Protective Effect of Ganoderma atrum Polysaccharide on Acrolein-Induced Apoptosis and Autophagic Flux in IEC-6 Cells

This study was designed to explore the beneficial effect and mechanism of Ganoderma atrum (G. atrum) polysaccharide (PSG-1) on acrolein-induced IEC-6 cells. Our results indicated that PSG-1 significantly reduced the impairment of acrolein on cell viability, decreased oxidative stress, and enabled normal expression of tight junction (TJ) proteins that were inhibited by acrolein in IEC-6 cells. Furthermore, PSG-1 attenuated the elevation of microtubule-associated proteins light chain 3 (LC3) and Beclin 1-like protein 1 (Beclin 1) and increased the protein levels of phospho-mTOR (p-mTOR) and phospho-akt (p-akt), indicating that PSG-1 activated the mammalian target of rapamycin (mTOR) signaling pathway and alleviated acrolein-induced autophagy in IEC-6 cells. Moreover, PSG-1 markedly attenuated the acrolein-induced apoptosis, as evidenced by the increase in mitochondrial membrane potential (MMP) and B-cell lymphoma 2 (Bcl-2) expression, and the decrease in cysteine aspartate lyase (caspase)-3 and caspase-9. In addition, autophagy the inhibitor inhibited acrolein-induced TJ and apoptosis of IEC-6 cells, while the apoptosis inhibitor also inhibited acrolein-induced TJ and autophagy, suggesting that autophagy and apoptosis were mutually regulated. Taken together, the present study proved that PSG-1 could protect IEC-6 cells from acrolein-induced oxidative stress and could repair TJ by inhibiting apoptosis and autophagic flux, where autophagy and apoptosis were mutually regulated.


Introduction
Acrolein is an electrophilic, unsaturated aldehyde, a colorless, flammable, volatile, pure liquid, known for its pungent odor and strong irritation of mucous membranes [1], which comes from food, the environment, and the human body itself [2,3]. Acrolein in food is mainly produced by oil fumes or fried and baked foods, which produce acrolein mainly through the Maillard reaction and lipid oxidation [4]. Acrolein is neurotoxic, genotoxic, and potentially carcinogenic, and the question of how to mitigate the side effects of acrolein has generated scholars' attention [5]. Therefore, finding active substances that can reduce or antagonize the toxic effects of acrolein in food is important to protect humans from its harm.
Programmed cell death (PCD) is a self-regulatory mechanism of the organism, mainly including autophagy, apoptosis, and necrosis, the first two of which can play an indispensable role in the life activities of the organism by working closely together. Autophagy is a highly conserved intracellular degradation pathway, a protective mechanism for cells in response to various of hazardous stimuli, which maintains intracellular homeostasis

Apoptosis Rate Detection
The IEC-6 cells were collected after washing with PBS and digesting with trypsin, centrifuged, and 195 µL binding buffer was added to keep the cells in suspension; 5 µL annexin V-FITC and 10 µL PI solutions were added and mixed. The cells were then cultured at 20-25 • C for 10-20 min, protected from light, observed by flow cytometry, and detected within 1 h.

Assay for Mitochondrial Membrane Potential (MMP)
Cells in 6-well plates were washed once with PBS, then 1 mL of cell culture solution and 1 mL of JC-1 staining buffer were added and mixed thoroughly. Cells were cultured in an incubator at 37 • C for 20 min. Following staining, the cells were polished twice with JC-1 staining buffer and observed by fluorescence microscopy.

Inhibitor Experiments
According to the instructions, cells induced by acrolein were pretreated with configured autophagy inhibitor 3-MA and the apoptosis inhibitor Z-DEVD-FMK, respectively. Cellular proteins were extracted after a certain period of time, and the proteins expression were examined by Western blot method to explore the interaction between TJ and autophagy, TJ and apoptosis, and the autophagy and apoptosis pathways.

Statistical Analysis
Statistical analysis was carried out using Graphpad Prism 6.01 software. Results were analyzed by a one-way ANOVA analysis of variance and expressed as the mean ± SD. p < 0.05 was considered statistically significant.

PSG-1 Increased the IEC-6 Cells Viability Exposed to Acrolein
As shown in Figure 1A, acrolein inhibited the viability of IEC-6 cells in a dosedependent manner. Besides, when acrolein concentration was 40 µM, the cell viability was about 50%. Thus, we chose an acrolein concentration of 40 µM for subsequent experiments to detect the potential cytoprotective capacity of PSG-1 (p < 0.01).

Statistical Analysis
Statistical analysis was carried out using Graphpad Prism 6.01 software. Results were analyzed by a one-way ANOVA analysis of variance and expressed as the mean ± SD. p < 0.05 was considered statistically significant.

PSG-1 Increased the IEC-6 Cells Viability Exposed to Acrolein
As shown in Figure 1A, acrolein inhibited the viability of IEC-6 cells in a dosedependent manner. Besides, when acrolein concentration was 40 μM, the cell viability was about 50%. Thus, we chose an acrolein concentration of 40 μM for subsequent experiments to detect the potential cytoprotective capacity of PSG-1 (p < 0.01). Acrolein treatment caused a significant decrease in cell viability, while different concentrations of PSG-1 significantly inhibited the toxicity of acrolein. As can be seen in Figure 1B, all 4 concentration groups were effective, and cell viability tended to enhance and then diminish with increasing PSG-1 concentration, especially at a dose of 80 μg/mL, which almost restored cell viability to normal levels. The differences between the groups were not significant (p < 0.01).

PSG-1 Attenuated Acrolein-Induced Oxidative Damage of IEC-6 Cells
As shown in Figure 2A, the cellular SOD activity declined after acrolein treatment, the addition of PSG-1 restored the SOD activity to different degrees, and the PSG-1 dose group with 160 μg/mL had the highest effect on the increase in SOD activity.  Acrolein treatment caused a significant decrease in cell viability, while different concentrations of PSG-1 significantly inhibited the toxicity of acrolein. As can be seen in Figure 1B, all 4 concentration groups were effective, and cell viability tended to enhance and then diminish with increasing PSG-1 concentration, especially at a dose of 80 µg/mL, which almost restored cell viability to normal levels. The differences between the groups were not significant (p < 0.01).

PSG-1 Attenuated Acrolein-Induced Oxidative Damage of IEC-6 Cells
As shown in Figure 2A, the cellular SOD activity declined after acrolein treatment, the addition of PSG-1 restored the SOD activity to different degrees, and the PSG-1 dose group with 160 µg/mL had the highest effect on the increase in SOD activity.
analyzed by a one-way ANOVA analysis of variance and expressed as the mean ± SD. p < 0.05 was considered statistically significant.

PSG-1 Increased the IEC-6 Cells Viability Exposed to Acrolein
As shown in Figure 1A, acrolein inhibited the viability of IEC-6 cells in a dosedependent manner. Besides, when acrolein concentration was 40 μM, the cell viability was about 50%. Thus, we chose an acrolein concentration of 40 μM for subsequent experiments to detect the potential cytoprotective capacity of PSG-1 (p < 0.01). Acrolein treatment caused a significant decrease in cell viability, while different concentrations of PSG-1 significantly inhibited the toxicity of acrolein. As can be seen in Figure 1B, all 4 concentration groups were effective, and cell viability tended to enhance and then diminish with increasing PSG-1 concentration, especially at a dose of 80 μg/mL, which almost restored cell viability to normal levels. The differences between the groups were not significant (p < 0.01).

PSG-1 Attenuated Acrolein-Induced Oxidative Damage of IEC-6 Cells
As shown in Figure 2A, the cellular SOD activity declined after acrolein treatment, the addition of PSG-1 restored the SOD activity to different degrees, and the PSG-1 dose group with 160 μg/mL had the highest effect on the increase in SOD activity.  In Figure 2B, the MDA level significantly increased after the addition of acrolein, while PSG-1 treatment significantly decreased the MDA contents.
The significant decrease in GPx viability was caused by acrolein. Under the protection of PSG-1, GPx significantly increased especially in the 160 µg/mL dose group ( Figure 2C), indicating that PSG-1 dramatically prevented the inhibition of acrolein-induced oxidative stress (p < 0.05).

Effects of PSG-1 on TJ Proteins Damaged by Acrolein in IEC-6 Cells
TJ mainly includes transmembrane proteins and intracellular plaque proteins, such as Zonula occludens (ZO), different claudin-1 family members, and occludin. As Figure 3 shows, acrolein-induced expression of ZO-1, claudin-1, and occludin were strikingly reduced, indicating that acrolein treatment significantly impaired intestinal barrier function, and PSG-1 treatment clearly promoted the expression of the three proteins (p < 0.05), suggesting that PSG-1 was able to tightly link the reduction in proteins and thus protect the intestinal barrier.
In Figure 2B, the MDA level significantly increased after the addition of acrolein, while PSG-1 treatment significantly decreased the MDA contents.
The significant decrease in GPx viability was caused by acrolein. Under the protection of PSG-1, GPx significantly increased especially in the 160 μg/mL dose group ( Figure 2C), indicating that PSG-1 dramatically prevented the inhibition of acroleininduced oxidative stress (p < 0.05).

Effects of PSG-1 on TJ Proteins Damaged by Acrolein in IEC-6 Cells
TJ mainly includes transmembrane proteins and intracellular plaque proteins, such as Zonula occludens (ZO), different claudin-1 family members, and occludin. As Figure 3 shows, acrolein-induced expression of ZO-1, claudin-1, and occludin were strikingly reduced, indicating that acrolein treatment significantly impaired intestinal barrier function, and PSG-1 treatment clearly promoted the expression of the three proteins (p < 0.05), suggesting that PSG-1 was able to tightly link the reduction in proteins and thus protect the intestinal barrier.  Figure 4B results revealed that the LC3-II/I ratio was significantly higher in acroleintreated cells, while LC3 protein was significantly lower in PSG-1-treated cells than the model group. In addition, we examined key proteins of the PI3K/Akt/mTOR pathway and the Beclin 1 pathway, the phosphorylation levels of mTOR and akt were clearly lower and the level of Beclin 1 was significantly higher in acrolein-induced cells than in the control group. After treating IEC-6 cells with PSG-1, the levels of p-mTOR and p-akt were significantly enhanced, and the content of Beclin 1 was significantly lower than that of the acrolein group (p < 0.05) ( Figure 4C-E). Values are means ± SD (n = 3), * p < 0.05, ** p < 0.01 compared with the acrolein group. Figure 4B results revealed that the LC3-II/I ratio was significantly higher in acroleintreated cells, while LC3 protein was significantly lower in PSG-1-treated cells than the model group. In addition, we examined key proteins of the PI3K/Akt/mTOR pathway and the Beclin 1 pathway, the phosphorylation levels of mTOR and akt were clearly lower and the level of Beclin 1 was significantly higher in acrolein-induced cells than in the control group. After treating IEC-6 cells with PSG-1, the levels of p-mTOR and p-akt were significantly enhanced, and the content of Beclin 1 was significantly lower than that of the acrolein group (p < 0.05) ( Figure 4C-E).

Effect of PSG-1 on Acrolein-Induced Apoptosis
Double staining with annexin V and PI to detect apoptosis showed that the left lower quadrant had live cells and the right lower quadrant had apoptotic cells. Figure 5 showed that after acrolein induction, the apoptosis rate of cells increased significantly, reaching more than 70%, far exceeding that of normal controls. A decrease in the percentage of apoptotic cells could be clearly observed after PSG-1 treatment.

Effect of PSG-1 on Acrolein-Induced Apoptosis
Double staining with annexin V and PI to detect apoptosis showed that the left lower quadrant had live cells and the right lower quadrant had apoptotic cells. Figure 5 showed that after acrolein induction, the apoptosis rate of cells increased significantly, reaching more than 70%, far exceeding that of normal controls. A decrease in the percentage of apoptotic cells could be clearly observed after PSG-1 treatment.

Effect of PSG-1 on Acrolein-Induced Apoptosis
Double staining with annexin V and PI to detect apoptosis showed that the left lower quadrant had live cells and the right lower quadrant had apoptotic cells. Figure 5 showed that after acrolein induction, the apoptosis rate of cells increased significantly, reaching more than 70%, far exceeding that of normal controls. A decrease in the percentage of apoptotic cells could be clearly observed after PSG-1 treatment.

Effects of PSG-1 on MMP Induced by Acrolein in IEC-6 Cells
The process of apoptosis is often accompanied by a disruption of the MMP, which is thought to be one of the earliest events in the apoptotic process. The change of MMP was detected by JC-1 fluorescent probe. It can be seen that cells after acrolein induction have depolarized mitochondrial transmembrane potential and impaired mitochondrial permeability, while PSG-1 treatment restored cell morphology with enhanced red fluorescence intensity and weaker green fluorescence intensity, implying that PSG-1 attenuated acrolein-induced loss of MMP in IEC-6 cells, thereby preserving mitochondria ( Figure 6).

Effects of PSG-1 on MMP Induced by Acrolein in IEC-6 Cells
The process of apoptosis is often accompanied by a disruption of the M thought to be one of the earliest events in the apoptotic process. The change detected by JC-1 fluorescent probe. It can be seen that cells after acrolein ind depolarized mitochondrial transmembrane potential and impaired m permeability, while PSG-1 treatment restored cell morphology with en fluorescence intensity and weaker green fluorescence intensity, implying attenuated acrolein-induced loss of MMP in IEC-6 cells, thereby preserving m ( Figure 6).

Effects of PSG-1 on Apoptotic Proteins Damaged by Acrolein in IEC-6 Cells
As depicted in Figure 7, acrolein significantly promoted the two posit proteins, caspase-3 and caspase-9, and significantly declined the level of a protein Bcl-2. The addition of PSG-1 decreased the expression of caspase-3 a proteins and increased the content of Bcl-2 protein (p < 0.05), which can indic 1 can inhibit acrolein-induced apoptosis.

Effects of PSG-1 on Apoptotic Proteins Damaged by Acrolein in IEC-6 Cells
As depicted in Figure 7, acrolein significantly promoted the two positively related proteins, caspase-3 and caspase-9, and significantly declined the level of anti-apoptotic protein Bcl-2. The addition of PSG-1 decreased the expression of caspase-3 and caspase-9 proteins and increased the content of Bcl-2 protein (p < 0.05), which can indicate that PSG-1 can inhibit acrolein-induced apoptosis.

Effects of Autophagy Inhibitor and Apoptosis Inhibitor on the TJ in IEC-6 Cells
After treating cells with autophagy inhibitor and apoptosis inhibitor, we separately inspected the expression of three TJ proteins, and it was found that the expression of TJ protein was markedly increased in IEC-6 cells, where the inhibitor co-interacted with acrolein, compared with acrolein alone (p < 0.05) (Figure 8).

Effects of Autophagy Inhibitor and Apoptosis Inhibitor on the TJ in IEC-6 Cells
After treating cells with autophagy inhibitor and apoptosis inhibitor, we separately inspected the expression of three TJ proteins, and it was found that the expression of TJ protein was markedly increased in IEC-6 cells, where the inhibitor co-interacted with acrolein, compared with acrolein alone (p < 0.05) (Figure 8).

Effects of Autophagy Inhibitor and Apoptosis Inhibitor on the TJ in IEC-6 Cells
After treating cells with autophagy inhibitor and apoptosis inhibitor, we separately inspected the expression of three TJ proteins, and it was found that the expression of TJ protein was markedly increased in IEC-6 cells, where the inhibitor co-interacted with acrolein, compared with acrolein alone (p < 0.05) (Figure 8).

The Effects of Autophagy Inhibitor on IEC-6 Cell Apoptosis
Autophagy inhibitor treatment of IEC-6 cells significantly decreased the increase in acrolein-induced autophagy-related protein LC3 ( Figure 9B). After that, we examined the expression of apoptosis-related proteins Bcl-2, caspase-3, and caspase-9, and the results demonstrated that the autophagy inhibitor reduced the overexpression of acroleininduced, apoptosis-related proteins caspase-3 and caspase-9 and increased the expression of anti-apoptotic protein Bcl-2 (p < 0.05) ( Figure 9C-E).

The Effects of Autophagy Inhibitor on IEC-6 Cell Apoptosis
Autophagy inhibitor treatment of IEC-6 cells significantly decreased the increase in acrolein-induced autophagy-related protein LC3 ( Figure 9B). After that, we examined the expression of apoptosis-related proteins Bcl-2, caspase-3, and caspase-9, and the results demonstrated that the autophagy inhibitor reduced the overexpression of acrolein-induced, apoptosis-related proteins caspase-3 and caspase-9 and increased the expression of antiapoptotic protein Bcl-2 (p < 0.05) ( Figure 9C-E).

The Effects of Autophagy Inhibitor on IEC-6 Cell Apoptosis
Autophagy inhibitor treatment of IEC-6 cells significantly decreased the increase in acrolein-induced autophagy-related protein LC3 ( Figure 9B). After that, we examined the expression of apoptosis-related proteins Bcl-2, caspase-3, and caspase-9, and the results demonstrated that the autophagy inhibitor reduced the overexpression of acroleininduced, apoptosis-related proteins caspase-3 and caspase-9 and increased the expression of anti-apoptotic protein Bcl-2 (p < 0.05) ( Figure 9C-E).

The Effects of Apoptosis Inhibitor on IEC-6 Cell Autophagy
In Figure 10B, treatment of IEC-6 cells with apoptosis inhibitors markedly inhibited the increase in acrolein-induced, apoptosis-related protein caspase-3. To verify the apoptosis inhibitor effect on cellular autophagy, we examined the expression of

The Effects of Apoptosis Inhibitor on IEC-6 Cell Autophagy
In Figure 10B, treatment of IEC-6 cells with apoptosis inhibitors markedly inhibited the increase in acrolein-induced, apoptosis-related protein caspase-3. To verify the apoptosis inhibitor effect on cellular autophagy, we examined the expression of autophagyrelated proteins Beclin 1, and the result showed that apoptosis inhibitor significantly reduced acrolein-induced overexpression of autophagy-related protein Beclin 1 (p < 0.05) ( Figure 10C).

Discussion
The intestinal epithelial cells (IECs) have roles in the functions of digestion absorption of nutrients [25], and the barrier function preventing toxic substances from entering the body, etc. [3,26]. They are the main component of the mechanical barrier o the intestinal mucosa and the first line of defense of the body against intestinal bacteria and toxins [27]. In this study, acrolein was found to cause a reduction in growth and viability of IEC-6 cells, and its cytotoxic action on IEC-6 cells was a concentration dependent action, with a concentration of 40 μM significantly impairing the viability Meanwhile, several concentrations of PSG-1 extracts were found to have cytoprotective effects against acrolein-induced damage in IEC-6 cells.
GPx is an essential peroxidase enzyme that is extensively present in living organisms [28]. MDA and SOD content is a vital indicator of the potential antioxidant capacity of the body [29,30]. Meanwhile, we found that acrolein induced oxidative stress in IEC-6 cells decreased the SOD activity and GPx activity, and increased the intracellular lipid peroxide MDA content. PSG-1 prevented acrolein-induced oxidative stress by improving GPx and SOD levels and reducing MDA content. Therefore, we hypothesized that PSG-1 altered the antioxidant activity in IEC-6 cells under the effect of acrolein to enhance the antioxidant capacity of cells and prevented oxidative stress damage to cells, which indirectly achieved cell protection. The mechanism may be due to the PSG-1 combining with radicals to form a stable radical, or combining with the radical ions, which are

Discussion
The intestinal epithelial cells (IECs) have roles in the functions of digestion, absorption of nutrients [25], and the barrier function preventing toxic substances from entering the body, etc. [3,26]. They are the main component of the mechanical barrier of the intestinal mucosa and the first line of defense of the body against intestinal bacteria and toxins [27]. In this study, acrolein was found to cause a reduction in growth and viability of IEC-6 cells, and its cytotoxic action on IEC-6 cells was a concentration-dependent action, with a concentration of 40 µM significantly impairing the viability. Meanwhile, several concentrations of PSG-1 extracts were found to have cytoprotective effects against acrolein-induced damage in IEC-6 cells.
GPx is an essential peroxidase enzyme that is extensively present in living organisms [28]. MDA and SOD content is a vital indicator of the potential antioxidant capacity of the body [29,30]. Meanwhile, we found that acrolein induced oxidative stress in IEC-6 cells, decreased the SOD activity and GPx activity, and increased the intracellular lipid peroxide MDA content. PSG-1 prevented acrolein-induced oxidative stress by improving GPx and SOD levels and reducing MDA content. Therefore, we hypothesized that PSG-1 altered the antioxidant activity in IEC-6 cells under the effect of acrolein to enhance the antioxidant capacity of cells and prevented oxidative stress damage to cells, which indirectly achieved cell protection. The mechanism may be due to the PSG-1 combining with radicals to form a stable radical, or combining with the radical ions, which are necessary for radical chain reaction, and then the reaction is terminated [18].
The TJ of IECs is the main component connecting IECs to the intestinal mucosal barrier, which regulates the permeability of barrier and is the first line of defense of cell bypass [31]. The TJ determines the electrolyte and water balance in the intestine, which is the key factor of intestinal integrity and mucosal barrier function, and it consists mainly of occludin, claudins, and other protein families, which are composed of the linker complex proteins (ZO-1), and cytoskeletal structures which form the TJ complex [32]. Three common TJ proteins-occludin, claudin, and ZO-1-were assayed. Acrolein was able to decrease the level of TJ proteins, while the PSG-1 treatment markedly enhanced the expression of these proteins, which is beneficial for maintaining the integrity of the intestinal mucosa. This is consistent with the oxidative damage index data. The experiment suggests that PSG-1 can maintain intestinal barrier function by up-regulating the expression level of TJ proteins in IEC-6 cells.
Autophagy, known as type II programmed cell death, is a self-protective mechanism widely present in eukaryotic cells [33]. To confirm autophagy is engaged in the protection of acrolein-damaged IEC-6 cells by PSG-1, the expression of key proteins of autophagy was inspected. The expression ratios of LC3-II and LC3-I can be used as important indicators for autophagy experiments. The ratio of LC3-II/I was significantly higher in IEC-6 cells treated with acrolein, suggesting that autophagy was promoted by acrolein exposure.
Autophagy is a complex signaling process with multi-step regulations. Studies have revealed that autophagy mainly consists of the PI3K/Akt/mTOR pathway and the Beclin 1 pathway, which is a key regulation protein of autophagy [34]. The mTOR signaling pathway is a critical regulation of autophagy initiation, activated mTOR inhibits autophagy [35]. After acrolein treatment, the expression of Beclin 1 increased, and the p-mTOR and p-akt content significantly decreased. Moreover, PSG-1 therapy significantly reduced LC3-II/I and Beclin 1 expression and promoted p-mTOR and p-akt expression. Experiments suggested that PSG-1 was able to inhibit acrolein-stimulated excessive autophagy, which may be achieved by promoting the mTOR pathway.
Apoptosis is a fundamental and important biological phenomenon that plays an essential role in the clearance of abnormal cells [36]. It was found that acrolein caused apoptosis of IEC-6 cells at pathological concentrations. To investigate whether apoptosis has a function in acrolein-induced IEC-6 cells, we first looked at the rate of apoptotic cell death. The experiments suggested that the apoptosis rate of cells remarkably elevated after acrolein induction and decreased significantly after the addition of PSG-1, which suggested that acrolein can induce IEC-6 cells apoptosis and PSG-1 can protect IEC-6 cells by reducing the number of apoptotic cells. Various studies have shown that mitochondria are closely related to apoptosis and that apoptosis is irreversible once the transmembrane potential of the mitochondrial membrane collapses. Therefore, the change of MMP was detected, the result showed that IEC-6 cells treatment with acrolein caused MMP loss and PSG-1 treatment significantly improved this situation.
Afterwards, the expression of apoptosis-related proteins was also examined. Bcl-2 is located upstream of mitochondria and an important regulation of the altered permeability of mitochondrial membranes. Caspase-3 and caspase-9 play an irreplaceable role in apoptosis. Caspase-9 involves the initiation of apoptosis. Caspase-3 is the most critical apoptosis executor downstream of the caspase cascade reaction. We found that after acrolein induction of IEC-6 cells, the expression of Bcl-2 was significantly decreased, the expression of two apoptotic proteins, caspase3 and caspase-9, was significantly increased, and PSG-1 restored the expression of these three proteins to a certain extent. The above data suggested that acrolein-induced IEC-6 cells led to MMP loss, increased expression of caspase 3 and caspase 9 and decreased expression of the Bcl-2 protein, which indicated that PSG-1 effectively restored acrolein-induced apoptosis through the mitochondrial pathway. It had been established that the biological activity of mushroom polysaccharides were attributed to their chemical structure [14,37]. Thus, we speculated that the regulation of autophagy and apoptosis by PSG-1 was most likely related to the main structure β-(1 → 3, 1 → 6)-glucan, which was elucidated in our previous study [18,38].
Numerous studies have shown that autophagy and apoptosis play an invaluable function in the intestinal barrier and jointly maintain the integrity of the intestinal barrier. To explore the association between autophagy/apoptosis and TJ, we pretreated IEC-6 cells with specific inhibitors of autophagy and apoptosis pathways, respectively, examining the expression of occludin, claudin, and ZO-1 in each group. It was found that the expression of TJ proteins was increased after treatment of IEC-6 cells with two inhibitors, suggesting that acrolein-induced autophagy and apoptosis occur in IEC-6 cells could disrupt the integrity of the intestinal barrier.
It is well known that a complex interactive regulation exists between autophagy and apoptosis [39]. The relationship between autophagy and apoptosis in acrolein-induced IEC-6 cells by pathway inhibitors was explored in this experiment. It was revealed that the expression of autophagy-related proteins was reduced in autophagy inhibitor-treated IEC-6 cells, and that autophagy inhibitor inhibited the expression of key apoptotic proteins, suggesting that autophagy may be modulated by acrolein-induced apoptosis of IEC-6 cells. Apoptosis inhibitor-treated cells showed reduced expression of apoptosis-related proteins. In addition, the apoptosis inhibitor inhibited the expression of key autophagic proteins, suggesting that apoptosis may be modulated by acrolein-induced autophagy of IEC-6 cells.

Conclusions
This study proved that PSG-1 repaired oxidative stress in acrolein-induced IEC-6 cells, restored the expression of TJ proteins inhibited by acrolein, and attenuated acrolein-induced autophagy and apoptosis in IEC-6 cells. Furthermore, PSG-1 restored TJ by inhibiting apoptosis and autophagic flux, where autophagy and apoptosis were mutually regulated. The finding may provide a theoretical basis for the development and generation of mushroom β-(1 → 3, 1 → 6)-glucan as high value-added products.