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Basic Science Aspects

Novel Role of Resveratrol

Suppression of High-Mobility Group Protein Box 1 Nucleocytoplasmic Translocation by the Upregulation of Sirtuin 1 in Sepsis-Induced Liver Injury

Xu, Wei*; Lu, Yang; Yao, Jihong; Li, Zhenlu*; Chen, Zhao*; Wang, Guangzhi*; Jing, Huirong*; Zhang, Xinyuan; Li, Mingzhu; Peng, Jinyong; Tian, Xiaofeng*

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doi: 10.1097/SHK.0000000000000225
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Abstract

INTRODUCTION

Sepsis is a leading cause of disability, morbidity, and mortality in intensive care units and is associated with host responses, innate immunity, coagulation abnormalities, anti-inflammatory effects/immunosuppression, and organ dysfunction (1). In the pathogenesis of sepsis, damage-associated molecular patterns, such as extracellularly released high-mobility group protein box 1 (HMGB1), promote innate immune cell activation and antigen-presenting cell recruitment and activation. These cells are engaged in the host response, in which both proinflammatory and anti-inflammatory mechanisms contribute to the clearance of infection and tissue recovery on the one hand and organ injury and secondary infections on the other, and in tissue damage through pattern recognition receptors, triggering systemic inflammatory response syndrome and immunological homeostasis disorders that can cause sepsis (2, 3). However, the mechanism of HMGB1 translocation during sepsis is poorly understood. Previous studies have demonstrated that HMGB1 translocation from the nucleus to the cytoplasm is increased in acute liver failure (ALF) (4) and that inhibition of HMGB1 secretion attenuates systemic inflammatory response syndrome and sepsis-induced organ injury (5). In contrast, HMGB1 release from hepatocytes increases the risk of liver damage during sepsis. Therefore, inhibiting HMGB1 translocation and release should protect against liver injury during sepsis and may offer a wider therapeutic window for sepsis.

High-mobility group protein box 1 is a nuclear protein that binds DNA, stabilizes nucleosomes, and facilitates gene transcription (6). It functions as an alarmin, is secreted into the extracellular milieu, and mediates downstream inflammatory responses in endotoxemia, arthritis, and sepsis (7). Extracellular HMGB1 sustains inflammatory responses by stimulating innate immune cell migration and activation, facilitating the innate recognition of bacterial products (8, 9), and inhibiting the phagocytosis of apoptotic neutrophils by macrophages (10). Extracellular HMGB1 release also acts as an inflammatory mediator and is promoted by nucleocytoplasmic HMGB1 translocation. Acetylation is important for active HMGB1 translocation (11, 12), and histone acetyltransferase and the histone deacetylase (HDAC) isoforms HDAC1/4 (13), HDAC4/5 (14), and sirtuin 6 (SIRT6) (15) may control this process. Nucleocytoplasmic HMGB1 translocation has also been observed in hepatocytes in an ALF animal model generated using d-galactosamine and lipopolysaccharide (LPS) and in ALF patients (4). Nevertheless, the mechanism underlying HMGB1 translocation during sepsis-induced liver injury is still unknown.

The HDAC SIRT1 is constitutively expressed in most cells and is involved in signaling pathways regulating the cellular life span and responses to oxidative stress (16). Sirtuin 1 specifically removes acetyl groups from lysine residues and leads to histone and nonhistone protein deacetylation. A wide variety of proteins are SIRT1 substrates, including many transcription factors and cofactors (17). For example, SIRT1 inhibits adipocyte differentiation and suppresses inflammation by targeting the peroxisome proliferator-activated receptor γ and nuclear factor κB transcription factors (18) and regulates genomic stability, cellular stress resistance, and senescence by deacetylating its target proteins (19). As HMGB1 is actively released from the nucleus to the cytoplasm after acetylation (11, 20), SIRT1 may regulate HMGB1 translocation via deacetylation.

Resveratrol (3,5,4′-trihydroxy-trans-stilbene; RESV), a polyphenol that is best known as a constituent of red wine and several plants, exerts positive effects on various disease states (21). Resveratrol is also a potent SIRT1 activator (22) with anti-inflammatory properties that can upregulate SIRT1 and attenuate severe liver injury after sepsis. Therefore, we hypothesized that RESV inhibits nucleocytoplasmic HMGB1 translocation by activating SIRT1. In turn, SIRT1-mediated HMGB1 repression may be a new target for preventing sepsis-induced liver injury.

MATERIALS AND METHODS

Ethics Statement

All procedures were conducted according to the Institutional Animal Care and Use Committees of Dalian Medical University and were approved by the Institutional Ethics Committee of Dalian Medical University.

Drugs

Resveratrol (RESV) (CAS 501-36-0, purity >99%, molecular formula: C14H12O3, molecular weight: 228.24) was purchased from Shanghai Winherb Medical Science Co, Ltd (Shanghai, China) and dissolved in olive oil. For the cell culture experiments, RESV was dissolved in dimethyl sulfoxide.

Nicotinamide (NAM) (CAS 98-92-0, purity ≥99%, molecular formula: C6H6N2O, molecular weight: 122.13) was purchased from the Shanghai Source Leaf Biological Technology Co, Ltd (Shanghai, China) and dissolved in saline.

Experimental model

Male Sprague-Dawley rats (180–220 g) were obtained from the Animal Center of Dalian Medical University (Dalian, China). Thirty-six rats were randomly divided into six groups, including the (i) control group, (ii) cecal ligation and puncture (CLP) group, (iii) control + RESV (60 mg/kg per day) pretreatment group, (iv) CLP + RESV (60 mg/kg per day) pretreatment group, (v) control + NAM (60 mg/kg per day) pretreatment group, and (vi) CLP + NAM (60 mg/kg per day) pretreatment group.

Rats were treated with CLP to induce sepsis. Rats were anaesthetized under light isoflurane. A small midline incision was made to allow for exteriorization of the cecum. The cecum was carefully ligated to ensure unobstructed movement of digested food through the gastrointestinal tract. Double puncture of the cecum was performed using a 22-gauge needle. The cecum was gently squeezed to extrude a small amount of feces into the peritoneal cavity. The cecum was carefully placed back into the peritoneal cavity, and the incision closed. Animals received prewarmed normal saline (20 mL/kg body weight) subcutaneously after the surgical procedure to prevent dehydration. Sham animals received all surgical manipulations with the exception of ligation and puncture of the cecum. Resveratrol or NAM was administered by gavage for the 3 days leading up to the surgery. The doses and administration methods for RESV or NAM were previously described (23, 24). The rats in the control and CLP groups were given an equal volume of olive oil as a vehicle control. At 24 h after surgery, blood and liver samples were collected and preserved for the subsequent analysis.

Serum alanine aminotransferase and aspartate aminotransferase levels

Blood samples were taken from the abdominal aorta and centrifuged at 3,000 rpm for 15 min, after which the serum was collected. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured according to the manufacturer’s protocols (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Histological examination and terminal deoxynucleotidyl transferase dUTP nick end-labeling staining

The middle lobe of the right liver was excised for histopathology. Consecutive sections (5 μm) of paraffin-embedded liver tissues were prepared for hematoxylin-eosin staining and were subsequently evaluated by light microscopy. Hepatic injury scores were measured with the liver morphologic criteria (25): spotty necrosis, capsular inflammation, portal inflammation, ballooning degeneration, and steatosis. We graded spotty necrosis and scored it as follows: 0 = none, 1 = one focus or less per 10× objective, 2 = two to four foci per 10× objective, 3 = five to 10 foci per 10× objective, and 4 = more than 10 foci per 10× objective. We graded capsular inflammation and scored it in each ×10 area after magnification for the presence of capsular inflammation, as follows: 0 = none, 1 = capsular inflammation in 1 × 10 magnification area, 2 = capsular inflammation in 2 × 10 magnification areas, and 3 = capsular inflammation in 3 × 10 magnification areas. We scored portal inflammation as follows: 0 = none; 1 = mild, some, or all portal areas; 2 = moderate, some, or all portal areas; 3 = marked, all portal areas. We scored ballooning degeneration as follows: 0 = none, 1 = ballooning degeneration in one third of hepatic lobule, 2 = ballooning degeneration in two thirds of hepatic lobule, and 3 = ballooning degeneration in all parts of hepatic lobule. We scored steatosis as follows: 0 = none, 1 = less than 30% hepatocytes containing fat, 2 = 30% to 70% hepatocytes containing fat, and 3 = more than 70% hepatocytes containing fat. The liver injury severity score ranged from 0 (none) to 16 (severe). Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining was performed using an in situ Cell Death Detection Kit (Roche, Nutley, NJ).

Measurement of the serum cytokines and malondialdehyde activity

The serum levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and HMGB1 were measured using enzyme-linked immunosorbent assay kits from the BOSTER Bio-engineering Limited Company (Wuhan, China) according to the manufacturer’s protocols. The level of malondialdehyde (MDA) in the liver tissues was quantified with a lipid peroxidation MDA assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions.

Cell culture and treatment

Human liver L02 cell lines (HL-7702) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science Cell Bank (Shanghai, China). L02 cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, Calif) supplemented with 10% fetal bovine serum. The cells were kept at 37°C in a humidified atmosphere with 5% CO2. To simulate sepsis in vitro, the L02 cells were stimulated with LPS (400 ng/mL) for 24 h (26). For pretreating the L02 cells with RESV and NAM, the cells were incubated with 10 μM of RESV or 10 mM of NAM for 6 h, and the cells were then subjected to LPS stimulation as needed.

siRNA transfection

siRNAs, both gene-specific and nonspecific control, were obtained from Shanghai GenePharma Co (Shanghai, China). The SIRT1 siRNA sequences were sense 5′-CCCUGUAAAGCUUUCAGAA(dTdT)-3′ and antisense 5′-UUCUGAAAGCUUUACAGGG(dTdT)-3′. The transient siRNA transfection was performed as previously described (27). After 48 h of siRNA transfection, the cells were treated with 10 μM RESV or 10 mM NAM for an additional 6 h and then stimulated with 400 ng/mL LPS for 24 h. The cells were then collected for the protein and immunofluorescence analyses.

Western blotting

Proteins were extracted from the liver tissues or L02 cells using a cytoplasm and nucleus protein extraction reagent or total protein extraction reagent (KeyGEN Biotech, Nanjing, China) according to the manufacturer’s instructions. Protein concentrations were determined using a BCA protein assay kit (Beyotime Biotech, Hangzhou, China). Sample proteins (30 μg/lane) were separated by 10% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, Calif); afterward, the samples were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Calif). After blocking, the membranes were probed overnight at 4°C with the following primary antibodies: anti-SIRT1, anti-HMGB1, antihistone H3 (Santa Cruz Biotechnology, Santa Cruz, Calif), and anti–β-actin (Santa Cruz Biotechnology). After a triple wash in Tween-20 tris buffered saline, the membranes were incubated in relevant horseradish peroxidase–conjugated secondary antibodies for 2 h at 37°C. Proteins were detected using chemiluminescence-plus reagents from the Beyotime Institute of Biotechnology (Beyotime Biotech). The emitted light was captured by a BioSpectrum-410 multispectral imaging system with a Chemi HR camera 410, and the images were analyzed using Gel-Pro Analyzer (version 4.0; Media Cybernetics, Rockville, Md).

Coimmunoprecipitation

Immunoprecipitation was performed with antibodies against acetyl-lysine (Abcam Ltd, Cambridge, UK), SIRT1, or HMGB1 in the cytoplasm and nucleus lysate protein in IP buffer (50 mM HEPES, 0.5% Nonidet P-40, 150 mM NaCl, 10% glycerol, and 1 mM EDTA). Normal immunoglobulin G (IgG) was used as a negative control. The samples were first precleared with a nonspecific IgG antibody. The precleared lysates were then incubated overnight with an antibody to anti–acetyl-lysine, anti-SIRT1, or anti-HMGB1; the samples were then incubated for 2 h with protein A/G agarose. The samples were washed four times with phosphate-buffered saline and subjected to Western blot analysis.

Statistical analysis

All statistical analyses were performed using the SPSS16.0 statistical software package (SPSS Inc, Chicago, Ill). Comparisons between the groups were made using the Kruskal-Wallis test for nonnormal distributions or a one-way analysis of variance followed by Tukey test for normal distributions. P < 0.05 was considered statistically significant, and the results are expressed as the mean ± SD.

RESULTS

RESV inhibits HMGB1 release and attenuates sepsis-induced acute liver injury

Sepsis-induced liver injury activates Küpffer cells, the resident macrophages in the liver, to release proinflammatory cytokines and chemokines (28). After CLP, the serum levels of ALT and AST were increased relative to those in the control group, indicating liver damage. Interleukin 6, TNF-α, and HMGB1 levels increased relative to those in the control group, indicating that inflammatory imbalance occurs with sepsis (Figs. 1 and 2A). Pretreatment with the RESV reduced the elevated serum transaminase, proinflammatory cytokines, and HMGB1 levels. However, pretreating CLP rats with NAM (an SIRT1 inhibitor) increased the levels of these factors during sepsis as compared with the CLP group, indicating that NAM, unlike RESV, aggravates liver injury during sepsis. The liver injury caused by sepsis and the RESV and NAM effects were confirmed by histological analyses. As shown in Fig. 2, B and C, CLP caused marked pathological changes and neutrophil influx, which were prevented by RESV. Resveratrol effectively inhibits HMGB1 release and protects against CLP-induced acute liver injury.

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Fig. 1:
Resveratrol attenuates acute liver injury and inflammation in septic rats. Sepsis was induced by the surgical perforation and CLP. A, Serum ALT levels. B, Serum AST levels. C, Serum TNF-α levels. D, Serum IL-6. Values are expressed as the means ± SD, n = 6. *P< 0.05 vs. control group; **P < 0.01 vs. control group; #P < 0.05 vs. CLP group; ##P < 0.01 vs. CLP group.
F2-9
Fig. 2:
Liver histopathologic and serum HMGB1 level changes in the septic rats from different groups. A, Alterations in the HMGB1 serum levels. B, The liver injury severity was scored as described above. C, Hematoxylin-eosin staining of representative liver sections (original magnification ×400). Large arrow: hepatic sinusoid; small arrow: neutrophil influx. Results are given as the means ± SD, n = 6. *P< 0.05 vs. control group; **P< 0.01 vs. control group; #P < 0.05 vs. CLP group; ##P < 0.01 vs. CLP group.

RESV protects hepatocytes from sepsis-induced oxidative stress and apoptosis

Apoptosis and liver oxidation were assessed by TUNEL staining (Fig. 3, A and B) and measuring MDA (Fig. 3C). Malondialdehyde is a degradation product of polyunsaturated fatty acids and reacts with free radicals; therefore, MDA is an indirect marker of the presence of reactive oxygen species in hepatocytes. As shown in Figure 3, the rats displayed a immediate increases in apoptosis and MDA after CLP. However, RESV reversed the high levels of MDA and apoptosis in CLP rats. Rats that were pretreated with the SIRT1 inhibitor NAM also displayed increased MDA levels and increased apoptosis. These results indicate that RESV protects rats against CLP-induced oxidative stress and apoptosis, which may be associated with SIRT1.

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Fig. 3:
Resveratrol inhibits apoptosis and oxidative stress during sepsis. Rats were pretreated with RESV or NAM before CLP. A, Representative TUNEL staining after CLP (original magnification ×400). B, Quantification of hepatic TUNEL staining. The results are given as the means ± SEM of 10 to 12 frames per group from three or four animals per group. C, Malondialdehyde content (n = 6). Values are given as the means ± SD. *P< 0.05 vs. control group; **P< 0.01 vs. control group; #P < 0.05 vs. CLP group; ##P < 0.01 vs. CLP group.

RESV protects against CLP-induced liver injury through the upregulation of SIRT1

Sirtuin 1 upregulation inhibits apoptosis and protects against oxidative stress, ameliorating hepatic steatosis and acute liver injury (29). In a previous study, RESV protected the heart, lung, and kidneys from injury during sepsis by limiting energy metabolism, oxidation, and apoptosis (23, 30). Because RESV is an SIRT1 activator, we investigated whether SIRT1 upregulation is associated with RESV-induced protection from sepsis. In our study, the CLP group had lower hepatic SIRT1 expression than did the control group (Fig. 4A). However, RESV upregulated SIRT1 expression, and RESV treatment prevents CLP-induced downregulation of SIRT1 expression in liver and mitigated liver injury. The SIRT1 inhibitor NAM aggravated the reduction in liver SIRT1 expression caused by CLP and exacerbated the liver injury. These results suggest that SIRT1 plays an important role in the RESV-induced amelioration of CLP-induced liver injury.

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Fig. 4:
Sirtuin 1 represses the nucleocytoplasmic translocation of HMGB1 in the CLP rat liver. Sepsis reduces SIRT1 expression, and RESV increases SIRT1 expression in the CLP rat liver. After 24 h of CLP, HMGB1 was released from the nucleus into the cytoplasm. Resveratrol inhibited HMGB1 translocation from the nucleus to the cytoplasm in the control and CLP groups, whereas NAM enhanced the HMGB1 translocation during CLP. A, Expression of the SIRT1 protein. B, Expression of the HMGB1 protein in the cytoplasm (cyt). C, Expression of the HMGB1 protein in the nucleus (nu). D, Expression of the SIRT1 protein. Values are given as the means ± SD, n = 3. *P< 0.05 vs. control group; **P< 0.01 vs. control group; #P < 0.05 vs. CLP group; ##P < 0.01 vs. CLP group; &P < 0.05 vs. CLP + RESV group; &&P < 0.01 vs. CLP + RESV group.

Overexpression of SIRT1 represses the nucleocytoplasmic translocation of HMGB1 in vivo

After sepsis, necrotic and inflammatory cells release HMGB1, leading to the induction of immune and inflammatory responses through RAGE (receptor for advanced glycation end-products) and TLR4 signaling pathways (31). As shown in Fig. 4B, the CLP group had higher HMGB1 expression in the cytoplasm than did the sham-treated group. Resveratrol treatment reduced the CLP-induced increase in the cytoplasmic HMGB1 expression as compared with the CLP group; the RESV-treated group had higher HMGB1 in the cell nuclei as well as higher SIRT1 expression in the liver (Fig. 4, A and C). The overexpression of SIRT1 downregulates the HMGB1 levels in hepatocyte cytoplasm. The protective effects of RESV against CLP-induced liver injury may be related to the SIRT1-mediated inhibition of HMGB1 translocation from the nucleus to the cytoplasm.

To test the effect of SIRT1 on HMGB1 translocation in vivo, the CLP model rats were pretreated with the SIRT1 inhibitor NAM. We found that NAM increased the levels of HMGB1 in the cytoplasm and reduced the levels of HMGB1 in the nucleus in vivo. There was also a reduction in SIRT1 expression in the liver. We also explore whether NAM can prevent RESV-induced increase in SIRT1 expression. As shown in Fig. 4D, the RESV-induced upregulation of SIRT1 expression in liver was abrogated by NAM.

Overexpression of SIRT1 represses the nucleocytoplasmic translocation of HMGB1 in vitro

To test the effect of SIRT1 on HMGB1 translocation in vitro, SIRT1 was knocked down by siRNA transfection in human liver L02 cells. Consistent with a previous study showing that trichostatin A, an HDAC inhibitor, induces HMGB1 translocation and release (32), we found that SIRT1 siRNA transfection increased the levels of HMGB1 in the cytoplasm and reduced the levels of HMGB1 in the nucleus in vitro. The regulatory effect of SIRT1 on HMGB1 nucleocytoplasmic translocation was confirmed with Western blot analysis (Fig. 5A). As shown in Fig. 5B, the LPS group had higher HMGB1 expression in the cytoplasm than did the control group. Resveratrol treatment reduced the LPS-induced increase in the cytoplasmic HMGB1 expression as compared with the LPS group. The RESV-treated group had higher HMGB1 in the cell nuclei and higher SIRT1 expression in the L02 cell. The overexpression of SIRT1 downregulates the HMGB1 levels in L02 cell cytoplasm. Taken together, these results indicate that RESV reduces the translocation of HMGB1 from the nucleus to the cytoplasm by upregulating SIRT1 and that this mechanism may contribute to the RESV-induced prevention of sepsis-induced liver injury.

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Fig. 5:
Sirtuin 1 represses the LPS-induced HMGB1 nucleocytoplasmic translocation in L02 cells. A, Protein expression of SIRT1 and HMGB1 in the L02 cells (a, protein expression of SIRT1; b, protein expression of HMGB1 in the nucleus; and c, protein expression of HMGB1 in the cytoplasm). Cells were transfected with SIRT1 siRNA or control siRNA before RESV administration. Following cell fractionation, the SIRT1 and HMGB1 content in the cytoplasmic or nuclear fraction was determined by Western blot analysis. Values are given as the means ± SD, n = 3. *P< 0.05 vs. si-control group; **P< 0.01 vs. si-control group; #P < 0.05 vs. si-control + RESV group; ##P < 0.01 vs. si-control + RESV group. B, Protein expression of SIRT1 and HMGB1 in the LPS-induced L02 cells (d, protein expression of SIRT1; e, protein expression of HMGB1 in nucleus; and f, protein expression of HMGB1 in cytoplasm). L02 cells were transfected with the nontargeting control or SIRT1 siRNA. After 48 h, the transfected cells were exposed to 10 μM of RESV or 10 mM of NAM for an additional 6 h, after which they were stimulated with 400 ng/mL of LPS for 24 h and monitored for SIRT1 and HMGB1 cytoplasmic translocation by Western blot analysis after cell fractionation. Values are given as the means ± SD, n = 3. *P< 0.05 vs. control group; **P< 0.01 vs. control group; #P < 0.05 vs. LPS group; ##P < 0.01 vs. LPS group.

Interaction between SIRT1 and HMGB1 and their cellular localization

We speculated that SIRT1 interacts with HMGB1 by deacetylating HMGB1. As shown in Fig. 6, A and B, a tissue extract was immunoprecipitated with anti-HMGB1 antibody, which was followed by immunoblotting with an anti-SIRT1 antibody. Sirtuin 1 coprecipitated with HMGB1, indicating a positive interaction between these two proteins (Fig. 6A). The same result was obtained when the tissue extract was immunoprecipitated with an anti-SIRT1 antibody followed by immunoblotting with an anti-HMGB1 antibody, confirming the interaction between these two proteins (Fig. 6B). Nuclear and cytosolic extracts of liver tissue were also immunoprecipitated with anti-IgG or anti-SIRT1 antibody, which was followed by immunoblotting with an anti-HMGB1 antibody. The physical interaction between SIRT1 and HMGB1 occurs in both the cytoplasm and the nucleus, but there is a stronger interaction in the nucleus (Figs. 6, C and D).

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Fig. 6:
Cellular localization of SIRT1 and HMGB1 and their interaction. A and B, Coimmunoprecipitation of the (a) IgG control group, (b) control group, and (c) CLP group. Samples subjected to immunoprecipitation with the anti-HMGB1 antibody followed by immunoblotting analysis with the anti-SIRT1 antibody. Samples subjected to immunoprecipitation with the anti-SIRT1 antibody followed by immunoblotting analysis with the anti-HMGB1 antibody. C, Immunoprecipitation with the anti-SIRT1 antibody followed by immunoblotting analysis with the anti-HMGB1 antibody in the nucleus. D, Immunoprecipitation with the anti-SIRT1 antibody followed by immunoblotting analysis with the anti-HMGB1 antibody in the cytoplasm.

SIRT1 regulates HMGB1 translocation by deacetylating HMGB1 in septic rats

High-mobility group protein box 1 is a conservative nonhistone protein, and SIRT1 is a well-known deacetylase enzyme of HDAC class III that deacetylates nonhistone proteins (33). Previous studies have demonstrated that highly acetylated HMGB1 translocates from the nucleus to the cytoplasm in macrophages and inflammatory cells, and this phenomenon has also been observed in nonimmune parenchymal cells. To clarify whether the translocation of HMGB1 is mediated by its SIRT1-mediated deacetylation in the liver after CLP, we analyzed the levels of acetylated HMGB1 in both the nuclei and cytoplasm of hepatic cells. As shown in Figs. 4A and 7A, the CLP group had lower SIRT1 expression, with higher levels of acetylated HMGB1 in the cytoplasm than the control group. Resveratrol treatment increased the expression of SIRT1 and reduced the levels of acetylated HMGB1 in the cytoplasm as compared with the levels in the CLP group; RESV also decreased the levels of acetylated HMGB1 in the nucleus (Fig. 7B). However, the SIRT1 inhibitor NAM had the opposite effect on the levels of acetylated HMGB1 in the nucleus and cytoplasm. These results indicate that SIRT1-mediated HMGB1 nucleocytoplasmic translocation is regulated by the deacetylation of HMGB1 in the septic liver.

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Fig. 7:
Sirtuin 1 regulates HMGB1 translocation by deacetylation in septic rats. A, Expression of acetylated HMGB1 protein in the nucleus (nu). B, Expression of acetylated HMGB1 protein in the cytoplasm (cyt). Values are given as the means ± SD, n = 3. *P< 0.05 vs. control group; **P< 0.01 vs. control group; #P < 0.05 vs. CLP group; ##P < 0.01 vs. CLP group. C, Suppression of HMGB1 nucleocytoplasmic translocation by the upregulation of SIRT1 in sepsis-induced liver injury. Sepsis induced the acetylation of HMGB1. The acetylation of HMGB1 promoted its translocation from the nucleus to the cytoplasm, and the extracellular release of HMGB1 increased, which caused inflammation and aggravated organ damage. In contrast, SIRT1 upregulation reduced HMGB1 acetylation. The decrease in acetylated HMGB1 caused its nuclear accumulation. Also, the extracellular release of HMGB1 decreased, which attenuated inflammation and organ damage. Therefore, the SIRT1-HMGB1 pathway may be a novel therapeutic target for the prevention of sepsis-induced organ injury.

DISCUSSION

Sepsis is a life-threatening medical condition characterized by the dysregulation of inflammation, dysfunction of blood coagulation, and multiple organ injuries (1). Sepsis-induced liver injury is often cryptic and neglected in the clinical context, even though this liver injury increases the risk of mortality in sepsis. Therefore, identifying the mechanism of sepsis-induced liver injury and new therapeutic targets are urgently required to improve the survival outcomes of these patients. In this study, we focused on sepsis-induced liver dysfunction via the SIRT1-HMGB1 signaling pathway. We found that HMGB1 upregulation plays a central role in the pathogenesis of sepsis-induced liver injury. Also, the RESV-mediated inhibition of excessive HMGB1 translocation from the nucleus to the cytoplasm attenuated the liver injury, which was, at least in part, via the upregulation of SIRT1. To the best of our knowledge, this is the first study to examine the relationship between SIRT1 and HMGB1 in sepsis-induced liver injury.

Resveratrol is a stilbenoid, a type of natural phenol. Previous studies have demonstrated that RESV has therapeutic effects in sepsis (23, 30, 34), but the mechanism of its protective effects in sepsis-induced organ injury has not previously been characterized. In this study, pretreatment with RESV clearly attenuated inflammatory cell infiltration and protected hepatocytes against apoptosis/oxidative stress. Pretreatment with RESV also ameliorated sepsis-induced liver injury. In addition, we found that reduced SIRT1 expression was accompanied by the increased translocation of acetylated HMGB1 to the cytoplasm in septic rat livers.

Many studies have demonstrated that SIRT1 has an essential role in the deacetylation of proteins and the regulation of proinflammatory cytokine release, apoptosis, stress resistance, metabolism, senescence, differentiation, and aging. Howitz et al. (22) reported that RESV is a small-molecule activator of SIRT1, which regulates the deacetylation of histone and nonhistone proteins. In this study, sepsis reduced SIRT1 expression, whereas RESV pretreatment reversed the sepsis-induced downregulation of SIRT1. To determine the specific effects of SIRT1 in sepsis-induced liver injury, we inhibited SIRT1 in septic rats and showed that pretreatment with the SIRT1 inhibitor NAM clearly reduced hepatic SIRT1 expression and aggravated liver damage, apoptosis, and oxidative stress. Resveratrol pretreatment markedly attenuated the increase in the inflammatory factor levels (TNF-α and IL-6) induced by sepsis, whereas NAM had the opposite effect. We also explore whether NAM can prevent RESV-induced increase in SIRT1 expression and showed that the RESV-induced upregulation of SIRT1 expression in liver was abrogated by NAM. These data indicate that the protective effects of RESV against sepsis-induced liver injury may be related to the upregulation of SIRT1.

High-mobility group protein box 1 is a late-stage proinflammatory cytokine that plays an important role in the pathogenesis of sepsis. It is stored in the nucleus because it contains two lysine-rich nuclear localization sequences (11, 35); it is actively secreted by innate immune cells and passively released by damaged or virus-infected cells. Lacking a leader signal sequence, HMGB1 cannot actively be secreted via the classical secretory pathway (from the endoplasmic reticulum through the Golgi complex). Previous research has demonstrated that HMGB1 release is regulated by its deacetylation; therefore, we speculated that SIRT1 regulates the deacetylation and release of HMGB1. We found that pretreatment with RESV induced SIRT1 expression and consequently inhibited HMGB1 acetylation as well as its translocation to the cytoplasm. In contrast, the SIRT1 inhibitor NAM and SIRT1 siRNA knockdown produced the opposite effects, which is consistent with a published study in which a deacetylase inhibitor, trichostatin A, caused the relocalization of HMGB1 from the nucleus to the cytoplasm in vitro (11). The NAM and SIRT1 siRNA knockdown experiments confirmed that RESV increases the SIRT1-mediated repression of HMGB1 nucleocytoplasmic translocation in vivo and in vitro. Our immunoprecipitation results further showed that SIRT1-mediated HMGB1 deacetylation mainly occurs in the nucleus. Collectively, these data indicate that SIRT1 plays an important role in protecting against sepsis-induced liver injury, which involves SIRT1-mediated HMGB1 deacetylation and nucleocytoplasmic translocation (Fig. 7C).

The inhibition of HMGB1 release is an important phenomenon protecting against sepsis-induced liver injury. The RESV-mediated upregulation of SIRT1 downregulates the acetylation of HMGB1 and suppresses HMGB1 relocalization, which attenuates the sepsis-induced liver injury. The SIRT1-HMGB1 pathway may provide a new target for treating sepsis-induced injury in the liver and other organs.

The principal limitation of this treatment in our study was that we did not explore the specific effect of RESV after sepsis and how to use it in patients. Resveratrol can mediate any number of physiological processes. Further studies are warranted to clarify other potential pathways of RESV in septic hosts, including the suppression of inflammation by targeting the peroxisome proliferator-activated receptor γ or nuclear factor κB transcription factors. Because the available therapeutic strategies for sepsis are limited, RESV may provide promising benefits for septic patients.

ACKNOWLEDGMENT

This work was supported by grants from the Chinese National Natural Science Foundation (No. 81372037).

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Keywords:

SIRT1; HMGB1; resveratrol; nucleocytoplasmic translocation; deacetylation; sepsis; liver injury; ALT — alanine aminotransferase; ALF — acute liver failure; AST — aspartate aminotransferase; CLP— cecal ligation and puncture; HDAC — histone deacetylase; HMGB1 — high-mobility group protein box 1; IL-6 — interleukin 6; LPS — lipopolysaccharide; MDA — malondialdehyde; NAM — nicotinamide; NF-κB — nuclear factor κB; RESV — resveratrol; SIRT1 — sirtuin 1; TNF-α — tumor necrosis factor α

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