1,25(OH)2D3 downregulates the Toll-like receptor 4-mediated inflammatory pathway and ameliorates liver injury in diabetic rats

Abstract

Background: Fatty acid deposition in the liver can activate a number of pro-inflammatory signaling pathways such as the Toll-like receptor 4 (TLR4) pathway, which may be important in the pathogenesis of nonalcoholic steatohepatitis. 1,25(OH)2D3 downregulates the expression of TLR4 and may represent a novel treatment strategy for reducing hepatocyte injury. Therefore, in this study, we investigated the protective effects of 1,25(OH)2D3 on diabetic liver injury in vivo.

Methods: Streptozotocin (STZ)-induced diabetic rats were randomly divided into five groups and treated with low-dose 1,25(OH)2D3 (0.025 μg/kg/day), medium-dose 1,25(OH)2D3 (0.15 μg/kg/day), high-dose 1,25(OH)2D3 (0.3 μg/kg/day), insulin (protamine zinc insulin 16 U/kg/day, subcutaneous injection), or no intervention (the control group). Sixteen weeks later, the rats were killed, and blood samples were obtained to test lipid profiles and hepatic function. The infiltration of inflammatory cells, the level of fibrosis, and the expression levels of TLR4, nuclear factor-kappa B (NF-κB), and tumor necrosis factor-α (TNF-α) in the liver were analyzed. The hepatocytes were treated with vehicle control, LPS (100 ng), high fat [DMEM + FFA (0.1 mM: palmitic acid, oleic acid, 1:2)], LPS + high fat, vehicle + 1,25(OH)2D3 (10(-7) M), LPS + 1,25(OH)2D3, high fat + 1,25(OH)2D3, or LPS + high fat + 1,25(OH)2D3. RNA and protein were extracted to detect the expression of TLR4 and downstream inflammatory factors such as NF-ΚB, TNF-α, and IL-6. Groups of data were compared by single factor variance analysis.

Results: High-dose 1,25(OH)2D3 administration for 16 weeks downregulated the expression of TLR4, NF-κB, and TNF-α in the liver tissue of diabetic rats and attenuated hepatic inflammation and fibrosis, as shown by immunohistochemical staining, hematoxylin and eosin staining, Masson's trichrome staining, reverse transcription polymerase chain reaction (RT-PCR), and western blotting. In vitro, hepatocytes treated with high fat or LPS exhibited significantly increased expression of TLR4, NF-κB, and downstream inflammatory factors (P < 0.05). Intervention with 1,25(OH)2D3 decreased the expression of TLR4, NF-κB, and inflammatory factors (P < 0.05).

Conclusions: 1,25(OH)2D3 exhibited protective effects against diabetes-related liver injury, possibly through downregulation of components of the TLR4 signaling pathway.

Keywords: 1,25(OH)2D3; Diabetes; Hepatocytes; Inflammation; Liver injury; Toll-like receptor 4.

Fig. 1

1,25(OH)₂D₃ exhibited protective effects in livers from diabetic rats. a H&E staining (×200) showed obvious inflammatory cell infiltration around hepatocytes and diffuse infiltration around the portal vein in diabetic rats. Infiltration of inflammatory cells with steatosis of hepatocytes was observed in diabetic rats treated with insulin. Alleviation of the infiltration of inflammatory cells was observed in diabetic rats treated with medium- and high-dose 1,25(OH)₂D₃. b Masson’s trichrome staining (×400) showed colorization around perisinusoidal spaces, cells, and the portal area, and the structure of the hepatic lobule was abnormal in diabetic rats. The level of interstitial fibrosis decreased, and the structure of the liver was intact in diabetic rats treated with high-dose 1,25(OH)₂D₃

Fig. 2

1,25(OH)₂D₃ administration downregulated TLR4, NF-κB, TNF-α, and CD68 proteins in the liver (magnification, ×200). a TLR4 staining was observed in the cytoplasm of hepatocytes and Kupffer cells in diabetic rats, with reduced staining observed in rats treated with high-dose 1,25(OH)₂D₃. b NF-κB staining was observed in the nuclei of hepatocytes and Kupffer cells in diabetic rats. However, minimal, discrete staining was observed in Kupffer cells in the hepatic sinusoids in rats treated with high-dose 1,25(OH)₂D₃. c TNF-α staining showed brown deposition on the surfaces of hepatocytes and Kupffer cells in diabetic rats. The number of positively stained cells decreased, and the staining became lighter in rats treated with high-dose 1,25(OH)₂D₃. d CD68 staining showed that there were more and larger Kupffer cells in diabetic control (untreated) rats than in rats treated with insulin or medium- or high-dose 1,25(OH)₂D₃

Fig. 3

1,25(OH)₂D₃ downregulated the expression of components of the TLR4 signaling pathway. The relative mRNA expression levels of TLR4, NF-κB, and TNF- α were measured in all groups of rats, and changes in expression relative to Group C were determined (a–c). Western blotting was used to assess the levels of TLR4, phospho-NF-κB, and TNF-α in all groups of rats (d). *Compared with Group C, P < 0.05; ^#compared with Group D, P < 0.05; ^▼compared with Group I, P < 0.05

Fig. 4

TLR4, NF-κB, IL-6, and TNF-α mRNA levels in hepatocytes. a–d The mRNA expression levels of TLR4, NF-κB, IL-6, and TNF-α were examined for the different treatment groups. e Protein expression of TLR4 and TNF-α and phosphorylation levels of NF-κB 65 were examined by western blotting