Twenty-four-week-old male C57BL6 mice, weighing approximately 12-16 g, were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Science. Mice were housed in a clean grade barrier systems laboratory in the Medical Laboratory Animal Center of Shanghai Jiao Tong University. Animals were randomized into four groups: the control group (n = 5), high-fat (HF) diet group (n = 5), HF + LPS group (n = 5), and HF + LPS + IMD (IKK2 inhibitor) group (n = 5). The mice in the control group were given a normal diet (ND) and the HF group animals were fed with an HF diet for 10 wk. The ND chow was supplied by the Animal Center of the Medical College of Subsidiary Basic Medical of Shanghai Jiao Tong University. The HF diet (50% fat, pork fat 18%, yolk 12%, sugar 8% and basal diet 62%) was supplied by SLAC Precision Equipment Inc. Mice in the intervention group were intraperitoneally injected with 30 mg/kg IKK2 IMD 0354 (Tocris Bioscience, Bristol, United Kingdom) for 14 d, and at the end of 12 wk this was combined with intraperitoneal injection of 10 mg/kg LPS (Sigma-Aldrich, St Louis, MO, United States). Mice were then sacrificed after fasting for 12 h. Subsequently, 1 mL eyeball blood was obtained and all mice were killed by cervical dislocation. The liver tissue was fast fixed and lightly washed in ice-cold phosphate buffered solution (PBS). Then, part of the liver was placed into 10% formalin fixation solution, while the other part of the liver was quickly stored at -70 °C for cryopreservation. The blood was sent to the laboratory of Renji Hospital for liver enzyme assays. Some liver tissue was embedded in paraffin for 24 h and was then observed by hematoxylin and eosin (HE) staining, Masson staining and immunohistochemistry (IHC). Other liver tissue was saved under an ultra low temperature for Western blotting and polymerase chain reaction (PCR) procedures.

Serum was collected to analyze alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using automatic biochemical instrumentation at Renji Hospital Lab, Shanghai, China.

For HE and Masson staining, 4-μm liver tissue sections were cut from the same position and embedded in paraffin after being stabilized in 10% formalin. Changes in liver tissues were observed under a light microscope.

Evaluation of inflammation activity: scores of inflammation activity were in accordance with chronic liver disease activity[20], and were divided into four parts; i.e., portal area inflammation (P), lobular inflammation (L), patch necrosis (PN) and bridging necrosis (BN, including lobular necrosis). Every item was recorded as 1, 2, 3 or 4 based on degrees of pathological changes. The scores counting formula was: P + L + 2 × (PN + BN).

Fatty hepatic fibrosis: fibrosis scores were divided into four stages according to the degree of fibrosis in three areas of the liver; i.e., the liver acinus, the portal vein, and bridging fibrosis, as well as the presence or absence of liver cirrhosis. S1 indicated perisinusoidal space fibrosis of three areas of the local or extensive liver acinus; S2 indicated the above pathological changes with local or extensive periportal fibrosis; S3 indicated S2 pathological changes with local or extensive bridging fibrosis; and S4 indicated fatty liver cirrhosis, forming fibrous septa that divided lobuli hepatic and central veins to the portal area and formed false lobules.

Liver tissue sections (4 μm) were prepared for IL-6 immunohistochemical study. The glass was treated by polylysine to promote cell attachment. Microwave antigen repairing was carried out with 0.01 mol/L citrate buffer solution (pH 6.0). After blocking with rabbit serum, the sections were incubated overnight at 4 °C with monoclonal primary antibody against mouse IL-6 (PPMX, Tokyo, Japan). On the next day, liver sections were taken out and washed with PBS three times and were incubated with the second antibody for 1 h at room temperature. Coloration with freshly prepared diaminobenzidine (DAB) was performed, and then the tissues were counterstained with hematoxylin, dewatered, and then mounted with neutral gum. The second antibody in the ElivisionTM plus Polyer HRP (Mouse/Rabbit) IHC Kit and DAB developer were supplied by Manxin Bio Co^®, Fuzhou, China. PBS taking the place of the primary antibody was considered as the negative control. We chose 10 views of each section under light microscopy to obtain the average positive absorbance using ImageProplus2.0.

Liver (1 g) was placed in PBS with 0.1 mmol/L phenylmethyl sulfonylfluoride (PMSF, Sigma) and was then manually homogenized, centrifuged at 100 000 r/min for 15 min at 4 °C, and the supernatant was removed. Double antibody enzyme linked immunosorbent assay (ELISA) (ELISA kit TNF-α, BD Bioscience, Franklin Lakes, NJ, United States) was used for detection, and the procedure was strictly based on the guidelines provided by the manufacturer.

Western blotting analysis of NF-κB p65, TGF-β1 andα-SMA

Liver tissue was saved in a refrigerator at -80 °C after homogenization, and the tissue protein extract solution was prepared by centrifugation. Based on the operating instructions of the bicinchoninic acid protein quantitation kit (Sunbio, Beijing, China), the concentration of protein was detected. After denaturation, each tissue protein was sampled at 50 μg, and reducibility was performed with sodium dodecyl sulfate polyacrylamide gel electrophoresis cataphoresis in an 8% polyacrylamide gel. Sampling after denaturation was performed and electrophoresis was started. The electrophoresis voltage of the condensed glue and separation gel was 80 V and 120 V, respectively. The electrophoresis terminated after bromophenol blue electrophoresis moved to the bottom of the glue, and a damp-dry transmembrane (polyvinylidene fluoride membrane) was applied with a constant 50 mA current for 90 min. The membrane was sealed at room temperature with 5% defatted milk powder prepared with Tris-buffered saline Tween-20 (TBST) and primary antibodies (NF-κB p65, TGF-β1, α-SMA and β-actin, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, United States) and was incubated overnight in a swing bed at 4 °C. After the film was washed with TBST buffer solution in the swing bed, the second antibody 1:3000 (rabbit polyclonal antibody, Manxin Bio, Fuzhou, China) was incubated at room temperature for 1 h. After being repeatedly washed in TBST buffer solution, DAB staining was performed. After proper staining, the reaction was terminated by water. In a dark room, a nitrocellulose filter was put into a brightening agent with sufficient contact, and was then exposed to light with an X-ray device. The image was developed and fixed. Simultaneous determination of the expression level of β-actin in the same filter was carried out as an internal control. Separate analyses were performed for each sample and the experiment was repeated three times. We obtained the integrated density value by Microsoft BandScan, and the ratio of the target bands to β-actin substantiated the presence of the proteins NF-κB p65, TGF-β1 and α-SMA.

RNA extraction and analysis of mRNA expression of typesIand III collagen,α-SMA and TGF-β1

Total RNA was isolated from snap-frozen liver tissue using Trizol reagent (Invitrogen, Carlsbad, CA, United States) and the ratio between the absorbance values at 260 nm and 280 nm gave an estimate of RNA purity. Real-time (RT)-PCR was performed using a one-step RT-PCR kit from the Shanghai Daweike Biotechnology Company. Two micrograms of the total RNA was chosen and reverse transcription was performed. Its reaction product was placed into a 50-μL PCR reaction system. α-SMA, TGF-β1, typesIand III collagen, and the specific primer of the internal reference β-actin was used in PCR amplification, and agarose electrophoresis was performed. Electrophoresis results were scanned with a BioSens GelImaging System. For the PCR primer sequences and fragment lengths (Table 1). The real-time survey meter (7500 Sequence Detection System) was obtained from ABI, United States. The PCR conditions were: predegeneration for 2 min at 50 °C, denaturation for 20 s at 95 °C, annealing for 45 s at 60 °C, and extension for 30 s at 72 °C, with a total of 40 cycles; an internal reference of β-actin was used with predegeneration for 3 min at 94 °C, denaturation for 20 s at 95 °C, annealing for 20 s at 60 °C, extension for 30 s at 72 °C, with a total of 35 cycles, and extension again for 10 min after the cycles, and then termination at 4 °C.

Data were expressed as mean ± SD. Statistical analysis was performed with a one-way analysis of variance using SPSS17.0 software, followed by Scheffe’s test, and comparisons between groups was performed using the Mann-Whitney test. P < 0.05 was considered to be statistically significant.

The levels of ALT and AST for each group are shown in Figure 1. In Figure 1A, the ALT levels of the HF and HF + LPS groups were significantly increased compared to the control group (P < 0.05). After treatment with the IKK2 inhibitor (IMD0354), the level of serum ALT in the mice was significantly decreased compared to the HF group (P < 0.01), as well as in the HF + LPS group (P < 0.05), but was still higher than that of the control group (P < 0.05). Figure 1B shows that the change in serum AST was not as significant as that of ALT. The level of serum AST in the HF + LPS group was significantly increased compared to the control group (P < 0.01). The level of serum AST when treated with IMD0354 was significantly decreased compared to that of the HF + LPS group (P < 0.05).

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Figure 1 IKK2 inhibitor prevented HF + LPS-induced liver injury, as determined by serum ALT and AST levels. The normal values for ALT and AST were 45 U/L and 160 U/L. Serum ALT (A) and AST (B) were measured in different groups (control group, HF group, LPS-induced HF group and IMD-treated group), data are expressed as mean ± SD. A: ^aP < 0.05 vs control group, ^cP < 0.05 vs the HF and LPS + HF groups; B: ^bP < 0.01 vs the control group, ^eP < 0.05 vs the LPS + HF group. HF: High-fat; LPS: Lipopolysaccharide; IMD: IKK2 inhibitor; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase.

HE staining results showed that pathological changes in the mice were in line with the diagnostic gold standard of chronic NASH (Figure 2A). At the same time, typical hepatic fibrosis was observed with Masson staining (Figure 2B). In the control group, the structure of the hepatic lobules was clear without inflammatory cell infiltration in the portal area and without fibrotic tissue hyperplasia. The liver sections of the HF and HF + LPS groups showed that the normal structure of the hepatic lobules was lost, the structure of the blood vessels in the liver was disordered with severe liver cell degeneration, patch necrosis and bridging necrosis, and there were many inflammatory cell infiltrates in the portal area. There was also light to moderate hyperplasia of the broglia fibrils and fibrous septa were formed occasionally. Inflammation and fibrosis scores showed significant differences compared with the control group (P < 0.05). In the IMD-treated group, the structure of the hepatic lobules was normal, liver cell degeneration was significantly decreased, and liver cell necrosis and inflammatory cell infiltration were significantly improved. Fibrillar collagen sediment still existed, which was significantly reduced compared with the controls, and its inflammation score was also significantly decreased (P < 0.05), but was still higher than that of the control group (P < 0.05, Table 1). The results of the fibrosis scores were analyzed by Mann-Whitney statistical methods, and the results showed that there was a significant difference between the HF + LPS + IMD and HF + LPS groups (Z = -2.35, P = 0.018, P < 0.05, Table 1). There were no differences among the other groups.

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Figure 2 Hematoxylin and eosin stain and Masson staining in sections of (a) control group; (b) HF group; (c) HF + LPS group; and (d) HF + LPS + IMD group. A: Hematoxylin and eosin stain. Macrovesicular steatosis, lobular inflammation and balloon degeneration of hepatocytes were observed in liver sections of HF-treated mice and HF + LPS + treated mice with a significantly large amount of inflammatory cell infiltration surrounding the centrilobular veins of the liver. Significant amelioration was observed in the group treated with IMD (d); B: Masson staining. A thin lining of collagen was observed in the HF group, HF + LPS group and HF + LPS + IMD group. With LPS treatment, there was an increase in the amount of collagen accumulated along the central vein with the presence of collagen in the pericellular area. Treatment with IMD reduced LPS-induced collagen accumulation. HF: High-fat; LPS: Lipopolysaccharide; IMD: IKK2 inhibitor.

Previous studies have demonstrated that many cell factors, such as TNF-α and IL-6, play important roles in the NF-κB dependent signaling pathway[21]. Some evidence has indicated that TNF-α, as well as IL-6, participated in the formation of hepatic fibrosis and had a positive correlation with the level of serum hyaluronic acid, laminin type IV, for example, suggesting that TNF-α, as well as IL-6, not only mediated inflammatory reactions but also participated in the formation of hepatic fibrosis during the promotion of extracellular matrix (ECM) synthesis. In this study, the average absorbance values (A) of liver cell positive immunity of mice in each group were analyzed by Image-pro plus 6.0 and statistical analysis was performed, showing that there was a small amount of IL-6 expression in the control and HF groups, and the expression of IL-6 was significantly increased in the HF group after being activated by LPS (P = 0.013, P < 0.05). IL-6 expression was mainly concentrated in the liver cell cytoplasm around the central veins and portal area, appearing as brown and grainy, and was also expressed in the sinus hepaticus and parts of monocytes. In the HF + LPS group treated with the intervention of IMD, the expression of IL-6 was significantly decreased (P = 0.012, P < 0.05). There was no significant difference when compared with the HF group (P = 0.70, P > 0.05), and the expression of IL-6 was higher than the control group (Figure 3).

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Figure 3 Interleukin-6 expression was assessed by immunohistochemistry. A: Positive staining was observed in hepatocytes in the control group (a), HF group (b), LPS-induced HF group (c) and IMD-treated group (d). B: The optical density (OD) of interleukin-6 (IL-6)-positive areas was measured with ImageProplu6.0 (^aP < 0.05). HF: High-fat; LPS: Lipopolysaccharide; IMD: IKK2 inhibitor.

IKK2 inhibitor (IMD) inhibited an LPS-induced increase in the pro-inflammatory cytokine levels of TNF-αin mice livers

Recent research has shown that the IKK2-NF-κB signal pathway participate in insulin resistance, and cell factors of TNF-α and IL-6 play important roles dependent on NF-κB signals[21], especially in the pathological process of hepatic fibrosis[17,22]. Therefore, TNF-α was the key pro inflammatory factor, which was likely to induce the formation and development of hepatic fibrosis. The protein levels of TNF-α in mouse livers were detected by ELISA. With the development of liver injury, an increased expression of TNF-α was shown in the liver[23]. The levels of TNF-α in mouse livers of the HF and HF + LPS groups were significantly increased compared to the control group (P < 0.05). After intervention with the IKK2 inhibitor, the level of TNF-α was significantly reduced compared to the non-intervention group (P < 0.05, Figure 4).

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Figure 4 The levels of nuclear factor-κB-dependent pro inflammatory cytokines and tumor necrosis factor-alpha were measured in livers obtained from the control group, HF group, HF + LPS group and HF + LPS + IMD group. ^aP < 0.05, compared with the control group. ^cP < 0.05, significant compared with both the HF and LPS + HF exposed groups. HF: High-fat; LPS: Lipopolysaccharide; IMD: IKK2 inhibitor; TNF-α: Tumor necrosis factor-alpha.

IKK2 inhibitor (IMD) decreased nuclear translocation of NF-κB p65 in mice livers in response to LPS and HF exposure

When combined with IκBα in the cytoplasm, NF-κB was inactive. IKK2, as the major subunit of promotion, activated NF-κB and its subunit with phosphorylation, while IMD could inhibit the activation of NF-κB p65 and its nuclear transcription[24]. Therefore, expressions of NF-κB p65 and P-IκBα in the livers in each group was detected to determine the inhibitory action of IMD. Western blotting showed that the expression of NF-κB p65 in the HF group was increased compared to that of the normal diet group. In the HF group, LPS promoted the expression of NF-κB p65, and P-IκBα increased simultaneously, while intervention by the IKK2 inhibitor reduced the pro-inflammatory role of LPS and significantly reduced the expression of NF-κB p65 and its subunit (Figure 5).

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Figure 5 IKK2 inhibitor decreased lipopolysaccharide-induced nuclear translocation of nuclear factor-κB p65 and P-IκBα in livers. Nuclear levels of the p65 subunit of nuclear factor-κB (NF-κB) were measured by Western blotting in different groups (A: Control; B: HF group; C: HF + LPS group; D: HF + LPS + IMD group). β-actin was used as a loading control. Administration of IMD at 30 mg/kg doses decreased the DNA binding activity of NF-κB, which was induced by HF and LPS in mice livers. HF: High-fat; LPS: Lipopolysaccharide; IMD: IKK2 inhibitor.

IKK2 inhibitor (IMD) decreased protein levels of TGF-β1 andα-SMA related to fibrosis in livers

TGF-β1 is an important inflammatory factor that stimu-lates accumulation in the ECM and tissue fibrosis. Our results showed that TGF-β1 expression in the liver was increased under the condition of the HF diet and stimulation of LPS. While α-SMA was a marker of HSC activation, its variation tendency was similar to TGF-β1. In the mouse liver model group, the expression of α-SMA was significantly increased compared to the control group. After application of the IKK2 inhibitor, the protein expression of TGF-β1 in livers decreased, and the expression of α-SMA was reduced accordingly. Correspondingly, activation of HSCs and the formation of collagen decreased, leading to effectively preventing hepatic fibrosis (Figure 6).

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Figure 6 Western blotting analysis of tumor growth factor-beta1, alpha-smooth muscle actin proteins were measured that were involved in IKK2-nuclear factor-κB pathways in the liver in different groups (A: Control; B: HF group; C: HF + LPS group; D: HF + LPS + IMD group). β-actin was used as a loading control. The levels of tumor growth factor-beta1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) measured in livers were increased in the HF and HF + LPS groups. IKK2 inhibitor significantly inhibited LPS and HF-induced expression of TGF-β1 and α-SMA in mouse livers. The ratio of TGF-β1 and α-SMA/β-actin in the liver was increased in other groups, compared with the control group, ^aP < 0.05. IKK2 inhibitor normalized TGF-β1 and α-SMA significantly compared with the HF or LPS + HF groups. ^cP < 0.05. HF: High-fat; LPS: Lipopolysaccharide; IMD: IKK2 inhibitor.

IKK2 inhibitor IMD inhibitedα-SMA, TGF-β1, typesI(αI) and III collagen and mRNA expression in LPS-stimulated mice

The formula described previously was used to measure mRNA relative expression (amount = 2^-Δct× 100%), and the relative expression levels of α-SMA, typeI(αI) collagen, type III collagen and TGF-β1 mRNA were obtained. RT-PCR was performed with the mouse primers shown in Table 2. The results showed that the expression of TGF-β1 mRNA in the HF group and the HF + LPS group was higher than the control group (P < 0.05), which was significantly decreased after intervention of IMD. In addition, the fibrosis indexes of α-SMA, typeIcollagen and type III collagen in the model group were also increased. In addition, the contents of typeIcollagen in the HF diet group and type III collagen in the HF + LPS group were significantly increased, independently, compared to the control group (P < 0.01, P < 0.05, respectively). After intervention with IMD, the levels of α-SMA, typeI(αI) collagen and III collagen were significantly decreased compared to the HF + LPS group (P < 0.05). However, there was no significant difference in α-SMA and type III collagen with intervention of IMD and the normal group (P > 0.05, Figure 7).

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Figure 7 The IKK2 inhibitor inhibited LPS and HF-induced increases in pro inflammatory cytokine levels in mouse livers. The level of tumor growth factor-beta1 (TGF-β1) was measured in the livers of mice in the control, HF, HF + LPS and HF + LPS + IMD groups. Also, expression of the fibrosis index, such as alpha-smooth muscle actin (α-SMA), typeIcollagen and type III collagen, were detected in the four groups by real-time polymerase chain reaction. The level of TGF-β1 measured in livers was increased in the HF and HF + LPS groups, compared with the control group, ^aP < 0.05. The mRNA content of typeIcollagen in the HF group and type III collagen in the HF + LPS group were significantly higher, ^aP < 0.05, compared with the control group. Intraperitoneally administered IKK2 inhibitor normalized the expression of TGF-β1, as well as the contents of α-SMA, typeIand type III collagen mRNA, compared with the HF or LPS + HF groups. ^aP < 0.05, compared with the control group. ^cP < 0.05, compared with both HF group and LPS + HF exposed group. HF: High-fat; LPS: Lipopolysaccharide; IMD: IKK2 inhibitor.

The nuclear factor-κB (NF-κB) signaling pathway improves insulin resistance and fat accumulation in the development of nonalcoholic fatty liver disease (NAFLD). Inhibition of NF-κB activation by an IKK2 inhibitor could effectively suppress the expression of many kinds of inflammatory factors, and even improve hepatic fibrosis. Therefore, the authors of this study investigated the potential therapeutic prospects of the IKK2-NF-κB signaling pathway in the reversion of fibrosis in NAFLD.

The study is believed to be the first to evaluate the role of IKK2-NF-κB signals in NAFLD. The potential effect of an IKK2 inhibitor is likely to block inflammation through inhibiting NF-κB activation. The IKK2 inhibitor may play an important role in the occurrence and development of NAFLD.

This study explored one of the possible mechanisms of inflammation, which could produce a potentially facilitative effect in the occurrence and development of NAFLD.

This study provides an experimental basis for future studies on the role of IKK2 in NAFLD. Control of the expression level of IKK2 in the liver may become a new possibility for therapy of NAFLD.

In the present study, the authors tested the effect of IKK2 in NAFLD in mice, and found a facilitative effect on the occurrence and development of NAFLD.

The paper should be accepted with some minor revisions. IκB kinase-beta inhibitor attenuates hepatic fibrosis in mice liver injury and its potential mechanisms.