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NF-kB signaling pathways
Biochemical and Biophysical Research Communications xxx (2017) 1e7
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Chronic intermittent hypoxia induces liver fibrosis in mice with dietinduced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways Hyeon Hui Kang, In Kyoung Kim, Hye in Lee, Hyonsoo Joo, Jeong Uk Lim, Jongmin Lee, Sang Haak Lee, Hwa Sik Moon* Department of Internal Medicine, College of Medicine, St. Paul's Hospital, The Catholic University of Korea, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2017 Accepted 12 June 2017 Available online xxx
Obstructive sleep apnea (OSA) is associated with nonalcoholic fatty liver disease (NAFLD), and causes chronic intermittent hypoxia (CIH) during sleep. Inflammation is associated with the development of metabolic complications induced by CIH. Research suggests that innate immune mechanisms are involved in the pro-inflammatory pathways of liver fibrosis. The purpose of this study was to investigate whether innate immune responses induce liver fibrosis, and to evaluate mechanisms underlying hepatic inflammation related to CIH in a murine diet-induced obesity (DIO) model. Inflammatory and oxidative stress markers, TLR4, MyD88, Toll/interleukin-1-receptor-domain-containing adaptor-inducing interferon-b (TRIF), I-kB, NF-kB, p38 MAPK, c-JNK, and ERK activation, were measured in the serum and liver. As a result, a1(I)-collagen mRNA was significantly higher in DIO mice exposed to CIH than in the control groups. CIH mice exhibited liver fibrosis and significantly higher protein expression of TLR4, MyD88, phosphorylated (phospho-) I-kB, and phospho-ERK1/2 activation in the liver, and higher expression of NF-kB than that in the controls. TRIF, p38 MAPK, and JNK activation did not differ significantly between groups. We conclude that CIH in DIO mice leads to liver fibrosis via TLR4/MyD88/MAPK/NF-kB signaling pathways. © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Obstructive sleep apnea Chronic intermittent hypoxia Obesity Liver fibrosis Toll like receptor 4
1. Introduction The already high prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing with the obesity epidemic, varying from 6 to 33% in the general population [1]. Although its pathophysiology is incompletely understood, insulin resistance, inflammation, oxidative stress, and lipotoxicity are involved [2]. Although inflammation occurs in most patients with hepatic fibrosis and correlates with fibrosis progression [3], the molecular correlation between hepatic inflammation and fibrogenesis remains elusive. Obstructive sleep apnea (OSA) is characterized by repetitive partial (hypopnea) or complete (apnea) upper airway collapse during sleep, inducing chronic intermittent hypoxia (CIH) resulting in low-grade inflammation, sympathetic overactivity, and oxidative stress [4]. OSA is associated with obesity in 60% of cases [5], and causes significant cardiovascular and metabolic morbidity and
* Corresponding author. Department of Internal Medicine, College of Medicine, St. Paul's Hospital, The Catholic University of Korea, Wangsanro 180, Dongdaemungu, Seoul, 130-709, Republic of Korea. E-mail address: [email protected] (H.S. Moon).
mortality [6]. OSA and NAFLD have similar risk factors, including obesity, and OSA is thought to contribute to the pathogenesis and exacerbation of NAFLD [7,8]. Although evidence suggests that OSA is associated with NAFLD pathogenesis and progression, few studies have investigated the mechanism of OSA-induced liver injury. Toll like receptors (TLRs) are pattern recognition receptors that perceive pathogens and bacteria-derived molecules, leading to proinflammatory cytokine production [9]. TLRs regulate innate and adaptive immune responses [10], which are also associated with noninfectious inflammatory liver diseases [11]. Of the 13 mammalian TLRs, only TLR2, TLR4, TLR5, and TLR9 are associated with NAFLD [12,13]. TLR4 triggers two signaling pathways: the MyD88-dependent pathway that leads to the rapid activation of NF-kB and increased TNF-a production, and the MyD88independent pathway that requires the Toll/interleukin-1 receptor (TIR)-containing adaptor molecule. Both pathways ultimately lead to proinflammatory cytokine, chemokine, and type I interferon production [9]. TLR4 might be involved in OSA, as monocytes from patients with OSA have higher TLR4 surface expression [14]. TLR4 deletion in mice prevented CIH-induced insulin resistance and
http://dx.doi.org/10.1016/j.bbrc.2017.06.047 0006-291X/© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Abbreviations OSA NAFLD CIH DIO TRIF NFD HFD RA
obstructive sleep apnea nonalcoholic fatty liver disease chronic intermittent hypoxia diet-induced obesity Toll/interleukin-1-receptor-domain-containing adaptor-inducing interferon-b normal fat diet high fat diet room air
1 beta Quantikine ELISA Kit (MLB00C; R&D Systems, Minneapolis, MN, USA). The sensitivities of the assays were 1.5 ng/mL and 4.8 pg/ mL, respectively. Chemiluminescence was detected using a Multimode Microplate Reader (Infinite® 200 PRO; Life Science, Horsham, PA, USA) at 450 nm. The concentrations of superoxide dismutase (SOD), myeloperoxidase (MPO), and catalase (CAT) were measured in the tissue lysates, using the following kits: SOD Assay Kit (S311; Dojindo Molecular Technologies, Kumamoto, Japan), the Myeloperoxidase (MPO) Activity Colorimetric Assay Kit (K744-100; BioVision, Milpitas, CA, USA), and the EnzyChromTM Catalase Assay Kit (ECAT-100; Bioassay Systems, Hayward, CA, USA), respectively, according to the manufacturers' instructions. Chemiluminescence was detected at 450 nm, 412 nm, and 570 nm, respectively. 2.4. Western blot analysis
morphological and inflammatory changes in epididymal fat and aorta [15]; however, the relationship between TLR4 and liver injury has not been explored in OSA, and little is known about the processes that propagate TLR signaling downstream during CIH. Here, we investigated the innate immune responses associated with liver fibrosis in CIH, and evaluated the molecular mechanisms underlying hepatic responses to CIH in a murine diet-induced obesity (DIO) model. 2. Materials and methods 2.1. Animals Seven-to-eight week-old male, C57BL/6 J mice were purchased from DBL (Chungcheongbuk-do, Korea). The study was approved by the Ethical Committee on Animal Experiments of the Catholic University of Korea. All animals were maintained in a pathogenfree environment at 22e24 C with a 12:12 h light:dark cycle; lights on at (09:00) with free access to food and water. 2.2. Experimental design and animal model of CIH The mice were divided into two groups and fed a normal fat diet (NFD group) or a high-fat diet (HFD group) for 12 weeks. Each group was then divided into two additional groups then exposed to chronic intermittent hypoxia (CIH, n ¼ 10) or control conditions (room air; RA, n ¼ 10) for 4 weeks. A gas control delivery system was designed to regulate air, nitrogen, and oxygen flow into the cages. During periods of CIH, the fractional inspired O2 (FiO2) was reduced from RA levels to approximately 7± 0.5% within 30 s, followed by reoxygenation to RA levels within the subsequent 30 s (Supplementary Fig. 1A). Exposure proceeded from 09:00 to 21:00 daily to coincide with mouse sleep cycles, and the duration of exposure was 4 weeks. The oxygen level was measured with a ProOx 110 analyzer (Biospherix, Redfield, NY, USA). The HFD group was fed a high-fat, high-cholesterol diet (D12492; Research Diets, Inc. New Brunswick, NJ, USA; 34.9 gm% Fat, 300.8 mg/kg Cholesterol) and the NFD group was fed regular chow for 12 weeks. The NFD þ CIH and HFD þ CIH groups were then moved into the intermittent hypoxia (IH) chamber for 4 weeks with their corresponding feed. The NFD þ RA and HFD þ RA groups were exposed to RA during the same period (Supplementary Fig. 1B). Body weight was monitored once per week for each mouse. 2.3. ELISA The concentration of albumin was measured using Albumin Mouse ELISA Kit (ab108792; Abcam, Burlingame, CA, USA) and the concentration of interleukin (IL)-1b was measured using Mouse IL-
Total protein was isolated from the liver by homogenization in cell lysis buffer containing a mixture of protease and phosphatase inhibitors (GenDEPOT, Katy, TX, USA), and then centrifuged at 13,000 rpm for 30 m at 4 C. The proteins (40 mg/mL) were separated by 10% SDS-PAGE, in a Tris-glycine-SDS buffer. Separated proteins were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk (Sigma, St. Louis, MO, USA) for 2 h at room temperature, and then incubated with a 1:200 dilution of the individual primary antibodies antiTLR4, anti-TRIF, anti-MyD88, anti-I-kB, anti-p-I-kB, anti-NF-kB p65, anti-Lamin B, and anti-b-actin) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), or a 1:1000 dilution of the primary antibodies anti-ERK, anti-p-ERK, anti-c-JNK, anti-p-JNK, anti-p38 MAPK, antip-p38 MAPK, anti-c-Jun, or anti-p-c-Jun (Cell Signaling Technology, Danvers, MA., USA). The antibodies were diluted in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), and applied overnight at 4 C. Membranes were washed and incubated with HRP-conjugated secondary antibodies (goat anti-mouse/rabbit, rabbit anti-goat IgG-HRP Secondary Antibody (Santa Cruz Biotechnology, Inc.). Protein bands were detected using the Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc.). 2.5. Quantitative real-time polymerase chain reaction (RT-PCR) Total RNA was extracted from lung tissue according to the protocol for Trizol according to the manufacturer's instructions (Takara, Japan). First-strand cDNA was synthesized from total RNA using the Super Script II first-strand synthesis system (Invitrogen, Carlsbad, CA, USA). The primer sequences are provided as follows: a1(I)-collagen (forward 50 -CTGGCGGTTCAGGTCCAA-30 , reverse 50 GCTTCCCCATCATCATCTCCATTC-30 ); TGFb1 (forward 50 -GGAACTCTACCAGAAATATAGCAACAATTC-30 , reverse 50 TGTATTCCGTCTCCTTGGTTCAG-30 ); PDGFRb (forward 50 -CCGGAACAAACACACCTTCT-30 , reverse 50 -TATCCATGTAGCCACCGTCA-30 ); caspase 3 (forward 50 - TGTCATCTCGCTCTGGTACG-30 , reverse 50 AAATGACCCCTTCATCACCA-30 ); and b-actin (forward 50 -ACAGGAAGTCCCTTGCCATC-30 , reverse 50 -AGGGAGACCAAAAGCCTTCA30 ). Quantitative RT-PCR was performed using the QuantiFast SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). The threshold cycle (Ct) value for each gene was normalized to that of b-actin. The relative mRNA expression was calculated using the 2DDCT method. 2.6. Tissue histology The liver tissues in each group were fixed with 10% formalin, embedded in paraffin wax, and cut into 3 mm sections. Deparaffinized tissue sections were subjected to H&E staining and then observed under a light microscope to make precise pathological diagnoses. Separate sections were stained using the standard
Please cite this article in press as: H.H. Kang, et al., Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.047
H.H. Kang et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Masson's trichrome staining protocol for identifying reactive fibrosis and its distribution.
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(23.28 ± 3.52 g) than in NFD-fed mice (9.75 ± 1.90 g). Body weight in the HFD þ CIH group (17.68 ± 5.94 g) was considerably lower (24.1%) than that in the HFD þ RA group (23.28 ± 3.52 g).
2.7. Hydroxyproline analysis The collagen concentration was measured in peripheral portions of the liver using a hydroxyproline assay kit (Biovision Inc., Milpitas, CA., USA), according to the manufacturer's instructions. Briefly, liver tissue was homogenized in dH2O, using 300 mL of H2O per 30 mg of tissue. The samples were added to 300 mL of 12 N HCl at 120 C, overnight. After centrifugation (10,000g for 3 min at room temperature), 100 mL of the chloramine T reagent was added to 10 mL of each hydrolyzed sample, the mixtures were incubated at room temperature for 5 m and then 100 mL of the DMAB reagent was added to each well. The samples were then incubated for 90 min at 60 C, and were analyzed at 560 nm using a spectrophotometer (multimode microplate reader (Infinite® 200 PRO). 2.8. Statistical analysis The data were analyzed using one-way ANOVA followed by the Dunnett's multiple range test using GraphPad Prism (version 5.00; GraphPad Software, San Diego, CA, USA). All data are expressed as means ± S.E.M, and in all cases, differences were considered statistically significant at p < 0.05. 3. Results 3.1. Mouse characteristics and change in body weight The weight of the mice increased over time. The weight of the NFD þ CIH group did not increase following IH. Fig. 1B demonstrates that weight gain was greater in HFD-fed mice
3.2. Markers of oxidative stress and inflammation in liver tissues and serum CIH resulted in a trend toward increases in serum albumin and IL-1b levels, though they were not statistically significant (Fig. 2A and B). Liver SOD activity was markedly higher in the NFD þ CIH, HFD þ RA, and HFD þ CIH groups than in the NFD þ RA group (p < 0.05, p < 0.001, and p < 0.001, respectively) (Fig. 2C). The levels of MPO in the liver were slightly higher in the HFD þ RA and HFD þ CIH groups than in the NFD þ RA group (Fig. 2D). The catalase levels in the liver were lower in the HFD þ RA group than in the NFD þ RA group (p < 0.05) (Fig. 2E). 3.3. CIH induces liver fibrosis in HFD-fed mice Liver histopathology demonstrated marked hepatic macrovesicular and microvescicular steatosis using H&E stain on HFD group sections (Fig. 3A). Masson trichrome staining demonstrated that the NFD þ CIH, HFD þ RA, and HFD þ CIH groups displayed hyperplasia in periportal collagen deposition when compared with the NFD þ RA group (Fig. 3B). In particular, the HFD þ CIH group had more severe periportal collagen accumulation with mild perisinusoidal fibrosis. Quantitative analysis of collagen concentrations showed that CIH also led to upregulated hydroxyproline levels when compared with those of the NFD þ RA group (p < 0.05), which was consistent with the hepatic fibrosis observed by histopathology (Fig. 3C). In addition, in the HFD þ CIH group, a1(I)collagen mRNA was significantly higher than that in the NFD þ RA group (p < 0.05) (Fig. 3D). CIH increased TGFb1, PDGFRb, and
Fig. 1. Effect of obesity and CIH on body weight (BW). (A) BW of NFD- or HFD-fed mice in RA or under conditions of CIH (n ¼ 5 mice/group). BW was recorded weekly. (B) Comparison of weight gain in mice. (DBW ¼ BW at 16 weeks e BW at 1 week). (C) Representative images of NFD and HFD mice under RA or CIH conditions, after 16 weeks. (*, p < 0.05, ***, p < 0.001 vs. NFD þ RA; ##, p < 0.01, ###, p < 0.001 vs. NFD þ CIH).
Please cite this article in press as: H.H. Kang, et al., Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.047
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Fig. 2. Effect of obesity and CIH on proinflammatory cytokine expression and oxidative stress. The levels of (A) albumin and (B) IL-1b were measured in the serum; and (C) SOD, (D) MPO and (E) catalase (CAT) were measured in liver lysates. Data are expressed as the mean ± S.E.M. (n ¼ 5 mice/group) (*, p < 0.05, ***, p < 0.001 vs. NFD þ RA).
caspase 3, which were augmented in the HFD group (Fig. 3E, F, and G), though the difference was not statistically significant. 3.4. CIH increases the expression of TLR4 and MyD88 with up regulating ERK-dependent MAPK signaling pathway in HFD-fed mice We assayed the induction of TLR4, MyD88, and TRIF, a downstream molecule of TLR4, using western blot analysis, ELISA, and quantitative RT-PCR (Fig. 4). TLR4 and MyD88 protein expression was significantly higher in the HFD þ CIH group. In addition, an upregulation in TLR4 mRNA was observed in the HFD groups, which was augmented in the CIH group (Fig. 4B). TRIF protein expression did not differ significantly among groups. In the HFD þ CIH group, the protein expression of phospho-I-kB and the NF-kB p65 subunit was significantly higher than that in the NFD þ RA group (Fig. 4C). There was a statistically insignificant trend toward an increase in the expression of TGFb1, which was consistent with the result obtained using RT-PCR. These results suggest that in the HFD group, CIH increases the mRNA and protein expression of TLR4 and the protein expression of MyD88 via the TLR4 receptor, which is not involved in the TRIF-dependent pathway. To determine whether the MAPK signaling pathway, including ERK, p38, and JNK, is involved in CIH-induced hepatic injury, we examined the activation of MAPK signaling proteins using western blots (Fig. 4D). Phospho-ERK1/2 was significantly higher in the HFD þ CIH group than in the NFD þ RA group, while the levels of phospho-p38 MAPK, JNK, and c-jun did not differ among groups. Thus, in the HFD þ CIH group, activation of the MAPK signaling pathway is involved in the ERK-dependent pathway. 4. Discussion We demonstrated, using a murine CIH model with a HFD to investigate liver fibrogenesis, that noninfectious TLR4 signaling was
involved in the pathophysiological process. Following 4 weeks of IH, liver fibrosis was significantly higher than in the normoxic controls, and was augmented following high-fat and highcholesterol diet administration. This suggests that CIH leads to collagen deposition in the liver, regardless of whether the subjects are obese. We also observed that exposing DIO mice to CIH induces a significant increase in a1(I)-collagen mRNA and trends toward upregulation of pro-fibrotic mediators compared with levels in the RA-exposed groups. CIH significantly increased protein expression of TLR4, MyD88, phospho-I-kB, phospho-ERK1/2, and the nuclear fraction of NF-kB in liver, all of which were augmented in DIO mice. Since there were no changes in TRIF, p38 MAPK, or JNK activation in any group, our data suggest possible involvement of the TLR4/ MyD88/MAPK/NF-kB signaling pathway in liver fibrosis caused by CIH in DIO mice. OSA and NAFLD likely share common intermediary mechanisms; OSA might therefore be involved in the development and progression of NAFLD [7,8]. The pathophysiology of OSA-induced metabolic consequences in the liver remains poorly understood. Evidence demonstrates that TLR4-induced innate immunity and noninfectious inflammatory responses play critical roles in the cardiovascular, respiratory, and hepatic systems [16]. Endogenous molecules, such as heat-shock protein 70, high-mobility group box1, and myeloid-related protein 8/14, activators of TLR4, were also detected in patients with OSA [17e19]. Similarly, OSA is associated with enhanced TLR4 expression and signaling downstream of TLR4 in circulating monocytes [14]. Furthermore, 8 weeks of continuous positive airway pressure (CPAP) treatment downregulated TLR4 expression and reduced inflammatory cytokine release. However, few studies have investigated TLR4-induced liver damage in patients with OSA and NAFLD. A study in children suggested that OSA increases intestinal permeability, resulting in elevated systemic lipopolysaccharide secretion, which led to TLR4 upregulation in hepatocytes, Kupffer cells, and hepatic stellate cells (HSC) [20]. This suggests that gut microbiota might be involved in OSA-related
Please cite this article in press as: H.H. Kang, et al., Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.047
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Fig. 3. Effect of obesity and CIH in fibrosis. Representative photomicrographs of liver sections derived from mice fed a NFD or a HFD for 16 weeks, stained with (A) H&E as a measure of hepatic steatosis or lobular inflammation, and (B) Masson trichrome as a measure of collagen accumulation (phase contrast microscopy, 200 magnification). After mice were sacrificed, (C) livers were collected from each mouse for use in the hydroxyproline assay. (DeG) RT-PCR was used to measure the mRNA expression of a1-collagen, TGFb1, PDGFRb, and caspase 3. The mRNA levels were normalized to b-actin. Data are expressed as mean ± S.E.M. (n ¼ 5 mice/group) (*, p < 0.05 vs. NFD þ RA).
NASH pathogenesis. We observed trends toward higher induction of inflammatory genes, such as IL-1b, and pro-fibrotic mediators, such as PDGFRb and TGFb1, in the livers of CIH-exposed mice than in normoxic controls. We also determined that TLR4 protein and mRNA levels were higher in the livers of these mice than in those of the normoxic control and HFD groups. These results suggest that CIH upregulates the inflammatory response and fibrogenesis in the liver through TLR4 signaling. TLR4 signaling occurs through MyD88-and TRIF-dependent pathways [16]. Researchers have evaluated the correlation between the TLR4/MyD88 pathway and myocardial injury in animal models of CIH/reoxygenation [21]. The repetitive process of short desaturation cycles followed by rapid reoxygenation in patients with OSA is similar to the ischemic/reperfusion injury observed in myocardial tissue injury. Pro-inflammatory cytokine release and tissue infiltration by inflammatory mediators is observed during ischemia, and is exacerbated upon reperfusion, which likely activates progressive inflammatory signal transduction. Endogenous molecule release from ischemic cells is thought to activate innate
immunity in rodents [22]. Chong et al. observed that ischemia activates the TLR4/MyD88-dependent pathway, which results in the cardiac release of innate cytokines and leads to the activation of NFkB. This ischemic/reperfusion injury also occurs in the liver; however, unlike with myocardial ischemia/reperfusion injury, the association between CIH and liver injury has been postulated as a “two hit” process [2]. Obesity, dyslipidemia, and other factors cause hepatic lipid accumulationdthe “first hit.” The “second hit” results from the reactive oxygen species-induced oxidative stress [23]. It initiates lipid peroxidation, produces inflammation and fibrosis, and causes NAFLD [24]. Savransky et al. revealed that IH alone did not cause liver fibrosis, while steatosis, caused by a highcholesterol diet, constituted the “first hit.” Based on this model, repeated hypoxia/reoxygenation is the “second hit,” which might cause fibrosis in susceptible livers [25]. We provide the first evidence that CIH contributes to the pathogenesis of liver fibrosis in DIO, acting via the TLR4-mediated MyD88/NF-kB signaling pathway. The MAPK family includes ERK1/2, JNK, and p38 MAPK. Upon
Please cite this article in press as: H.H. Kang, et al., Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.047
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Fig. 4. Effect of obesity and CIH on the expression of TLR4 and MAPK signaling pathway. (A) The protein expression of TLR4, MyD88, TRIF, and phospho-I-kB were analyzed by western blotting. The expression of NF-kB p65 was analyzed in the nuclear fraction of the liver tissue. Images are representative of three independent experiments. (B) Quantitative RT-PCR analysis of TLR4. The TLR4 mRNA level was normalized to that of b-actin. (C) Levels of phospho-NF-kB were measured by ELISA in the liver tissues of NFD- or HFD-fed mice. (D) The activation of ERK, JNK, p38 MAPK, and c-Jun in liver tissues was analyzed by western blotting. Images are representative of three independent experiments. b-actin was used as a control. (*, p < 0.05, **, p < 0.01, ***, p < 0.001 vs. NFD þ RA; #, p < 0.05, ###, p < 0.001 vs. NFD þ CIH; $$, p < 0.01 vs. HFD þ CIH).
activation, these molecules recruit Ras, which stimulates the transcription of proliferative and profibrogenic factors [26]. MAPKs are crucial for HSC activation and collagen synthesis [27]. Several studies have assessed the timing and location of MAPK activation in various tissues under conditions of stress [28]. Zhao et al. demonstrated that, under IH, ERK1/2, p38 MAPK, and JNK were activated at all time-points assessed (2, 4, 6, and 8 weeks). In contrast, under continued hypoxia, the levels of phospho-ERK1/2 in brain tissues peaked at 4 weeks and then declined, whereas phospho-p38 MAPK and JNK were detected only in late stages [29]. In the HFD þ CIH group, our study indicated that phospho-ERK1/2 was significantly higher than in the NFD þ RA group, while levels of phospho-p38 MAPK, JNK, and c-jun did not differ among groups, suggesting that MAPK signaling pathways might be selectively activated under conditions of IH with a variable timeline. p38 MAPK and JNK are similar in function, and activation of either has a negative regulatory role, leading to cell injury and death [30]. In contrast, ERK1/2 has a dual effect, which may promote short-term cell survival and proliferation with appropriate activation, but may also cause cell death with excessive activation in the long term [31]. The activation of ERK1/2 might upregulate the protective anti-oxidant system and facilitate tissue survival [32]. ERK1/2 activation may have had a protective effect on the DIO mice upon IH, increasing hypoxiainduced compensatory mechanisms of the liver. ERK1/2 activation during the early stages might have increased hypoxic tolerance for a short time. In conclusion, our study indicated that in DIO mice, CIH induces liver fibrosis, and this phenomenon is associated with TLR4mediated MAPK and NF-kB signaling. This suggests that TLR4 is associated with the pathogenesis of liver fibrosis in CIH; TLR4 inhibition might thus be a strategy for preventing hepatic fibrosis in patients with OSA.
Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.06.047. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.06.047. References [1] Z.M. Younossi, A.B. Koenig, D. Abdelatif, et al., Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence and outcomes, Hepatology 64 (2016) 73e84. [2] C.P. Day, O.F. James, Steatohepatitis: a tale of two “hits”? Gastroenterology 114 (1998) 842e845. [3] E. Seki, S. De Minicis, C.H. Osterreicher, et al., TLR4 enhances TGF-b signaling and hepatic fibrosis, Nat. Med. 13 (2007) 1324e1332. [4] C. Arnaud, M. Dematteis, J.-L. Pepin, et al., Obstructive sleep apnea, immunoinflammation, and atherosclerosis, Semin. Immunopathol. 31 (2009) 113e125. [5] T. Young, E. Shahar, F.J. Nieto, et al., Predictors of sleep-disordered breathing in community-dwelling adults: the sleep heart health study, Arch. Intern Med. 162 (2002) 893e900. [6] J.-P. Baguet, G. Barone-Rochette, R. Tamisier, et al., Mechanisms of cardiac dysfunction in obstructive sleep apnea, Nat. Rev. Cardiol. 9 (2012) 679e688. pin, Nonalcoholic fatty liver disease and [7] J. Aron-Wisnewsky, K. Clement, J.L. Pe obstructive sleep apnea, Metabolism 65 (2016) 1124e1135. pin, New insights in the pathophysiology of chronic [8] J. Aron-Wisnewsky, J.L. Pe intermittent hypoxia-induced NASH: the role of guteliver axis impairment,
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Chronic intermittent hypoxia induces liver fibrosis in mice with dietinduced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways Hyeon Hui Kang, In Kyoung Kim, Hye in Lee, Hyonsoo Joo, Jeong Uk Lim, Jongmin Lee, Sang Haak Lee, Hwa Sik Moon* Department of Internal Medicine, College of Medicine, St. Paul's Hospital, The Catholic University of Korea, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2017 Accepted 12 June 2017 Available online xxx
Obstructive sleep apnea (OSA) is associated with nonalcoholic fatty liver disease (NAFLD), and causes chronic intermittent hypoxia (CIH) during sleep. Inflammation is associated with the development of metabolic complications induced by CIH. Research suggests that innate immune mechanisms are involved in the pro-inflammatory pathways of liver fibrosis. The purpose of this study was to investigate whether innate immune responses induce liver fibrosis, and to evaluate mechanisms underlying hepatic inflammation related to CIH in a murine diet-induced obesity (DIO) model. Inflammatory and oxidative stress markers, TLR4, MyD88, Toll/interleukin-1-receptor-domain-containing adaptor-inducing interferon-b (TRIF), I-kB, NF-kB, p38 MAPK, c-JNK, and ERK activation, were measured in the serum and liver. As a result, a1(I)-collagen mRNA was significantly higher in DIO mice exposed to CIH than in the control groups. CIH mice exhibited liver fibrosis and significantly higher protein expression of TLR4, MyD88, phosphorylated (phospho-) I-kB, and phospho-ERK1/2 activation in the liver, and higher expression of NF-kB than that in the controls. TRIF, p38 MAPK, and JNK activation did not differ significantly between groups. We conclude that CIH in DIO mice leads to liver fibrosis via TLR4/MyD88/MAPK/NF-kB signaling pathways. © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Obstructive sleep apnea Chronic intermittent hypoxia Obesity Liver fibrosis Toll like receptor 4
1. Introduction The already high prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing with the obesity epidemic, varying from 6 to 33% in the general population [1]. Although its pathophysiology is incompletely understood, insulin resistance, inflammation, oxidative stress, and lipotoxicity are involved [2]. Although inflammation occurs in most patients with hepatic fibrosis and correlates with fibrosis progression [3], the molecular correlation between hepatic inflammation and fibrogenesis remains elusive. Obstructive sleep apnea (OSA) is characterized by repetitive partial (hypopnea) or complete (apnea) upper airway collapse during sleep, inducing chronic intermittent hypoxia (CIH) resulting in low-grade inflammation, sympathetic overactivity, and oxidative stress [4]. OSA is associated with obesity in 60% of cases [5], and causes significant cardiovascular and metabolic morbidity and
* Corresponding author. Department of Internal Medicine, College of Medicine, St. Paul's Hospital, The Catholic University of Korea, Wangsanro 180, Dongdaemungu, Seoul, 130-709, Republic of Korea. E-mail address: [email protected] (H.S. Moon).
mortality [6]. OSA and NAFLD have similar risk factors, including obesity, and OSA is thought to contribute to the pathogenesis and exacerbation of NAFLD [7,8]. Although evidence suggests that OSA is associated with NAFLD pathogenesis and progression, few studies have investigated the mechanism of OSA-induced liver injury. Toll like receptors (TLRs) are pattern recognition receptors that perceive pathogens and bacteria-derived molecules, leading to proinflammatory cytokine production [9]. TLRs regulate innate and adaptive immune responses [10], which are also associated with noninfectious inflammatory liver diseases [11]. Of the 13 mammalian TLRs, only TLR2, TLR4, TLR5, and TLR9 are associated with NAFLD [12,13]. TLR4 triggers two signaling pathways: the MyD88-dependent pathway that leads to the rapid activation of NF-kB and increased TNF-a production, and the MyD88independent pathway that requires the Toll/interleukin-1 receptor (TIR)-containing adaptor molecule. Both pathways ultimately lead to proinflammatory cytokine, chemokine, and type I interferon production [9]. TLR4 might be involved in OSA, as monocytes from patients with OSA have higher TLR4 surface expression [14]. TLR4 deletion in mice prevented CIH-induced insulin resistance and
http://dx.doi.org/10.1016/j.bbrc.2017.06.047 0006-291X/© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Abbreviations OSA NAFLD CIH DIO TRIF NFD HFD RA
obstructive sleep apnea nonalcoholic fatty liver disease chronic intermittent hypoxia diet-induced obesity Toll/interleukin-1-receptor-domain-containing adaptor-inducing interferon-b normal fat diet high fat diet room air
1 beta Quantikine ELISA Kit (MLB00C; R&D Systems, Minneapolis, MN, USA). The sensitivities of the assays were 1.5 ng/mL and 4.8 pg/ mL, respectively. Chemiluminescence was detected using a Multimode Microplate Reader (Infinite® 200 PRO; Life Science, Horsham, PA, USA) at 450 nm. The concentrations of superoxide dismutase (SOD), myeloperoxidase (MPO), and catalase (CAT) were measured in the tissue lysates, using the following kits: SOD Assay Kit (S311; Dojindo Molecular Technologies, Kumamoto, Japan), the Myeloperoxidase (MPO) Activity Colorimetric Assay Kit (K744-100; BioVision, Milpitas, CA, USA), and the EnzyChromTM Catalase Assay Kit (ECAT-100; Bioassay Systems, Hayward, CA, USA), respectively, according to the manufacturers' instructions. Chemiluminescence was detected at 450 nm, 412 nm, and 570 nm, respectively. 2.4. Western blot analysis
morphological and inflammatory changes in epididymal fat and aorta [15]; however, the relationship between TLR4 and liver injury has not been explored in OSA, and little is known about the processes that propagate TLR signaling downstream during CIH. Here, we investigated the innate immune responses associated with liver fibrosis in CIH, and evaluated the molecular mechanisms underlying hepatic responses to CIH in a murine diet-induced obesity (DIO) model. 2. Materials and methods 2.1. Animals Seven-to-eight week-old male, C57BL/6 J mice were purchased from DBL (Chungcheongbuk-do, Korea). The study was approved by the Ethical Committee on Animal Experiments of the Catholic University of Korea. All animals were maintained in a pathogenfree environment at 22e24 C with a 12:12 h light:dark cycle; lights on at (09:00) with free access to food and water. 2.2. Experimental design and animal model of CIH The mice were divided into two groups and fed a normal fat diet (NFD group) or a high-fat diet (HFD group) for 12 weeks. Each group was then divided into two additional groups then exposed to chronic intermittent hypoxia (CIH, n ¼ 10) or control conditions (room air; RA, n ¼ 10) for 4 weeks. A gas control delivery system was designed to regulate air, nitrogen, and oxygen flow into the cages. During periods of CIH, the fractional inspired O2 (FiO2) was reduced from RA levels to approximately 7± 0.5% within 30 s, followed by reoxygenation to RA levels within the subsequent 30 s (Supplementary Fig. 1A). Exposure proceeded from 09:00 to 21:00 daily to coincide with mouse sleep cycles, and the duration of exposure was 4 weeks. The oxygen level was measured with a ProOx 110 analyzer (Biospherix, Redfield, NY, USA). The HFD group was fed a high-fat, high-cholesterol diet (D12492; Research Diets, Inc. New Brunswick, NJ, USA; 34.9 gm% Fat, 300.8 mg/kg Cholesterol) and the NFD group was fed regular chow for 12 weeks. The NFD þ CIH and HFD þ CIH groups were then moved into the intermittent hypoxia (IH) chamber for 4 weeks with their corresponding feed. The NFD þ RA and HFD þ RA groups were exposed to RA during the same period (Supplementary Fig. 1B). Body weight was monitored once per week for each mouse. 2.3. ELISA The concentration of albumin was measured using Albumin Mouse ELISA Kit (ab108792; Abcam, Burlingame, CA, USA) and the concentration of interleukin (IL)-1b was measured using Mouse IL-
Total protein was isolated from the liver by homogenization in cell lysis buffer containing a mixture of protease and phosphatase inhibitors (GenDEPOT, Katy, TX, USA), and then centrifuged at 13,000 rpm for 30 m at 4 C. The proteins (40 mg/mL) were separated by 10% SDS-PAGE, in a Tris-glycine-SDS buffer. Separated proteins were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk (Sigma, St. Louis, MO, USA) for 2 h at room temperature, and then incubated with a 1:200 dilution of the individual primary antibodies antiTLR4, anti-TRIF, anti-MyD88, anti-I-kB, anti-p-I-kB, anti-NF-kB p65, anti-Lamin B, and anti-b-actin) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), or a 1:1000 dilution of the primary antibodies anti-ERK, anti-p-ERK, anti-c-JNK, anti-p-JNK, anti-p38 MAPK, antip-p38 MAPK, anti-c-Jun, or anti-p-c-Jun (Cell Signaling Technology, Danvers, MA., USA). The antibodies were diluted in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), and applied overnight at 4 C. Membranes were washed and incubated with HRP-conjugated secondary antibodies (goat anti-mouse/rabbit, rabbit anti-goat IgG-HRP Secondary Antibody (Santa Cruz Biotechnology, Inc.). Protein bands were detected using the Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc.). 2.5. Quantitative real-time polymerase chain reaction (RT-PCR) Total RNA was extracted from lung tissue according to the protocol for Trizol according to the manufacturer's instructions (Takara, Japan). First-strand cDNA was synthesized from total RNA using the Super Script II first-strand synthesis system (Invitrogen, Carlsbad, CA, USA). The primer sequences are provided as follows: a1(I)-collagen (forward 50 -CTGGCGGTTCAGGTCCAA-30 , reverse 50 GCTTCCCCATCATCATCTCCATTC-30 ); TGFb1 (forward 50 -GGAACTCTACCAGAAATATAGCAACAATTC-30 , reverse 50 TGTATTCCGTCTCCTTGGTTCAG-30 ); PDGFRb (forward 50 -CCGGAACAAACACACCTTCT-30 , reverse 50 -TATCCATGTAGCCACCGTCA-30 ); caspase 3 (forward 50 - TGTCATCTCGCTCTGGTACG-30 , reverse 50 AAATGACCCCTTCATCACCA-30 ); and b-actin (forward 50 -ACAGGAAGTCCCTTGCCATC-30 , reverse 50 -AGGGAGACCAAAAGCCTTCA30 ). Quantitative RT-PCR was performed using the QuantiFast SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). The threshold cycle (Ct) value for each gene was normalized to that of b-actin. The relative mRNA expression was calculated using the 2DDCT method. 2.6. Tissue histology The liver tissues in each group were fixed with 10% formalin, embedded in paraffin wax, and cut into 3 mm sections. Deparaffinized tissue sections were subjected to H&E staining and then observed under a light microscope to make precise pathological diagnoses. Separate sections were stained using the standard
Please cite this article in press as: H.H. Kang, et al., Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.047
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Masson's trichrome staining protocol for identifying reactive fibrosis and its distribution.
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(23.28 ± 3.52 g) than in NFD-fed mice (9.75 ± 1.90 g). Body weight in the HFD þ CIH group (17.68 ± 5.94 g) was considerably lower (24.1%) than that in the HFD þ RA group (23.28 ± 3.52 g).
2.7. Hydroxyproline analysis The collagen concentration was measured in peripheral portions of the liver using a hydroxyproline assay kit (Biovision Inc., Milpitas, CA., USA), according to the manufacturer's instructions. Briefly, liver tissue was homogenized in dH2O, using 300 mL of H2O per 30 mg of tissue. The samples were added to 300 mL of 12 N HCl at 120 C, overnight. After centrifugation (10,000g for 3 min at room temperature), 100 mL of the chloramine T reagent was added to 10 mL of each hydrolyzed sample, the mixtures were incubated at room temperature for 5 m and then 100 mL of the DMAB reagent was added to each well. The samples were then incubated for 90 min at 60 C, and were analyzed at 560 nm using a spectrophotometer (multimode microplate reader (Infinite® 200 PRO). 2.8. Statistical analysis The data were analyzed using one-way ANOVA followed by the Dunnett's multiple range test using GraphPad Prism (version 5.00; GraphPad Software, San Diego, CA, USA). All data are expressed as means ± S.E.M, and in all cases, differences were considered statistically significant at p < 0.05. 3. Results 3.1. Mouse characteristics and change in body weight The weight of the mice increased over time. The weight of the NFD þ CIH group did not increase following IH. Fig. 1B demonstrates that weight gain was greater in HFD-fed mice
3.2. Markers of oxidative stress and inflammation in liver tissues and serum CIH resulted in a trend toward increases in serum albumin and IL-1b levels, though they were not statistically significant (Fig. 2A and B). Liver SOD activity was markedly higher in the NFD þ CIH, HFD þ RA, and HFD þ CIH groups than in the NFD þ RA group (p < 0.05, p < 0.001, and p < 0.001, respectively) (Fig. 2C). The levels of MPO in the liver were slightly higher in the HFD þ RA and HFD þ CIH groups than in the NFD þ RA group (Fig. 2D). The catalase levels in the liver were lower in the HFD þ RA group than in the NFD þ RA group (p < 0.05) (Fig. 2E). 3.3. CIH induces liver fibrosis in HFD-fed mice Liver histopathology demonstrated marked hepatic macrovesicular and microvescicular steatosis using H&E stain on HFD group sections (Fig. 3A). Masson trichrome staining demonstrated that the NFD þ CIH, HFD þ RA, and HFD þ CIH groups displayed hyperplasia in periportal collagen deposition when compared with the NFD þ RA group (Fig. 3B). In particular, the HFD þ CIH group had more severe periportal collagen accumulation with mild perisinusoidal fibrosis. Quantitative analysis of collagen concentrations showed that CIH also led to upregulated hydroxyproline levels when compared with those of the NFD þ RA group (p < 0.05), which was consistent with the hepatic fibrosis observed by histopathology (Fig. 3C). In addition, in the HFD þ CIH group, a1(I)collagen mRNA was significantly higher than that in the NFD þ RA group (p < 0.05) (Fig. 3D). CIH increased TGFb1, PDGFRb, and
Fig. 1. Effect of obesity and CIH on body weight (BW). (A) BW of NFD- or HFD-fed mice in RA or under conditions of CIH (n ¼ 5 mice/group). BW was recorded weekly. (B) Comparison of weight gain in mice. (DBW ¼ BW at 16 weeks e BW at 1 week). (C) Representative images of NFD and HFD mice under RA or CIH conditions, after 16 weeks. (*, p < 0.05, ***, p < 0.001 vs. NFD þ RA; ##, p < 0.01, ###, p < 0.001 vs. NFD þ CIH).
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Fig. 2. Effect of obesity and CIH on proinflammatory cytokine expression and oxidative stress. The levels of (A) albumin and (B) IL-1b were measured in the serum; and (C) SOD, (D) MPO and (E) catalase (CAT) were measured in liver lysates. Data are expressed as the mean ± S.E.M. (n ¼ 5 mice/group) (*, p < 0.05, ***, p < 0.001 vs. NFD þ RA).
caspase 3, which were augmented in the HFD group (Fig. 3E, F, and G), though the difference was not statistically significant. 3.4. CIH increases the expression of TLR4 and MyD88 with up regulating ERK-dependent MAPK signaling pathway in HFD-fed mice We assayed the induction of TLR4, MyD88, and TRIF, a downstream molecule of TLR4, using western blot analysis, ELISA, and quantitative RT-PCR (Fig. 4). TLR4 and MyD88 protein expression was significantly higher in the HFD þ CIH group. In addition, an upregulation in TLR4 mRNA was observed in the HFD groups, which was augmented in the CIH group (Fig. 4B). TRIF protein expression did not differ significantly among groups. In the HFD þ CIH group, the protein expression of phospho-I-kB and the NF-kB p65 subunit was significantly higher than that in the NFD þ RA group (Fig. 4C). There was a statistically insignificant trend toward an increase in the expression of TGFb1, which was consistent with the result obtained using RT-PCR. These results suggest that in the HFD group, CIH increases the mRNA and protein expression of TLR4 and the protein expression of MyD88 via the TLR4 receptor, which is not involved in the TRIF-dependent pathway. To determine whether the MAPK signaling pathway, including ERK, p38, and JNK, is involved in CIH-induced hepatic injury, we examined the activation of MAPK signaling proteins using western blots (Fig. 4D). Phospho-ERK1/2 was significantly higher in the HFD þ CIH group than in the NFD þ RA group, while the levels of phospho-p38 MAPK, JNK, and c-jun did not differ among groups. Thus, in the HFD þ CIH group, activation of the MAPK signaling pathway is involved in the ERK-dependent pathway. 4. Discussion We demonstrated, using a murine CIH model with a HFD to investigate liver fibrogenesis, that noninfectious TLR4 signaling was
involved in the pathophysiological process. Following 4 weeks of IH, liver fibrosis was significantly higher than in the normoxic controls, and was augmented following high-fat and highcholesterol diet administration. This suggests that CIH leads to collagen deposition in the liver, regardless of whether the subjects are obese. We also observed that exposing DIO mice to CIH induces a significant increase in a1(I)-collagen mRNA and trends toward upregulation of pro-fibrotic mediators compared with levels in the RA-exposed groups. CIH significantly increased protein expression of TLR4, MyD88, phospho-I-kB, phospho-ERK1/2, and the nuclear fraction of NF-kB in liver, all of which were augmented in DIO mice. Since there were no changes in TRIF, p38 MAPK, or JNK activation in any group, our data suggest possible involvement of the TLR4/ MyD88/MAPK/NF-kB signaling pathway in liver fibrosis caused by CIH in DIO mice. OSA and NAFLD likely share common intermediary mechanisms; OSA might therefore be involved in the development and progression of NAFLD [7,8]. The pathophysiology of OSA-induced metabolic consequences in the liver remains poorly understood. Evidence demonstrates that TLR4-induced innate immunity and noninfectious inflammatory responses play critical roles in the cardiovascular, respiratory, and hepatic systems [16]. Endogenous molecules, such as heat-shock protein 70, high-mobility group box1, and myeloid-related protein 8/14, activators of TLR4, were also detected in patients with OSA [17e19]. Similarly, OSA is associated with enhanced TLR4 expression and signaling downstream of TLR4 in circulating monocytes [14]. Furthermore, 8 weeks of continuous positive airway pressure (CPAP) treatment downregulated TLR4 expression and reduced inflammatory cytokine release. However, few studies have investigated TLR4-induced liver damage in patients with OSA and NAFLD. A study in children suggested that OSA increases intestinal permeability, resulting in elevated systemic lipopolysaccharide secretion, which led to TLR4 upregulation in hepatocytes, Kupffer cells, and hepatic stellate cells (HSC) [20]. This suggests that gut microbiota might be involved in OSA-related
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Fig. 3. Effect of obesity and CIH in fibrosis. Representative photomicrographs of liver sections derived from mice fed a NFD or a HFD for 16 weeks, stained with (A) H&E as a measure of hepatic steatosis or lobular inflammation, and (B) Masson trichrome as a measure of collagen accumulation (phase contrast microscopy, 200 magnification). After mice were sacrificed, (C) livers were collected from each mouse for use in the hydroxyproline assay. (DeG) RT-PCR was used to measure the mRNA expression of a1-collagen, TGFb1, PDGFRb, and caspase 3. The mRNA levels were normalized to b-actin. Data are expressed as mean ± S.E.M. (n ¼ 5 mice/group) (*, p < 0.05 vs. NFD þ RA).
NASH pathogenesis. We observed trends toward higher induction of inflammatory genes, such as IL-1b, and pro-fibrotic mediators, such as PDGFRb and TGFb1, in the livers of CIH-exposed mice than in normoxic controls. We also determined that TLR4 protein and mRNA levels were higher in the livers of these mice than in those of the normoxic control and HFD groups. These results suggest that CIH upregulates the inflammatory response and fibrogenesis in the liver through TLR4 signaling. TLR4 signaling occurs through MyD88-and TRIF-dependent pathways [16]. Researchers have evaluated the correlation between the TLR4/MyD88 pathway and myocardial injury in animal models of CIH/reoxygenation [21]. The repetitive process of short desaturation cycles followed by rapid reoxygenation in patients with OSA is similar to the ischemic/reperfusion injury observed in myocardial tissue injury. Pro-inflammatory cytokine release and tissue infiltration by inflammatory mediators is observed during ischemia, and is exacerbated upon reperfusion, which likely activates progressive inflammatory signal transduction. Endogenous molecule release from ischemic cells is thought to activate innate
immunity in rodents [22]. Chong et al. observed that ischemia activates the TLR4/MyD88-dependent pathway, which results in the cardiac release of innate cytokines and leads to the activation of NFkB. This ischemic/reperfusion injury also occurs in the liver; however, unlike with myocardial ischemia/reperfusion injury, the association between CIH and liver injury has been postulated as a “two hit” process [2]. Obesity, dyslipidemia, and other factors cause hepatic lipid accumulationdthe “first hit.” The “second hit” results from the reactive oxygen species-induced oxidative stress [23]. It initiates lipid peroxidation, produces inflammation and fibrosis, and causes NAFLD [24]. Savransky et al. revealed that IH alone did not cause liver fibrosis, while steatosis, caused by a highcholesterol diet, constituted the “first hit.” Based on this model, repeated hypoxia/reoxygenation is the “second hit,” which might cause fibrosis in susceptible livers [25]. We provide the first evidence that CIH contributes to the pathogenesis of liver fibrosis in DIO, acting via the TLR4-mediated MyD88/NF-kB signaling pathway. The MAPK family includes ERK1/2, JNK, and p38 MAPK. Upon
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Fig. 4. Effect of obesity and CIH on the expression of TLR4 and MAPK signaling pathway. (A) The protein expression of TLR4, MyD88, TRIF, and phospho-I-kB were analyzed by western blotting. The expression of NF-kB p65 was analyzed in the nuclear fraction of the liver tissue. Images are representative of three independent experiments. (B) Quantitative RT-PCR analysis of TLR4. The TLR4 mRNA level was normalized to that of b-actin. (C) Levels of phospho-NF-kB were measured by ELISA in the liver tissues of NFD- or HFD-fed mice. (D) The activation of ERK, JNK, p38 MAPK, and c-Jun in liver tissues was analyzed by western blotting. Images are representative of three independent experiments. b-actin was used as a control. (*, p < 0.05, **, p < 0.01, ***, p < 0.001 vs. NFD þ RA; #, p < 0.05, ###, p < 0.001 vs. NFD þ CIH; $$, p < 0.01 vs. HFD þ CIH).
activation, these molecules recruit Ras, which stimulates the transcription of proliferative and profibrogenic factors [26]. MAPKs are crucial for HSC activation and collagen synthesis [27]. Several studies have assessed the timing and location of MAPK activation in various tissues under conditions of stress [28]. Zhao et al. demonstrated that, under IH, ERK1/2, p38 MAPK, and JNK were activated at all time-points assessed (2, 4, 6, and 8 weeks). In contrast, under continued hypoxia, the levels of phospho-ERK1/2 in brain tissues peaked at 4 weeks and then declined, whereas phospho-p38 MAPK and JNK were detected only in late stages [29]. In the HFD þ CIH group, our study indicated that phospho-ERK1/2 was significantly higher than in the NFD þ RA group, while levels of phospho-p38 MAPK, JNK, and c-jun did not differ among groups, suggesting that MAPK signaling pathways might be selectively activated under conditions of IH with a variable timeline. p38 MAPK and JNK are similar in function, and activation of either has a negative regulatory role, leading to cell injury and death [30]. In contrast, ERK1/2 has a dual effect, which may promote short-term cell survival and proliferation with appropriate activation, but may also cause cell death with excessive activation in the long term [31]. The activation of ERK1/2 might upregulate the protective anti-oxidant system and facilitate tissue survival [32]. ERK1/2 activation may have had a protective effect on the DIO mice upon IH, increasing hypoxiainduced compensatory mechanisms of the liver. ERK1/2 activation during the early stages might have increased hypoxic tolerance for a short time. In conclusion, our study indicated that in DIO mice, CIH induces liver fibrosis, and this phenomenon is associated with TLR4mediated MAPK and NF-kB signaling. This suggests that TLR4 is associated with the pathogenesis of liver fibrosis in CIH; TLR4 inhibition might thus be a strategy for preventing hepatic fibrosis in patients with OSA.
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Please cite this article in press as: H.H. Kang, et al., Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.06.047