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Heme Oxygenase-1 Protects the Liver from Septic Injury by Modulating TLR4-Mediated Mitochondrial Quality Control in Mice

Park, Jin-Sook; Choi, Hyo-Sun; Yim, So-Yeon; Lee, Sun-Mee

doi: 10.1097/SHK.0000000000001020
Basic Science Aspects
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ABSTRACT Mitochondrial dysfunction is involved in the pathogenesis of sepsis-induced multiple organ dysfunction syndrome (MODS). Mitochondrial quality control (QC) is characterized by self-recovering mitochondrial damage through mitochondrial biogenesis, mitophagy, and fission/fusion. Heme oxygenase (HO)-1 acts as a signaling molecule to modulate inflammation. The present study elucidated the cytoprotective mechanisms of HO-1 in sepsis, particularly focusing on toll-like receptor (TLR)4-mediated mitochondrial QC. Mice were subjected to sepsis by cecal ligation and puncture (CLP). The mice were injected intraperitoneally with hemin (10 mg/kg) at 12 h before CLP or zinc protoporphyrin IX (ZnPP; 30 mg/kg) at 2 h before CLP. The serum and tissues were collected 6 h after CLP. Mortality, MODS, and proinflammatory cytokines increased in septic mice. These increases were augmented by ZnPP but attenuated by hemin. Hemin decreased mitochondrial lipid peroxidation and mitochondrial dysfunction. Hemin enhanced mitochondrial biogenesis, as indicated by increased levels of peroxisome proliferator-activated receptor-γ coactivator 1α, nuclear respiratory factor 1, and mitochondrial transcription factor A (TFAM). Hemin also enhanced mitophagy, as indicated by decreased PTEN-induced putative kinase 1 (PINK1) level and increased Parkin level. Hemin decreased fission-related protein, dynamin-related protein 1 (DRP1), and increased fusion-related protein, mitofusin 2. Hemin attenuated the increased TLR4 expression. TAK-242, a TLR4 antagonist, attenuated mortality, inflammatory response, and impaired mitochondrial QC. Our findings suggest that HO-1 attenuates septic injury by modulating TLR4-mediated mitochondrial QC.

School of Pharmacy, Sungkyunkwan University, Suwon, Gyonggi-do, Republic of Korea

Address reprint requests to Sun-Mee Lee, PhD, School of Pharmacy, Sungkyunkwan University, Seobu-ro 2066, Jangan-gu, Suwon, Gyeonggi-do 16419, Republic of Korea. E-mail: sunmee@skku.edu

Received 17 August, 2017

Revised 6 September, 2017

Accepted 2 October, 2017

J-SP and H-SC contributed equally to this work.

This work was supported by the Mid-Career Researcher Program through a National Research Foundation (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) in Korea (NRF-2016R1A2B4009880).

All authors have read the journal's policy on disclosure of potential conflicts of interest and the journal's authorship agreement, and they have nothing to declare. All authors have read the journal's authorship agreement, and the manuscript was reviewed and approved by all named authors.

The authors report no conflicts of interest.

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INTRODUCTION

Sepsis is a systemic inflammatory response induced by infection, leading to multiple organ dysfunction syndrome (MODS) and death. Growing evidence indicates that mitochondrial dysfunction is a possible mechanism of sepsis, which disrupts adenosine triphosphate (ATP) synthesis and promotes reactive oxygen species (ROS) production and release of inflammatory molecules and apoptotic proteins, resulting in cell death. Mitochondrial ultrastructural changes in the liver of patients who died from multiple organ dysfunction with sepsis have been reported (1). Maintaining optimal function of the mitochondria is important for the recovery of the cell and organ function in sepsis.

Mitochondrial quality control (QC), a highly exquisite mechanism, maintains a population of optimally-functioning mitochondria and has been intimately implicated in cell recovery by mitochondrial biogenesis (formation of mitochondria), mitophagy (selective engulfment of mitochondria), and mitochondrial dynamics (maintaining mitochondrial morphology). Recent studies have uncovered critical links between mitochondrial QC and human diseases such as inflammatory and neurodegenerative diseases (2). Deficiency of mitogen-activated protein kinase kinase 3 increased mitochondrial biogenesis and mitophagy and improved lipopolysaccharide (LPS)-induced lung injury (3). Excessive mitochondrial fragmentation with increased fission or impaired fusion is a hallmark of many neurodegenerative diseases (4).

Heme oxygenase (HO)-1 is a cytoprotective enzyme that is upregulated under cellular stress. HO-1 overexpression suppressed inflammatory response and improved survival in sepsis (5). In E coli-induced septic mice, HO-1 system upregulated anti-inflammatory cytokines and synchronously activated mitochondrial biogenesis by regulating multiple transcriptional elements such as nuclear respiratory factor 1 (NRF1) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) (6). Although the cytoprotective effects of HO-1 are associated with mitochondrial health, the precise molecular mechanism of its function remains under intensive investigation.

Toll-like receptor (TLR) signaling plays an important role in the initiation of the inflammatory response in sepsis. Riquelme et al. (7) reported that HO-1/carbon monoxide down-modulated TLR4/myeloid differentiation factor 2 expression on innate immune cells and reduced endotoxic shock susceptibility. TLR4 knockout mice were protected from disruption of mitochondrial homeostasis and biogenesis in endotoxin-induced acute renal injury (8).

The aim of this study was to clarify the mechanisms underlying the protective effects of HO-1 against septic injury, particularly focusing on mitochondrial QC associated with TLR4 activation.

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MATERIALS AND METHODS

Animals

All animals received care in compliance with the Principles of Laboratory Animal Care formulated by the National Institutes of Health (NIH publication No. 86-23, revised 1985) and guidelines of the Sungkyunkwan University Animal Care Committee. Male C57BL/6 mice weighing 23 g to 25 g were supplied by Orient Bio Inc (Seongnam, Korea) and acclimatized to the laboratory conditions at Sungkyunkwan University.

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Experimental procedures

Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) as described by Chaudry et al. (9). Zinc protoporphyrin IX (ZnPP; Sigma-Aldrich, St Louis, Mo) and hemin (Sigma-Aldrich) were prepared under subdued light by dissolving each compound in 0.2 M NaOH, adjusting the pH to 7.4 with 1 M HCl, and diluting the solution to the final volume with normal saline. Mice intraperitoneally received vehicle (normal saline), hemin (10 mg/kg, 12 h before CLP), or ZnPP (30 mg/kg, 2 h before CLP). The dose and time point of hemin and ZnPP administration were selected based on previously published reports (10, 11) and our preliminary study. Mortality was recorded for up to 7 days after CLP, and survivors were monitored for additional 3 weeks to ensure that no late mortalities occurred (each group, n = 10). For biochemical assays and histology, animals were randomly divided into six groups as follows (each group, n = 6–8): vehicle-treated sham (Sham), hemin-treated sham (Hemin), ZnPP-treated sham (ZnPP), vehicle-treated CLP (CLP), hemin-treated CLP (Hemin + CLP), and ZnPP-treated CLP (ZnPP + CLP). To investigate the involvement of TLR4 in septic injury, mice intravenously received vehicle (10% dimethylsulfoxide in saline) or TAK-242 (TLR4 selective antagonist; 3 or 10 mg/kg, 1 h after CLP; Takeda Pharmaceutical, Osaka, Japan). The animals were randomly divided into five groups as follows (survival test for each group, n = 10; biochemical assays for each group, n = 6–8): vehicle-treated sham (Sham), TAK-242-treated sham (TAK-242), vehicle-treated CLP (CLP), 3 mg/kg TAK-242-treated CLP (CLP + TAK 3 mg/kg), 10 mg/kg TAK-242- treated CLP (CLP + TAK 10 mg/kg). On the basis of survival test, a dose of 10 mg/kg of TAK-242 was chosen for further biochemical assays. Under anesthesia, blood samples from the inferior vena cava and liver, lung, heart, and kidney tissues were collected 6 h after CLP and immediately stored at −75°C until biochemical analyses. Serum was separated by centrifugation at 10,000 × g for 10 min at 4°C.

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Isolation of liver mitochondrial fractions

The liver mitochondrial fraction was prepared based on the previous report (12). The protein content of the mitochondrial fractions was determined using a bicinchoninic acid (BCA) Protein Assay kit (Pierce Biotechnology, Rockford, Ill).

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HO enzyme activity

Liver tissue was homogenized in 1.15% KCl (w/v; 4°C) containing protease inhibitors and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was centrifuged at 100,000 × g for 60 min at 4°C to obtain the microsomal fraction, and the microsomal fractions were suspended in 0.1 M potassium phosphate buffer, pH 7.4. The HO activities of isolated microsomes and mitochondria were assayed by detecting the formation of bilirubin at 465 nm and 530 nm.

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Western blot analysis

Liver tissue was homogenized in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea) for total protein samples. The protein concentrations were determined using a BCA Protein Assay kit (Pierce Biotechnology). Protein samples (16–20 μg) were loaded on 7.5% to 15% polyacrylamide gels, separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, Mass) using the Semi-Dry Trans-Blot Cell (Bio-Rad Laboratories, Hercules, Calif). After the transfer, the membranes were blocked for 1 h with 5% (w/v) skim milk in Tris-buffered saline with 0.1% Tween-20 at room temperature. Blots were incubated with primary antibodies overnight at 4°C. Primary antibodies were used as follows: HO-1 (1:2,500; Enzo Life Sciences, Farmingdale, NY); cytochrome c oxidase IV (COX IV), superoxide dismutase 2 (SOD2), NRF1, mitochondrial transcription factor A (TFAM), B-cell lymphoma 2 (Bcl2)/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), mitofusin 2 (MFN2) (1:2,500; Abcam, Cambridge, UK); PGC1α, PTEN-induced putative kinase 1 (PINK1), Parkin, dynamin-related protein 1 (DRP1) (1:2,500; Santa Cruz Biotechnology, Santa Cruz, Calif), TLR4 (1:2,500; Novus Biologicals, Littleton, Colo); and β-actin (1:5,000; Sigma-Aldrich). Bands were detected using an enhanced chemiluminescence detection system (iNtRON Biotechnology) according to the manufacturer's instructions. The intensities of immunoreactive bands were evaluated using Total-Lab TL120 software (Nonlinear Dynamics, Newcastle, UK). Signals were standardized to those of β-actin or COX IV.

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Histological analysis

For histological analysis, the liver, lung, heart, and kidney tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, sliced into 5 μm sections, and then stained with hematoxylin and eosin. Histological changes were evaluated in random and nonconsecutive fields at ×200 magnification (Olympus BX51/Olympus DP71, Tokyo, Japan).

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Measurement of organ damage

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine, and lactate dehydrogenase (LDH) levels were measured using a Hitachi 7600 automatic analyzer (Hitachi, Tokyo, Japan).

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Serum cytokine levels

Serum interleukin (IL)-6 and IL-1β levels were quantified using enzyme-linked immunosorbent assay with commercial IL-6 and IL-1β kits (BD Biosciences, San Jose, Calif) according to the manufacturer's instructions.

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Mitochondrial lipid peroxidation

Following the method by Buege and Aust (13), the level of malondialdehyde (MDA) in hepatic mitochondrial fractions was analyzed by measuring the level of substances reactive to thiobarbituric acid spectrophotometrically at 535 nm.

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Mitochondrial swelling

The rate of mitochondrial swelling, which indicates the level of the mitochondrial permeability transition (MPT), was determined from the change in the absorbance of a mitochondrial suspension at 520 nm according to the procedure reported by Elimadi et al. (14).

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Serum glutamate dehydrogenase activity

The serum glutamate dehydrogenase (GDH) activity was spectrophotometrically measured by the method of Ellis and Goldberg (15).

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Hepatic ATP level

Hepatic ATP levels were measured with a commercial ATP colorimetric/fluorometric assay kit (BioVision, San Francisco, Calif) according to the manufacturer's instructions.

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Statistical analysis

Survival data were prescribed by the Kaplan–Meier curve and analyzed by log-rank test. All other data were analyzed by the one-way analysis of variance, and the Bonferroni test was used for multiple comparisons. Statistical differences between the groups were considered significant at P < 0.05. Results are presented as mean ± standard error of the mean (S.E.M).

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RESULTS

HO-1 protects mice against sepsis-induced mortality and organ injury

To investigate the role of HO-1 in CLP-induced mortality, survival rate was monitored for 7 days. In the CLP group, the survival rate on the first day of observation was 70% and reached a stable 20% on the fourth day after CLP. Pretreatment of hemin improved survival rate compared with that of the CLP group (P = 0.0421), while ZnPP had the opposite effect (Fig. 1A). We then evaluated whether HO-1 affects MODS 6 h after CLP. Serum ALT and AST levels were 29.4 ± 1.4 U/L and 56.0 ± 2.1 U/L in the sham group, respectively. CLP significantly increased serum ALT and AST levels by 2.7- and 4.9-fold compared with those of the sham group, respectively. Serum LDH levels were 893.4 ± 97.0 U/L in the sham group and significantly increased by 4.7-fold compared with that of the sham group. Serum BUN and creatinine levels were 23.4 ± 2.2 U/L and 0.40 ± 0.02 U/L in the sham group, respectively. CLP significantly increased serum BUN and creatinine levels by 1.4- and 1.6-fold compared with those of the sham group, respectively. Pretreatment of hemin attenuated change in ALT, AST, LDH and creatinine (Table 1). Histological evaluations revealed normal cell structures in the liver, lung, kidney, and heart of sham. At 6 h after CLP, significant histopathological changes (such as inflammatory cell infiltration and cell death) were observed. These pathological changes were attenuated by hemin and augmented by ZnPP (Fig. 1, B–E).

Fig. 1

Fig. 1

Table 1

Table 1

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CLP increases HO activity and HO-1 protein expression in whole liver and mitochondria

We assessed HO activity and HO-1 protein expression during sepsis. Hemin and ZnPP alone did not affect the HO activity and HO-1 protein expression. Hepatic microsomal HO activity in the sham group remained at 70.1 ± 1.7 pmol/min/mg protein in the sham groups. CLP significantly increased hepatic microsomal HO activity and whole liver HO-1 protein expression by 1.2- and 13.5-fold compared with those of the sham group, respectively. These increases were augmented by hemin and attenuated by ZnPP (Fig. 2A). Similar to these results, CLP significantly increased hepatic mitochondrial HO activity and HO-1 protein expression by 2.0- and 3.5-fold compared with those of the sham group, respectively. These increases were augmented by hemin and attenuated by ZnPP (Fig. 2B).

Fig. 2

Fig. 2

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HO-1 decreases CLP-induced proinflammatory cytokines levels

To assess the role of HO-1 in inflammatory responses, we measured the serum inflammatory cytokines. Hemin and ZnPP alone did not affect the serum cytokine levels. Serum IL-6 and IL-1β levels were 53.0 ± 5.9 pg/mL and 3.3 ± 0.6 pg/mL in the sham group, respectively. CLP significantly increased serum IL-6 and IL-1β levels by 194.9- and 42.2-fold compared with those of the sham group, respectively. Hemin attenuated these increases, while ZnPP augmented the CLP-induced IL-6 and IL-1β levels (Fig. 3, A and B).

Fig. 3

Fig. 3

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HO-1 decreases CLP-induced mitochondrial oxidative damage and dysfunction

Oxidative stress plays a critical role in the pathogenesis of sepsis. In the sham group, the hepatic mitochondrial MDA level was 0.25 ± 0.04 nmol/mg protein. CLP significantly increased this level by 2.4-fold compared with that of the sham group, and treatment with hemin attenuated this increase (Fig. 4A). We also assessed the level of SOD2 protein expression, a representative mitochondrial antioxidant enzyme. CLP significantly decreased the level of SOD2 protein expression to 88.5% of that in the sham group. This decrease was attenuated by hemin (Fig. 4B). To determine the change in mitochondrial membrane potential, we measured the rate of mitochondrial swelling. The rate of mitochondrial swelling was 0.003 ± 0.001 (ΔA × 10–2/min mg of protein) in the sham group. CLP significantly increased this level by 7.6-fold compared with that of the sham group. This increase was attenuated by hemin (Fig. 4C). Furthermore, to investigate the role of HO-1 in mitochondrial dysfunction, we measured serum GDH activity and hepatic ATP level. Serum GDH activity was 4.7 ± 0.4 U/L in the sham group. CLP significantly increased by 4.9-fold compared with that of the sham group. This increase was attenuated by hemin (Fig. 4D). Hepatic ATP level markedly decreased to 77.5% of that in the sham group. This decrease was attenuated by hemin and ZnPP did not affect the CLP-induced change in the hepatic mitochondrial MDA level, SOD2 protein expression, the rate of mitochondrial swelling, serum GDH activity, and hepatic ATP level. Hemin and ZnPP alone did not affect the mitochondrial oxidative damage and dysfunction (Fig. 4E).

Fig. 4

Fig. 4

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HO-1 increases mitochondrial biogenesis and restores impaired mitophagy

To investigate whether HO-1 may affect mitochondrial homeostasis, we first examined the expressions of molecules involved in mitochondrial biogenesis. CLP significantly decreased the hepatic levels of PGC1α and TFAM protein expressions to 70.5 and 54.7% of those in the sham group, respectively. These decreases were attenuated by hemin. CLP did not affect the NRF1 protein expression, and hemin increased the level of NRF1 protein expression. ZnPP did not affect the CLP-induced change in hepatic mitochondrial biogenesis (Fig. 5A). Next, we measured the levels of mitophagy-related proteins. CLP significantly increased the hepatic level of PINK1 protein expression by 1.5-fold compared with that of the sham group. This increase was attenuated by hemin and augmented by ZnPP. CLP decreased the levels of Parkin and BNIP3 protein expressions to 79.1% and 76.0% of those in the sham group, respectively. These decreases were attenuated by hemin. ZnPP did not affect the CLP-induced change in the levels of Parkin and BNIP3 protein expression (Fig. 5B).

Fig. 5

Fig. 5

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HO-1 suppresses fission and increases fusion

Mitochondrial dynamics consists of fission and fusion. CLP significantly increased the hepatic level of DRP1 protein expression by 1.3-fold compared with the sham group. This increase was attenuated by hemin. CLP decreased the level of MFN2 protein expression to 74.8% of that in the sham group. This decrease was attenuated by hemin. ZnPP did not affect the CLP-induced changes in fission and fusion (Fig. 6).

Fig. 6

Fig. 6

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Inhibition of TLR4 attenuates sepsis-induced mortality, inflammation, and impairment of mitochondrial QC

To assess the role of HO-1 in TLR4 activation, we measured the hepatic TLR4 protein expression. Hemin and ZnPP alone did not affect the TLR4 protein. The hepatic level of TLR4 protein expression significantly increased by 1.6-fold compared with that of the sham group 6 h after CLP. This change was attenuated by hemin, and ZnPP did not affect the CLP-induced change in hepatic TLR4 protein expression (Fig. 7A). Moreover, to confirm the involvement of TLR4 activation in septic injury, we treated with TAK-242, a TLR4 antagonist, 1 h after CLP. In the CLP group, the survival rate on the first day observation was 70% and reached a stable 20% on the fourth day after CLP. Post-treatment of 10 mg/kg TAK-242 improved survival rate compared with that of the CLP group (P = 0.0402), while post-treatment of 3 mg/kg TAK-242 did not affect in survival rate (Fig. 7B). Serum IL-6 was 48.3 ± 1.3 pg/mL in the sham group. CLP significantly increased serum IL-6 level by 165.6-fold compared with that of the sham group, and 10 mg/kg TAK-242 attenuated this increase (Fig. 7C). Serum GDH activity was 6.4 ± 0.8 U/L in the sham group. CLP significantly increased by 2.3-fold compared with that of the sham group, and this increase was attenuated by 10 mg/kg TAK-242 (Fig. 7D). CLP decreased the level of TFAM protein expression to 60.0% of that in the sham group, and this decrease was attenuated by TAK-242 (Fig. 7E). CLP increased the level of PINK1 protein expression by 1.3-fold compared with that of the sham group and decreased the level of Parkin protein expression to 76.7% of that in the sham group, and TAK-242 attenuated these changes (Fig. 7F). CLP increased the level of DRP1 protein expression by 1.7-fold compared with that of the sham group. TAK-242 attenuated the increase in the level of DRP1 protein expression (Fig. 7G).

Fig. 7

Fig. 7

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DISCUSSION

HO-1 is located in the endoplasmic reticulum; however, it can translocate to the mitochondria under stress conditions such as inflammation and oxidative stress. The localization of mitochondrial HO-1 attenuated cigarette smoke-induced ATP depletion and cell death (16). In indomethacin-induced gastritis, mitochondrial HO-1 attenuated mitochondrial oxidative stress and subsequent gastric mucosal injury (17). Indeed, we also observed that the efficacious anti-inflammatory mediator HO-1 is upregulated in whole livers and translocated to mitochondria during sepsis, possibly in insufficient quantities. Previous studies have demonstrated that pretreatment of hemin effectively attenuated mortality in in vivo sepsis models (18). Although clinicians are involved in the treatment rather than prevention, attenuation of septic injury with pretreatment of hemin could be meaningful for hospitalized patients after serious surgical procedures and elderly people with high risk of sepsis (19, 20). To confirm the role of HO-1 in septic injury, we compared the HO activity and protein expression of HO-1, in hemin- or ZnPP-treated animals. We demonstrated here that induction of HO-1 markedly reduced the inflammatory response and damage in liver and inhibition of HO-1 aggravated these changes. This phenomenon was supported by histological examinations. Our results suggest that induction of HO-1 is responsible for attenuating the inflammatory response, MODS, and in improving survival in murine sepsis.

Mitochondrial dysfunction is a major cause of sepsis-induced organ failure, which is closely related to mortality. Mitochondria are the main source of ROS and also target organelles for oxidative stress. Accordingly, many studies have focused on the critical relevance of mitochondrial dysfunction in sepsis, in which antioxidant therapeutics that specifically target mitochondrial damage have been found to ameliorate septic injury in experimental models (21). The MPT is highly sensitive to the redox state of mitochondria, and oxidative stress triggers permeability transition pore opening, which leads to the destruction of the mitochondrial membrane potential, and mitochondria swelling, resulting in inhibition of ATP synthesis. To prevent oxidative stress, tissues utilize antioxidant defense mechanisms such as SOD, GSH, and catalase. SOD2, an antioxidant enzyme that is located in the mitochondrial matrix, constitutes an essential defense against molecular damage. In this study, CLP significantly increased mitochondrial swelling and lipid peroxidation, while it decreased the level of SOD2 protein expression. Hemin attenuated these changes. GDH, a mitochondrial matrix enzyme, is an indicator of mitochondrial damage. During hepatic ischemia/reperfusion (I/R), mitochondrial injury was indicated by increased serum GDH activity (22). In our study, CLP increased the serum GDH activity, and hemin attenuated this increase. Indeed, the hepatic ATP level, a marker of mitochondrial function, was higher in hemin-treated mice than in the CLP group. Collectively, our results indicate that induction of HO-1 inhibits the mitochondrial oxidative damage and improves mitochondrial dysfunction during sepsis.

Mitochondrial QC is a critical process in recovery from organ damage through return to highly dynamic equilibrium among mitochondrial biogenesis, mitophagy, mitochondrial fission, and fusion. In mammalian cells, mitochondrial biogenesis is regulated by several transcription factors, including PGC1α, NRF1, and TFAM. PGC1α acts as a master transcription regulator of mitochondrial biogenesis by activating NRF1. PGC1α and NRF1 activate TFAM, which in turn regulates the replication of mitochondrial deoxyribonucleic acid and maintenance of mitochondrial density. During the progress of sepsis, PGC1α and TFAM mRNA decreased between 6 and 18 h after CLP in mice hearts (23). In contrast, Staphylococcus aureus-induced septic mice increased mitochondrial biogenesis in liver (24). This discordance may be due to the use of various animal models and differences in response to mitochondrial biogenesis. Recently, tissue-specific HO-1 overexpression induced mitochondrial biogenesis that protected the heart against doxorubicin-induced dilated cardiomyopathy (25). During hepatic I/R, cilostazol induced nuclear factor erythroid-derived 2-dependent HO-1 upregulation, activated mitochondrial biogenesis, and attenuated liver injury (26). In our study, CLP decreased the levels of PGC1α and TFAM protein expression. Hemin attenuated these decreases, suggesting that HO-1 activates mitochondrial biogenesis.

Mitophagy plays an important role in eliminating dysfunctional mitochondria. Deficient mitophagy can lead to accumulation of damaged mitochondria in a chronic obstructive pulmonary disease model (27). In most mammalian cells, PINK1 and Parkin play a major role in mediating the canonical mitophagy pathway. PINK1 is a sensor for mitochondrial damage, and its accumulation on the outer membrane of damaged mitochondria induces translocation of Parkin from the cytosol to damaged mitochondria. Indeed, increased PINK1 protein expression was observed in septic patients compared with non-septic controls (3). PARK2 deficiency impaired mitochondrial metabolic function and cardiac contractility in LPS-induced sepsis (28). BNIP3, a Bcl2-related protein with an atypical Bcl2 homology 3 domain, localizes to the mitochondria where it induces mitochondrial dysfunction and subsequent cell death. BNIP3 could also induce mitophagy by disrupting the interaction between Beclin1 and Bcl2/B-cell lymphoma-extralarge (29). Recently, BNIP3 was shown to interact with PINK1 to suppress its cleavage, leading to increased Parkin recruitment and enhanced mitochondrial clearance (30). In our study, CLP increased the level of hepatic PINK1 protein expression. In contrast, the levels of hepatic Parkin and BNIP3 protein expressions decreased. These changes were attenuated by hemin. These results suggest that HO-1 enhances Parkin- and BNIP3-mediated mitophagy.

Mitochondrial fission and fusion are the major processes that maintain mitochondrial morphology. Abnormal mitochondrial fission and fusion balance contributed to the progression of experimental sepsis. DRP1-mediated mitochondrial fission inhibited mitochondrial electron transport chain complexes activities, and induced liver injury in LPS-injected rats (31). MFN2 mediates mitochondrial fusion by the tethering of adjacent mitochondria and is associated with mitochondrial biogenesis. In our study, CLP markedly increased the level of DRP1 protein expression and decreased the level of MFN2 protein expression. Hemin attenuated these changes. Our findings indicate that HO-1 regulates mitochondrial dynamics by suppressing fission and increasing fusion.

TLRs are regarded as important upstream mediators that trigger excessive inflammatory response and drive mitochondrial damage. It has been well known that TLR4 signaling is regulated by HO-1. Induction of HO-1 diminished TLR4-mediated inflammatory response and ameliorated lung inflammation in LPS-induced lung injury (32). Deficiency of TLR4 improved respiratory control ratio of complex I and complex II of the electron transport chain in LPS-treated mice (33). Recently, septic mice limited mitochondrial biogenesis and induced renal dysfunction through TLR4-dependent extracellular signal-regulated kinase activation (8). In taurocholate-treated pancreatic acinar cells, deficiency of TLR4 decreased the mitochondrial fission but increased mitochondrial fusion (34). In our study, CLP increased the level of TLR4 protein expression, and hemin attenuated this increase. Inhibition of TLR4 by TAK-242 decreased serum GDH activity and IL-6 level, and improved survival rate. TAK-242 restored the impairment of mitochondrial biogenesis and mitophagy and reduced mitochondrial fission induced by sepsis. Our data suggests that HO-1 regulates TLR4-mediated mitochondrial QC during sepsis.

In conclusion, our study suggests that HO-1 ameliorates sepsis-induced mitochondrial dysfunction through the regulation of TLR4-mediated mitochondrial QC. Induction of HO-1 might be a useful pharmacological maneuver for attenuating septic injury. Also, modulation of mitochondrial QC is emerging as a new therapeutic strategy for inflammatory diseases.

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Keywords:

Heme oxygenase-1; mitochondrial biogenesis; mitochondrial dynamics; mitophagy; sepsis; toll-like receptor 4; ALT; alanine aminotransferase; AST; aspartate aminotransferase; ATP; adenosine triphosphate; BCA; bicinchoninic acid; Bcl2; B-cell lymphoma 2; BNIP3; Bcl2/adenovirus E1B 19-kDa interacting protein 3; BUN; blood urea nitrogen; COX IV; cytochrome c oxidase IV; CLP; cecal ligation and puncture; DRP1; dynamin-related protein 1; GDH; glutamate dehydrogenase; HO; heme oxygenase; IL; interleukin; I/R; ischemia and reperfusion; LDH; lactate dehydrogenase; LPS; lipopolysaccharide; MDA; malondialdehyde; MFN2; mitofusin 2; MODS; multiple organ dysfunction syndrome; MPT; mitochondrial permeability transition; NRF1; nuclear respiratory factor 1; PGC1α; peroxisome proliferator-activated receptor gamma coactivator 1α; PINK1; PTEN-induced putative kinase 1; QC; quality control; ROS; reactive oxygen species; S.E.M; standard error of the mean; SOD2; superoxide dismutase 2; TFAM; mitochondrial transcription factor A; TLR; toll-like receptor; ZnPP; zinc protoporphyrin IX

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