Alleviating Effects of Black Soybean Peptide on Oxidative Stress Injury Induced by Lead in PC12 Cells via Keap1/Nrf2/TXNIP Signaling Pathway

Abstract

Many researchers have found that Pb exposure can cause oxidative stress damage to the body’s tissue. Black soybean peptide (BSP) has a variety of physiological functions, especially in terms of oxidative stress. Nevertheless, the mitigation function of BSPs on Pb-induced oxidative stress damage in PC12 cells has not been clearly defined. In this study, cell viability was detected by CCK8. Oxidative stress indicators, such as ROS, GSH/GSSG, MDA, SOD, CAT, GPx, and GR, were tested with biochemical kit. Protein expression of Keap1, Nrf2, and TXNIP was measured by Western blot. Compared with the control group, Pb reduced the cell viability of PC12 cells. However, BSP treatment significantly increased the viability of PC12 cells induced by lead exposure (p < 0.05). Lead can enrich the contents of MDA and ROS, but decrease the amount of CAT, SOD, GR, GPx, and GSH/GSSG in PC12 cells, while BSP can alleviate it (p < 0.05). Lead can enhance the expression of Keap1 and TXNIP proteins, but reduce Nrf2 expression. In contrast, BSPs reversed this phenomenon (p < 0.05). BSPs can alleviate oxidative stress injury induced by lead in PC12 cells through the Keap1/Nrf2/TXNIP signaling pathway.

Keywords: Keap1/Nrf2/TXNIP; PC12 cells; Pb; black soybean peptide; oxidative stress.

Figure 1

Mechanism of action of BSP1, BSP3, and BSP4 in mitigating oxidative stress due to lead exposure.

Figure 2

Cell viability assay. (A) Effects of lead on cell viability in PC12 cells. PC12 cells were incubated with lead (0–160 μM) for 24 h. (B–D) Effects of BSP1, BSP3, and BSP4 cell viability in PC12 cells. PC12 cells were incubated with (B) BSP1, (C) BSP3, and (D) BSP4 for 4 h. (E) Effects of BSP1–5 on cell viability in lead-exposed PC12 cells. PC12 cells were preincubated with BSP1–5 (200 μM) for 4 h, then treated with 10 μM lead for 24 h. The value of the bars indicate the means ± SD (n = at least 3). The same letter means there is no significant difference, and different letters mean there is a significant difference (p < 0.05).

Figure 3

Effects of BSP1, BSP3, BSP4, and VC on intracellular ROS in PC12 cells. (A) PC12 cells were incubated with BSP1, BSP3, BSP4, and VC for 4 h. (B) PC12 cells were preincubated with BSP1, BSP3, BSP4, and VC for 4 h, then treated with lead for 24 h. The value of the bars indicate the means ± SD (n = at least 3). The same letter means there is no significant difference, and different letters mean there is a significant difference (p < 0.05).

Figure 4

Effect of BSP1, BSP3, BSP4, and VC on SOD/CAT/MDA in PC12 cells. (A–C) PC12 cells were incubated with BSP1, BSP3, BSP4, and VC for 4 h. (D–F) PC12 cells were preincubated with BSP1, BSP3, BSP4, and VC for 4 h, then treated with lead for 24 h. SOD results are shown in (A,D). CAT results are shown in (B,E). MDA results are shown in (C,F). The value of the bars indicates the means ± SD (n = at least 3). The same letter means there is no significant difference, and different letters mean there is a significant difference (p < 0.05).

Figure 5

Effect of BSP1, BSP3, BSP4, and VC on GPx/GR/GSH/GSSG in PC12 cells. (A–C) PC12 cells were incubated with BSP1, BSP3, BSP4, and VC for 4 h. (D–F) PC12 cells were preincubated with BSP1, BSP3, BSP4, and VC for 4 h, then treated with lead for 24 h. The results of GPx are shown in (A,D). The results of GR are shown in (B,E). The results of GSH/GSSG are shown in (C,F). The value of the bars indicates the means ± SD (n = at least 3). The same letter means there is no significant difference, and different letters mean there is a significant difference (p < 0.05).

Figure 6

Related protein expression of Keap1/Nrf2/TXNIP signaling pathway. (A) WB results for Keap1, Nrf2 and TXNIP; (B) change in Keap1 protein expression relative to β-actin in different groups; (C) change in Nrf2 protein expression relative to β-actin in different groups; (D) change in TXNIP protein expression relative to β-actin in different groups. The value of the bars indicates the means ± SD (n = at least 3). The same letter means there is no significant difference, and different letters mean there is a significant difference (p < 0.05).