2.1 Cell culture

The Alpha Mouse Liver 12 (AML-12) cell line, derived from mouse normal hepatocytes, was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a humidified incubator (37 °C, 5% carbon dioxide (CO₂)) using Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with fetal bovine serum (FBS, 10%). To create an in vitro AFL model, cells were treated with ethyl alcohol (EtOH, 250 mmol/L) and palmitic acid (PA, 0.25 mmol/L) for 24 hours. This was achieved by treating cells with Sigma-Aldrich product #P0500 from St. Louis, MO, USA.

2.2 Cell transfection

GenePharma, a company located in Shanghai, China, provided the siRNAs used to target GBP5 (si-GBP5) and a negative control (si-NC). The siRNAs were transfected into AML-12 cells by Lipofectamine 2000 (11668019), which is a product manufactured by Invitrogen located in Carlsbad, CA, USA. Plasmids containing si-GBP5 and si-NC were utilized in the transfection process.

2.3 Western blot

Proteins extracted from AML-12 cells were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%) and transferred onto polyvinylidene difluoride (PVDF) membranes obtained from Beyotime, located in Shanghai, China. After blocking, the membranes were incubated overnight (4 °C) with primary antibodies against GBP5 (1:1000; ab96119; Abcam, Shanghai, China), B-cell lymphoma-2-associated X (Bax) (1:2000; ab182733), B-cell lymphoma-2 (Bcl-2) (1:1000; ab196495), phosphorylation-proteinp65 (p-p65) (1:1000; ab76302), proteinp65 (p65) (0.5 µg/mL; ab16502), phosphorylation-Inhibitory subunit of NF kappa B alpha (p-IκBα) (1:10,000; ab133462), Inhibitory subunit of NF kappa B alpha (IκBα) (1:1000; ab32518), and β-actin (1 µg/mL; ab8226; the internal reference). Subsequently, appropriate secondary antibodies (1:2000; ab7090) were added to the membranes for another 2 hours. Finally, the blots were visualized and analyzed.

2.4 Oil Red O staining

To prepare the AML-12 cells for analysis, they were first fixed using paraformaldehyde solution (24 h). Afterward, the cells were washed with phosphate Buffered Saline (PBS) and stained with saturated Oil Red O for a duration of 15 minutes. The cells were then subjected to differentiation in alcohol, which involved treating them with 75% alcohol three times. Images of the cells were captured using an optical microscope-the Nikon Eclipse E100 model (Nikon, Tokyo, Japan).

2.5 TG and aspartate aminotransferases (AST)/alanine aminotransferases (ALT) activity assay

To quantify the levels of TG, AST and ALT, the corresponding assay kits were utilized. These kits were purchased from Jiancheng Biology Institution PeproTech, a company located in Nanjing, Jiangsu, China.

2.6 RT-qPCR

RNA isolation from AML-12 cells was performed using TRIzol reagent (15596018, Invitrogen, Carlsbad, CA, USA) from Invitrogen. Subsequently, cDNA was synthesized using the PrimeScript RT Reagent Kit (RR037A, Takara, Dalian, China). RT-qPCR was performed using the SYBR Premix Ex Taq™ II commercial kit (RR820A, Takara, Dalian, China). The calculation of the relative expressions was performed by the 2^-ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as an internal reference.

Tumor Necrosis Factor-alpha (TNF-α),

F: GAA ACA CAC GAG ACG CTG AA;

R: AGG GAG GCC TGA GAC ATC TT.

Interleukin-1 beta (IL-1β),

F: TAC AGG CTC CGA GAT GAA CAA C;

R: TTT GAG GCC CAA GGC CAC AG.

Interleukin-6 (IL-6),

F: CCA GAA ACC GCT ATG AAG TTC C;

R: GTT GGG AGT GGT ATC CTC TGT GA.

GADPH,

F: CC ACA GTC CAT GCC ATC AC;

R: TCC ACC ACC CTG TTG CTG TA.

2.7 ELISA

To quantify the levels of IL-6 (ab222503), IL-1β (ab197742), and TNF-α (ab208348), commercial ELISA kits were employed. The ELISA kits (ab222503, ab197742 and ab208348) used were purchased from Abcam, which is located in Shanghai, China. The protocols provided by the manufacturer were followed in carrying out the assays.

2.8 Flow cytometry

The Annexin V-FITC Apoptosis Detection Kit (C1062S, Beyotime, Shanghai, China), which was obtained from Beyotime located in Shanghai, China, was used to evaluate cell apoptosis. The analysis was conducted using a BD FACS Calibur instrument (BD FACSCalibur, BD Biosciences, San Jose, CA, USA). Prior to analysis, the AML-12 cells were washed with PBS and then stained with Annexin V-FITC and propidium iodide (PI) in a dark environment. Finally, flow cytometry was employed to examine the extent of cell apoptosis using a BD Biosciences instrument located in San Jose, CA, USA.

2.9 Statistical analysis

All experiments were independently performed three times, and the resulting data was presented as means ± SD. Statistical analysis was performed using IBM Corp.’s SPSS 22.0 statistical software, which is located in Armonk, NY, USA. To compare the various groups, one-way Analysis of Variance (ANOVA) and Student’s t-test were used. A p-value less than 0.05 was deemed to be statistically significant.

3.1 Knockdown of GBP5 suppressed EtOH-induced lipid accumulation

GBP5 protein expression was increased in the EtOH group, but reduced after GBP5 knockdown (Fig. 1A). Additionally, lipid accumulation was increased after EtOH induction, but this change was attenuated after silencing of GBP5 (Fig. 1B). Moreover, the levels of TG, AST and ALT were all strengthened after EtOH treatment, but these changes were reversed after GBP5 inhibition (Fig. 1C). Taken together, knockdown of GBP5 suppressed EtOH-induced lipid accumulation.

Fig. 1.Knockdown of Guanylate binding protein 5 (GBP5) suppressed Ethyl Alcohol (EtOH)-induced lipid accumulation. Groups were divided into Control, EtOH, EtOH + si-NC and EtOH + si-GBP5 groups. (A) GBP5 protein expression was examined through western blot. (B) The lipid accumulation level was confirmed through Oil Red O staining assay. (C) The Triglyceride (TG), aspartate aminotransferases (AST), and alanine aminotransferases (ALT) levels were measured through the corresponding commercial kits. ^**p < 0.01. si-GBP5: siRNAs used to target GBP5; si-NC: siRNAs used to a negative control.

3.2 Silencing of GBP5 reduced EtOH-mediated inflammation

As shown in Fig. 2A, the mRNA expressions of TNF-α, IL-1β and IL-6 were all elevated following EtOH treatment, but this effect was attenuated following GBP5 suppression. In addition, as confirmed by ELISA assay, TNF-α, IL-1β and IL-6 levels were also heightened following EtOH treatment, but these changes were attenuated following GBP5 inhibition (Fig. 2B). These results demonstrated that silencing of GBP5 reduced EtOH-mediated inflammation.

Fig. 2.Silencing of GBP5 reduced EtOH-mediated inflammation. The mRNA expressions (A) and levels (B) of Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1 beta (IL-1β) and Interleukin-6 (IL-6) were confirmed through reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immuno sorbent assay (ELISA), respectively. ^*p < 0.05, ^**p < 0.01. GBP5: Guanylate binding protein 5; EtOH-Ethyl Alcohol; si-GBP5: siRNAs used to target GBP5; si-NC: siRNAs used to a negative control.

3.3 Suppression of GBP5 inhibited EtOH-triggered cell apoptosis

Next, it was discovered that cell apoptosis was increased following EtOH induction, but this change was counteracted after GBP5 suppression (Fig. 3A, B). Furthermore, the enhancement of Bax protein expression and the decrease in Bcl-2 protein expression mediated by EtOH treatment were reversed after GBP5 knockdown (Fig. 3C). In general, suppression of GBP5 inhibited EtOH-triggered apoptosis.

Fig. 3.Suppression of GBP5 inhibited EtOH-triggered apoptosis. (A, B) Cell apoptosis was assessed through flow cytometry. (C) The protein expressions of B-cell lymphoma-2-associated X (Bax) and B-cell lymphoma-2 (Bcl-2) were evaluated through western blot. ^*p < 0.05, ^**p < 0.01. GBP5: Guanylate binding protein 5; EtOH-Ethyl Alcohol; si-GBP5: siRNAs used to target GBP5; si-NC: siRNAs used to a negative control.

3.4 Knockdown of GBP5 retarded the EtOH-stimulated NF-κB pathway

The protein expressions of p-p65 and p-IκBα were enhanced, and IκBα was reduced in response to EtOH stimulation, but these effects were rescued following GBP5 inhibition (Fig. 4). These data illustrated that knockdown of GBP5 retarded the EtOH-stimulated NF-κB pathway.

Fig. 4.Knockdown of GBP5 retarded the nuclear factor kappa-B (NF-κB) pathway. The protein expressions were examined through western blot. ^*p < 0.05, ^**p < 0.01. GBP5: Guanylate binding protein 5; EtOH-Ethyl Alcohol; si-GBP5: siRNAs used to target GBP5; si-NC: siRNAs used to a negative control.