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. 2021 Jun 18;12(1):77.
doi: 10.1186/s40104-021-00595-x.

Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

Caiyun Huang  1 , Zijuan Fan  1 , Dandan Han  1 , Lee J Johnston  2 , Xi Ma  1   3 , Fenglai Wang  4
Affiliations

Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

Caiyun Huang et al. J Anim Sci Biotechnol. .

Abstract

Background: Oxidative stress is a main cause of piglet gut damage and diarrhea. Pyrroloquinoline quinone (PQQ), is a novel redox cofactor with antioxidant properties. However, the effect and mechanism that PQQ supplementation decreases oxidative injury in weaned pigs is not understood. Therefore, the aim of this study is to confirm the effect of PQQ on regulating redox status in weaned pigs and the mechanism for antioxidant function by porcine intestinal epithelial cell line (IPEC-J2) challenged with H2O2.

Results: Experiment 1, 144 Duroc × Landrace × Yorkshire pigs (weaned at 28 d) were allocated to four groups: received a basal diet (control) and diets supplemented with 0.15%, 0.30% and 0.45% PQQ, respectively. On d 28, growth performance, diarrhea incidence and redox factors were measured. Experiment 2, IPEC-J2 were treated with or without PQQ in the presence or absence of H2O2 for indicated time points. Experiment 3, IPEC-J2 were transfected with or without Nrf2 siRNA, then treated according to Experiment 2. The cell viability, redox factors, protein of tight junctions and Nrf2 pathway were determined. In vivo, PQQ supplementation demonstrated dose-related improvements in average daily gain, and gain to feed ratio (Linear P < 0.05). During d 0-28, compared to controls, 0.45% PQQ supplementation for pigs decreased diarrhea incidence and MDA content in liver and jejunum, and increased concentration of SOD in liver; 0.3% PQQ supplementation decreased ileal and liver MDA concentration; and 0.15% PQQ supplementation decreased ileal MDA concentration (P < 0.05). In vitro, compared to cells cultured with H2O2, pre-treatment with PQQ increased cell viability, tight junction proteins expression including ZO-1, ZO-2, Occludin and Claudin-1; and decreased ROS concentration and level of Caspase-3 (P < 0.05); as well as upregulated the ratio of Bcl-2 to Bax and protein expression of nuclear Nrf2, HO-1. Notably, Nrf2 knockdown by transfection with Nrf2 siRNA largely abrogated the positive effects of PQQ pretreatment on H2O2-induced intracellular changes.

Conclusions: PQQ administration attenuated oxidative stress in weaned pigs which is associated with activation of Nrf2/HO-1 pathway.

Keywords: H2O2; IPEC-J2; Nrf2; Oxidative stress; Pyrroloquinoline quinone; Weaned pig.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effects of PQQ and H2O2 on cell viability in vitro. a, Cells were incubated with H2O2 for 2, 6, 18 and 24 h at concentrations of 0, 200 μmol/L, 500 μmol/L, 800 μmol/L, 1 mmol/L, 1.5 mmol/L and 2 mmol/L; b, Cells were incubated with PQQ at concentrations of 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 μmol/L, 10 μmol/L and 100 μmol/L for 6, 18, 36 and 48 h; c, Cells were pretreated with PQQ for 6 h at concentrations of 1 nmol/L, 10 nmol/L, or 100 nmol/L and were then treated with both H2O2 (200 μmol/L) and PQQ for another 2 h. Cell viability in all treatment groups were evaluated with a CCK-8 assay. CTRL, cells cultured in basal medium; H2O2, cells cultured with 200 μmol/L H2O2; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both 200 μmol/L H2O2 and 10 nmol/L PQQ for another 2 h. * denotes a significant difference (P < 0.05) with respect to the CTRL group. # denotes a significant difference (P < 0.05) with respect to the H2O2 group. n = 6
Fig. 2
Fig. 2
Effects of PQQ on oxidative cytokines and tight junction proteins in cells cultured with H2O2. a-c, Levels of SOD, GSH-Px and MDA in IPEC-J2 cells. d, Western blot analysis of ZO-1, ZO-2, ZO-3, occludin, and claudin-1. Densitometric values were normalized to those of Tubulin. e, Statistical analysis of the data in D. CTRL, cells cultured in basal medium; H2O2, cells cultured with 200 μmol/L H2O2; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both 200 μmol/L H2O2 and 10 nmol/L PQQ for another 2 h. * denotes a significant difference (P < 0.05) with respect to the CTRL group. # denotes a significant difference (P < 0.05) with respect to the H2O2 group. n = 3
Fig. 3
Fig. 3
Effects of PQQ on levels of ROS and ROS-regulating proteins in cells cultured with H2O2. a, Level of ROS in IPEC-J2 cells. b, Western blot analysis of bax, bcl-2 and Caspase-3. Densitometric values were normalized to those of Tubulin. CTRL, cells cultured with basal medium; H2O2, cells cultured with 200 μmol/L H2O2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both 200 μmol/L H2O2 and 10 nmol/L PQQ for another 2 h. * denotes a significant difference (P < 0.05) with respect to the CTRL group. # denotes a significant difference (P < 0.05) with respect to the H2O2 group. n = 3
Fig. 4
Fig. 4
Effects of PQQ on proteins expression of Nrf2 and HO-1 in cells cultured with H2O2. a, Immunofluorescence staining of cytoplasmic and nuclear Nrf2 in IPEC-J2 cells. The scale bar represents 50 μm. b, Western blot analysis of nuclear Nrf2, cytoplasmic Nrf2, and HO-1. Densitometric values were normalized to those of PCNA or β-actin, as appropriate; c-e, Statistical analysis of the data in B. CTRL, cells cultured with basal medium; H2O2, cells cultured with 200 μmol/L H2O2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both 200 μmol/L H2O2 and 10 nmol/L PQQ for another 2 h. n = 3
Fig. 5
Fig. 5
Effects of PQQ on levels of Nrf2 and HO-1 in cells treated with siRNA. a Nrf2 knockdown efficiency in cells transfected with three different sequences of Nrf2 siRNA or with NC siRNA. b and c mRNA expression levels of Nrf2 and HO-1 in IPEC-J2 cells transfected with or without Nrf2 siRNA-2. d Western blot analysis of Nrf2 and HO-1 in cells transfected with or without Nrf2 siRNA-2. e Statistical analysis of the data in D. CTRL, cells cultured with basal medium; H2O2, cells cultured with 200 μmol/L H2O2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H2O2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H2O2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H2O2 for another 2 h. * denotes a significant difference (P < 0.05) with respect to the CTRL group. # denotes a significant difference (P < 0.05) with respect to the H2O2 group. n = 3
Fig. 6
Fig. 6
Effects of PQQ on levels of ROS and ROS-regulated proteins in cells treated with siRNA. a Level of ROS in IPEC-J2 cells treated with or without Nrf2 siRNA-2. b-c Ratio of Bcl-2/Bax mRNA levels and mRNA expression levels of Caspase-3 in IPEC-J2 cells treated with or without Nrf2 siRNA-2. d Western blot analysis of cytoplasmic Bax, Bcl-2 and Caspase-3. Densitometric values were normalized to those of β-actin. e Statistical analysis of the data in D. f Statistical analysis of the Bcl-2/Bax protein ratio. CTRL, cells cultured with basal medium; H2O2, cells cultured with 200 μmol/L H2O2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H2O2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H2O2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H2O2 for another 2 h. n = 3
Fig. 7
Fig. 7
Effects of PQQ on tight junctions expression and cell viability in cells treated with siRNA. a Western blot analysis of ZO-1, ZO-2, Occludin and Claudin-1 in IPEC-J2 cells treated with or without Nrf2 siRNA-2. The densitometric values were normalized to those of β-actin. n = 3. b Viability of IPEC-J2 cells treated with or without Nrf2 siRNA-2 and incubated in the presence or absence of H2O2 or PQQ. CTRL, cells cultured with basal medium; H2O2, cells cultured with 200 μmol/L H2O2 for 2 h; PQQ, cells cultured with 10 nmol/L PQQ for 6 h and then incubated with both H2O2 (200 μmol/L) and PQQ (10 nmol/L) for another 2 h. NC siRNA, cells transfected with NC siRNA and then pretreated with PQQ for 6 h prior to incubation with both PQQ and H2O2 for another 2 h. Nrf2 siRNA, cells transfected with Nrf2 siRNA-2, pretreated with PQQ for 6 h, and then incubated with both PQQ and H2O2 for another 2 h. n = 6. * denotes a significant difference (P < 0.05) with respect to the CTRL group. # denotes a significant difference (P < 0.05) with respect to the H2O2 group
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