Despite advances of critical care medicine, sepsis remains the leading cause of death among patients in intensive care units (1). Sepsis is characterized by initial presentation of systemic inflammatory responses due to infection, followed by dysfunction of multiple organs such as the liver and heart. If not treated promptly, it may develop to septic shock with severe hemodynamic derangement. Lipopolysaccharide (LPS), a component from outer membrane of Gram-negative bacteria, plays a key role in the pathogenesis of septic shock (2). Although multiple clinical trials exploring various agents that neutralized LPS have been performed, none of them has met with success (3). As a result, hospital mortality from sepsis remains high, ranging from 25% to 80%, over the last few decades (4).
Heat shock proteins (HSPs) are a family of highly conserved proteins that play an important role in protecting cells from stress (5). The 70-kd HSP family includes the inducible HSP70 (also termed HSP72) and the constitutive heat shock cognate protein 70 (HSC70, also termed HSP73). HSP70 and HSC70 are highly homologous, but HSC70 is more abundantly expressed in most cell types (6). Although HSP has been conventionally regarded as the intracellular chaperone that contributes to protein folding and transport (7), it was recently demonstrated with extracellular function capable of conveying immunomodulatory effects similar to cytokines (8).
Emerging research efforts have been focused on the role of extracellular HSP70 in sepsis. In septic patients, plasma HSP70 levels are increased and associated with clinical outcome (9). Similarly, increased extracellular HSP70 levels correlate with disease severity in critically ill patients with liver disease and heart failure (10,11). Notably, recent animal studies have shown that pretreatment with exogenous HSP70 can improve septic shock and decreased mortality (12,13) and thus shed some light in the novel pharmacologic strategy in the prophylaxis of sepsis.
The mechanisms underlying the prophylactic effects of extracellular HSP70 in sepsis remain elusive, but some in vitro studies suggest that its anti-inflammatory actions to induce endotoxin tolerance may play important roles. For example, in macrophages, HSP70 preconditioning can decrease LPS-induced productions of nitric oxide (NO) (13). In mononuclear cells, HSP70 can induce endotoxin tolerance by inhibition of inflammatory mediators including nuclear factor κB (NF-κB) and tumor necrosis factor α (TNF-α) (14).
While most previous studies are focused on HSP70, there is a paucity of publications regarding the protective effects and mechanisms of HSC70 in sepsis. A recent animal study suggests that HSC70 pretreatment may convey cardioprotection by ameliorating LPS-induced activation of the NF-κB pathway (15). However, whether these prophylactic effects of extracellular HSC70 can be extrapolated in hepatic tissue is not investigated. In addition, whether it can regulate other NF-κB–related inflammatory mediators such as inducible NO synthase (iNOS), cyclooxygenase 2 (COX-2), and matrix metalloproteinase 9 (MMP-9) and MAPK signaling pathway is also unknown.
We hypothesize that HSC70 has prophylactic effects on septic cardiomyopathy and hepatic dysfunction with anti-inflammatory actions due to endotoxin tolerance. In this study, we examined (1) whether HSC70 pretreatment can attenuate cardiac and hepatic dysfunction in rats with endotoxemia; (2) the effects of HSC70 on inflammatory mediators including TNF-α, iNOS, COX-2, and MMP-9; and (3) the effects of HSC70 on NF-κB activation and the MAPK signaling pathway.
MATERIALS AND METHODS
Materials and reagents
Recombinant bovine HSC70 was purchased from Enzo Life Sciences (Farmingdale, NY). The recombinant HSC70 was a low-endotoxin–containing preparation, with endotoxin less than 5.0 pg/μg protein (<0.05 EU/μg protein), as measured by Limulus assay. Bacterial LPS (Escherichia coli serotype 026:B6, L8274) and antibody against iNOS were obtained from Sigma Aldrich Inc (St Louis, Mo). Antibodies against COX-2 and ERK1/2 were products of Upstate Biotechnology (Lake Placid, NY), whereas antibodies of p38 and phosphorylated p38 were provided by Santa Cruz Biotech (Santa Cruz, Calif). Antibodies against phospho-ERK, phosphor–inhibitor of nuclear factor κB (IκBα), IκBα, and MMP-9 were supplied by Cell Signaling Technology (Beverly, Mass). The antibody against GAPDH was obtained from Millipore (Temecula, Calif).
Adult male Wistar rats weighing 250 to 350 g were purchased from the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan. They were housed under conditions of constant temperature and controlled illumination (light on between 7:30 AM and 7:30 PM). Food and water were available ad libitum. All protocols were approved by the Animal Care and Use Committee of the Kaohsiung Medical University.
The rats were anesthetized by i.p. injection of urethane (1.2 g/kg). After surgical tracheotomy, a shortened 14- gauge i.v. cannula was secured in the trachea, through which the lungs were ventilated (Harvard Apparatus Rodent Respirator, model 680; Harvard Apparatus, Holliston, Mass). The left femoral artery was cannulated with a polyethylene-50 catheter and connected to a pressure transducer (MLT0380/D; ADInstruments, Sydney, Australia), attached to an amplifier (ML118; ADInstruments) and an A/D interface (Power Lab SP4269; AD Instruments) for the measurement of mean arterial pressure (MAP) and heart rate (HR). The left femoral vein was cannulated for the administration of drugs. A Micro-Tip pressure catheter (SPR-407, Millar Instruments, Houston, Tex) was inserted into the right carotid artery and advanced into the left ventricle (LV) while the LV pressure was digitally (2 kHz) recorded. The LV systolic pressure (LVSP), maximal slope of systolic pressure increment (max dP/dt), and the diastolic pressure decrement (min dP/dt) in LV were then calculated. After the completion of the surgery, all cardiovascular parameters were allowed to stabilize for 30 to 60 min.
Animals were randomly assigned into three groups: (1) control group, (2) LPS group, (3) HSC70 + LPS group, and (4) HSC70 group. The control group received vehicle injection. The LPS group received an i.v. injection of 10 mg/kg E. coli LPS. The HSC 70 + LPS group received an i.v. injection of 20 μg/kg HSC70 at 10 min prior to LPS administration. The HSC70 group received 20 μg/kg HSC70 alone. The number of experiments was six for each group. Changes in blood pressure and HR were monitored for 4 h in all groups. The number of experiments was six for each group. At the end of each experiment, blood samples were drawn from the femoral artery, collected in heparinized tubes, and kept at 4°C, followed by centrifugation at 2,000g for 15 min. Plasma samples were stored at −70°C until analysis. During the course of experiments, rectal temperature was maintained in the range of 36.5°C to 37.5°C.
Analysis of liver function, plasma levels of glucose and lactate dehydrogenase, TNF-α, and nitrite/nitrate
Blood samples were collected at 4 h after the injection of LPS. Liver function was determined based on changes in plasma levels of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). Plasma levels of glucose and lactate dehydrogenase (LDH) were measured using Spotchem EZ SP 4430 (ARKRAY Inc, Kyoto, Japan). Plasma levels of TNF-α were measured using enzyme-linked immunoadsorbent assay provided by Pierce Biotechnology (Rockford, Ill) according to the manufacturer’s instructions. The plasma level of nitrite/nitrate (NOx), an indirect marker of NO production, was determined by Griess reaction (Cayman Chemical, Ann Arbor, Mich).
Western blot analysis
Tissues of heart and liver were obtained from rats treated with or without LPS and homogenized on ice with a polytron PT MR 3000 homogenizer (Kinematic, Littau, Germany) in a buffer composed of (in mM): Tris-HCl 50, EDTA 0.1, EGTA 0.1, 2-mercaptoethanol 12, and phenylmethylsulfonyl fluoride 1 (pH 7.4). The homogenized tissues were centrifuged at 10,000g for 30 min, and the supernatant stored at −70°C until further analysis. Aliquots of tissue homogenates were used for protein assay (Bio-Rad protein assay reagent, Hercules, Calif, USA) and Western blot analysis. Proteins in the whole-cell lysate were resolved on 7.5% to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene fluoride membrane. After blocking, the membrane was incubated overnight at 4°C with various primary antibodies specific for iNOS, COX-2, MMP-9, IκBα, ERK1/2, p38, and GAPDH. The membranes were washed twice and incubated with secondary anti–mouse or rabbit immunoglobulin G horseradish peroxidase–conjugated antibodies for 1 h. After intensive washing, the membranes were subjected to an ECL chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer’s directions and exposed to x-ray films (Fuji Photo Film, Tokyo, Japan).
Assessment of MMP-9 activity by gelatin zymography
Matrix metalloproteinase 9 activity was detected by gelatin zymography on premade 8% polyacrylamide gels containing 0.1% gelatin using 10 mL serum-free media from treated cultures. After electrophoresis, the gel was removed and incubated in 1× zymogram renaturing buffer for 30 min at room temperature with gentle agitation. The gel was equilibrated for 30 min with 1× zymogram developing buffer and then incubated with fresh 1× zymogram developing buffer overnight. The bands were visualized by staining for 30 to 60 min with a solution containing 0.1% Coomassie R-250 in 40% ethanol and 10% acetic acid, followed by destaining for 2 h at room temperature in a solution containing 10% ethanol and 7.5% acetic acid. The images were taken by using UVP Biochemi EC3 imaging system (Quansys Biosciences, Logan, Utah, USA).
Evaluation of NF-κB p65 activity
Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Waltham, Mass, USA). Nuclear factor κB activity was measured in nuclear protein extracts (15 μg) by the TransAM NF-κB p65 protein assay (Active Motif, Carlsbad, Calif), an enzyme-linked immunosorbent assay–based method designed to specifically detect and quantify NF-κB p65 subunit activation, with high sensitivity and reproducibility. Briefly, nuclear extracts were incubated in 96-well plates coated with immobilized oligonucleotide containing a consensus binding site for p65 NF-κB. Nuclear factor κB binding to the target oligonucleotide was detected by a primary antibody specific for the activated form of p65, visualized by anti–immunoglobulin G horseradish peroxidase conjugate, and quantified at a wavelength of 450 nm.
Data are expressed as means ± SEM. Statistical differences were estimated by one-way analysis of variance followed by Dunnett test. P < 0.05 was considered significant.
Effects of HSC70 on MAP and HR
In the control group and HSC70 group, there was no significant change of HR and MAP compared with baseline (see Table, Supplemental Digital Content 1, at http://links.lww.com/SHK/A231). Figure 1 shows that MAP decreased rapidly after i.v. administration of 10 mg/kg of LPS (Fig. 1A). Pretreatment of HSC70 (20 μg/kg, i.v.) 10 min before LPS injection attenuated LPS-induced hypotension at the period of 1 to 4 h (P < 0.05 vs. LPS group). Similarly, the HR increased significantly 1 h after i.v. injection of LPS (Fig. 1B). Pretreatment of HSC70 prevented LPS-induced tachycardia at 2 to 4 h (P < 0.05 vs. LPS group). The data demonstrate that hypotension and tachycardia, the hallmarks of septic shock, were induced by LPS in our animal model and were both attenuated by HSC70 pretreatment.
Effects of HSC70 on left ventricular function
Ventricular performance was determined by LVSP, max dP/dt, and min dP/dt. In the control group and HSC70 group, there was no significant change of these parameters compared with baseline (see Table, Supplemental Digital Content 1, at http://links.lww.com/SHK/A231). Figure 2 shows that LVSP was reduced by 50% in LPS-treated rats at 4 h compared with baseline value, and HSC pretreatment attenuated the LPS-induced decrease in LVSP at 3 to 4 h (P < 0.05) (Fig. 2A). In addition, LPS impaired LV contraction as shown by decrease in max dP/dt at 3 to 4 h, and this decrease was prevented by HSC70 at 3 to 4 h when compared with LPS group without HSC pretreatment (P < 0.05) (Fig. 2B). In parallel, LPS impaired LV relaxation as shown by decrease in min dP/dt at 3 to 4 h, whereas the impaired min dP/dt was prevented by HSC70 at 1 to 4 h following LPS administration (P < 0.05) (Fig. 2C).
Effects of HSC70 on liver function and plasma levels of glucose, LDH, NOx, and TNF-α
As shown in Figures 1A and 3B, liver function was assessed by plasma levels of GOT and GPT at the time point of 4 h. Lipopolysaccharide injection caused significant increase in GOT compared with control (P < 0.05), and HSC70 prevented this elevation (P < 0.05 vs. LPS group) (Fig. 1A). Similarly, HSC70 prevented the elevation of GPT caused by LPS at 4 h (P < 0.05 vs. LPS group) (Fig. 1B). In addition, blood glucose was decreased, whereas LDH was elevated at 4 h after LPS injection in LPS-treated rats (P < 0.01 vs. control group), whereas their levels were preserved in those with pretreatment of HSC70 (P < 0.01 vs. LPS group) (Figs. 1C and 3D). Plasma levels of NOx and TNF-α were significantly increased in LPS-treated rats (P < 0.01), whereas HSC70 pretreatment attenuated the LPS-induced elevations of NOx and TNF-α (P < 0.01 vs. LPS group) (Figs. 1E and 3F).
Effects of HSC70 on the expression of iNOS and COX-2 in the heart and liver
Figure 2 depicts Western blotting of iNOS and COX-2 in cardiac and hepatic tissues. We found that compared with control group, iNOS and COX-2 were upregulated in both organs at 4 h following LPS injection (P < 0.01), whereas the LPS-induced upregulations of iNOS and COX-2 were attenuated in HSC70 pretreatment group (P < 0.01 vs. LPS group).
Effects of HSC70 on MMP-9 in the heart and liver
Protein expression and activity of MMP-9, an important mediator of inflammatory responses in sepsis, were determined at the 4-h time point in cardiac and hepatic tissue (Fig. 3). Compared with the control group, protein expression and activity of MMP-9 were both upregulated in cardiac and hepatic tissues in LPS-treated rats (P < 0.05), whereas the LPS-induced MMP-9 activations in both organs were inhibited by HSC70 pretreatment (P < 0.05 vs. LPS group) (Fig. 3).
Effects of HSC70 on expression of IκBα and p65 in the heart and liver
Protein expression of IκBα and activity of p65 in cardiac and hepatic tissue were examined at 4 h. We found that compared with the control group, the expression of IκBα and the activity of p65 were both increased in cardiac and hepatic tissues 4 h following LPS administration (P < 0.05), whereas the LPS-induced activations of IκBα and p65 in both organs were attenuated by HSC70 pretreatment (P < 0.05 vs. LPS group) (Fig. 4).
Effects of HSC70 on the expressions of ERK1/2 and p38 in the heart and liver
We finally examined the MAPK pathway including ERK1/2 and p38 by Western blotting. Compared with the control group, ERK1/2 and p38 were both upregulated in cardiac and hepatic tissues 4 h following LPS injection (P < 0.05), whereas the LPS-induced upregulations of ERK1/2 and p38 in both organs were attenuated by HSC70 pretreatment (P < 0.05 vs. LPS group) (Fig. 5).
Cardiac dysfunction and hepatic injury are both characteristic manifestations of sepsis with prognostic implications. Sepsis-induced cardiac dysfunction is present in more than 40% of sepsis patients (16), and its appearance can increase the mortality rate to 70% (17,18). Likewise, hepatic injury at diagnosis was associated with early death in patients with sepsis as shown in a recent multicenter study (19). The present study showed that in rats with septic shock, exogenous HSC70 was capable of attenuating LPS-induced myocardial and hepatic dysfunction along with systemic derangements including hypotension, tachycardia, hypoglycemia, and increased plasma LDH. These protections against endotoxemia-induced cardiac and hepatic dysfunctions are associated with anti-inflammatory effects as shown by inhibitions of proinflammatory mediators including TNF-α, NO, COX-2, and MMP-9 via the MAPK/NF-κB pathway. To our knowledge, these pleiotropic protections of HSC70 have not been previously reported. Given the fact that the major contributing factor to the high morbidity and mortality of septic shock is lack of the effective treatment (20), our study may provide a basis for a novel prophylactic strategy for management of septic shock (Figs. 6 and 7).
Both HSP70 and HSC70 are conventionally regarded as intracellular proteins. They function as molecular chaperons, playing important roles in folding and transporting newly synthesized peptides in response to stress. Recently, emerging data implicate that extracellular HSP70 and HSC 70 play a protective role in sepsis. Rozhkova et al. (13) demonstrated that exogenous HSP70 inhibited LPS-induced production of reactive oxygen species in myeloid cells and NO expression in macrophages. These protective effects of extracellular HSP70 were later reported in rats with septic shock induced by lipoteichoic acid, another model of sepsis caused by Gram-positive bacteria (21). In addition, Su et al. (15) recently showed that HSC70 preconditioning induced cardiac tolerance to endotoxin through attenuation of inflammatory responses. Our study further demonstrates the important findings that the beneficial effects of HSC70 not only take place in myocardium but also occur in liver and systemic circulation.
Tolerance to bacterial endotoxin induced by prior exposure to endotoxin is a well-known phenomenon, which is characterized by a transient state of cellular hyporesponsiveness with decreased production of proinflammatory cytokines in response to LPS (22). Classically, LPS tolerance is achieved by a low concentration of LPS to induce the nonresponsive state followed by a subsequent challenge with a higher concentration of LPS. However, recent studies have demonstrated that other intracellular proteins, such as high-mobility group box protein 1 and HSP70, when present in the extracellular compartment, can also induce LPS tolerance (13,23). The precise mechanisms underlying endotoxin tolerance in the present study are not fully elucidated; however, we speculate that Toll-like receptor 4 (TLR4) and CD14 are potential targets of HSC70. It has been shown that in myocardium HSC70 can preactivate TLR4 to induce endotoxin tolerance (15). Asea et al. (8) have initially identified CD14 as a necessary receptor in myeloid cell for HSP70. Likewise, a recent study demonstrated that in fibroblasts, HSC70-mediated immunomodulatory effects are dependent on CD14 and TLR4 (24). Therefore, our study strengthens the notion that the nonnoxious agent such as HSC70 can also achieve endotoxin tolerance.
In response to endotoxemia, the organism releases proinflammatory mediators including TNF-α and NO into surrounding tissues, thereby causing tissue damage and organ failure (25). In the present study, we found that HSC70 pretreatment attenuated LPS-induced cardiac and hepatic dysfunction with the associated decreases in the production of circulating proinflammatory mediators (TNF-α and NO). These findings were in line with a recent study showing that extracellular HSC70 induced cardiac tolerance to endotoxin with associated inhibition of TNF-α production (15). Furthermore, we demonstrate novel findings that not only in the heart, but also in the liver, there are inhibitory effects of HSC70 on expressions of proinflammatory mediators (iNOS, COX-2, and MMP-9).
Nuclear factor κB is an important transcription factor for proinflammatory mediators such as iNOS, COX-2, MMP-9, and TNF-α (26). Lipopolysaccharide elaborates the nuclear translocation of NF-κB through IκB degradation. Inhibitor of κB kinase can phosphorylate IκBα. Phosphorylated IκBα is subsequently ubiquitinated and degraded by the 26S proteasome. In addition, NF-κB activation has been implicated as a possible mechanism for the induction of endotoxin tolerance in an in vitro LPS-preconditioning model (27). To test the hypothesis that HSC70-induced LPS tolerance is associated with deactivation of the NF-κB pathway, we determined if HSC70 pretreatment inhibits phosphorylation and subsequent degradation of IκBα. We found that HSC70 pretreatment significantly attenuated LPS-induced IκBα phosphorylation in both myocardium and liver. Similarly, we found that HSC70 decreased LPS-induced phosphorylation of NF-κB subunit p65, a downstream marker of NF-κB activation and nuclear translocation. These observations implicate the role of NF-κB activation cascade in HSC70-induced cardiac and hepatic tolerance to endotoxin.
The signaling pathways responsible for sepsis-induced NF-κB activation are not fully elucidated. Certain evidence suggests that MAPKs have an essential role (28,29). There are three well-characterized subfamilies of MAPKs: ERK1/2, JNK, and p38 MAPK. All of these subfamilies have been implicated as cell signaling components involved in the generation of inflammatory mediators by a variety of cells (30–32). Intriguingly, our results demonstrated that in both cardiac and hepatic tissue, both ERK1/2 and p38 MAP kinase, but not JNK, were activated by LPS, and these activations were inhibited by HSC70 pretreatment. These data are in line with a recent study by Yang et al. (33) showing that p38 MAP kinase and ERK1/2, but not JNK, were activated in cardiomyocytes treated with septic plasma. Their study also indicates the central role of p38 MAP kinase/NF-κB signaling pathway in the sepsis-induced conversion of cardiomyocytes to a proinflammatory phenotype. Similarly, in hepatocytes, it has also been found that p38 MAPK pathway mediated endotoxin-induced leukocyte recruitment and hepatocellular injury (30). Taken together, our data provide novel evidence suggesting that p38 MAPK and ERK1/2 participate in the regulation of HSC70-induced cardiac and hepatic tolerance to endotoxin.
The limitation of this study is that only prophylactic but not therapeutic effects were determined because HSC70 was administered prior to but not after initiation of sepsis. In addition, LPS-injection model is used instead of cecal ligation and puncture, which more mimics the clinical sepsis. However, given that interactions with TLR4 and CD14, two receptors essential for LPS-induced NF-κB signaling (26), have been hypothesized as mechanistic targets of HSC70, our study design was to examine the preconditioning effects by HSC70 pretreatment and to investigate endotoxin tolerance using the LPS model. Therefore, future studies are necessary to determine therapeutic effects of HSC70 and substantiate these results in other models of sepsis such as cecal ligation and puncture or lipoteichoic acid.
In conclusion, the present study shows that in rats with septic shock, exogenous HSC70 pretreatment attenuated LPS-induced myocardial and hepatic dysfunction along with systemic hemodynamic derangements, hypoglycemia, and increased plasma LDH. These protections against endotoxemia-induced cardiac and hepatic dysfunctions are associated with inhibitions of TNF-α, NO, COX-2, and MMP-9 and potentially through the MAPK/NF-κB pathway. Further animal and clinical studies are needed to substantiate these important findings.
This research was supported by the grant from the National Science Council of Taiwan (NSC 98-2314-B-037-005-MY3), and by grants from the Kaohsiung Medical University Hospital, Taiwan (KMUH99-9R24, KMUH100-0R31 and KMUH101-1R32).
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Heat shock protein; sepsis; shock; HSC70 — heat shock cognate protein 70; LPS — lipopolysaccharide; LVSP — left ventricular systolic pressure; max dP/dt — maximal slope of systolic pressure increment; min dP/dt — maximal slope of diastolic pressure decrement; GOT — glutamic-oxaloacetic transaminase, GPT — glutamic-pyruvic transaminase; LDH — lactate dehydrogenase; TNF-α — tumor necrosis factor α; NOx — nitrite/nitrate; iNOS — inducible nitric oxide synthase; COX-2 — cyclooxygenase 2; MMP-9 — matrix metalloproteinase 9; NF-κB — nuclear factor κB; IκBα — inhibitor of nuclear factor κB; MAPK — mitogen-activated protein kinase
Supplemental Digital Content
© 2014 by the Shock Society