Foxa2 attenuates steatosis and inhibits the NF-κB/IKK signaling pathway in nonalcoholic fatty liver disease

Animals

Twenty-four male C57BL/6 mice (four-week-old), obtained from GemPharmatech Co. Ltd. (Nanjing, China), were used to build the NAFLD mouse model. They were maintained in a controlled room with the temperature at 22–25 °C, a humidity of 55% ± 5%, and 12 h light/dark cycle. One week later, mice were randomly divided into four groups (n = 6): control, high-fat diet (HFD), HFD + overexpression negative control (oe-NC), and HFD + oe-Foxa2. Control mice were fed a normal chow and the rest were fed HFD (consisting of 60% fat, 20% protein, and 20% carbohydrate) (Qiao et al., 2018). After eight weeks, the HFD-fed mice received tail-vein injections of 1  × 10⁷ TU/mL of lentiviral-packed oe-Foxa2 (NM_001291065.1) or oe-NC (Vector ID: VB010000-9389rbj; VectorBuilder, Guangzhou, China) every two weeks, totaling two injections (Wang et al., 2017). Four weeks post-injection, the mice were anesthetized with sodium pentobarbital (50 mg/kg) for blood collection from the abdominal aorta. They were then sacrificed by cervical dislocation and liver tissue was collected for the following experiments. The animal study protocol was approved by Ethics Committee of the 940th Hospital of Joint Support Force (2020KYLL004).

Body and liver weight

After the feeding experiment, the weight of individual mice was weighed. The liver of each animal was removed and weighed, and the ratio of liver weight to body weight was calculated.

Histological examination

The histological changes in liver tissues were observed using a hematoxylin-eosin (HE) kit (#C0105S; Beyotime, Shanghai, China) according to manufacturer’s instruction. Simply, liver tissue samples were fixed in 10% formaldehyde for 48 h before undergoing a graded ethanol dehydration series (50%–100%). The samples were then cleared in xylene and embedded in paraffin. Sections of 4 µm thickness were prepared using a microtome (Leica, Wetzlar, Germany) after pre-cooling the tissue blocks at −20 °C. These sections were affixed to slides and baked at 65 °C for adhesion. Deparaffinization and rehydration followed, utilizing xylene and a descending alcohol series (100%–75%), respectively. Staining was conducted using hematoxylin and eosin solutions for cellular morphology and inflammation assessment. The slides were then cleared in xylene and sealed with neutral balsam. Microscopic imaging was subsequently performed to evaluate the tissue samples. Images were captured using a microscope (Olympus, Tokyo, Japan).

Cell culture and treatment

Human HepG2 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) under 5% CO₂ at 37 °C. HepG2 cells were transfected with oe-Foxa2 (NM_021784.5) or oe-NC (VectorBuilder) using HighGene transfection reagent (ABclonal, Wuhan, China) for 48 h. Then, the medium was changed and supplemented with oleic acid (OA; 0.5 mM) to induce the cellular NAFLD model. After 16 h, cells were harvested for subsequent analysis. After transfected with oe-Foxa2 and treated with OA, cells were treated with NF-κB/IKK pathway activator lipopolysaccharide (LPS, 1 µg/mL) for 12 h to validate the effect of the NF-κB/IKK pathway in hepatic steatosis.

Oil red O staining

Oil red O staining was performed on an oil red O staining kit (#C0157S; Beyotime, Shanghai, China) according to the manufacturer’s illustration. For animals, frozen liver tissues were cut into 15 µm slices. For HepG2 cells, harvested cells were fixed with 4% paraformaldehyde. Then, slices or cells were dipped in oil red O solution for 20 min, washed with phosphate-buffered saline (PBS), and took photos with a microscope (Olympus, Tokyo, Japan). Afterward, the oil red O reagent was dissolved by 100% isopropanol. The absorbance value was examined at 490 nm.

Biochemical and ELISA analysis

The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were detected using a biochemical auto-analyzer (BIOBASE, Jinan, China).

Liver tissues were first homogenized in a protein extraction buffer containing protease inhibitors. HepG2 cells were washed with PBS and then lysed with RIPA lysis buffer supplemented with protease and phosphatase inhibitors. After homogenization or cell lysis, the samples were centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was then collected for further analysis. The levels of total cholesterol (TC; #YFXEM00593; Yifei Xue Biotechnology, Nanjing, China) and triglyceride (TG; #ml037202; mIbio, Shanghai, China) in liver tissues or HepG2 cells were measured following ELISA kits instruction.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells using the TRIzol reagent (Invitrogen, Waltham, MA, USA) and the RNA concentration was measured by the Nano-Drop 8000 (Thermo, Waltham, MA, USA). FastKing gDNA Dispelling RT reagent Kit (TIANGEN, Beijing, China) was used to synthesis cDNA from 1 µg of total RNA. qRT-PCR was performed on a CFX96 Touch instrument (Bio-Rad, Hercules, CA, USA) with SYBR Green PCR Master Mix (Roche Applied Sciences, Barcelona, Spain). Each well (20 µL PCR reaction volume) included 10 µL of SYBR Green PCR Master Mix, 1 µL of cDNA, 2 µL of primer pair mix (10 µM each primer) and 7 µL of DNAse/RNAse-free H2O. Each sample was prepared in three replicates. The reaction conditions were as follows: 95 °C for 3 min, 40 cycles of 95 °C for 12 s and 62 °C for 40 s. The relative expression of Foxa2 was calculated by a 2^−ΔΔCt method and normalized to GAPDH. The primer sequences are as follows: Foxa2 (NM_021784.5) forward 5′-TGCACTCGGCTTCCAGTATG-3′ and reverse 5′-CGT GTT CAT GCC GTT CAT CC-3′, PCR product length 143 bp, and GAPDH (NM_001357943.2) forward 5′-CAT GGG TGT GAA CCA TGA GAA-3′ and reverse 5′-GGC ATG GAC TGT GGT CAT GAG-3′, PCR product length 145 bp.

Western blotting

Collected liver tissues and cells were lysed in RIPA buffer (Beyotime, Shanghai, China). After gel electrophoresis, samples were transferred onto membranes, blocked with 5% fat-free milk for 1 h, and incubated with primary antibodies at 4 °C for 12 h. After washing, membranes were incubated with secondary antibody (1:2000; ab205718; Abcam, Cambridge, UK) at 25 °C for 1 h. Protein bands were observed using an enhanced chemiluminescence kit (APPLYGEN, Beijing, China). The band intensities were quantified using ImageJ software. The quantification was normalized to the intensity of the GAPDH band to account for potential loading variations. The primary antibodies including carnitine palmitoyl transferase 1α (CPT1α; 1:1000; DF12004), Foxa2 (1:1000; DF13363), NF-κB (1:2000; AF5006), p-NF-κB (1:2000; AF2006), IKK (1:2000; AF6014), and p-IKK (1:2000; AF3013) were purchased from Affinity Biosciences (JIangsu, China); acetyl-CoA carboxylase (ACC; 1:1000; 3676) and GAPDH (1:1000; 2118) were purchased from Cell Signaling Technology (Waltham, MA, USA); fatty acid synthase (FAS; 1:1000; E-AB-40063) was purchased from Elabscience Biotechnology Co., Ltd (Wuhan, China).

Statistical analysis

Results data were exhibited as mean with standard deviations. Statistical analyses were carried out using GraphPad Prism 8.0 software. For comparisons involving only two groups, an unpaired t-test was performed. For comparisons among multiple groups, one-way analysis of variance followed by Tukey’s test were conducted. P < 0.05 indicates results data significantly different.

Foxa2 expression is decreased in NAFLD mice

Foxa2, an insulin-sensitive transcriptional regulator, has a role in regulating hepatic lipid metabolism and ketogenesis (Liu et al., 2022; Wolfrum et al., 2004). In addition, a previous study showed that Foxa2 activation ameliorates hepatic steatosis (Liu et al., 2022). In this study, we built an HFD-induced NAFLD mouse model to examine whether Foxa2 overexpression could ameliorate NAFLD. HFD significantly increased body and liver weight and the ratio of liver/body weight as well as fasting blood glucose (FBG) of mice, whereas decreased Foxa2 expression (P < 0.01). However, these changes were effectively mitigated by overexpressing Foxa2 (P < 0.05; Figs. 1A–1C). These results suggest that Foxa2 may be a potential therapeutic target for ameliorating NAFLD symptoms.

Foxa2 overexpression reduces hepatic steatosis in NAFLD mice

Hepatic steatosis is a key histological feature of NAFLD (Pan et al., 2021). The serum levels of AST and ALT in NAFLD mice were higher than those in control mice (P < 0.01). Similar trends of TG and TC were also observed in the liver tissues of NAFLD mice (P < 0.01). In contrast, oe-Foxa2 transfection suppressed the levels of AST, ALT, TG, and TC in HFD-stimulated NAFLD mice (P < 0.01; Figs. 2A–2B). HE staining revealed that the hepatocytes of NAFLD mice were filled with a large number of white vacuoles, hepatocyte ballooning, and inflammatory cell aggregation. oe-Foxa2 treatment resulted in a significantly smaller number of white vacuoles, occasional small vesicular fat vacuoles, and no inflammatory cell aggregation in mice. Oil Red O staining showed that the accumulation of lipid drops was reduced after oe-Foxa2 treatment (Fig. 2C), suggesting that Foxa2 could suppress hepatic steatosis in NAFLD mice.

Foxa2 regulates hepatic lipid metabolism in NAFLD mice

To explore the mechanism by which Foxa2 regulates lipid metabolism in NAFLD mice, the expression of lipid synthesis proteins FAS and ACC and fatty acid β-oxidation protein CPT1α were measured by western blotting. The results showed that Foxa2 overexpression reduced HFD-stimulated FAS and ACC upregulation and CPT1α downregulation (P < 0.05; Fig. 3), indicating that Foxa2 inhibits lipid synthesis and facilitates fatty acid β-oxidation, which in turn reduces lipid deposition in the liver.

Foxa2 overexpression inhibits lipid accumulation in OA-induced HepG2 cells

To further investigate the effect of Foxa2 on NAFLD, we constructed an OA-induced NAFLD cellular model. Western blotting and qRT-PCR showed that OA significantly decreased Foxa2 protein and mRNA expression, which was reversed by Foxa2 overexpression (P < 0.01; Figs. 4A–4B). Oil red O staining exhibited that lipid accumulation was obvious in OA-induced HepG2 cells. Also, oe-Foxa2 transfection significantly reduced the elevated lipid content induced by OA treatment (P < 0.01; Fig. 4C). Next, the levels of TG and TC were enhanced in OA-induced HepG2 cells, which were suppressed by transfecting oe-Foxa2 (P < 0.05; Fig. 4D). Finally, the upregulation of FAS and ACC and the downregulation of CPT1α in OA-stimulated HepG2 cells were reversed after Foxa2 overexpression (P < 0.05; Fig. 4E). The in vitro results corroborate the in vivo findings and indicate that Foxa2 is a critical regulator of lipid metabolism in NAFLD.

Foxa2 may ameliorate hepatic steatosis via inhibiting NF-κB/IKK pathway

NF-κB/IKK signaling pathway has been reported involved in steatosis (Heida et al., 2021; Heo et al., 2019). To verify that Foxa2 attenuates steatosis through the NF-κB/IKK pathway, OA-induced HepG2 cells were treated with oe-Foxa2 and LPS (NF-κB/IKK pathway activator). Western blotting suggested that OA significantly increased phosphorylation levels of NF-κB and IKK (P < 0.01). oe-Foxa2 transfection markedly downregulated the expression of p-NF-κB/NF-κB and p-IKK/IKK in OA-treated HepG2 cells, which was reversed by LPS administration. (P < 0.01; Figs. 5A–5B). Subsequently, western blotting also showed that oe-Foxa2-induced downregulation of FAS and ACC was reversed after LPS addition (P < 0.01). Conversely, LPS addition attenuated oe-Foxa2-induced CPT1 α upregulation (P < 0.05; Fig. 5C). The above results suggest that Foxa2 may ameliorate hepatic steatosis by inhibiting the NF-κB/IKK pathway.