*3.8. 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).

**Figure 8.** *Cont*.

**Figure 8.** Autophagy and apoptosis inhibitor attenuated TJ. (**A**) The protein levels of occludin, claudin, and ZO-1, "+" indicates addition of acrolein or PSG-1 and "-" means not added. (**B**–**G**) Relative intensities of these proteins. Values are means ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 compared with the acrolein group.

#### *3.9. 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).

**Figure 9.** Autophagy inhibitor attenuated apoptosis. (**A**) The protein levels of LC3, caspase-9, caspase-3, and Bcl-2. (**B**–**E**) Relative intensities of these proteins. Values are means ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 compared with the acrolein group.

#### *3.10. 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).

**Figure 10.** Apoptosis inhibitor attenuated autophagy. (**A**) The protein levels of caspase-3 and Beclin 1 proteins. (**B**,**C**) Relative intensities of these proteins. Values are means ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 compared to ACR group.

#### **4. 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.

#### **5. 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.

**Author Contributions:** Conceptualization, Q.Y.; formal analysis, Y.W.; funding acquisition, Q.Y.; investigation, Y.W.; methodology, Y.W.; project administration, Y.C., X.H. (Xiaobo Hu) and Q.Y.; resources, B.Z.; software, Y.C. and X.H. (Xiaobo Hu); supervision, J.X. and J.S.; visualization, Y.C. and X.H. (Xiaobo Hu); writing—original draft preparation, X.C.; writing—review and editing, X.C., J.X., J.S., X.H. (Xiaoyi Hu), X.D. and Q.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (81760737).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

