1,25-dihydroxyvitamin D3 improves non-alcoholic steatohepatitis phenotype in a diet-induced rat model

Abstract

We studied the potential protective effects of 1,25-dihydroxyvitamin D3 (1,25 VD3) supplementation on liver damage induced by a choline-deficient (CD) diet in rats, where impaired liver function leads to decreased 25-hydroxyvitamin D3 levels, the precursor for the active 1,25 VD3. The CD diet reduced serum 25 VD3 levels and increased liver enzymes, indicative of liver damage. Conversely, 1,25 VD3 supplementation alleviated liver damage, reducing liver enzymes and improving histopathological features characteristic of non-alcoholic steatohepatitis (NASH). Oxidative stress and inflammation were mitigated by 1,25 VD3, as evidenced by decreased malondialdehyde and nuclear factor kappa B (NF-κB) expression, and increased total antioxidant capacity (TAOC). 1,25 VD3 also enhanced fatty acid metabolism by increasing peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase-1 (CPT-1) expression, promoting lipid transport and oxidation. Additionally, 1,25 VD3 supplementation modulated inflammation by increasing PPARγ expression, reducing NF-κB expression, and decreasing pro-inflammatory cytokines (TNF-α, IL-1β). Anti-inflammatory cytokines (IL-10, IL-4) were increased, and macrophage polarization was shifted towards an anti-inflammatory M2 phenotype. Moreover, 1,25 VD3 upregulated CYP2J3, a cytochrome P450 epoxygenase that converts arachidonic acid to anti-inflammatory epoxyeicosatrienoic acids (EETs) and decreased soluble epoxide hydrolase activity, likely contributing to increased EET levels. Correlation studies revealed positive associations between 1,25 VD3 supplementation, CYP2J3 expression, EETs, as well as negative correlations with NF-κB and TNF-α. PPARα expression positively correlated with TAOC and CPT-1, while PPARγ expression negatively correlated with inflammatory markers. These findings demonstrate the therapeutic potential of 1,25 VD3 in alleviating NASH through regulation of fatty acid metabolism, inflammation, and oxidative stress.

Keywords: CYP450; inflammation; macrophage; non-alcoholic steatohepatitis; vitamin D3.

Figure 1

A schematic diagram representing study design.

Figure 2

Effects of 1,25 VD3 supplementation on serum biomarkers and liver histopathology in a rat model of NASH. (A) Serum ALT and (B) AST activities were measured using Automatic Biochemical Analyzer. (C) 25VD₃ levels were measured using Roche Electrochemiluminescence method in the indicated groups. (D) Representative H&E-stained liver sections (5 μM) from the indicated groups. (E) Histopathological scores for steatosis, lobular inflammation, ballooning degeneration, activity, and SAF scores were determined following the protocol as described in Table 1 . Data are presented as mean ± SEM, n=6. Statistical significance: A-C - ***p < 0.001 vs. CG; ##p < 0.01, ###p < 0.001 vs. CDG; +++p < 0.001 vs. CDVDG. D-E - ***p <0.001 vs. CG; ###p < 0.001 vs. CDG; ++p < 0.01, +++p < 0.001 vs. CDG. CG: Normal rat chow diet; CVDG: Normal diet + 1,25 VD3; CDG: Choline-deficient diet; CDVDG: Choline-deficient diet + 1,25 VD3.

Figure 3

Effects of 1,25 VD3 supplementation on hepatic lipid peroxidation, inflammation and fatty acid metabolizing genes in NASH rats. To assess oxidative stress, hepatic (A) MDA was measured using a thiobarbituric acid kit and (B) TAOC levels measured using the FRAP method. (C) NF-κB expression in liver tissue, analyzed by Western blotting and normalized to β-actin (left panel showing representative blot and right panel showing quantification by densitometry of 65 kDa band). Antibodies used and their dilutions: primary antibodies against NF-κB (anti-rabbit, 1:1000) and β-actin (anti-rabbit, 1:1000), followed by HRP-conjugated secondary antibody (1:2000). (D) PPARα and (E) CPT-1 mRNA levels were quantified by qPCR in liver tissues of indicated groups, and expressed as relative mRNA levels following normalization to GAPDH mRNA levels. Data are presented as mean ± SEM, n=6. *p < 0.05, **p<0.01 and ***p < 0.001 vs CG; ##p < 0.01 and ###p < 0.001 vs CDG.

Figure 4

Effects of 1,25 VD3 supplementation on macrophage polarization and PPARγ expression in NASH rats. (A) M1-type KCs identified by double immunofluorescence staining; green arrows indicate CD68-positive cells, red arrows indicate CD11c-positive cells, and yellow arrows indicate CD11c/CD68 double-positive cells (M1KCs). (B) M2-type KCs identified by double immunofluorescence staining; green arrows indicate CD68-positive cells, red arrows indicate CD163-positive cells, and yellow arrows indicate CD163/CD68 double-positive cells (M2KCs). Antibodies were diluted as follows: anti-CD68 (1:100), anti-CD11c (1:100), and anti-CD163 (1:200). (C) The M1/M2 macrophage ratio was determined by quantifying the proportion of M1 and M2 KCs in liver tissue, as identified through double immunofluorescence staining. (D) PPARγ levels in liver tissue of the indicated groups were determined by qPCR and expressed as relative mRNA levels following normalization to GAPDH mRNA levels. Data are presented as mean ± SEM, n=6. *p < 0.05 and ***p < 0.001 vs CG; ##p < 0.01 and ###p < 0.001 vs CDG.

Figure 5

Effects of 1,25 VD3 supplementation on hepatic cytokine production in NASH rats. The proinflammatory, (A) TNF-α and (B) IL-1β levels, and the anti-inflammatory (C) IL-10 and (D) IL-4 levels in liver tissue lysates of indicated groups were measured using specific ELISA kits. Data are presented as mean ± SEM, n=6. *p,0.05 and ***p < 0.001 vs CG; #p < 0.05 and ###p < 0.001 vs CDG; +++p < 0.001 vs CG.

Figure 6

Effects of 1,25 VD3 supplementation on CYP450 and COX-2 pathways in liver of NASH rats. (A) CYP2J3 mRNA level was quantified by qPCR and expressed as relative mRNA levels following normalization to GAPDH mRNA levels. LC-MS/MS was used to measure (B) EETs, (C) sEH, and (D) DHETs levelss in livers of indicated groups. Data are presented as mean ± SEM, n=6. *p < 0.05 and **p < 0.01 vs CG; ###p < 0.001 vs CDG; ++p < 0.01.