*2.3. Cell Viability*

A 96-well microplate format was used. After 24 h of incubation, treating IEC-6 cells with acrolein (0–80 μM) for 20 h, a solution of CCK-8 was added to each well. Finally, the optical absorbance was read at 450 nm with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Under the same cultural conditions as above, PSG-1 at concentrations of 20, 40, 80, and 160 (μg/mL) were added to each group, while a blank control group was set up. The effect of PSG-1 on IEC-6 cells viability was detected.

#### *2.4. Antioxidant Enzyme Assays*

After cell culture, cells were washed once with phosphate-buffered saline (PBS), cells were digested with trypsin, and they were collected by centrifugation for 10 min at 1000× *g*. Protein concentrations were determined using the BCA protein assay kit, superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GPx) (Beyotime Biotechnology Nanjing, China) were estimated by referring to the instructions.

#### *2.5. 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.

#### *2.6. 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.

#### *2.7. Western Blot Analysis*

Protein samples were prepared by treating IEC-6 cells with acrolein and PSG-1 in 6 well plates, after 10% sodium dodecyl sulfate (SDS)-PAGE electrophoresis, and transported to a PVDF membrane (Millipore Co., Belford, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h. Membrane were incubated with primary antibodies (1:1000): ZO-1, claudin-1, occludin, LC3B, Beclin 1, phospho-mammalian target of rapamycin (p-mTOR), phospho-Protein Kinase B (p-akt), caspase-3, caspase-9, Bcl-2, and beta actin monoclonal antibody (β-actin) (CST, Boston, MA, USA) at 4 ◦C overnight under shaking and incubated with horseradish peroxidase-conjugated secondary antibodies (ZSGB Biotechnology, Beijing, China) for 1 h at room temperature. After incubation in ECL detection reagent, the fluorescent signal was detected using the Molecular Imager ChemiDoc™ XRS Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

#### *2.8. 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.

#### *2.9. 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.

#### **3. Results**

#### *3.1. 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).

#### *3.2. 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.

**Figure 2.** PSG-1 regulated acrolein-induced SOD, MDA, and Gpx. The oxidative state of IEC-6 cells, following acrolein (40 μM) and PSG-1 (20, 40, 80, and 160 μg/mL) treatment, was determined by measuring SOD (**A**), MDA (**B**) and GPx (**C**).Values are means ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 versus the acrolein group.

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).
