Peroxiredoxin 6 mediates the protective function of curcumin pretreatment in acute lung injury induced by serum from patients undergoing one-lung ventilation in vitro

Abstract

Background: Curcumin has attracted much attention due to its wide range of therapeutic effects. In this study, we used serum collected from patients undergoing one-lung ventilation (OLV) to establish an in vitro acute lung injury (ALI) model to explore the potential protective mechanism of curcumin on ALI. Our study provides a new reference for the prevention and treatment of ALI induced by OLV.

Methods: A549 cells were treated with 20% serum from patients undergoing OLV to establish an in vitro ALI model. Curcumin, at a dose of 40 μg/ml, was administered two hours prior to this model. The levels of inflammation and oxidative stress markers were observed by Western blot, qRT-PCR, ELISA and reactive oxygen species assay. Additionally, the expression of peroxiredoxin 6 (Prdx6) and proteins involved in the NF-κB signaling pathway was evaluated.

Results: Twenty percent of serum collected from patients undergoing OLV downregulated the expression of Prdx6, leading to the activation of the NF-κB signaling pathway, which was associated with the subsequent overproduction of inflammatory cytokines and reactive oxygen species. Pretreatment with curcumin restored Prdx6 downregulation and inhibited NF-κB pathway activation by suppressing the nuclear translocation of P65, eventually reducing inflammation and oxidative stress damage in A549 cells.

Conclusions: Prdx6 mediated the protective function of curcumin by inhibiting the activation of the NF-κB pathway in ALI in vitro.

Keywords: Curcumin; Lung injury; NF-κB signaling; One-lung ventilation; Peroxiredoxin 6.

Fig. 1

Twenty percent OLV_after serum induced the most obvious inflammation and oxidative stress in A549 cells. A Representative image of cell morphology after treatment with different concentrations of serum. Scale bar = 100 μm. B Western blot analysis images of IL-6 in each group. C The relative protein levels of IL-6 were calculated. D qRT–PCR analysis of IL-6 in each group. E ROS levels were measured by DCFH-DA staining, and the relative DCF-DA fluorescence intensities were normalized and summarized. Scale bar = 100 μm. The results are shown as the means ± SD of 3 individual experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

Different concentrations of curcumin alleviate inflammation and oxidative stress in A549 cells. A Western blot analysis images of IL-6 in each group. B The relative protein levels of IL-6 were calculated. C qRT–PCR analysis of IL-6 in each group. D ROS levels measured by DCFH-DA staining and the relative DCF-DA fluorescence intensities were normalized and summarized. Scale bar = 50 μm. The results are shown as the means ± SD of 3 individual experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

The protective effect of curcumin on OLV-induced ALI is related to Prdx6. A Western blot analysis of representative inflammatory factor levels, among which IL-6, IL-1β, and TNF-α are the classic proinflammatory factors and IL-10 is the anti-inflammatory factor in each group. B ELISA analysis of MDA levels in each group. C ELISA analysis of SOD levels in each group. D ROS levels were measured by DCFH-DA staining in each group. Scale bar = 100 μm. E Western blot analysis images of Prdx6 in each group. F qRT–PCR analysis of Prdx6 in each group. G Immunofluorescence analysis of Prdx6 in each group. DAPI is shown in blue and represents the nucleus. Scale bar = 100 μm. In the control group (control): cells were pretreated with the DMSO vehicle alone for 2 h, then changed to a serum-free medium for 48 h; in the curcumin group (cur): cells were pretreated with 40 μg/ml curcumin + DMSO for 2 h, then changed to serum-free medium for 48 h; in the serum group (serum): the cells were intervened with 20% OLV_after serum for 48 h; in the curcumin + serum group (cur + serum): the cells were first pretreated with 40 μg/ml curcumin and DMSO, then changed to 20% OLV_after serum for 48 h. The results are shown as the means ± SD of 3 individual experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

Prdx6 mediated the protective function of curcumin in OLV-induced ALI by regulating the NF-κB pathway. A The levels of NF-κB signaling markers in each group were determined via Western blot. B Western blot analysis of Prdx6 expression in A549 cells transfected with either Prdx6-siRNA or NC-siRNA. C qRT–PCR analysis of Prdx6 expression in A549 cells transfected with either Prdx6-siRNA or NC-siRNA. D The expression of representative inflammatory factors and P65 in Prdx6-siRNA- or NC-siRNA-transfected cells with or without SC75741. E ROS levels were measured by DCFH-DA staining in cells transfected with either Prdx6-siRNA or NC-siRNA. Scale bar = 100 μm. F ELISA analysis of oxidative stress factor levels (MDA and SOD) in cells transfected with either Prdx6-siRNA or NC-siRNA. G The protein levels of NF-κB signaling markers in cells transfected with either Prdx6-siRNA or NC-siRNA. In the control group (control): the cells were pretreated with the DMSO vehicle alone for 2 h, then changed to a serum-free medium for 48 h; curcumin group (cur): the cells were pretreated with 40 μg/ml curcumin + DMSO for 2 h, then changed to serum-free medium for 48 h; serum group (serum): the cells were intervened with 20% OLV_after serum for 48 h; curcumin + serum group (cur + serum): the cells were first pretreated with 40 μg/ml curcumin and DMSO, then changed to 20% OLV_after serum for 48 h. The results are shown as the means ± SD of 3 individual experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

Prdx6 inhibits activation of the NF-κB signaling pathway by suppressing the nuclear translocation of P65. A Immunofluorescence analysis of P65 in cells transfected with either Prdx6-siRNA or NC-siRNA. DAPI is shown in blue and represents the nucleus. Scale bar = 100 μm. B Nuclear (N) and cytosolic/membrane (C + M) proteins of the NF-κB pathway in Prdx6-siRNA- or NC-siRNA-transfected cells. The cells in all groups were first pretreated with 40 μg/ml curcumin and DMSO and then changed to 20% OLV_after serum for 48 h. The results are shown as the means ± SD of 3 individual experiments. *P < 0.05; **P < 0.01; ***P < 0.001