Evaluation of Serum L-FABP as a Biomarker and Hepatoprotective Effect of L-FABP Using Wild-Type and Human L-FABP Chromosome Transgenic Mice
Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are the major prevalent liver diseases and growing public health problems worldwide. Because MASLD/MASH is known as a risk for progression to cirrhosis and development of hepatocellular carcinoma, therapeutic approaches and biomarkers that reflect the presence and progression of the disease are needed. In recent years, the usefulness of serum L-FABP levels has been reported for monitoring of hepatocellular damage in various liver diseases including MASLD/MASH in humans. Furthermore, it is reported that hepatic L-FABP is a potential therapeutic target. The purpose of this study was to validate the usefulness of serum L-FABP as a liver damage biomarker in the mouse model of MASLD/MASH and to evaluate the function of L-FABP in the pathogenesis of MASLD/MASH. First, we evaluated the changes in serum L-FABP as a liver damage biomarker using a mouse model of MASLD/MASH fed a choline-deficient, methionine-lowered, amino acid-defined, high-fat diet. The results demonstrated that serum L-FABP levels in the MASLD/MASH model continuously increased with the progression of steatosis and correlated with histopathologic changes. Serum L-FABP may be a useful biomarker for liver disease with respect to translational research bridging between animal models and human clinical research. Further, we showed that in human L-FABP chromosomal transgenic mice L-FABP had a suppressive effect on the gene expression associated with oxidative stress, fibrosis, and inflammation in the MASLD/MASH model. L-FABP is not only a biomarker in the blood but also has the functional aspect of hepatoprotection against MASLD/MASH.
Introduction
Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are liver diseases associated with comorbidities such as obesity, diabetes mellitus, and metabolic syndrome. MASLD is characterized primarily by steatosis, which further progresses to MASH with hepatocellular damage, inflammation, and fibrosis. MASLD/MASH are major prevalent liver diseases, and represent growing public health problems worldwide. The prevalence of MASLD/MASH and related mortality are expected to increase at least until 2030, globally.1 Further, MASLD/MASH is known as a risk for progression to cirrhosis and development of hepatocellular carcinoma (HCC), and new therapeutic approaches, such as resmetirom, which was recently approved by the FDA, are needed.2
Biomarkers that can reflect the presence and progression of liver disease are important in drug development as indicators of the pharmacological response to therapeutic intervention. Liver-type fatty acid binding protein (L-FABP) is a 14-kDa soluble protein abundantly found in the liver.3 L-FABP is an important factor for fatty acid uptake and intracellular transport, and it also has an important role as an endogenous cytoprotectant in reducing hepatocyte damage induced by oxidative stress.4,5 In humans, serum L-FABP levels are used for monitoring fibrosis and hepatocellular damage during MASLD/MASH,6–8 infection with hepatitis C virus,9 and liver surgery.10,11 L-FABP is suggested as a useful biomarker in screening for drug-induced liver injury, including that induced by acetaminophen.12,13 Furthermore, serum L-FABP has possibile utility as a prognostic factor in chronic liver diseases ranging from chronic hepatitis to cirrhosis and HCC patients.14,15 However, there are few research reports on the changes in L-FABP in the blood as a biomarker of liver injury in animals compared with humans.16–18 The changes in serum L-FABP as a biomarker in MASLD/MASH using animal models and the functional utility of L-FABP in MASLD/MASH in vivo have not been well studied.
In this study, we evaluated the changes in serum L-FABP using a wild-type (WT) mouse model of MASLD/MASH fed a choline-deficient, methionine-lowered, L-amino acid-defined, high-fat diet (CDAA-HFD),19,20 with respect to translational research designed to bridge between animal model and human clinical research. The mouse model fed CDAA-HFD develops steatosis, steatohepatitis, and hepatic fibrosis; and long-term feeding leads to disease progression toward HCC. Thus, CDAA-HFD can be used to produce the MASLD/MASH mouse model.19,20
In addition to WT mice, we used human L-FABP chromosomal transgenic (hL-FABP Tg) mice, which have human L-FABP genomic DNA including its promoter region (hL-FABP is not forced expression in these mice).21 The hL-FABP Tg mouse was originally developed to evaluate the utility of urinary L-FABP as a renal disease biomarker because L-FABP expression is suppressed in the kidneys of WT mice, making it difficult to evaluate as a renal disease biomarker.21 Since the hL-FABP Tg mouse was first reported almost 20 y ago, it has been used in many studies evaluating the utility of urinary L-FABP and has formed the scientific basis for the utility of urinary L-FABP as a marker of renal disease.21–25 hL-FABP Tg mice express human L-FABP in the liver as well as the kidney.21 That is, it is an internal environment in which both mouse L-FABP and human L-FABP are expressed in the liver of hL-FABP Tg mice, unlike in the kidney. In basic science, L-FABP knockout mice are frequently used in studies evaluating the role of L-FABP in MASLD/MASH,26 and some studies27,28 reported that high-fat diet-induced hepatic steatosis was attenuated in L-FABP knockout mice. However, the limitation of L-FABP knockout mice is that it is difficult to evaluate the function of L-FABP after the MASLD/MASH pathology is established, because the knocking out of L-FABP, which is involved in fatty acid uptake and transport, can suppress pathogenesis itself. In the present study, we fed CDAA-HFD to hL-FABP Tg mice to induce MASLD/MASH and compared the change in biomarkers and gene expression levels with those of WT mice to examine the add-on effect of L-FABP.
Materials and Methods
Animals.
C57BL/6 mice (n = 28) were purchased from Japan SLC (Shizuoka, Japan). C57BL/6 background human L-FABP chromosomal transgenic (hL-FABP Tg) mice (n = 28) were generated as described previously.21 Seven-week-old male mice were used for the study. The breeding colony was determined to be free from Helicobacter hepaticus, Pseudomonas aeruginosa, Citrobacter rodentium, Salmonella spp., Salmonella typhimurium, Pasteurella pneumotropica, Corynebacterium kutscheri, Clostridium piliforme, Mycoplasma pulmonis, mouse hepatitis virus, Sendai virus, intestinal protozoa, ectoparasites, and pinworm. The microbiologic tests were conducted using various detection methods including PCR, bacterial culture, agglutination test, enzyme-linked immunosorbent assay, and microscopy. These mice were fed CDAA-HFD consisting of 45 kcal% fat and 0.1% methionine by weight (A06071309; Research Diets) for 2 (n = 4), 13 (n = 5), and 26 (n = 5) weeks, and a standard diet (SD; CE-2; CLEA Japan) was fed for 2 (n = 4), 13 (n = 5), and 26 (n = 5) weeks to a control group. Mice were randomly assigned to the treatment groups. Food consumption was measured once per week. The mice were maintained at 23 ± 3 °C on a 12-h/12-h light-dark cycle with ad libitum access to these diets and tap water. This study was approved by the IACUC of the Tokyo University of Agriculture (approval no. 300050). All the experiments were performed following the guidelines of the Tokyo University of Agriculture, Japan, which are equivalent to those described in the Guide for the Care and Use of Laboratory Animals.29
Measurement of biologic parameters.
Blood samples were collected from the abdominal aorta of mice euthanized by isoflurane overdose, and urine samples were collected using a metabolic cage before each day of euthanasia at 2, 13, and 26 wk to determine the value of biomarkers, such as ALT, AST, serum L-FABP, urinary L-FABP, and urinary creatinine. These biomarkers were measured using commercial kits [ALT and AST: DRI CHEM NX500V (Fujifilm); serum mouse L-FABP: Mouse/Rat FABP1/L-FABP Quantikine ELISA Kit (R&D Systems); serum human L-FABP and urinary human L-FABP: High Sensitivity Human L-FABP ELISA Kit (CMIC Holdings); and urinary creatinine: Creatinine Assay Kit QuantiChrom (BioAssay Systems)].
mRNA quantification with real-time quantitative PCR.
The liver tissues were collected from euthanized mice at 2, 13, and 26 wk, and these were immediately quick-frozen with liquid nitrogen for mRNA analyses using quantitative RT-PCR. These liver tissues were stored at −80 °C until RNA extraction. Total RNA was extracted and purified using Sepasol-RNA I Super G (Nacalai Tesque) according to the manufacturer’s instructions. Reverse transcription and cDNA generation were performed with extracted total RNA and PrimeScript RT Master Mix (Takara Bio). Quantitative RT-PCR was performed with TB Green PremixEx Taq II (Takara Bio) using a Thermal Cycler Dice Real-Time System (Takara Bio). Primers were as follows: collagen type-1a (collagen-1a): forward 5′-GAACTGGACTGTCCCAACCC-3′ and reverse 5′-CTTGGGTCCCTCGACTCCTA-3′; transforming growth factor-β (TGF-β): forward 5′-GCAACATGTGGAACTCTACCAGA-3′ and reverse 5′-GACGTCAAAAGACAGCCACTCA-3′; P67phox: forward 5′-CTGGCTGAGGCCATCAGACT-3′ and reverse 5′-AGGCCACTGCAGAGTGCTTG-3′; monocyte chemoattractant protein-1 (MCP-1): forward 5′-CCCACTCACCTGCTGCTACT-3′ and reverse 5′-ATTTGGTTCCGATCCAGGTT-3′; and TNF-α: forward 5′-TCGTAGCAAACCACCAAGTG-3′ and reverse 5′-AGATAGCAAATCGGCTGACG-3′. The expression level of each gene was normalized to the mRNA levels of genes encoding cyclophilin: forward 5′-TGGCTCACAGTTCTTCATAACCA-3′ and reverse 5′-ATGACATCCAGTGGCTTGTC-3′.
Histology and immunohistochemistry.
For histopathologic analysis, the livers and kidneys were immediately fixed in 10% paraformaldehyde. After resection, the tissues were paraffin-embedded by standard techniques and sectioned (4 µm). The sections of the liver were stained with hematoxylin and eosin or Sirius red. Immunohistochemistry was performed for human L-FABP using anti-human L-FABP mouse monoclonal antibody (CMIC Holdings). The samples were all examined histopathologically in a blind manner, and the findings were graded from normal to severe; normal (0), slight change (0.5+; the histologic change was observed in 1% to 5% area in a tissue section), mild (1+; the histologic change was observed in 6% to 33% area of the tissue section), moderate (2+; the histologic change was observed in 34% to 66% area of the tissue section), and severe (3+; the histologic change was observed in 67% to 100% area of the tissue section). The MASLD activity score was calculated by adding the values of fatty change score, hepatocyte hypertrophy score, and inflammatory cell infiltration score. The histopathologic specimens were examined by Katsuhiro Miyajima, who is a member of the Japanese College of Veterinary Pathologists and is a diplomate of the Japanese Society of Toxicologic Pathology.
Statistical analysis.
Data analysis was expressed as the mean ± SE. Differences among each level of urinary L-FABP in the same group were analyzed by the Steel-Dwass test. The Mann-Whitney U test was used for comparisons between the 2 groups. The correlation was determined using single regression analysis or Spearman rank-sum test. The statistical analyses were performed using SatFlex, version 7 (Artech). Differences were defined as significant at P < 0.05. A priori power analysis was performed using G*Power 3.1.9.7. Assuming a 2-tailed test for the population correlation coefficient with an expected correlation coefficient of 0.5, a significance level of 5%, and a power of 80%, the required sample size was calculated to be 26 mice.
Results
Histopathological changes in CDAA-HFD- or SD-fed mice.
Liver tissues stained with hematoxylin and eosin and Sirius red staining were observed by light microscopy, and both WT and Tg mice showed characteristic and significant histologic changes associated with MASLD/MASH after CDAA-HFD feeding (Figure 1A). Fatty change score, hepatocyte hypertrophy score, fibrosis score, inflammatory cell infiltration, and MASLD activity score increased in the CDAA-HFD group and were significantly higher than in the SD group (Table 1). No significant differences in food intake were observed between WT and hL-FABP Tg mice (Figure 1B). Nodular regenerative hyperplasia was observed in one hL-FABP Tg mouse fed CDAA-HFD in this study, and it was excluded from the data.
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-003
| 2 wk | 13 wk | 26 wk | ||||
|---|---|---|---|---|---|---|
| SD | CDAA-HFD | SD | CDAA-HFD | SD | CDAA-HFD | |
| WT | ||||||
| Fatty change score | 0.00 ± 0.000 | 2.25 ± 0.479* | 0.00 ± 0.000 | 2.60 ± 0.400** | 0.00 ± 0.000 | 2.60 ± 0.245** |
| Hepatocyte hypertrophy score | 0.00 ± 0.000 | 0.50 ± 0.204 | 0.00 ± 0.000 | 1.00 ± 0.000** | 0.10 ± 0.100 | 1.00 ± 0.000** |
| Fibrosis score | 0.00 ± 0.000 | 0.38 ± 0.125 | 0.00 ± 0.000 | 1.40 ± 0.245** | 0.20 ± 0.122 | 2.60 ± 0.400** |
| Inflammatory cell infiltration | 0.00 ± 0.000 | 0.38 ± 0.125 | 0.00 ± 0.000 | 1.10 ± 0.245** | 0.00 ± 0.000 | 2.40 ± 0.400** |
| MASLD activity score | 0.00 ± 0.000 | 3.13 ± 0.774* | 0.00 ± 0.000 | 4.70 ± 0.200** | 0.10 ± 0.100 | 6.00 ± 0.316** |
| hL-FABP Tg | ||||||
| Fatty change score | 0.00 ± 0.000 | 2.50 ± 0.580* | 0.00 ± 0.000 | 2.40 ± 0.550** | 0.10 ± 0.200 | 2.40 ± 0.800** |
| Hepatocyte hypertrophy score | 0.00 ± 0.000 | 0.63 ± 0.480 | 0.00 ± 0.000 | 0.80 ± 0.270** | 0.10 ± 0.200 | 0.70 ± 0.240* |
| Fibrosis score | 0.00 ± 0.000 | 0.13 ± 0.250 | 0.00 ± 0.000 | 2.00 ± 0.000** | 0.00 ± 0.000 | 1.80 ± 0.750** |
| Inflammatory cell infiltration | 0.00 ± 0.000 | 0.63 ± 0.250* | 0.00 ± 0.000 | 1.60 ± 0.550** | 0.00 ± 0.000 | 1.80 ± 0.750** |
| MASLD activity score | 0.00 ± 0.000 | 3.75 ± 0.479* | 0.00 ± 0.000 | 5.60 ± 0.400** | 0.20 ± 0.200 | 4.90 ± 0.367** |
Values are average ± SE. *, P ≤ 0.05; **, P ≤ 0.01.
Serum mouse L-FABP and human L-FABP levels in CDAA-HFD- or SD-fed mice.
Serum mouse L-FABP levels significantly increased in CDAA-HFD-fed WT mice compared with SD-fed mice and remained elevated throughout the study period (Figure 2A). Further, serum mouse L-FABP levels significantly increased in CDAA-HFD-fed hL-FABP Tg mice compared with SD-fed mice and remained elevated throughout the study period, same as WT (Figure 2B); and serum human L-FABP levels significantly increased in CDAA-HFD-fed hL-FABP Tg mice and remained high throughout the study period (Figure 2C). These values of L-FABP were significantly correlated with MASLD activity score (WT-mouse L-FABP: r = 0.7503, P ≤ 0.01; hL-FABP Tg-mouse L-FABP: r = 0.7834, P ≤ 0.01; hL-FABP Tg-human L-FABP: r = 0.811, P ≤ 0.01) (Figure 2D–F). The correlation of serum L-FABPs was especially high with fatty change score (WT-mouse L-FABP: r = 0.783, P ≤ 0.01; hL-FABP Tg-mouse L-FABP: r = 0.8556, P ≤ 0.01; hL-FABP Tg-human L-FABP: r = 0.8549, P ≤ 0.01) compared with hepatocyte hypertrophy score, fibrosis score and inflammatory cell infiltration score in each group (Figure 3A–L). For immunohistochemical staining of L-FABP, the protein expression distributed surrounding the portal vein of the liver in SD-fed mice. In contrast, the distribution of L-FABP in CDAA-HFD-fed mice was localized around lipid droplets (Figure 4). Urinary human L-FABP levels remained low throughout the experiment period differing from serum L-FABP levels (Figure 5A). Histopathological changes in the kidney in CDAA-HFD-fed mice were not observed (Figure 5B).
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-003
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-003
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-003
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-003
Changes of serum ALT and AST as traditional biomarkers for liver injury in CDAA-HFD- or SD-fed mice.
Serum ALT and AST levels were significantly higher in CDAA-HFD-fed WT mice and hL-FABP Tg mice than the levels in SD-fed mice (Figure 6A and B; P ≤ 0.05). Notably, ALT and AST levels decreased after 13 wk differing from serum L-FABP levels, and the decrease in the levels of ALT was marked (Figure 6A and B; P ≤ 0.05 compared with the values at 2 wk on each strain). In comparison between WT mice and hL-FABP Tg mice fed CDAA-HFD, serum ALT and AST levels were significantly suppressed in hL-FABP Tg mice compared with WT mice at 2 and 26 wk (Figure 6A and B; P ≤ 0.05).
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-003
The change in mRNA expression in CDAA-HFD- or SD-fed mice.
The mRNA expression of p67 phox (oxidative stress marker), TGF-β and collagen-1α (fibrosis markers), and TNF-α and MCP-1 (inflammation markers) in CDAA-HFD-fed mice were significantly higher than in SD-fed mice (Figure 6C–G; P ≤ 0.05). The mRNA expression of p67 phox and TGF-β in CDAA-HFD-fed hL-FABP Tg mice were significantly lower at 2 wk than in CDAA-HFD-fed WT mice (Figure 6C and D; P ≤ 0.05). TNF-α in CDAA-HFD-fed hL-FABP Tg mice was significantly lower at 13 wk than in CDAA-HFD-fed WT mice (Figure 6E; P ≤ 0.05). MCP-1 and collagen-1α levels in CDAA-HFD-fed hL-FABP Tg mice tended to be lower at 2 and 26 wk (Figure 6F and G; P ≤ 0.05).
Discussion
In the study described here, we found that feeding CDAA-HFD to WT mice resulted in a significant increase in pathologic histologic change scores consistent with MASLD/MASH. Mouse L-FABP levels in the blood as a biomarker remained significantly higher than those in the SD-fed group throughout the study period. Furthermore, mouse L-FABP levels in the blood showed a significant correlation with fatty change score, hepatocyte hypertrophy score, fibrosis score, inflammatory cell infiltration, and MASLD activity score. In particular, the correlation coefficient with the fatty change score was the highest. The accumulation of lipids is one of the main causes of oxidative stress and inflammation leading to hepatic steatosis.30 In contrast, ALT and AST transiently increased, with a peak at 2 wk, but then decreased, unlike L-FABP. The decrease in ALT was particularly marked after 13 wk. Although ALT and AST are frequently used as biomarkers of liver disease, it is well known in humans that ALT and AST decrease at the end stage of liver disease; thus, ALS and AST are unsuitable for long-term monitoring of patients with liver disease.15 Our results demonstrate that serum L-FABP is a biomarker reflective of histologic changes typical of MASLD/MASH, with elevated levels through the study period in the mouse model, establishing consistency with changes reported in humans.6–8,14 These results suggest an advantage of serum L-FABP as a biomarker compared with ALT and AST.
We found that feeding CDAA-HFD to hL-FABP Tg mice resulted in persistently elevated mouse L-FABP blood levels throughout the study period with an increase similar to that observed in WT mice. Pathologic evaluation demonstrated a significant correlation between mouse blood L-FABP level in hL-FABP Tg mice with scores associated with MASLD/MASH. As with WT mice, the correlation coefficient was highest with the fatty change score. In addition to mouse L-FABP, hL-FABP Tg mice showed a significant increase in human L-FABP levels in the blood, which remained elevated throughout the study period, and these also showed a significant relationship with scores associated with MASLD/MASH. Notably, there were no obvious lesions in the kidney and no significant increase in urinary L-FABP levels, suggesting that increased serum L-FABP levels are not associated with the changes in urinary L-FABP in this model.
The expression of human L-FABP in liver tissues was confirmed by immunostaining using anti-human L-FABP antibody, and L-FABP positivity was observed only in hL-FABP Tg mice. These data indicate that the human L-FABP ELISA kit can specifically measure only human L-FABP. L-FABP was distributed around the portal vein in the SD-fed group, the same as in previous reports.31 In the CDAA-HFD-fed group, the distribution area of L-FABP changed in localization to surround fat droplets. L-FABP is known to have fatty acid transport and lipid metabolism functions, antioxidant activity, and an important role in VLDL secretion,4,5,26,32 but the mechanism by which L-FABP is secreted into the blood from the liver cells has not been determined. Intracellular L-FABP in the proximal tubules of the kidney has been reported across species to be released with cytotoxic lipids into the tubular lumen subsequent to ischemia and oxidative stress,33–35 and it was speculated that such a mechanism may contribute to increased L-FABP levels in the blood with the progression of liver disease.
Interestingly, the liver injury markers ALT and AST tended to be lower in hL-FABP Tg mice compared with WT mice in this study, and the difference was significant at 2 and 26 wk. To explore the factors contributing to the differences in ALT and AST between WT and hL-FABP Tg mice, we examined gene expression and found a significant decrease in P67 phox and TGF-β at 2 wk and a significant decrease in TNF-α at 13 wk. L-FABP has been reported to have hepatoprotective effects due to its antioxidant properties.4,5 Further, we previously showed L-FABP to be protective in the kidney.23,24,35 Our results in this study may indicate that L-FABP has the ability to suppress the degree of oxidative stress, fibrosis, and inflammation at least in the early to midstage of MASLD/MASH pathogenesis. Recently, attempts to suppress L-FABP expression at an early stage of MASLD/MASH to reduce disease progression have been reported,26,36,37 but this may lead to loss of the antioxidant and anti-inflammatory effects of L-FABP. In the initiation of L-FABP-targeted therapy in MASLD/MASH, careful identification of eligible patients may be necessary to determine the optimal timing for maximizing the therapeutic effect. In the future, the combination of L-FABP and other biomarkers may find use as diagnostic tools to determine when to initiate treatment with therapeutic agents.
Although nodular regenerative hyperplasia was observed in one hL-FABP Tg mouse fed CDAA-HFD that was excluded from the analysis, the mouse showed a very high blood L-FABP level (serum mouse L-FABP: 969.1 ng/mL; serum human L-FABP: 495.8 ng/mL; urinary L-FABP: 3.4 μg/g Cre). L-FABP levels in the blood have been reported to be elevated in patients with hepatocellular carcinoma,14,15 and the mouse with nodular regenerative hyperplasia may reflect such a change. In addition, it was reported that L-FABP promotes angiogenesis and migration of hepatocellular carcinoma.38 Further studies using hL-FABP Tg mice to focus on carcinoma may help further define this process.
In conclusion, serum L-FABP levels in WT mice were shown to be a biomarker reflective of tissue changes consistent with MASLD/MASH, similar to that reported in humans as an aspect of translational study.6–8,14 In hL-FABP Tg mice, both mouse and human L-FABP in the blood reflected histologic changes, and L-FABP was shown to have a suppressive effect on the gene expression associated with oxidative stress, fibrosis, and inflammation in the MASLD/MASH model. However, this is the first report of a liver disease model using hL-FABP Tg mice, and further analysis is required to determine the add-on effects of L-FABP. In addition, the function of human L-FABP in lipid metabolism in mice needs to be evaluated. Continued work is needed to evaluate not only the usefulness of serum L-FABP as a biomarker but also its utility in the preclinical evaluation of therapeutic drugs targeting human L-FABP in various liver disease models using hL-FABP Tg mice.
(A) Histopathological changes in liver tissues of WT and hL-FABP Tg mice after CDAA-HFD or SD feeding for 26 wk. (SR, Sirius red staining; HE, hematoxylin and eosin staining). Steatosis, fibrosis, hepatocyte hypertrophy, and inflammatory cell infiltration were observed in both WT and hL-FABP Tg mice after CDAA-HFD feeding. Bar = 100 μm. (B) Changes in food consumption in CDAA-HFD or SD fed mice (w, week).
Changes in serum L-FABP levels: (A) WT-serum mouse L-FABP; (B) hL-FABP Tg-serum mouse L-FABP; (C) hL-FABP Tg-serum human L-FABP. Data of CDAA-HFD fed mice are shown in the gray boxes, and data of SD fed mice are shown in the white boxes (W, week). Correlation of serum L-FABPs with MASLD activity score: (D) WT-serum mouse L-FABP; (E) hL-FABP Tg-serum mouse L-FABP; (F) hL-FABP Tg-serum human L-FABP. Lines in the figure indicate regression lines and the CI. *, P ≤ 0.05; **, P ≤ 0.01.
Correlation of serum L-FABPs with fatty change score, hepatocyte hypertrophy score, fibrosis score, and inflammatory cell infiltration: (A–D) WT-serum mouse L-FABP; (E–H) hL-FABP Tg-serum mouse L-FABP; (I–L) hL-FABP Tg-serum human L-FABP. Lines in the figure indicate regression lines and the CI.
Immunohistological staining of liver tissues at 26 wk using anti-human L-FABP antibody in CDAA-HFD or SD fed hL-FABP Tg mice. Bar = 100 μm.
(A) Changes in urinary human L-FABP in CDAA-HFD- or SD-fed hL-FABP Tg mice. (B) Histopathological changes in kidney tissues of hL-FABP Tg mice after CDAA-HFD or SD feeding for 26 wk (H&E staining). Bar = 100 μm.
Changes in ALT and AST in serum in CDAA-HFD- or SD-fed WT and hL-FABP Tg mice: (A) ALT; (B) AST. Changes in gene expression in the liver in CDAA-HFD- or SD-fed WT and hL-FABP Tg mice: (C) p67 phox; (D) TGF-β; (E) TNF-α; (F) MCP-1; (G) collagen-1a. Data of hL-FABP Tg mice are shown in the gray boxes, and data of WT mice are shown in the white boxes. *, P ≤ 0.05 WT compared with hL-FABP Tg mice. †, P ≤ 0.05, compared with SD fed mice. §, P ≤ 0.05, compared with the values at 2 wk for each strain.
Contributor Notes
These authors contributed equally to this study.