OP-D was obtained from Shanghai Yuanye Biotechnology Co., Ltd. Palmitic acid (PA) was purchased from Sigma-Aldrich (Merck KGaA).

A total of 15 male C57BL/6J mice (age, 3-4 weeks; weight, 17-20 g) were purchased from Shanghai Model Organisms Centre, Inc. Mice with similar body weights were allowed to adjust to the environment for one week prior to the experiments. Subsequently, mice (age, 4-5 weeks) were randomly divided into the following three groups: Normal chow diet (ND); HFD; and HFD + OP-D group. Mice were given free access to food and distilled water. Mice fed with ND (cat. no. D12450B; 10% fat, 70% carbohydrates and 20% protein; Research Diets, Inc.) or HFD (cat. no. D12492; 60% fat, 20% carbohydrate and 20% protein; Research Diets, Inc.). Following feeding for 12 weeks, HFD-induced obese mice were randomly allocated to the test groups for pharmacological studies. Mice in the HFD + OP-D group continued to be fed with HFD and were intragastrically administered with 5 mg/kg/day OP-D (dissolved in 0.5% of carboxymethyl cellulose) for the following 4 weeks, according to preliminary experiments and previous studies (14,15). Mice in the other groups were treated with the same volume of 0.5% sodium carboxyl methyl cellulose solution (in order to protect the gastric mucosa of mice) according to a previous study (16,17). At the end of the experiment (16 weeks), all mice were sacrificed by cervical dislocation. All mice were housed in specific pathogen-free facility under a 12-h light/dark cycle, a relative humidity of 50% and a controlled temperature of 22±1˚C. All animal procedures were approved by the Medical Ethics Committee of Wuhan University of Science and Technology (Wuhan, China; approval number, 2022139).

At the end of the experiment, mice were fasted overnight and blood samples from the tail were collected to detect blood glucose levels using a blood glucose meter (Bayer AG). The mice were then sacrificed, and blood samples were collected by aortic puncture with centrifugation (1,300 x g for 10 min at 4˚C) to obtain serum, and stored at -80˚C for the blood chemistry measurements. The levels of insulin, IL-6, IL-1β and TNFα in serum were detected using the corresponding commercially available ELISA kits (insulin: cat. no. KCD-E1025; IL-6: cat. no. KCD-E1002; IL-1β: cat. no. KCD-E10010; TNFα: cat. no. KCD-E1018; Shanghai Kechuangda Biomedical Technology Co., Ltd.), according to the manufacturer's instructions. The serum levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automatic biochemical analyzer (Hitachi, Ltd.). In addition, the serum levels of free fatty acids (FFA) were detected by a commercially available colorimetric assay kit (cat. No. A042-1-1, Nanjing Jiancheng Bioengineering Institute). The homeostatic model assessment for insulin resistance (HOMA-IR) index was calculated as follows: HOMA-IR=[fasting serum glucose (mmol/l) x fasting serum insulin (µU/ml)]/22.5.

Liver tissues from mice in each group were collected and fixed in 10% formaldehyde neutral buffer solution for 24 h at 4˚C, followed by washing with tap water, dehydration in alcohol and embedding in paraffin. Subsequently, the 5-µm thick transversal sections were obtained, deparaffinized, dehydrated in ethanol (50-100%) and cleared with xylene. The sections were stained using hematoxylin and eosin (H&E) solution for 5 min at room temperature (Wuhan Servicebio Technology Co., Ltd.), according to the manufacturer's protocols. For Oil-red O staining, liver tissues were frozen in optimal cutting temperature compound at -20˚C and 10-µm thick sections were obtained, followed by staining with Oil-red O solution (Wuhan Servicebio Technology Co., Ltd.) for 10 min at room temperature, according to the manufacturer's protocols. Images were observed using a NIKON imaging workstation (NIKON digital sight DS-FI2; Nikon Corporation).

The commercially available Triglyceride Assay Kit (cat. no. ab65336; Abcam) was used to assess the levels of liver TG by measuring the optical density at a wavelength of 570 nm using spectrophotometry. The index of OS, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) levels, was determined using the corresponding commercially available kits (MDA: cat. no. A003-1-1; SOD: cat. no. A001-1-1; CAT: cat. no. A007-1-1; GSH-Px: cat. no. A005-1-1. Nanjing Jiancheng Bioengineering Institute).

Primary hepatocytes (PMHs) were isolated from mice. Briefly, 6-8-week-old male C57BL/6J mice (n=6; weight, 18-22 g) were purchased from Shanghai Model Organisms Centre, Inc. Mice were given free access to food and distilled water, and were housed in a specific pathogen-free facility under a 12-h light/dark cycle, a relative humidity of 50% and a controlled temperature of 22±1˚C. Mice were anesthetized using intraperitoneal injections of sodium pentobarbital (50 mg/kg). After the liver tissue was separated, the mice were sacrificed by cervical dislocation. The liver tissue was then collected, minced and filtered after portal vein perfusion. PMHs were purified and cultured in DMEM (cat. no. G4510; Wuhan Servicebio Technology Co., Ltd.) supplemented with 1% penicillin/streptomycin solution and 10% FBS (cat. no. G0004; Wuhan Servicebio Technology Co., Ltd.). To assess cell viability, PMHs (5x10³) were seeded into 96-well plates for 24 h at 37˚C. The following day, when plates reached ~80% confluency, cells were treated with various concentrations (0-20 µmol/l) of OP-D for an additional 24 h at 37˚C. Cell viability was assessed using a Cell Counting Kit-8 assay (Wuhan Servicebio Technology Co., Ltd.). Briefly, 10 µl CCK-8 solution added into each well, which were then incubated at 37˚C with 5% CO₂ for 2 h. The optical density was measured at a wavelength of 450 nm. To establish an in vitro model of lipid accumulation, hepatocytes were treated with PA (400 µmol/l) for 24 h at 37˚C. Then the cells were treated with or without 10 mol/l OP-D for another 24 h. Cultured cells were collected, isopropyl alcohol was added into 2x10⁶ cells according to the proportion of 200 µl homogenizing medium, and centrifugation was performed at 4˚C and 10,000 x g for 10 min to obtain the lysed cells. The content of TG in lysed cells was measured using the TG Colorimetric Assay kit (cat. no. E-BC-K261-M; Elabscience Biotechnology, Inc.) following manufacturer's protocols. The optical density was measured at a wavelength of 510 nm.

The levels of TNF-α, IL-1β and IL-6 in PMH supernatants were determined using ELISA, according to the manufacturer's instructions (Shanghai Kechuangda Biomedical Technology Co., Ltd.).

Total RNA was extracted from liver tissues or cells and isolated using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the protocol provided by the manufacturer. The isolated RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR was performed using SYBR Green PCR master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primer sequences used are listed in Table SI. All reactions were conducted in triplicate and the thermal cycling conditions were as follows: 30 sec at 95˚C, followed by 50 cycles of 95˚C for 5 sec and 60˚C for 30 sec. The mRNA expression levels of target genes were calculated using the 2^-ΔΔCq method and normalized to those of GAPDH (18).

Liver tissues or cells were lysed with RIPA buffer containing protease inhibitors (Roche Applied Science). A BCA kit (Beyotime Institute of Biotechnology) was used to detect protein concentrations. The extracted proteins (20 µg of each protein) were loaded onto 10% SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (MilliporeSigma) after electrophoresis. Blots were blocked with 5% bovine serum albumin (cat. no. GC305006; Wuhan Servicebio Technology Co., Ltd.) at 4˚C for 2 h and then incubated with primary antibodies against GAPDH (1:2,000; cat. no. 5174), P65 (1:1,000; cat. no. 8242), phosphorylated (p-)P65 (Ser468) (1:1,000; cat. no. 3039), IκBα (1:1,000; cat. no. 9242), p-IκBα (1:1,000; cat. no. 2859), TNFα (1:1,000; cat. no. 3707) (all CST Biological Reagents Co., Ltd.) at 4˚C overnight, The membranes were then incubated with HRP-conjugated anti-IgG secondary antibodies (cat. no. A0192; 1:2,000; Beyotime Institute of Biotechnology) at room temperature for 45 min. GAPDH was used as an internal reference. The intensity of protein bands was assessed using the ImageJ software (version 1.8.0; National Institutes of Health).

IF staining was carried out according to standard procedures (19). Briefly, cells were first fixed with 95% ethanol for 15 min at -20˚C. The cells were then incubated and blocked with blocking solution (5% BSA/PBS) for 30 min at room temperature, followed by being incubated overnight with a primary antibody against p65 (1:200; cat. no. 8242; Cell Signaling Technology, Inc.) at 4˚C. Subsequently, incubation with Alexa Fluor^® 647-conjugated goat anti-rabbit IgG (1:400; cat. no. ab150079; Abcam) at 4˚C for 1 h was performed. The coverslips were mounted on microscope slides with fluorescence reagent containing 10 µg/ml DAPI (Thermo Fisher Scientific, Inc.). IF images were captured using the Fluoview FV1000 confocal microscope (Olympus Corporation).

Data are presented as the mean ± SEM. All analyses were performed using GraphPad Prism 8.0 (GraphPad Software; Dotmatics) or SPSS 22.0 (IBM Corp.) software. Data among groups were compared using one-way ANOVA followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

OP-D is a pharmacological compound of Ophiopogon japonicus, a traditional Chinese medicine herb (14). The molecular structure of OP-D is illustrated in Fig. 1A. The current study aimed to explore whether OP-D administration could inhibit body weight gain in mice fed with HFD. The results showed that the body weight of mice in the HFD + OP-D group was notably reduced compared with that in the HFD group at weeks 14-16 (Fig. 1B). Additionally, OP-D administration significantly reduced liver weight in HFD-treated mice compared with those in the HFD group (Fig. 1C).

Subsequently, the present study investigated whether OP-D could regulate glucose and insulin homeostasis in HFD-fed mice. Consistent with previous studies (20,21), HFD-treated mice exhibited elevated glucose and insulin levels compared with ND control mice. The effect of the HFD on the levels of fasting blood glucose (FBG) and insulin was significantly reduced by OP-D treatment (Fig. 2A and B). Furthermore, the HOMA-IR index suggested that mice in the HFD + OP-D group displayed significantly reduced IR compared with those in the HFD group (Fig. 2C). These findings indicated that OP-D could exert a significant role in improving glucose homeostasis and IR in HFD-induced obese mice.

To further explore whether OP-D could prevent hepatic steatosis, H&E staining was first carried out. H&E staining showed widespread vacuolation in the liver tissue of HFD mice. However, OP-D treatment alleviated hepatic steatosis (Fig. 3A). In addition, mice in the HFD + OP-D group displayed a smaller area of hepatic lipid droplets compared with mice in the HFD group (Fig. 3A). Treatment with OP-D significantly reduced TG levels in the liver compared with the HFD group, thus suggesting that OP-D could attenuate lipid accumulation (Fig. 3B). Furthermore, TC, TG, FFA and LDL-C levels were significantly reduced in the serum of HFD mice treated with OP-D compared with mice treated with vehicle (Fig. 3C-F). Subsequently, the mRNA expression levels of lipid metabolism-related genes were detected. The data showed that the expression levels of lipid droplet formation-related genes [3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), fat storage-inducing transmembrane protein (Fitm)1 and Fitm2], lipid uptake-related genes [fatty acid-binding protein 1 (Fabp1), fatty acid transporter 1 (Fatp1) and lipoprotein lipase (Lpl)] and lipogenesis-related genes [fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1) and peroxisome proliferator-activated receptor (PPAR)-γ] were significantly decreased by treatment of HFD-fed mice with OP-D (Fig. 3G-I). By contrast, the expression of fatty acid β-oxidation-related genes [PPAR-α, peroxisomal acyl-coenzyme A oxidase 1 (Acox1) and carnitine O-palmitoyl transferase 1α (Cpt1α)] were significantly upregulated after OP-D treatment compared with the HFD group (Fig. 3J). Furthermore, the HFD-induced increased levels of ALT and AST in HFD mice were significantly reduced by OP-D (Fig. 3K and L). Taken together, these results suggested that OP-D could exert a protective role in the development of NAFLD.

Lipid peroxidation, OS and inflammation serve a critical role in the progression of NAFLD (22). Therefore, the levels of OS in the liver tissue were determined in the present study. The results revealed that the hepatic levels of MDA, an oxidative marker, were significantly reduced following treatment of HFD-induced NAFLD mice with OP-D (Fig. 4A). Inversely, the levels of the antioxidative markers SOD, CAT and GSH-Px were significantly increased in the livers of mice in the HFD + OP-D group compared with the HFD group that did not receive OP-D treatment (Fig. 4B-D). Furthermore, the mRNA expression levels of nuclear respiratory factor 1 and transcription factor A mitochondria, two mitochondrial regulators, were significantly increased in the HFD + OP-D group compared with the HFD group (Fig. 4E). Subsequently, the inflammatory response in mice treated with or without OP-D was investigated. As shown in Fig. 4F-H, the serum levels of the cytokines TNFα, IL-6 and IL-1β were significantly increased in HFD mice compared with ND mice. However, the secretion levels of these inflammatory mediators were restored following OP-D administration. Additionally, OP-D administration significantly reversed the increased mRNA expression levels of TNFα, IL-6 and Il-1β after induction by HFD (Fig. 4I-K). Furthermore, the significantly increased gene expression levels of the inflammatory mediators, including matrix metalloproteinase 9 (MMP9), monocyte chemoattractant protein 1 (MCP1) and vascular cell adhesion protein 1 (VCAM1), were also significantly reduced in the HFD + OP-D group compared with the HFD group (Fig. 4L). These findings suggested that OP-D could reduce OS and inflammation in HFD-induced NAFLD mice.

The current study aimed to explore whether OP-D could exert a direct effect on PMHs. Therefore, the changes in fat deposition were evaluated in PMHs treated with different concentrations of PA for different time points (data not shown). Consistently, treatment with 400 µmol/l PA for 24 h was the optimum condition, as previously described (23). Since the CCK8 result showed that 0-20 µmol/l OP-D did not affect the cell viability of PMHs, the concentration of 10 µmol/l OP-D for 24 h was selected as the optimal condition (Fig. 5A). Treatment with OP-D significantly reduced TG levels in lysed PMHs compared with the PA-only treated group (Fig. 5B). The mRNA expression levels of lipid synthesis-related genes, such as acetyl-CoA carboxylase 1 (ACC1), FAS and SCD1, were significantly reduced in cells treated with OP-D compared with the PA group (Fig. 5C). Consistent with previous studies (11,24), PA significantly increased the levels of TNFα, IL-1β and IL-6 from PMHs, while OP-D reversed this effect (Fig. 5D-F). Additionally, OP-D could significantly reduce the mRNA expression levels of TNFα, IL-6 and IL-1β compared with the PA-only treated group (Fig. 5G). Collectively, these results indicated that OP-D could alleviate lipogenesis and inflammation in vitro.

The results suggested that OP-D could alleviate inflammatory responses, which have a significant effect on the progression of HFD-induced NAFLD in mice. Therefore, the present study further explored the mechanisms underlying the effect of OP-D on NAFLD in PMHs. It has been reported previously that the inflammatory NF-κB signaling pathway plays a crucial role in regulating inflammatory responses in NAFLD (25). In the current study, western blot analysis demonstrated that cell treatment with OP-D significantly reduced the PA-induced protein expression levels of p-P65, p-IκBα and TNFα in hepatocytes (Fig. 6A-D). OP-D also inhibited the PA-induced nuclear translocation of P65 (Fig. 6E). These results suggested that the NF-κB signaling pathway could mediate the beneficial effects of OP-D on NAFLD.