Eighty male C57BL/6J mice (Animal Experiment Center of Air Force Medical University, Xi’an, Shaanxi, China), aged 4–5 weeks, were randomly and equally divided into three groups and subjected to the following treatments: (1) normal diet group (n = 10): the mice were fed a standard chow diet. (2) HFD group (n = 10): the mice were provided with a high-fat diet. (3) HFD + VK2 group (n = 10): in addition to the high-fat diet, VK2 was daily administered via oral gavage at a dose of 120 mg/kg/day for the last eight weeks. The dosage of VK2 intervention was based on our previous research [10]. At the end of the experiment, mice in each group were deeply anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and diazepam (5 mg/kg). Subsequently, liver tissues and blood samples were collected for subsequent analysis.

H&E staining, Oil Red O staining, and immunofluorescence staining with anti-CD11b antibody (GB11058, 1:100 dilution; Servicebio Technology, Wuhan, China) were conducted on paraffin-embedded and (OCT) compound-embedded frozen liver sections, as previously described in references [11, 12].

Liver tissues were lysed with radioimmuno-precipitation assay (RIPA) buffer containing 1 mM PMSF (Beyotime Technology, Shanghai, China). Subsequently, the lysates were centrifuged at 14,000 g for 5 minutes. The total protein concentration was quantified using a BCA Protein Assay Kit (Beyotime Technology, Shanghai, China). Loading buffer was added to the protein extract at a final concentration of 5 µg/µL, and the mixture was boiled at 100 °C for 10 minutes to denature the proteins. Equal amounts of protein were separated by electrophoresis on 10% SDS-page gels and then transferred to a PVDF membrane. The membrane was blocked with non-fat milk and then incubated overnight at 4 °C with primary antibodies against SIRT3 (1:1000, 10099-1-AP, Proteintech, Wuhan, Hubei, China), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1$\alpha$) (1:1000, #2178, CST, Beverly, MA, USA), $\beta$-Tubulin (1:1000, 80713-1-RR, Proteintech, Wuhan, China), Catalase (1:1000, 21260-1-AP, Proteintech, Wuhan, Hubei, China), and superoxide dismutase 2 (SOD2) (1:1000, 24127-1-AP, Proteintech, Wuhan, Hubei, China). The membranes were washed three times with Tris-buffered saline + Tween 20 (TBST) buffer, followed by a 2-hour incubation with HRP conjugated secondary anti-rabbit (1:1000, #7073, CST) or anti-mouse antibodies (1:1000, #7076, CST). After another three washes with TBST buffer, the blots were visualized using an ECL kit [10].

Total RNA was isolated using the Magen HiPure Universal RNA Kit (R4130-02, Magen Biotechnology, Guangzhou, Guangdong, China), and the RNA concentration was measured with a NanoDrop 2000C spectrophotometer. gDNA was removed from 500 ng of RNA using the gDNA eraser (RR047Q, Takara, Kusatsu, Japan), and then the RNA was reverse-transcribed using the PrimerScript RT Reagent Kit. qPCR targets were normalized to $\beta$-Actin, and the relative mRNA levels were determined by calculating the average expression in the control group. Primer sequences were obtained from PrimerBank.

Donor feces were collected, diluted in deoxygenated saline, and filtered for inoculation into mice. Pseudo-germ-free mice were pre-treated with an antibiotic cocktail for two weeks. Subsequently, these mice were randomly divided into two groups: (1) HFD-HFD group: feces from the HFD group were administered to pseudo-germ-free HFD-fed mice; (2) VK2-HFD group: feces from the VK2 group were administered to pseudo-germ-free mice. The recipient mice were then fed a HFD for an additional 4 weeks [13].

Twenty HFD-fed mice were intraperitoneally injected with AAV-shSirt3 (1 $\times$ 10¹² vg/mL) for three consecutive weeks to establish the shSirt3 mouse model. Ten HFD-fed mice were intraperitoneally injected with Saav-shControl (1 $\times$ 10¹² vg/mL) for three consecutive weeks to establish the shControl mouse model. The shSirt3 mice were then divided into the following two groups: (1) shSirt3 + HFD group (n = 10); (2) shSirt3 + VK2 group (n = 10).

Immediately after sacrifice, mouse liver tissues were rinsed in cold PBS and homogenized. Mitochondria were isolated through a series of dual centrifugation steps. Freshly isolated liver mitochondria were suspended in mitochondrial assay solution (MAS) [14]. One to three micrograms of mitochondria were transferred to a XFe24 microplate, which was then filled with MAS to a final volume of 500 µL, containing either glutamate/malate or succinate/rotenone. The OCR was measured in response to the sequential injection of ADP, oligomycin, FCCP and antimycin A. The respiratory rates were reported as oxygen flux per unit mass, and all readings were normalized to the micrograms of protein in the mitochondria [10, 15].

Liver tissue sections (1 mm $\times$ 1 mm) were fixed in a 2.5% glutaraldehyde buffer solution at 4 °C for 3 hours. Subsequently, the fixation solution was replaced with a cacodylate buffer containing 0.1 M glutaraldehyde and kept at 4 °C. After washing, dehydration, and resin embedding, the liver tissues were sectioned using an ultramicrotome. The sections were observed under a transmission electron microscope (JEM 1400, JEOL Ltd., Tokyo, Japan), and images were captured at magnifications of 10,000$\times$ and 84,000$\times$ for the further analysis of mitochondrial density, number, and area [10].

The contents of ATP and SOD2 were determined using an ATP Assay Kit (Beyotime, S0026) based on the luciferin/luciferase method and a Superoxide Dismutase Assay Kit with NBT (Beyotime, S0101S), following the manufacturer’s instructions. All data were measured with multimode microplate readers (Tecan Infinite 200 PRO, Tecan Life Sciences, Männedorf, Switzerland). Briefly, liver tissue homogenates were centrifuged at 12,000 g for 5 minutes at 4 °C. The supernatant was collected and used for the detection of ATP and SOD2 levels. The luminescence was measured using multimode microplate readers (Tecan Infinite 200 PRO).

Mitochondria were extracted from fresh liver tissues, and the activities of mitochondrial respiratory complexes I and IV were measured using the corresponding kits from Abcam MitoSciences (Cambridge, UK) according to the manufacturer’s instructions.

Appropriate statistical methods were employed using SPSS software (Version 22, IBM Corp., Armonk, NY, USA). One-way and two-way ANOVA were conducted for multiple comparisons, while Student’s t-test was utilized for comparisons between two groups. A p value of less than 0.05 was considered statistically significant.

To investigate the effects of VK2 on MASLD, C57BL/6J mice were fed a HFD for 16 weeks, followed by oral administration of VK2 (120 mg/kg/day) while continuing the HFD for an additional 8 weeks (Fig. 1A). Compared with the control group, HFD-fed mice exhibited a significant increase in body weight over time. Both liver weight (LW) and the LW-to-body weight (LW/BW) ratio were significantly lower in the VK2-treated group when compared to the HFD group (Fig. 1B,C). Hepatic triglyceride (TG) and total cholesterol (TC) levels were also significantly reduced in VK2-treated mice (Fig. 1D,E). Given that lipotoxicity can lead to hepatocyte injury or death [16], serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were elevated after 24 weeks of HFD feeding. However, VK2 treatment significantly lower serum ALT and AST levels compared to the HFD group (Fig. 1F,G). Hepatic histology revealed that VK2 reduced HFD-induced hepatic ballooning and lipid droplet accumulation (Fig. 1H–J). VK2 treatment suppressed the expression of inflammatory genes (IL-1$\beta$, Cxcl2, Cxcl10, Ccl2, Ccl5) (Fig. 1K). Additionally, CD11b immunofluorescence staining showed that VK2 significantly decreased inflammatory cell infiltration in the liver tissues of HFD-fed mice (Fig. 1L,M). Collectively, these findings indicate that VK2 treatment ameliorates the progression of HFD-induced MASLD.

Fig. 1.

VK2 attenuates HFD-induced MASLD. (A) Schematic of VK2 gavage intervention. (B,C) LW and LW/BW ratios in all groups. n = 10 per group. (D,E) TG and TC levels in hepatic tissues. n = 10 per group. (F,G) Serum ALT and AST levels. n = 10 per group. (H–J) Representative images of H&E staining, Oil Red O staining, and MASLD activity score to evaluate hepatic steatosis and lipid accumulation. n = 6 per group. Scale bar = 100 µm. (K) Relative mRNA expression levels of inflammatory genes (IL-1$\beta$, Cxcl2, Cxcl10, Ccl2, Ccl5) determined by RT-qPCR, normalized to the $\beta$-actin-encoding gene Actb. n = 6 per group. (L,M) Immunofluorescence staining for CD11b (red, inflammatory cells) in liver tissues, with nuclei counterstained with DAPI (blue). n = 4 per group. Scale bar = 50 µm. Data were analyzed by one-way ANOVA, followed by Tukey’s post-hoc test. Significant differences were noted between the control and HFD groups, and between the HFD and VK2-treated groups. *p 0.05; **p 0.01; ***p 0.001; ****p 0.0001. VK2, Vitamin K2; HFD, high-fat diet; MASLD, metabolic dysfunction-associated steatotic liver disease; LW, liver weight; BW, body weight; TG, triacylglycerols; TC, total cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; RT-qPCR, real-time quantitative PCR.

Mitochondrial dysfunction contributes to MASLD progression. Increased free fatty acid (FFA) influx into hepatocytes induces metabolic changes that impair mitochondrial function [17]. VK2 treatment significantly reduced HFD-induced reactive oxygen species (ROS) production, as measured by DCFH-DA staining (Fig. 2A,B). Furthermore, VK2 treatment significantly enhanced ATP content, mitochondrial-DNA (mtDNA) gene expression, and SOD2 enzyme activity, suggesting that VK2 alleviated oxidative stress in HFD-fed mice (Fig. 2C–E). To further assess mitochondrial function, we measured bioenergetic parameters. Compared with the HFD group, VK2 treatment enhanced overall mitochondrial activity in the liver, restoring mitochondrial dysfunction and improving basal respiration and spare respiratory capacity in HFD-fed mice (Fig. 2F–K). Transmission electron microscopy (TEM) revealed that HFD-fed mice exhibited abnormal mitochondria with displaced myofibrils, swelling, and vacuole-like degeneration compared to the control group (Fig. 2L). In contrast, VK2-treated mice showed a significant increase in the total number and a decrease in average mitochondria area (Fig. 2M,N). Consistently, VK2 treatment upregulated the expression of mitochondrial biogenesis genes (Fig. 2O). Collectively, these findings indicate that VK2 ameliorates mitochondrial dysfunction and oxidative stress.

Fig. 2.

VK2 improves oxidative stress and mitochondrial function in HFD-fed mice. (A,B) Hepatic reactive oxygen species (ROS) levels measured by DCFH-DA staining and quantification of relative ROS production. n = 6 per group. Scale bar = 50 µm. (C–E) Hepatic superoxide dismutase 2 (SOD2) enzyme activity (C), ATP content (D), and mitochondrial DNA (mtDNA) copy number (mtDNA/nuclear DNA ratio) (E) in the liver tissue. n = 7 per group. (F–K) Oxygen consumption rate (OCR) of isolated liver mitochondria to assess mitochondrial bioenergetic. Basal respiration was measured under State 2 (F), State 3 (G), and State 3u (H) conditions. Electron transport chain complex activity was determined for complex Ⅰ (I), complex Ⅱ (J), and complex Ⅳ respiration (K). n = 6 per group. (L) Transmission electron microscopy (TEM) images of hepatic mitochondria (red triangle indicate mitochondrial structures). Images were acquired at 10,000$\times$ and 84,000$\times$ magnifications. Scale bars = 10 µm (low magnification) and 1 µm (high magnification). (M,N) Quantification of mitochondrial number and average area per cell using ImageJ software (Version 1.8.0.112, Madison, WI, USA). (O) Relative mRNA expression levels of mitochondrial biogenesis genes measured by RT-qPCR. n = 8 per group. Data are presented as mean $\pm$ standard deviation (SD). One-way ANOVA with Tukey’s post-hoc test was used for multiple-group comparisons. Significant differences were noted between the control and HFD groups, and between the HFD and VK2-treated groups. *p 0.05; **p 0.01; ***p 0.001; ****p 0.0001.

Given that the GM contributes to MASLD progression, we investigated the effect of VK2 treatment on GM composition. Compared with the HFD group, the VK2-treated group exhibited a significant increase in the Ace and Shannon indices (Fig. 3A,B), indicating that VK2 treatment restored reduced gut microbial diversity in HFD-fed mice. UniFrac-based principal coordinate analysis (PCoA) and partial least squares-discriminant analysis (PLS-DA) revealed distinct clustering of microbial communities among groups (Fig. 3C,D). The relative abundance of bacterial species Lactobacillus johnsonii and Lactobacillus murinues were significantly increased by VK2 treatment compared with the HFD group (Fig. 3E) [18]. To identify specific bacterial taxa altered by VK2 supplementation, linear discriminant analysis (LDA) effect size (LEfSe) was utilized to compare the fecal microbiota composition at the genus level. Compared with the control group, HFD group showed lower abundance of Dubosiella, norank_f__Muribaculaceae, unclassified_f__Erysipelotrichaceae Allobaculum, Bifidobacterium, Akkermansia, Corynebacterium, Parvibacter but a higher abundance of Rombountsia, Enterococcus, Clostridium_sensu_stricto_1, Christensenellanceae_R-7_group (Fig. 3F). Conversely, the HFD group enriched Ileibacterium, Enterococcus, Jeotgalicoccus, Staphylococcus, Anaeroplasma, Ruminococcus_torques_group, unclassified_f_Erysipelotrichaceae, ASF356, Negativibacillus, unclassified_c_Lactobacillales, unclassified_c_Bacilli, whereas VK2 treatment increased the abundance of Faecalibaculum, Macrococcus, norank_f__Clostridium_methylpentosum_group, Enterorhabdus, Pseudogracilibacillus, Bifidobacterium, Parvibacter in HFD-fed mice (Fig. 3G). At the genus level, the HFD group exhibited a significant decrease in Lactobacillus relative abundance compared with the control group, whereas VK2 treatment significantly restored Lactobacillus abundance (Fig. 3H). Redundancy analysis (RDA) showed that Lactobacillus was significantly negatively correlated with MASLD-associated parameters (hepatic TC, TG, serum ALT, AST, and NAS score) (Fig. 3I); suggesting that Lactobacillus enrichment may be mediate the protective effects of VK2 against MASLD. Collectively, these results indicate that VK2 reverses gut dysbiosis in HFD-induced MASLD.

Fig. 3.

VK2 mitigates gut dysbiosis in HFD-induced MASLD. Fresh fecal samples were collected from mice after 4 weeks of VK2 treatment for 16S rRNA gene sequencing analyses. (A) Ace and (B) Shannon diversity indices of the GM. (C,D) Weighted UniFrac PCoA analysis and PLS-DA analysis of microbial communities. (E) Relative abundance of bacterial genera at the genus level. (F,G) LEfSe was used to identify differentially abundant taxa in the GM. (H) Relative abundance of Lactobacillus across all groups. (I) RDA analysis of the correlation between Lactobacillus and MASLD-associated parameters. All data are expressed as mean $\pm$ SEM. p values shown in Fig. 3A,B,H were determined by one-way ANOVA with Dunnett’s post hoc test (comparison against the control group). Significant differences were noted between the control and HFD groups, and between the HFD and VK2-treated groups. *p 0.05; **p 0.01; ***p 0.001. GM, gut microbiota; PCoA, principal coordinate analysis; PLS-DA, partial least squares-discriminant analysis; LEfSe, linear discriminant analysis effect size; RDA, redundancy analysis.

To determine whether the beneficial effects of VK2 in HFD-fed mice are dependent on the GM, we transplanted fecal microbiota from VK2-treated HFD-fed donor mice to recipient HFD-fed mice via daily gavage. Recipient mice were pre-treated with broad-spectrum antibiotics in drinking water for 2 weeks to generate conditionally pseudo germ-free mice (Fig. 4A). Following the fecal microbiota transplantation (FMT), recipient mice that received VK2-treated microbiota exhibited significantly lower NAS scores (Fig. 4B,C), accompanied by reduced hepatic lipid levels (TC and TG) (Fig. 4D,E), decreased serum AST and ALT levels (Fig. 4F,G), and enhanced SOD2 activity (Fig. 4H) and mitochondrial respiratory complex Ⅰ and Ⅳ activity (Fig. 4I,J). These findings demonstrated that fecal microbiota transplantation (FTM) from VK2-treated donor recapitulated the beneficial effects of VK2, including reduced oxidative stress and improved mitochondrial function in the liver of HFD-fed mice. Collectively, these data indicate that VK2 ameliorates MASLD via a GM-dependent mechanism.

Fig. 4.

VK2 attenuates MASLD in a GM-dependent manner. (A) Schematic of the FTM study. (B,C) H&E staining and Oil Red O staining were used to determine hepatic steatosis and lipid accumulation, and the MASLD activity score. n = 6 per group. Scale bar = 100 µm. (D,E) Hepatic TG and TC levels. n = 10 per group. (F,G) Serum ALT and AST levels. n = 10 per group. (H) Hepatic SOD2 activity was measured. n = 7 per group. (I,J) Activity of hepatic mitochondrial respiratory complex Ⅰ and Ⅳ. n = 7 per group. One-way ANOVA was used to analyze data. Significant differences were observed between the control and HFD-fed groups, and between the HFD group and the groups treated with VK2 along with antibiotics plus HFD or the FMT group. *p 0.05; **p 0.01; ***p 0.001; ****p 0.0001.

Our previous studies have revealed that VK2 can significantly alleviate insulin resistance by improving mitochondrial dysfunction in HFD-fed mice via SIRT3 signaling. To determine whether SIRT3 mediates the effect of VK2 on mitochondrial function in vivo, we knocked down the gene encoding SIRT3 in the livers of mice by delivering shRNA using adeno-associated virus 9. SIRT3 knockdown reduced the effect of VK2 on the LW/BW ratio (Fig. 5A,B), activities of AST and ALT (Fig. 5C,D), and hepatic lipid levels (TG and TC) (Fig. 5E,F). Moreover, the ATP level and SOD2 enzyme activity were not significantly improved by VK2 treatment in HFD-fed mice with SIRT3 knockdown (Fig. 5G,H). Additionally, VK2 did not enhance the mitochondrial basal respiration and spare respiratory capacity in HFD-fed mice with SIRT3 knockdown (Fig. 5I–N). VK2 treatment increased the protein expression levels of SIRT3, SOD2, and catalase in the liver tissues of HFD-fed mice (Fig. 5O,P). However, SIRT3 knockdown abolished the effect of VK2 on mitochondrial function and the protein expression of SIRT3, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1$\alpha$), SOD2 and catalase (Fig. 5Q,R). These findings revealed that SIRT3 plays a critical role in the protective effects of VK2 on MASLD.

Fig. 5.

VK2 improves mitochondrial activity through SIRT3 signaling pathway. (A,B) LW and the LW/BW ratio. (C,D) Serum ALT and AST levels. n = 10 per group. (E,F) TG and TC levels. n = 8 per group. (G,H) Hepatic ATP contents and SOD2 enzyme activity. n = 7 per group. (I–N) OCR of isolated liver mitochondria to assess mitochondrial bioenergetic. Basal respiration was measured under State 2 (I), State 3 (J), and State 3u (K) conditions. Electron transport chain complex activity was determined for complex Ⅰ (L), complex Ⅱ (M), and complex Ⅳ (N). n = 6 per group. (O,P) Protein expression of SIRT3, SOD2 and catalase were determined in all groups. n = 7 per group. (Q,R) Protein expression of SIRT3, PGC-1$\alpha$, SOD2, and catalase in control and the shSIRT3 + HFD, and the VK2-treated shSIRT3 + HFD groups. n = 6 per group. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Significant differences were observed between the control and the shSIRT3 + HFD, and the VK2-treated shSIRT3 + HFD groups. *p 0.05; **p 0.001; ***p 0.001; ****p 0.0001. PGC-1$\alpha$, peroxisome proliferator-activated receptor gamma coactivator 1-alpha.