Sepsis is a serious public health issue and the leading cause of death in critically ill patients in intensive care units. Sepsis represents the host's systemic inflammatory response to a severe infection (1). There are not effective therapies since its mechanisms still remain unknown. Recent studies have indicated that inflammation and endoplasmic reticulum (ER) stress are involved in the pathogenesis of sepsis (2). The disturbances in ER homeostasis include hypoxia, glucose depletion, and oxidative stress. ER stress results in the activation of the unfolded protein response (UPR), an evolutionarily conserved cellular response regulated primarily by glucose-regulated protein 78KD (GRP78). The UPR involves the activation of multiple proapoptotic pathways that ultimately lead to cell death (3). Additionally, signaling from the UPR is correlated with major inflammatory pathway nuclear factor κB (NF-κB) (4). NF-κB is the main transcription factor in ER stress-mediated inflammation activated by tumor necrosis factor receptor-associated factor 2 (TRAF2) and C/EBP homologous protein (CHOP) (5, 6). Therefore, ER stress-mediated NF-κB inflammatory pathway could be a key process in sepsis development. The study of pathways involved in ER stress-induced inflammation may provide specific molecular targets with impacting the progression of sepsis.
Thioredoxin-1 (Trx-1) is a redox regulating protein with redox-active disulfide/dithiol within its conserved active-site sequence: -Cys-Gly-Pro-Cys- (7). Trx-1 has been reported as a modulator of ER stress (8). Our previous study showed that Trx-1 played a neuroprotective role by suppressing ER stress (9, 10). It has been reported that neutralization of endogenous Trx-1 decreases survival of septic mice, whereas treatment with recombinant Trx-1 strongly extends the survival of mice (11). However, the mechanism of Trx-1 regulating ER stress in sepsis has not been studied well.
In this study, we explored the roles of Trx-1 in extending survival of sepsis and regulating pathways on ER stress. Our results indicated that Trx-1 plays a protective role in sepsis, and this protective effect may be dependent, at least in part, by suppressing ER stress induced NF-κB inflammatory signal pathway.
MATERIALS AND METHODS
Antibodies, GRP78 (sc-1051), IRE1α (sc-20790), TRAF2 (sc-7346), CHOP (sc-7351), NF-κB (sc-372), inhibitors of NF-κBα (IκBα) (sc-847), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antimouse monoclonal antibody histone (7631s) and Trx-1(2298s) were supplied by Cell Signaling Technology (CST, Danvers, Mass). Anti-human Trx-1 rabbit polyclonal antibody was owned by our laboratory.
Male C57BL/6 wild-type (WT) and C57BL/6 human-Trx-1 transgenic (hTrx-1 Tg) mice, 8 weeks of age, were used in the experiments. Mice were housed in plastic cages and maintained on a 12-h light/dark cycle and had free access to food and water. The animals were cared for and used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57BL/6 human Trx-1 transgenic (Trx-1 Tg) mice were constructed by (Cyagen Biosciences Inc, Guangzhou, China). The pronuclei of fertilized eggs from hyperovulated C57BL/6 were microinjectioned with hTrx-1 cDNA construct. The presence of Trx-1 transgene also was confirmed by reverse transcription–PCR analysis. All animal experiments were approved by the local Committee on Animal Use and Protection (No. LA2008305).
Cecal ligation and puncture (CLP) protocol
Mice were divided into four groups: wild-type sham control group (group WC), human Trx-1 transgenic sham control group (group TC), wild-type sepsis group (group WS), and human Trx-1 transgenic sepsis group (group TS). Mice were anesthetized with isoflurane via a vaporizer at 1.5 to 2.5 minimum alveolar concentrations. Under sterile conditions, 1.5-cm incision was made in the lower abdominal region, and the cecum was exposed. The distal portion of the cecum was completely ligated 1 cm from the end with 3.0 silk suture, punctured once with 18-gauge needle, and then replaced in the peritoneal cavity. Then, the peritoneal wall and skin were closed with double sutures. Peritoneal injection of 1 mL sterile saline (0.9%) was administered to mice after surgery. Sham mice had abdominal incision and cecal exposure without ligation and puncture. After the procedure, mice had access to water and food (12, 13). Survival was monitored for 7 d (n = 15 per group), while for cytokines, and organ harvesting, mice were sacrificed after CLP for 12 h by cervical vertebra dislocation (n = 9 per group).
Measure of inflammatory cytokines in the serum
Mice were sacrificed by cervical vertebra dislocation, and blood samples were collected from eyeball. The levels of TNF-α and IL-1ß in the serum were determined by using an enzyme-linked immunosorbent assay (ELISA; Biolegend, San Diego, CA) in accordance with the manufacturer's instructions.
After drawing blood from eyeball, the lungs were excised from the mice by opening the chest via median sternotomy. The right inferior lobe was removed and fixed in 10% buffered formalin for 24 h. Fixed specimens were stained with H&E for light microscopic evaluation. Lung injury scores were assessed by a pathologist blinded to the study groups and scored in slides. We used a modified ventilator-induced lung injury histology scoring system as previously described to score lung injury (14). In short, four pathologic parameters were scored on a scale of 0 to 4: alveolar congestion, hemorrhage, leukocyte infiltration, and thickness of alveolar wall/hyaline membranes. A score of 0 represents normal lungs; 1, mild, less than 25% lung involvement; 2, moderate, 25% to 50% lung involvement; 3, severe, 50% to 75% lung involvement and 4, very severe, greater than 75% lung involvement. The total histological score was expressed as the sum of the scores for all parameters.
Water content of lung
The left lung was excised from each mouse. The wet weight of the lung was measured by using an electronic scale and then desiccated in an oven at 85°C for 48 h to determine the dry weight. The water content was obtained by using the following equation: Water content (%) = (wet weight − dry weight)/wet weight × 100%.
Real-time quantitative RT-PCR
Total RNA was extracted from 0.1 g lung tissue by using a Trizol reagent kit (CWBIO Corporation, Beijing, China) and converted to cDNA by using the Revert Aid TM First Strand cDNA Synthesis Kit (Fermentas, Walldorf Baden, Germany). The product was analyzed by using a Prism 7300 Sequence Detection System (Applied Biosystems, Foster, CA). The following primer pairs were selected for real-time polymerase chain reaction (real-time PCR): gene expression was calculated relative to the housekeeping gene mouse β-actin F: 5′-CAG TTC GCC ATG GAT GAC GAT-3′, R 5′-ATC TGG GTC ATC TTT TCA CGG TTG-3′; TNF-α F: 5’-GCCTCT TCT CAT TCC TGC TC-3’, TNF-α R: 5’- CCC ATT TGG GAA CTT CTC CT -3’; IL-1β F: 5’-TGCCACCTTTTGACAGTGATG-3’, R: 5’-TGATGTGCTGCTGCGAGATT-3’.
Preparation of nuclear protein extracts
Protein lysates were prepared by using hypotonic lysis buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1 mM MgCl2, 0.5% NP-40, 0.5 mM dithiothreitol, and complete protease inhibitor cocktail) on ice for 30 min. The nuclei were then pelleted by centrifugation and resuspended in high salt nuclear extraction buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1 mM MgCl2, 400 mM NaCl, 0.5 mM dithiothreitol, and complete protease inhibitor cocktail) on ice for 30 min. The nuclear proteins were harvested after removal of the nuclear debris by centrifugation.
Protein lysates were prepared using a solubilizing solution (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA,1 mM phenylmethanesulfonyl fluoride, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM β-glycerol phosphate, and 1 mg/mL leupeptin). Protein concentration was determined using the Bio-Rad protein assay reagent (Hercules, CA). Equal quantities of protein were separated by 12% (for GRP78, CHOP, IRE1α, TRAF2, NF-κb) or 15% (for Trx-1) SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore Corp, Billerica, MA). The membrane was soaked in 10% skim milk in phosphate buffered saline, pH 7.2, containing 0.1% Tween 20 or 3% bovine albumin V in Tris-buffered saline, pH 7.2, containing 0.1% Tween 20 overnight at 4°C and then incubated with primary antibodies (1:1,000) followed by peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:10,000) (KPL, Gaithersburg, MD). The epitope was visualized by an ECL Western blot detection kit (Millipore Corp). The blots were stripped and then stained for β-actin. Densitometry analysis was performed using Image J software.
Data were expressed as means ± SD values. Statistical analysis was performed by using SPSS software. The one-way ANOVA followed by a post hoc multiple comparison test was used to compare control and treated groups. Survival was analyzed using the Kaplan–Meier method. Statistical analyses were performed using the log-rank test. P < 0.05 was considered to indicate a statistically significant difference.
Human Trx-1 was expressed in transgenic mice and mice Trx-1was increased by CLP
We examined expression of hTrx-1 and mTrx-1 in the lung tissues at 12 h after CLP. The human Trx-1 was overexpressed in transgenic mice (Fig. 1A). The expression of mice Trx-1was increased in CLP group (Fig. 1B). We noticed that hTrx-1 was also slightly expressed in wild-type mice group, because antibody for human Trx-1 has low binding activity with mice Trx-1.
Trx-1 overexpression extended survival of sepsis
The previous study showed the impact of exogenous Trx-1 (11). In this study, we compared survival in sepsis by CLP between male C57BL/6 wild-type (WT) and C57BL/6 human-Trx-1 transgenic (hTrx-1 Tg) mice. Survival was monitored every 4 h for 7 days. We found that 73.33% (11 of 15) of the hTrx-1 Tg septic mice survived, however, the 33.33% (5 of 15) of WT septic mice survived (P < 0.05) (Fig. 2). This data suggests that the overexpression of Trx-1 extends the survival in sepsis.
Trx-1 overexpression reduced injury of lung in sepsis
Severe sepsis usually induces acute lung injury. Then, we examined morphologic manifestations in the lung by using H&E staining at 12 h after CLP. The pulmonary organizational structures of group WC and TC were normal. However, the histological sections of lung from septic mice showed signs of injury, including thickening of the alveolar septa, inflammatory cell infiltration, and interstitial edema. The signs of injury were attenuated in the TS group (Fig. 3A). The lung injury scores in group WS were more severe when compared with group TS (Fig. 3B).
Trx-1 overexpression attenuated the pulmonary edema in sepsis
Pulmonary edema results from acute lung injury. Then we excised left lung from each mouse to measure the water content in the lung. The wet weight of the lung was measured by using an electronic scale and then lung was desiccated in an oven at 85°C for 48 h to determine the dry weight. As shown in Fig. 4, the water content in the lung of the mice in group WS was increased as compared with group WC, which was decreased in group TS (P < 0.05).
Trx-1 overexpression decreased the levels of TNF-a and IL-1β in sepsis
The inflammatory response is induced in sepsis, we next detected the levels of the TNF-a and IL-1β in the plasma of septic mice by ELISA after CLP 12 h. The levels of TNF-a and IL-1β were increased in group WS, which were suppressed in Trx-1 TG mice (Fig. 5, Aand B). Then, we determined the mRNA levels of TNF-α and IL-1ß in the lung tissue by RT-PCR. The mRNA levels of TNF-a and IL-1β were also increased in group WS, which were suppressed in Trx-1 TG mice (Fig. 5, C and D). Our results showed that Trx-1 overexpression significantly decreased the levels of TNF-α and IL-1ß in plasma and lung tissue, which suggests that Trx-1 overexpression inhibits inflammatory response in the sepsis.
Trx-1 overexpression suppressed the expressions of GRP78, IRE1α, TRAF2, and CHOP by CLP
It has been reported that ER stress is involved in sepsis (2). We further examined the effect of Trx-1 overexpression on ER stress induced by sepsis. The expressions of GRP78, IRE1α, TRAF2, and CHOP by CLP were suppressed by overexpression of Trx-1 (Fig. 6, A–D). These data suggest that Trx-1 overexpression inhibits ER stress in sepsis through suppressing expressions of GRP78, IRE1α, TRAF2, and CHOP.
Trx-1 overexpression suppressed the expressions of NF-κB, IκB-α by CLP
A growing body of evidence suggests that the signalling pathways on the ER stress and inflammation are cross-linked through various mechanisms, including activation of the transcription factor NF-κB (6). We further examined that whether Trx-1 overexpression regulated NF-κB activity. Our result showed that the IκB-α expression was significantly decreased in sepsis, which was restored by Trx-1 overexpression (Fig. 7A). The expression of NF-κB in cytosol and nuclear was increased, which was suppressed by Trx-1 overexpression (Fig. 7, B and C). These data suggest that Trx-1overexpression inhibits NF-κB activation by restoring the expression of IκB-α.
Sepsis is a serious public health issue and the leading cause of death in critically ill patients in intensive care units. Sepsis results from the host's systemic inflammatory response to a severe infection (15, 16). There are not effective therapies since its mechanism still remains unknown. The oxidative stress is involved in the pathogenesis of a variety of injury types, including radiation combined injury and the associated sepsis (17, 18). Several studies have shown that extracellular levels of Trx-1 are increased by oxidative stress and inflammation (19–21). It has been reported that plasma levels of Trx-1 are significantly increased in patients with sepsis (22). In the present study, we first examined whether Trx-1 expression was changed or not in lung tissue. As shown in Fig. 1B, the data showed Trx-1 was increased by CLP. Our results showed that Trx-1 was increased in lung tissue in sepsis by CLP, which means that oxidative stress occurred in lung tissue. Importantly, we found that Trx-1 overexpression extended survival of experimental sepsis by CLP when compared with wild type (Fig. 2). This result is consistent with a study in which treatment with recombinant Trx-1 after CLP strongly enhanced the survival of mice (11). Since lung injury is usually induced in sepsis (23, 24), we examined the pathological manifestations by using H&E staining, the Trx-1 overexpression inhibited the lung damage by CLP (Fig. 3). The edema was also suppressed by Trx-1 overexpression (Fig. 4). Trx-1 is a redox regulating molecule which has various biology activities, such as anti-oxidative stress and apoptosis inhibition (25, 26). These data suggest that Trx-1 suppresses sepsis in the present study. In addition to its anti-oxidative effects, Trx-1 is known as an anti-inflammatory molecule (7). Thus, we also examined expressions of the TNF-α and IL-1β. We found that Trx-1 overexpression significantly suppressed the expressions of the TNF-α and IL-1β in sepsis (Fig. 5). However, clinical trials targeting TNF and IL-1β have failed in the treatment of sepsis. It is needed to examine the other inflammatory cytokines in future study.
ER stress is involved in sepsis. ER-resident chaperone GRP78 is a marker for ER stress that plays a crucial role in the regulation of the ER dynamic homeostasis (27). Under nonstressed conditions, GRP78 binds and represses ER transmembrane proteins, including IRE1, PERK, and ATF6. In response to ER stress, GRP78 dissociates from these transmembrane proteins, which leads to their activation and triggers the UPR (10). C/EBP homologous protein (CHOP) is a transcription factor and mediator of the UPR that plays a major role in inducing apoptosis (4). In the present study, we found that level of GRP78 was induced in sepsis, which was inhibited by overexpression of Trx-1. The expressions of IRE1, TRAF2, and CHOP were also induced in sepsis and inhibited by overexpression of Trx-1 (Fig. 6). This result may be explained by our previous study in which Trx-1 played a role in suppressing ER stress (10).
An increase in the ER stress has been shown to result in the activation of NF-κB. Accumulating evidence suggests that IRE1α and CHOP mediated NF-κB activation. In response to ER stress, the autophosphorylation of IRE1α induces a conformational change in its cytosolic domain, which can then bind to the adaptor protein TRAF2, the IRE1α–TRAF2 complex can recruit IκB kinase, which phosphorylates IκB, leading to the degradation of IκB and the nuclear translocation of NF-κB (28). Recent study demonstrated that CHOP could also modulate NF-κB activation by decreasing IκB degradation and p65 translocation (29). The degradation of IκB exposes a nuclear-localization signal in NF-κB, allowing NF-κB to translocate to the nucleus, where it induces the transcription of numerous inflammatory genes (30). It has reported that genetic deletion of CHOP protects against LPS and CLP-induced mortality in sepsis (31).
We further examined the expressions of NF-κB and IκB in the present study. We demonstrated that Trx-1 overexpression inhibited the NF-κB activation in sepsis induced by CLP (Fig. 7). This data suggested that Trx-1 overexpression was able to inhibit the NF-κB signaling pathway which may be related to the decreases of IRE1α, TRAF2, and CHOP by Trx-1 overexpression. NF-κB is a transcriptional regulator that has a central role in the onset of inflammation. This mechanism leads to a decrease in NF-κB target genes involved in inflammation (32). Thus, after inhibiting ER stress by Trx-1, the inflammatory response was decreased. However, in the present study, the differences for nuclear translocation of NF-κB are small. As Trx-1 has anti-apoptosis, anti-inflammation, and anti-oxidation property, Trx-1 may impact several signaling pathways. Its protective effect may not be solely through changes in NF-κB to regulating inflammatory cytokines. More studies will be needed to investigate the detail mechanism of Trx-1 in sepsis.
In summary, we reported that overexpression of Trx-1 increased survival in a model of experimental sepsis partly by reducing the inflammatory response and lung edema. Trx-1 overexpression suppressed ER stress by regulating the expressions of GRP78, IRE1α, TRAF2, and CHOP, sequentially inhibited activation of NF-κB. Trx-1 may be a potential target in sepsis (Fig. 8).
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