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Introduction

Peroxiredoxins (Prxs) are antioxidant enzymes that catalyse cellular hydrogen peroxide (H2O2) production, exhibit a protective role in cells and are required for cell metabolism and redox signalling (1–3); in addition, Prxs play essential roles in neurodegenerative diseases, cancer and inflammatory processes (4).

Peroxiredoxin I (Prx I), a member of the Prx family, is a multifunctional protein originally identified as an intracellular scavenger of H2O2. Prx I has also been shown to act as a molecular chaperone with the ability to modulate the actions of numerous molecules, as a regulator of transcription, and as a signal modulator (5). Prx I knockout shortens the lifespan of erythrocytes, resulting in haemolytic anaemia with increased cellular ROS levels, protein oxidation, and Heinz body formation, and several malignant cancers in mice (6), suggesting the important role of Prx I in the defence against to oxidative stress. Recombinant Prx I can also bind to Toll-like receptor 4 (TLR4) and stimulate TLR4-dependent cytokine secretion in macrophages and dendritic cells through CD14/MD2/MyD88-dependent signalling pathways (7). Prx I knockdown can modulate the secretion of inflammatory cytokines, such as interleukin (IL)-10, IL-1β and tumour necrosis factor (TNF)-α, in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages via the STAT3 signalling pathway (8). Furthermore, our previous study revealed that Prx I deficiency attenuates the macrophage phagocytic capacity for clearing erythrocytes in the mouse response to oxidative stimulation (9). All these reports suggest the involvement of Prx I in oxidation-related inflammatory processes.

LPS has been reported to stimulate the innate immune response via recognition of the TLR4 complex and triggering the cellular signalling pathways that produce inflammatory cytokines and organic septic shock (10,11). It is well-known that intraperitoneal or intravenous injections of high LPS concentrations into animals can induce fatal septic shock and subsequently leads to tissue damage, body temperature dysregulation, and lethality. However, the regulatory effect of Prx I on LPS-stimulated mouse death is not understood.

In this study, we performed experiments to understand the protective function of Prx I on LPS-induced lethal shock in wild-type and Prx I knockout mice. Our results revealed that Prx I deficiency increased LPS-induced mouse death, with more liver damage, TNF-α tissue accumulation and oxidative stress, which was marked by higher expression levels of the antioxidant enzymes superoxide dismutase 2 (SOD2), catalase and glutathione peroxidase (GPx).

Materials and methods

Mice and genotype analysis

C57BL/6J (wild-type and Prx I knockout) pathogen-free mice were kindly provided by the laboratory of Dr. Dae-Yeul Yu, Korea Research Institute of Bioscience and Biotechnology (KRIBB). The mice used for the LPS challenge were 8–10 weeks of age. The experimental groups were age and sex matched. Escherichia coli O26:B6 LPS (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was diluted in sterile PBS and injected intraperitoneally (i.p) into the animals. The primers used for the mouse genotype analysis were 5′-gct tgg gtg gag agg cta ttcg-3′ and 5′-gta aag cac gag gaa gcg gtc agcc-3′ for the neo gene and 5′-ctg gaa acc tgg cag tga ta-3′ and 5′-ctg tga ctg ata gaa gat tggt-3′ for the wild-type gene. All animals were housed in microisolator cages in laminar flow units under ambient light. The Institutional Animal Care and Use Committee (Heilong Bayi Agricultural University, Daqing, China) approved both the animal care and experiments.

ELISA and nitric oxide (NO) production assay

TNF-α, IL-6 and IL-10 ELISA assay kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA), and assays were completed according to the manufacturer's instructions. To determine the serum cytokine level in LPS-injected mice, the wild-type and Prx I knockout mice were i.p. injected with 20 mg/kg LPS, and the serum and tissue cytokine levels were detected at different time points post injection. NO production was assessed based on the accumulation of nitrite in the serum using a colorimetric reaction with Griess reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 0.1% sulfanilamide, and 2.5% H3PO4]. The absorbance at 540 nm was measured with a UV MAX kinetic microplate reader (Molecular Devices, Menlo Park, CA, USA).

Haematoxylin and eosin (H&E) staining

The livers and lungs were collected from wild-type and Prx I-deficient mice at the indicated times after the LPS-injections; then, the tissues were trans-cardiac perfusion-fixed with heparinized saline containing 3.7% formaldehyde. The tissue sections (4 µm in thickness) were stained with H&E. The experiments were performed with 10 wild-type and 10 Prx I-deficient mice.

Western blotting analysis

Tissues protein lysates were separated on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blotted with primary antibodies against Prx I, cleaved caspase-3, Bcl-2, SOD2, GPx, catalase (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and β-actin (Sigma-Aldrich; Merck KGaA) at 4°C overnight. The membranes were washed five times with 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl (Tris-buffered saline, TBS) and 0.2% Tween-20 and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich; Merck KGaA) or anti-mouse IgG (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. After the removal of excess antibodies by washing with TBS, specific binding was detected using a chemiluminescence detection system (Amersham, Berkshire, UK) according to the manufacturer's instructions.

Statistical analysis

The data are depicted as the means ± SEM. Student's t-tests were performed using GraphPad Prism 4.0 software (GraphPad Software, Inc., La Jolla, CA, USA), and P<0.05 was considered to indicate a statistically significant difference.

Results

Prx I deletion increased LPS-induced lethality in mice

The homozygous knockout mice used in the experiments were generated by heterozygous mice, the genotype was confirmed by PCR (Fig. 1Aa), and the Prx I protein level was confirmed by western blotting (Fig. 1Ab). To investigate the role of Prx I on LPS-induced mouse lethal shock, we performed experiments to assess whether Prx I deletion affected mouse susceptibility to endotoxic shock. Prx I+/+ and Prx I-/- mice (11 mice were used for each group) were injected i.p. with LPS (20 mg/kg body weight), and mouse survival was observed for 4 d. As shown in Fig. 1B, lethality was higher in the LPS-stimulated Prx I−/− mice than in the Prx I+/+ mice (approximately 54% for Prx I+/+ and 90% for Prx I−/−mice).

Figure 1. Prx I deficiency increased LPS-induced mouse death. (Aa) PCR analysis of the genomic DNA isolated from wild-type (lane 1), Prx I homozygous (lane 2), and Prx I heterozygous (lane 3) mice. The 700 and 250 bp arrows indicate the PCR products of Prx II−/− and Prx II+/+ mice, respectively. (Ab) Western blot analysis of Prx I expression in the liver tissue from Prx I+/+ (#1 and #2) and Prx I−/− (#3 and #4) mice. (B) The Prx I+/+ (n=12) and Prx I−/− (n=12) mice were i.p injected with (20 mg/kg body weight) LPS, and viability was observed at the indicated times. No additional mice died 70 h after the injection. Prx I, peroxiredoxin I; LPS, lipopolysaccharide.

Decreased serum IL-10 production in Prx I−/− mice after LPS injection

To examine the effect of Prx I deletion on serum cytokine production after LPS stimulation, Prx I+/+ and Prx I−/− mice were injected i.p. with LPS (20 mg/kg body weight), and at the indicated times, the peripheral serum was collected for analysing the NO, TNF-α, IL-6 and IL-10 levels with Griess reagent (for NO determination) and ELISA kits. As shown in Fig. 2, there was no remarkable difference in NO, IL-6 and TNF-α production between Prx I+/+ and Prx I−/− mice after the LPS injection, but IL-10 production was decreased in Prx I−/− mice after the LPS injection.

Figure 2. Prx I deficiency decreased serum IL-10 production after LPS injection. Prx I+/+ and Prx I−/− mouse peripheral sera were collected at the indicated times after the LPS (20 mg/kg body weight) was injected. The serum (A) NO, (B) TNF-α, (C) IL-6 and (D) IL-10 levels were analysed with Griess reagent and ELISA kits. n=6, *P<0.05 and **P<0.01. Prx I, peroxiredoxin I; LPS, lipopolysaccharide; IL, interleukin; NO, nitric oxide; TNF, tumour necrosis factor.

Increased liver damage and apoptosis in Prx I-deficient mice after LPS injection

To assess the effect of Prx I deletion on LPS-induced tissue damage, we performed histological experiments after the LPS injection in Prx I+/+ and Prx I−/− mice. LPS stimulation increased the liver and lung damage, with increasing inflammatory cell infiltration, liver cell death and diffuse alveolar haemorrhages in both Prx I+/+ and Prx I−/− mice (Fig. 3). However, severe liver damage was observed in Prx I−/− mice compared with the Prx I+/+ mice (Fig. 3A), but no differences in the lungs (Fig. 3B) and kidneys (data not shown) were observed. Furthermore, we also examined the tissue cytokine levels at the indicated times after the LPS injection. The results revealed that the liver tissue TNF-α levels were increased in the Prx I−/− mice compared with the Prx I+/+ mice, but there were no differences in IL-6 and IL-10 production levels (Fig. 4A-C). In addition, the pro-apoptotic cleaved caspase-3 protein levels were more up-regulated in the Prx I−/− mouse livers than in the Prx+/+ mouse livers, but Bcl-2 protein expression levels were not different between the mouse genotypes (Fig. 5E and F).

Figure 3. Increased immune cell infiltration in Prx I−/− mice after LPS injection. The H&E-stained sections from Prx I+/+ and Prx I−/− mice at the indicated times (0, 6, and 18 h) after the LPS injection (magnification, ×400) show signs of immune cell infiltration in the (A) liver (blue arrow, immune cell infiltration and vacuole changes) and (B) lungs (black arrow, immune cell infiltration) n=6. Prx I, peroxiredoxin I; LPS, lipopolysaccharide.

Figure 4. Increased liver TNF-α production in Prx I−/− mice after LPS injections. The liver cytokine levels were analysed by ELISA kits. The liver extracts, containing proteinase inhibitors, from Prx I+/+ and Prx I−/− mice at the indicated times after the LPS injection were centrifuged, and the supernatant was collected for detecting the (A) TNF-α, (B) IL-6 and (C) IL-10 levels by ELISA. The production levels of cytokines are represented as pg/mg protein. n=6 and *P<0.05. Prx I, peroxiredoxin I; LPS, lipopolysaccharide; TNF, tumour necrosis factor; IL, interlrukin.

Figure 5. Prx I deficiency up-regulated antioxidant enzyme and cleaved caspase-3 levels after LPS injection. The protein expression levels of (A) Prx I, (B) SOD2, (C) catalase and (D) GPx were analysed by western blotting after LPS injections in Prx I+/+ and Prx I−/− mice. (E and F) The protein expression levels of C-C3 and Bcl-2 were analysed by western blotting after LPS injections in both the Prx I+/+ and Prx I−/− mouse livers. The protein expression is presented as the means ± standard deviation. n=6 and *P<0.05, **P<0.01. C-C3, cleaved caspase-3; Prx I, peroxiredoxin I; LPS, lipopolysaccharide.

Prx I deletion increased the liver antioxidant protein expression level in response to LPS injection

To verify whether Prx I deficiency affected the liver oxidative stress response to LPS stimulation, we also observed the liver antioxidant enzyme protein expression levels at the indicated times after the LPS injection in both Prx I+/+ and Prx I−/− mice. Compared with the Prx I+/+ mice, SOD2, catalase and GPx protein levels were up-regulated in Prx I−/− mice after the LPS injection (Fig. 5A-D), suggesting more oxidative stress accumulation in Prx I−/− mice.

Discussion

In the present study, we showed that Prx I is a key regulator of LPS-induced mouse lethal shock, as Prx I deficiency leads to a higher mortality, caused by the acute liver damage that accompanies the increased TNF-α tissue accumulation and apoptosis, rapid immune cell accumulation and decreased serum IL-10 levels. Paradoxically, no expected proinflammatory cytokine outbursts have been classically-associated with septic death. Our study revealed the existence of an alternative mechanism for LPS-induced lethal shock independent of the severe inflammatory response, which is limited by Prx I.

LPS injections have been reported to markedly increase both the serum and macrophage cytokine production, such as TNF-α, IL-6 and NO, and accelerate mortality in Prx II (a member of the Prx families) knockout mice (12), indicating that the Prxs may play essential roles in endotoxin-induced septic lethality. On the other hand, Prx I knockdown in RAW264.7 macrophage cells decreased the IL-10 production stimulated by LPS (8), indicating that Prx I and Prx II have different regulatory roles in macrophage cells. In our i.p. toxin model (13,14), LPS-induced death in Prx I−/− mice did not have a surge in serum cytokine production but did decrease the serum IL-10 level (Fig. 2), suggesting that Prx I deletion may down-regulate the macrophage anti-inflammatory response to LPS stimulation. Furthermore, recombinant Prx I stimulated the secretion of proinflammatory cytokines, such as TNF-α and IL-6, by binding to toll-like receptor 4 (TLR4), and this binding was dependent on Prx I chaperone activity and independent of its peroxidase activities in macrophages and dendritic cells (7). These findings suggest that Prx I may be a key regulator of LPS-induced macrophage cytokine production, but the systemic mechanism by which Prx I affects LPS-stimulated septic lethality requires further study.

LPS/TLR4 signalling is strongly associated with chronic liver diseases, such as alcoholic/nonalcoholic fatty liver diseases, hepatic fibrosis and hepatocarcinoma (15). In addition, several reports have also shown that LPS-induced mouse lethal shock is related to acute liver damage (16–18), suggesting that the liver is a major target tissue in response to LPS-induced lethal shock. In our studies, rapid immune cell accumulation and increased liver TNF-α and cleaved caspase-3 protein expression levels were observed in the Prx I−/− mice compared with the Prx I+/+ mice after LPS administration (Figs. 3, 4 and 5E-F). These results suggest that Prx I deficiency leads to more damage in the liver in response to LPS stimulation, indicating a protective role for Prx I against endotoxin-induced injury.

Mammalian antioxidant enzymes, such as SOD, GPx and catalase, play essential roles against oxidative stress and DNA damage-induced cell death as well as endotoxin stimulation (19–22). In response to LPS, the protein expression levels of SOD2, GPx and catalase were up-regulated in the Prx I−/− mouse livers compared with those of the Prx I+/+ mice (Fig. 5A-D). These results revealed that LPS stimulation increased oxidative stress in the livers of Prx I−/− mice, resulting in the up-regulation of liver antioxidant enzyme levels. However, the potential mechanism by which antioxidant enzymes are up-regulated requires further study.

In summary, our results reveal the role of Prx I in response to LPS-induced mouse lethal shock. Prx I deletion was associated with accelerated mouse death and marked hepatitis, accompanied by a compensatory up-regulation of antioxidant enzyme levels. These findings provide insight into the function of Prx I against endotoxin-induced injury.

Acknowledgements

Not applicable.

Funding

This work was supported by the Natural Science Foundation of Heilongjiang Province of China (grant no. C2015036) and the Research Project of Heilongjiang Bayi Agricultural University (grant no. XA2014-04).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

HNS and LF constructed the model and wrote the manuscript. AGW, JYW and LL performed the mouse care and handling. MHJ, GNS, CHJ and DSL performed the data analysis. THK and YDC performed the image analysis. DYY and YHH provided substantial contributions to conception and design of the study.

Ethics approval and consent to participate

The Institutional Animal Care and Use Committee (Heilong Bayi Agricultural University, China) approved both the animal care and experiments.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References