI3K complex comprising Atg14L, Beclin1, Vps34, Vps15, and Ambra1 [35]. Under normal conditions, the ULK kinase complex is activated by the inhibition of mTOR, and formation of the isolation membrane using plasma membranes, the ER, and the Golgi apparatus is initiated [36,37]. The PI3K complex then continues to form a phagophore. Beclin-1, which contains a BH-3 domain, a central coiled-coil domain (CCD), and an evolutionarily conserved domain (ECD), plays a central role in regulating phagophore formation. Under normal conditions, the Bcl-2 (B-cell lymphoma 2) protein inhibits autophagy by interacting with the BH-3 domain in Beclin-1. However, when this interaction is disrupted, Beclin-1 alternatively binds with Vps34 and Vps15 through the CCD and ECD [38]. In the present study, OXY treatment increased the expression of genes related to phagophore formation. In particular, OXY significantly increased the Beclin-1 protein expression. Additionally, OXY increased mRNA expression of genes associated with autophagosomes. Autophagosome formation results from elongation of the phagophore, and this organelle is formed via conjugation of Atg5-Atg12-Atg16L1 and LC3B, a major autophagosomal marker. First, Atg12 conjugates with Atg5 with the aid of Atg7 and Atg10; next, Atg12-Atg5 forms the Atg5-Atg12-Atg16L1 complex located on the outer surface of the autophagosomal membrane. Second, phosphatidylethanolamine (PE)-conjugated LC3B-II, which is converted from LC3B-I, participates in the development of the inner and outer surfaces of the autophagosome, and autolysosome formation is completed by fusion with lysosomes. In this study, we demonstrate that OXY enhances mRNA expression of genes associated with each step of autophagy development. In particular, OXY significantly increased the expression of LC3B-II, which is widely used for the evaluation of autophagy activity in a dose-dependent and time-dependent manner. Overall, OXY activates autophagy from the initial step of autophagy induction, which was confirmed by using the autophagy inhibitor 3-MA.
Diverse signaling pathways participate in the induction of autophagy in mammalian cells [39], among which the MAPK pathway is notable. Three MAPK families, ERK, JNK, and p38 kinases, have been well characterized as being involved in many biological processes by regulating actions such as apoptosis, proliferation, and signaling cascades in mammalian cells [40]. In our previous study, OXY improved tight junction integrity through the MAPK signaling pathway in a colonic epithelial cell line [14]. Resveratrol and OXY upregulate autophagy via the MAPK signaling pathway. Resveratrol induces apoptosis and autophagy by inhibiting Akt/mTOR and activating the p38-MAPK pathway [41]. It is reported that resveratrol exerts neuroprotective effects by modulating mitochondrial dynamics and upregulating autophagic flux via the MEK/ERK pathway [42]. OXY also changes the activity of p38 MAPK and PI3K/AKT/mTOR, which induces apoptosis cell death independent of autophagy [34]. In our study, OXY stimulated the expression levels of genes related to MAPK kinases (ERK, JNK, and p38); however, when cells were cotreated with the respective inhibitors, except for the ERK inhibitor, gene expression was not inhibited. OXY promoted phosphorylation of the ERK1/2 protein, and the expression level of ERK decreased by cotreatment with OXY and the MAPK/ERK kinase inhibitor U0126. Interestingly, U0126 addition attenuated the effect of OXY on Atg5 expression at both transcriptional and translational levels; nonetheless, inhibition of MEK/ERK signaling did not decrease the LC3B level after OXY exposure. The results suggest that OXY-dependent Beclin-1, Atg7, and Atg5 stimulation may partially occur via the MEK/ERK signaling pathway but that OXY-induced autophagy might ultimately not be regulated by MAPK kinase. The results suggest that activated Atg5 functions in non-autophagic roles, as demonstrated previously [43,44]. Further studies are needed to explain why and how the MEK/ERK pathway is involved in only Atg5 expression but not in that of LC3B.
Our 2D analysis revealed five proteins (PHB, GANAB, HSPA5, HSP90B1, and HSP90AA1) corresponding to the KEGG pathway known as protein processing in the ER to be highly connected. In the ER, correctly folded proteins are packaged into transport vesicles followed by transferring to the Golgi complex, whereas misfolded proteins are not packaged. Accumulation of such misfolded proteins causes ER stress and activates the unfolded protein response (UPR) signaling pathway for the purpose of preventing further damage [45]. GRP78/BiP is a major ER chaperone protein in the ER stress signaling pathway. When GRP78 is released from the ER, three main branches involving IRE1α, PERK, and ATF6 are activated through interaction with the GRP78 protein. Among them, IRE1α is the only ER stress sensor in yeast and the most conserved branch for the UPR signaling pathway in animals [46]. When IRE1α is activated by interacting with GRP78, IRE1α is autophosphorylated, which generates XBP1s, the spliced form from unspliced XBP1. A transcription factor, XBP1s translocates to the nucleus, where it upregulates the expression of target genes that maintain homeostasis and promote cell survival [45,47,48]. The ER stress signaling pathway is closely connected to autophagy, and the IRE1α and XBP1-mediated ER stress signaling pathway induces autophagy by converting LC3B-I to LC3B-II and enhancing Beclin-1 expression [49]. OXY increased the expression levels of ER stress signaling pathway-related genes and the IRE1α protein. Unlike a MEK1/2 inhibitor, the ER stress inhibitor 4-PBA suppressed the effect of OXY in increasing protein expression of both Atg5 and LC3B. This finding indicates that OXY promotes autophagy via ER stress signaling, resulting in increased Atg5 and LC3B expression.
Mucin is known as the major component in the intestinal mucus layer, and the intestinal mucosal barrier plays a critical role in maintaining intestinal homeostasis and preventing external damage. Autophagy promotes expression of MUC2 [19,50,51], and OXY induces MUC2 expression in intestinal goblet cells [17]. In this study, we found that OXY activates autophagy and that OXY-induced MUC2 expression is decreased by an ER inhibitor. The results suggest that OXY activates autophagy, which might result in stimulation of MUC2 expression.

Conclusions
Autophagy is known to promote the production of mucins in goblet cells. OXY induces autophagy via ER stress signaling pathways, and OXY-induced autophagy increases MUC2 expression in human LS 174T goblet cells. These findings suggest that OXY may be used for the treatment of intestinal disorders caused by disruption of the intestinal mucus layer through regulation of the autophagy process in the intestinal epithelium.

Conflicts of Interest:
The authors declare that they have no competing interests.