*3.3. 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 3.** PSG-1 regulated the expression of TJ-related proteins in acrolein-induced IEC-6 cells. (**A**) The protein levels of occludin, claudin, and ZO-1. (**B**–**D**) Relative intensities of these proteins. Values are means ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 compared with the acrolein group.

#### *3.4. Effects of PSG-1 on Autophagic Proteins Damaged by Acrolein in IEC-6 Cells*

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).

**Figure 4.** PSG-1-regulated, acrolein-induced autophagy-related proteins. (**A**) The protein levels of LC3, Beclin 1, p-mTOR, and p-akt. (**B**–**E**) Relative intensities of these proteins. Values are means ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 compared with the acrolein group.

#### *3.5. 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.

**Figure 5.** PSG-1 attenuated acrolein-induced apoptosis. Apoptosis measured by flow cytometry. (**A**) control cells; (**B**) the cells treated with acrolein, (**C**–**F**) the cells treated with acrolein and PSG-1 at concentrations of 20, 40, 80and 160μg/mL, (**G**) Effect of PSG-1 on acrolein-induced apoptosis. Values are means ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 compared with the acrolein group.

#### *3.6. 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 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).

**Figure 6.** PSG-1 attenuated acrolein-inducedMMP reduction. MMP was detected by fluorescence microscopy.

#### *3.7. 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.

**Figure 7.** PSG-1 regulated the expression of apoptosis-related proteins in acrolein-induced IEC-6 cells. (**A**) The protein levels of caspase-9, caspase-3, and Bcl-2. (**B**–**D**) Relative intensities of these proteins. Values are means ± SD (*n* = 3), \* *p* < 0.05, \*\* *p* < 0.01 compared with the acrolein group.
