A novel AMPK activator improves hepatic lipid metabolism and leukocyte trafficking in experimental hepatic steatosis

Journal of Pharmacological Sciences 140 (2019) 153e161

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A novel AMPK activator improves hepatic lipid metabolism and leukocyte trafficking in experimental hepatic steatosis Xueying Peng a, Jin Li a, b, Minjie Wang a, c, Kai Qu a, Haibo Zhu a, * a

State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China b School of Public Health, The First Affiliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China c School of Basic Medical Sciences, The Center for Drug Screening, Center for New Drug Safety Evaluation and Research, Inner Mongolia Medical University, Inner Mongolia, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 February 2019 Received in revised form 22 May 2019 Accepted 24 May 2019 Available online 4 June 2019

A novel AMP-activated protein kinase (AMPK) activator, IMM-H007 (H007), has been reported to reduce serum lipid levels and inhibit lipid accumulation in the liver in hyperlipidemic animal models. However, how H007 ameliorates hepatic steatosis and inflammation remains unknown. In the present study, H007, at 200 mg/kg, reduced hepatic lipid levels and the levels of collagenous fiber in the liver in high-fat diet (HFD)-fed hamsters compared to those in the HFD group. Meanwhile, compared to the controls, H007 significantly inhibited sterol-regulatory element binding protein (SREBP)-1c and acetyl CoA carboxylase (ACC) expression by upregulating the AMPK activity, suppressing the saturated fatty acid accumulation and increasing polyunsaturated fatty acid synthesis in the liver. Compared to the controls, H007 treatment inhibited the expression of monocyte chemotactic protein (MCP-1) in fatty acid-treated HepG2 cells; suppressed leukocyte adherence and rolling on the liver microvasculature; and suppressed hepatic macrophage infiltration. H007 also suppressed the expression of nuclear factor-kB (NF-kB) p65 in fatty acid- and lipopolysaccharide-treated HepG2 cells compared to that in the controls by activating AMPK. These data suggested that H007 had a beneficial effect by improving the lipid composition in the liver and inhibiting inflammatory cell trafficking in the development of nonalcoholic fatty liver disease. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Keywords: AMPK activator Hepatic lipid metabolism Leukocyte trafficking Hepatic steatosis Inflammation

1. Introduction Nonalcoholic fatty liver disease (NAFLD) is a common metabolic disorder characterized by the accumulation of abnormal amounts of fat within the liver. Up to 40% of patients with NAFLD may develop nonalcoholic steatohepatitis (NASH), which is caused by liver cell damage and inflammation.1 The accumulation of hepatic fat causes insulin resistance and oxidative stress, leading first to hepatic steatosis and second to hepatic damage (“the second hit”).2 Steatosis-induced hepatic inflammation is a critical factor in the recruitment of leukocytes during the liver inflammatory response,3e5 which can progress to scarring and ultimately to cirrhosis.

* Corresponding author. E-mail address: [email protected] (H. Zhu). Peer review under responsibility of Japanese Pharmacological Society.

AMP-activated protein kinase (AMPK) is an enzyme that is essential for maintaining energy balance, which plays a major role in regulating hepatic lipid metabolism.6,7 The lipid-lowering statin drugs have shown an AMPK activation effect in the liver, which contributes to the improvement of lipid metabolism. Additionally, AMPK has a direct effect on suppressing hepatocyte and macrophage inflammatory responses.8,9 Some studies have shown that AMPK activation can prevent leukocyte rolling and adhesion and protect microvascular function.10 These studies suggest that AMPK activation not only improves liver fat metabolism disorders but also regulates the inflammatory response to reduce leukocyte adhesion, which may result in decreased inflammatory cell recruitment to steatotic livers. The molecule 20 ,30 ,50 -tri-O-acetyl-N6-(3-hydroxyphenyl) adenosine, a cordycepin derivative (IMM-H007, H007), is a new adenosine analog and anti-hyperlipidemic drug candidate that was developed in our laboratory. Our previous study showed the protective effect of H007 on liver steatosis in animal models. However,

https://doi.org/10.1016/j.jphs.2019.05.008 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. 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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it is still unclear how H007 affects immune cell behavior in fatty livers. Here, we hypothesized that H007 could improve liver steatosis and NASH by activating AMPK in high-fat diet (HFD)-fed hamsters. 2. Materials and methods 2.1. Animals and treatments LVG Golden Syrian Hamsters (100 g ± 10 g, male, outbred closed colony, strain code: 501) were purchased from Vital River Laboratory (Beijing, China). All animal treatment procedures described in this study were approved by the Animal Care and Use Committee at the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (PUMC, Beijing, China). Animals were housed in a light- and temperature-controlled environment. Hamsters were fed standard chow diet or HFD (containing 20% lard and 0.2% cholesterol) that were purchased from Beijing HFK Bioscience Co., Inc. (Beijing, China). IMM-H007 (Supplemental Fig. 1, chemical structure) was provided by Prof. Song Wu at the Institute of Material Medica, PUMC (Beijing, China, 99.86% purity by high performance liquid chromatography). For pharmacodynamics experiments, three doses (50 mg/kg, 100 mg/kg, and 200 mg/kg) of H007 were used to observe the lipid-lowering effect of the molecule in hamsters. Hamsters were randomly divided into 6 groups, with 15 hamsters in each group: the normal chow diet (NCD) plus vehicle (0.25% CMC) daily group; HFD plus vehicle daily group; HFD plus 3 mg/kg simvastatin (purchased from Merck & Co., Inc. Hangzhou, China) daily (HFD þ Sim) group; and HFD plus H007 daily (HFD þ H007) groups, which included three groups (50 mg/kg, 100 mg/kg, and 200 mg/kg). All drugs and vehicle were given by oral gavage for 12 weeks. A doseeresponse study showed that simvastatin and H007 could decrease the total cholesterol (TC) and total triglyceride (TG) levels in serum (Supplemental Fig. 2) compared to those in the controls. A dose of 200 mg/kg of H007 was the most effective dose to reduce serum lipid levels. H007 at different doses did not affect the body weight change (Supplemental Fig. 3) or food intake (Supplemental Fig. 4) compared to those in the HFD group. In this study, hamsters were randomly divided into four groups, with 15 hamsters in each group: the NCD plus vehicle (0.25% CMC) daily group; HFD plus vehicle daily; HFD plus 3 mg/kg simvastatin daily (HFD þ Sim) group; and HFD plus 200 mg/kg H007 daily (HFD þ H007) group. All drugs and vehicle were given by oral gavage for 12 weeks. 2.2. Serum lipid content assay Blood samples were collected from the retro-orbital vein at the end of the 12 week drug treatments. Serum was subjected to a standard enzymatic assay to determine TC, TG and high-density lipoprotein cholesterol (HDL-C) levels. Commercially available kits were purchased from Bio Sino Bio-technology and Science Inc. (Beijing, China). 2.3. Hepatic lipid extraction and analysis Livers were homogenized in PBS, and the protein concentration was determined according to the instructions of a BCA kit. A total of 300 mL of homogenate was extracted with 5 mL of chloroform/ methanol (2:1, v/v) and 0.5 mL of 0.1% sulfuric acid.11 Hepatic lipid contents were determined using commercially available kits which were purchased from Bio Sino Bio-technology and Science Inc. (Beijing, China).

2.4. Determination of free fatty acids by high performance liquid chromatography (HPLC) Free fatty acids (FFAs) were extracted from 100 mg of hamster liver and analyzed with an Agilent 1260. The mobile phase was acetonitrile/water (81:19) at l.0 mL/min with UV detection at 242 nm, and the column temperature was 55  C. The analytic column was an Agilent Eclipse Plus C18 (4.6  100 mm, 3.5 mm). Fatty acid peaks were identified via retention times in relation to the internal standard heptadecanoic acid. Fatty acid concentrations were determined by using the peak areas of the fatty acid standards reference.12 2.5. Histological and immunohistochemistry staining Liver samples were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and slides were stained with hematoxylin and eosin (H&E) or Sirius red. Immunohistochemistry was performed as described previously.13 The slides were incubated with a monocyte chemotactic protein 1(MCP-1) primary antibody (1:200, Cell Signaling Technology, Danvers, MA) and counterstained with hematoxylin. The colorimetric reactions were visualized by using a DAB kit (ZSGB-Bio technology and Science Inc. Beijing, China). Images were acquired on an Olympus CZ-41 microscope and analyzed by ImageJ (NIH). For F4/80 fluorescence immunohistochemistry, anti-F4/80 antibody (Abcam, Cambridge, MA) was used for detection. 2.6. Red blood cell (RBC) labeling with FITC The labeling procedure was modified from a previous method.14 Whole blood was drawn from a donor hamster and labeled with FITC buffer for 2 h. After washing the mixture twice with PBS, the RBCs were resuspended in a nine-fold volume of PBS for i.v. injection. 2.7. In situ intravital microscopy Intravital microscopy was performed as previously described with slight modifications.15 Hamsters were anesthetized with sodium pentobarbital (100 mg/kg, i.p.). The left lobe of the liver was gently excised and adhered to the outer rim of a chamber to stabilize the visible area. Labeled RBCs were injected for blood flow visualization. The leukocytes were labeled with 0.1 mL of Rhodamine 6G (purchased from Sigma, 0.05 mg/mL, i.v.) injection. Blood flow through the terminal portal venules (TPVs), sinusoids and terminal hepatic venules (THVs) were detected by an Olympus BXRFA reflected fluorescence microscope using a 20 objective. The number of rolling and adherent leukocytes was calculated in 1 min. The leukocytes moving at a significantly slower rate than the RBC velocity were considered rollers.16 The count was repeated three times for each vessel with the arbitrary line repositioned each time. Adherent leukocytes were defined as leukocytes that were firmly attached to the endothelium and did not change positions in 1 min. In all experiments, five fields were captured for each hamster by a Toupcam industrial digital camera. The vascular surface area for each vessel was calculated by using ImageJ software (NIH). 2.8. Cell culture and treatment HepG2 cells were purchased from the Cell Resource Center (IBMS, CAMS/PUMC, Beijing, China). We used the metabolite of H007, named H007-M1, which has been verified as the major metabolite in vivo after the oral administration of H007.17 Either simvastatin or H007-M1 (100 mM) was then added to the medium

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in the presence of the FFA mixture (palmitic acid:oleic acid ¼ 2:1, the total fatty acid concentration was 250 mM) for 12 h. A769662 (100 mM)18 was used as an AMPK activation positive control. Compound C (25 mM)19 was used as a pretreatment for AMPK activity suppression. To examine the NF-kB activation, LPS (10 mg/mL) and FFA mixtures were coincubated with these drugs for 12 h.

polyunsaturated fatty acids. In contrast, compared to the HFD group, the H007 group showed significantly lower levels of palmitic acid, oleic acid and stearic acid but higher levels of linoleic acid and arachidonic acid. Compared to the HFD group, simvastatin treatment decreased the oleic acid level in the liver of hamsters, but no difference was observed in the other lipid contents.

2.9. Western blot analysis

3.3. H007 treatment reduced leukocyte adherence on the hepatic microvasculature

The expression and phosphorylation of each protein were analyzed by Western blotting.20 Immunostaining to detect each protein was performed using a corresponding primary antibody including rabbit anti-NF-kB p65 (Abcam), rabbit anti-phosphorAMPK, anti-AMPK, anti-p-sterol-regulatory element binding protein (SREBP)-1c, anti-p-acetyl CoA carboxylase (ACC) and anti-bactin antibody (Cell Signaling Technology). Three independent experiments were performed for each condition, and the intensity of the immune-reactive bands was analyzed with ImageJ. 2.10. Statistical analyses All the data were analyzed by one-way analysis of variance (ANOVA), and the Bonferroni test was used for post hoc comparisons. The differences between each group were considered statistically significant at a p value < 0.05. The results are presented as the mean ± S.E.M. in the figures and as the mean ± S.D. in Table 1.

We used an in vivo cell tracking system to observe the circulating leukocyte movement in the liver microcirculation. FITClabeled RBCs (Fig. 3A, white arrows) were used to visualize the hepatic microcirculation, and leukocytes were labeled with Rhodamine 6G (Fig. 3A, yellow arrows) to examine leukocyte trafficking. No significant difference in FITC-labeled the RBC speed was observed among the groups (Fig. 3B). In the livers of HFD-fed hamsters, the number of firmly adhesive leukocytes that were visualized by Rhodamine 6G-positivity increased by 4-fold compared to that in hamsters on NCD. However, the number of leukocytes arrested to microvessels in the simvastatin and H007 groups decreased by 2-fold compared with that in the HFD group (Fig. 3C). Similarly, the number of rolling cells on the vessel wall was reduced in the H007-treated group compared with the HFDfed group (Fig. 3D). 3.4. H007 treatment reduced hepatic macrophage accumulation and MCP-1 expression

3. Results 3.1. H007 treatment improved dyslipidemia and liver steatosis Hamsters that were on HFD for 12 weeks showed significant increases in serum lipid levels compared to those of hamsters on normal chow diet (NCD) (Table 1). Simvastatin or H007 treatment significantly lowered the serum lipid levels and increased the serum HDL levels compared to those of HFD-fed hamsters. Based on the pathological observations in Fig. 1A, the liver from hamsters on HFD had more lipid vacuoles than those in the control group, while the treatment with simvastatin and H007 markedly reduced the lipid vacuoles and ameliorated the liver steatosis compared to those in the HFD group. Correspondingly, hepatic TC and TG contents from hamsters in the simvastatin and H007 groups were lower than those in the HFD-fed hamsters (Fig. 1BeC). Sirius red staining showed that HFD-fed hamsters had increased levels of positive collagenous fiber in the liver compared to those in hamsters on NCD (Fig. 1D). However, simvastatin- and H007-treated hamsters showed less collagenous fiber in the liver than that in HFD-fed hamsters. 3.2. H007 treatment changed the FFA profile in steatotic livers We examined six FFA components in the hamster liver, which are shown in Fig. 2. Compared to the NCD group, the HFD group had significantly increased palmitic acid, stearic acid and oleic acid contents in liver, but no difference was found in the

Fig. 4 shows that the number of macrophages that were positive for F4/80 increased in the liver of HFD-fed hamsters compared to that in NCD controls. H007 treatment reduced the number of hepatic macrophages compared to that in HFD-fed animals, corresponding to a reduction in leukocyte infiltration in the liver. MCP-1, a major chemokine secreted by macrophages that is critical for macrophage infiltration, is increased during liver inflammation. The results showed that the MCP-1-positive area was higher in the liver sections from hamsters in the HFD group than in the NCD group. After 12 weeks of treatment, H007 significantly reduced MCP-1 expression in the livers compared to that in HFD-fed hamsters (Fig. 4D). 3.5. H007 inhibited SREBP and ACC activity by activating AMPK H007 was previously reported to activate AMPK.20,21 Therefore, we examined sterol regulatory element-binding protein (SREBP) and acetyl-CoA carboxylase (ACC) phosphorylation in fatty acidladen HepG2 cells by Western blotting. AMPK directly phosphorylates SREBP-1c and ACC, leading to decreased activity. After incubation with FFAs for 12 h, HepG2 cells showed significantly lower levels of p-SREBP and p-ACC than those in the control group (Fig. 5). As expected, the increase of p-SREBP and p-ACC expression was correlated with the upregulation of AMPK phosphorylation by A769662, simvastatin and H007-M1 treatment. Furthermore, the increase in p-SREBP and p-ACC by simvastatin and H007-M1 could

Table 1 Serum lipid levels in NCD and HFD hamsters. Group

Dosage (mg/kg)

n

TG (mmol/L)

NCD HFD HFD þ Sim HFD þ H007

e e 3 200

10 11 11 11

1.99 12.9 8.29 5.21

± ± ± ±

0.55 5.67# 4.81 1.73*

TC (mmol/L) 3.65 13.2 6.20 7.33

± ± ± ±

0.64 3.09# 2.33* 3.18*

HDL (mmol/L) 1.58 1.87 2.05 2.16

± ± ± ±

0.22 0.79 0.62* 0.45*

N ¼ 10e11 hamsters/group. Data are presented as the mean ± S.D. #p < 0.05 compared with the NCD group. **p < 0.01 and *p < 0.05 compared with the HFD group.

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Fig. 1. H007 improved the liver pathological injury and reduced hepatic lipid content. (A) H&E staining of livers from hamsters after 12 weeks of treatment with a normal chow diet, high-fat diet (HFD), high-fat diet with simvastatin (HFD þ Sim) and high-fat diet with H007 (HFD þ H007). (BeC) Hepatic TG and TC levels of 100 mg liver tissue from hamsters in different treatment groups. (D) Sirius red staining for fibrosis detection in hamsters in different treatment groups. Scale bar, 100 mm. n ¼ 5 hamsters/group, **p < 0.01 and *p < 0.05 compared with the HFD group.

be abolished by an AMPK inhibitor (Compound C, CC), indicating that the AMPK pathway plays an important role in the H007-M1 regulation of lipid metabolism-related targets. 3.6. H007 suppressed NF-kB expression by activating AMPK We treated HepG2 cells with FFAs and lipopolysaccharides (LPS) to induce NF-kB activation in vitro and examined the effect of H007 on NF-kB expression. P65 is one of the five components that form NF-kB. P65 expression showed an increasing trend after FFA þ LSP incubation compared to that in the controls, indicating that HepG2 cells experienced pro-inflammatory stress. Simvastatin and H007M1 treatments reduced p65 expression in FFA þ LPS-treated HepG2 cells compared to the control group (Fig. 6A). As expected, the decrease of p65 expression was correlated with the upregulation of AMPK phosphorylation by simvastatin and H007-M1 treatment (Fig. 6B). Compound C abolished the effects of simvastatin and H007-M1 on NF-kB p65 inhibition. Proposed mechanism of action of H007 in improving the lipid composition and inhibiting inflammation in the liver is shown in Fig. 7. 4. Discussion In the HFD hamster model, H007 showed significant benefits in the reduction of hepatic lipid levels. AMPK activation inhibits ATP consumption (gluconeogenesis and fatty acid release in the liver) and supports ATP production (fatty acid oxidation in the liver),22 which may contribute to the lipid-lowering effect of H007 in fatty livers. AMPK was also reported as an upstream kinase that phosphorylates 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase,23 which results in cholesterol reduction in the liver. The results from HepG2 cell culture indicated that the AMPK

phosphorylation level was significantly increased after H007 treatment in HepG2 cells with FFA incubation compared to that in the controls. The function of ACC is to provide the malonyl-CoA substrate for the biosynthesis of FAs. In FFA-treated HepG2 cells, H007 had a significant effect on the activation of ACC via AMPK phosphorylation, and this effect could be diminished by Compound C compared to that in the controls. The TG-derived free fatty acid profile showed that the HFD group had higher saturated fatty acid (SFA) levels, especially palmitic acid, which could elevate the NF-kB transcriptional activity by binding to Toll-like receptor-2 (TLR-2) and 4, increasing the expression of pro-inflammatory cytokines such as interleukin-6 (IL6) and tumor necrosis factor (TNF)-a.24,25 In addition, livers in the HFD group had lower levels of unsaturated fatty acids that play roles in anti-inflammatory processes compared to those in the NCD group. Studies in different cell lines show that polyunsaturated fatty acids could downregulate NF-kB activity via the activation of peroxisome proliferator-activated receptor (PPAR) and inhibit the production of pro-inflammatory cytokines, which would help reduce inflammation.26,27 We also observed higher MCP-1 expression and more macrophage infiltration in the liver in the HFD group compared to the NCD group, indicating an increased inflammatory state in livers from hamster on HFD than in the NCD group. Based on the abovementioned evidence, we postulate that inflammation is induced in fatty livers. H007 increased the polyunsaturated fatty acid content and decreased the saturated fatty acid levels in the liver in hamsters on HFD. Dietary saturated fat diets can increase SREBP-1c expression and its downstream targets in mice, which may induce hepatic steatosis.28 The in vitro results showed that compared to the controls, p-SREBP-1c expression was inhibited by fatty acid treatment, but A769662 and H007-M1 could increase the p-SREBP-1c level. A

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Fig. 2. The effects of H007 on the hepatic FFA composition of HFD-fed hamsters. After 12 weeks of treatment, FFAs from the livers of hamsters in different groups were isolated and separated by fast HPLC. Palmitic acid [16:0], stearic acid [18:0], oleic acid [18:1] and linoleic acid [18:2], arachidonic acid [20:4] and linolenic acid [18:3] concentrations were determined and normalized to a standard reference. n ¼ 5 hamsters/group, *p < 0.05 compared with the HFD group.

surplus of FFAs in non-adipose cells may cause the activation of deleterious pathways, leading to cell dysfunction (lipotoxicity) and apoptotic cell death (lipoapoptosis). These processes are considered to be the ‘second hit’ in oxidative injury and subsequent necroinflammation.29,30 Based on the abovementioned evidence, we postulate that H007 may inhibit the development of inflammation in the fatty liver by reducing lipid accumulation through AMPK activation. Lipid accumulation in the liver drives macrophages to exhibit a pro-inflammatory phenotype, which may produce high levels of pro-inflammatory cytokines/chemokines that recruit lymphocytes into the liver.31 Our previous studies demonstrated that H007 could activate AMPK and inhibit NF-kB transcriptional activity, which induced the downregulation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and reduced J774A.1 macrophage lipid uptake.32 We observed that after H007 treatment, the number

of macrophages and the expression of MCP-1 decreased in the hamster liver compared to that in the controls. These results suggested that H007 and simvastatin might decrease the recruitment and infiltration of circulating inflammatory cells and improve hepatitis. The direct fluorescence imaging of the liver microcirculation showed remarkable changes, including in the decreased area of sinusoids and the dilated central venular diameter in HFD hamsters compared to the NCD group. These phenotypes were consistent with those in a chronic CCl4-exposure model of cirrhosis, which indicated sinusoid dysfunction.33 Ineffective sinusoidal perfusion could cause an increasing number of leukocytes to adhere to endothelial cells.34 The present model in HFD-fed hamsters also showed a significantly increased number of labeled leukocytes in the sinusoid branches with similar RBC velocities among the groups. The H007 and simvastatin treatments reduced the number

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Fig. 3. Leukocyte recruitment to the liver microcirculation in HFD-fed hamsters. RBCs and leukocytes were labeled with fluorescent dye and observed by intravital microscopy. (A) Images of the representative areas of the liver surface sinusoids with FITC-labeled RBCs and Rhodamine 6G-labeled leukocytes. White arrows point to RBCs labeled with FITC, and yellow arrows point to leukocytes labeled with Rhodamine 6G. (B) Mean velocities of RBCs in 5 selected fields on the liver surface. (C) The number of adherent leukocytes without detectable movement. (D) The number of rolling leukocytes with significantly lower velocities compared to those of RBCs. Magnification, 200. Five different fields were examined per animal. n ¼ 5 hamsters/group. *p < 0.05 compared with the HFD group.

Fig. 4. H007 treatment reduced the F4/80-positive cell number and inhibited MCP-1 expression. HFD hamsters were treated with Sim or H007 for 12 weeks. The liver was harvested and fixed. After antibody retrieval, the sections were stained with F4/80 or MCP-1 antibodies. Fluorescent or HRP-conjugated secondary antibodies were used for detection. (A) Representative images of F4/80-positive cells in hamster livers. (B) Immunohistochemical staining of MCP-1 in the liver. Magnification, 200. n ¼ 5, *p < 0.05 compared with the HFD group.

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Fig. 5. AMPK and related downstream regulators were activated by H007 treatment. Total protein was extracted from HepG2 cells after 12 h of incubation with FFAs and different drugs. The phosphorylation levels of AMPK, ACC and SREBP-1c were detected with specific antibodies. n ¼ 3, *p < 0.05 versus the control group.

Fig. 6. AMPK activation and NF-kB inhibition by H007 treatment. HepG2 cells were lysed after 12 h of incubation with FFA þ LPS and different drugs. The total proteins were resolved on SDS-polyacrylamide gels and subjected to Western blot assays. The phosphorylation levels of AMPK and NF-kB were detected with specific antibodies. n ¼ 3, *p < 0.05 versus the control group.

of adhering leukocytes and increased the speed of rolling leukocytes in the sinusoidal venule, indicating an improvement in leukocyte behavior. NF-kB is closely related to lipid accumulation and inflammation. The selective inhibition of hepatic NF-kB signaling efficiently prevents liver steatosis and inflammation when rodents are fed a

HFD.35e37 Increased NF-kB transcriptional activity induces elevated levels of the pro-inflammatory cytokine IL-6, which results in increased hepatocyte expression of SREBP-1c via the upregulation of suppressor of cytokine signaling (SOCS)-3.38 Hepatic expression of monocyte recruitment molecules is increased in diet-induced steatohepatitis, which is attributed to the upregulation of NF-kB,

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Fig. 7. Mechanism of H007 in improving the lipid composition and inhibiting inflammation in the liver. H007 improves the inflammation and accumulation of fatty lipids in the liver by activating liver AMPK. AMPK activation upregulates the expression of p-ACC and p-SREBP-1c, which are associated with lipid accumulation. In addition, AMPK activation also downregulates the expression of NF-kB, which reduces the F4/80-positive cell number and directly suppresses macrophage inflammatory responses.

and in turn, the activation and gathering of a considerable quantity of inflammatory cells.39 In the present study, we observed that H007 significantly inhibited NF-kB phosphorylation in FFA þ LPStreated hepatic cells compared to the controls. This result suggested that a reduction in FA levels could influence the liver NF-kB activity. Therefore, NF-kB inhibition could contribute to the downregulation of the expression of inflammatory factors and the accumulation of lipids in the liver. Our results from both HFD-fed hamsters and HepG2 cells indicated that H007 has a potential therapeutic application for the inflammation associated with hepatic steatosis by activating AMPK. Consequently, these effects may contribute to the reduced expression of inflammatory cytokines and the decreased accumulation of inflammatory cells in an experimental NAFLD model. Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. Funding This study was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2016-I2M-1-009), the Drug Innovation Major Project (2018ZX09711001-003-011), Fundamental Research Funds of Peking Union Medical College (2015CX06), and National Natural Science Foundation of China (81302827, 91539126, and 81703511). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jphs.2019.05.008.

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