| Objective:Non-alcoholic steatohepatitis (NASH) is recognized as the most severe form of non-alcoholic fatty liver disease (NAFLD), with likely progression to liver cirrhosis and hepatocellular carcinoma. It has been reported that Vitamin D3 supplementation ameliorated NAFLD/NASH progression in high fat diet, Thioacetamide and choline-deficient and iron-supplemented amino acid-defined diet induced NAFLD/NASH model. However, it is unclear whether exogenous 1,25(OH)2D3 (1,25-VD3) plays a role in NASH induced by choline deficient amino acid-defined (CDAA) diet. In this study, we investigated the protecting effect of different doses of 1,25-VD3 on the CDAA-diet induced NASH rat and their potential mechanisms.Methods:Six-week-old male Wistar rats (150-180 g) were randomly assigned into three groups:a control group (CG, n= 5) with adequate level of dietary choline content, a choline deficient group (CDG, n-5) which was kept on CDAA-diet, and a 1,25-VD3 supplement group (CDVDG, n= 15) with CDAA-diet plus 1,25-VD3 injection(IP). The animals in CDVDG group were randomly assigned into three subgroups:CDVDG1 (n= 5), CDVDG5 (n= 5) and CDVDG10 (n= 5), and subjected to an IP of 1,25-VD3 with a dosage of 1,5, or 10 μg/kg of body weight, respectively, twice a week (Tuesdays and Saturdays).The same amount of saline (10 mL/kg) was administrated by IP into the rats in the CG and CDG groups. At the end of the experimental period (12 weeks), the rats were fasted for 12-13 h and then weighed. Sacrificed under anesthesia with ether, blood was withdrawn by abdominal aorta. The entire liver was dissected out and weighed, and calculated the liver index. The serum levels of Alanine aminotransferase (ALT), Aspartate transaminase (AST), Alkaline phosphatase (ALP), Triglyceride (TG), Total cholesterol (TC), High density lipoprotein-cholesterol (HDL-C), Low density lipoprotein-cholesterol (LDL-C), Calcium(Ca), Phosphate(P), Blood urea nitrogen (BUN), Creatinine (CREA), Leptin (LEP), Cytokeratin (CK)18-M30,25(OH)D3, Fasting blood-glucose (FBG) and Fasting insulin (FINS) were determined, and calculated the Homeostasis model of assessment for insulin resistance index (HOMA-IR). The hepatic levels of Free fatty acid (FFA), TG, Thiobarbituric acid reactive substances (TBARS), Total antioxidant capacity (TAOC) and CK18-M30 were determined Hepatic histology was examined by Haematoxylin/eosin (H&E) and Sirius red (SR) staining, and evaluated the Nonalcoholic fatty liver disease activity score (NAS) and fibrotic score. The hepatic expression of levels of a-smooth muscle actin (a-SMA) and Transforming growth factor beta (TGF-β)-1 were determined by immunohistochemical staining, the hepatocyte apoptosis was determined by TUNEL staining, and the hepatic expression of levels of Tissue inhibitor of metalloproteinase (TIMP)-1 and Vitamin D receptor (VDR) were determined by Western-blot. Dose response curves of 1,25-VD3 were performed and were analysed by correlation analyses.Results:Compared to the CG group, CDG group rats showed that the liver index (CDG vs CG:3.48±0.26% vs 2.72±0.13%,p<0.01) and the liver weight (CDG vs CG: 15.80±0.96 g vs 12.60±0.18 g,p<0.05) were significantly increased; the serum levels of ALT (CDG vs CG:67.44±1.57 mmol/L vs 43.01±3.13 mmol/L,p<0.01) and AST (CDG vs CG:209.41±4.59 mmol/L vs 96.20±1.36 mmol/L, p<0.01) were significantly increased; the serum level of CHO (CDG vs CG:1.88±0.00 mmol/L vs 1.58±0.00 mmol/L,p<0.05) was increased; NAS (CDG vs CG:7.40±0.40 vs 0.00±0.00, p<0.01) and fibrosis scores (CDG vs CG:0.60±0.25 vs 0.00±0.00,p<0.01) were significantly increased; the hepatic levels of FFA (CDG vs CG:456.61±3.70 mmol/L/mg Liver vs 332.98±2.55 mmol/L/mg Liver,p<0.01) and TG (CDG vs CG: 1.29±0.00 mmol/L/mg Liver vs 0.37±0.04 mmol/L/mg Liver, p<0.01) were significantly increased; the serum levels of FINS (CDG vs CG:29.50±0.34 μIU/mL vs 26.49Π0.76 μIU/mL,p<0.05) and HOMA-IR (CDG vs CG:11.07±0.20 vs 9.07±0.34, p<0.05) were increased; the serum level of LEP (CDG vs CG:569.71±38.97 ng/L vs 408.24±43.71 ng/L,p<0.01) was increased; the hepatic levels of TBARS(CDG vs CG: 233.46±1.03 pg/mL/mg Liver vs 98.59±1.49 pg/mL/mg Liver, p<0.01) and TAOC (CDG vs CG:1.10±0.04 mmol/mg Liver vs 0.86±0.02 mmol/mg Liver, p<0.05) were significantly increased; the TUNEL positive cells (CDG vs CG:36.80±1.83 vs 3.60±0.51,p<0.01) were significantly increased; the serum level of CK18-M30 (CDG vs CG:179.45±15.01 ng/L vs 97.15±4.89 ng/L,p<0.01) was significantly elevated; the hepatic level of CK18-M30 (CDG vs CG:9.05±0.43μg/L/mg Liver vs 15.86±0.12 μg/L/mg Liver, p<0.01) was significantly down-regulated; the hepatic expression of levels of a-SMA (CDG vs CG:45.40±2.42 vs 2.80±0.25, p<0.01), TGF-β1 (CDG vs CG:62.80±2.92 vs 18.80±1.39, p<0.01) and TIMP-1 (CDG vs CG:2.69±0.02 vs 1.51±0.02,P<0.01) were significantly increased; the serum level of 25(OH)D3 (CDG vs CG:253.72±4.10 μg/L vs 416.51±2.65 μg/L,p<0.01) and the hepatic expression of level of VDR (CDG vs CG:2.07±0.01 vs 3.66±0.11,p<0.01) were significantly decreased.Compared to the CDG group, the liver index (CDVDG5 vs CDG:2.95±0.13% vs 3.48±0.11%,p<0.05) was decreased in CDVDG5 group; the serum levels of ALT (CDVDG1,5,10 vs CDG:54.63±2.25 mmol/L,42.83±4.45 mmol/L,47.82±3.92 mmol/L vs 67.44±1.57 mmol/L,p<0.05,p<0.011,p<0.01) and AST (CDVDG1,5,10 vs CDG:182.04±3.02 mmol/L,148.01±2.21 mmol/L,148.42±6.41 mmol/L vs 209.41 ±4.59 mmol/L, all p<0.01) were significantly decreased in CDVDG1,5 and 10 group; the serum level of TG (CDVDG5 vs CDG:1.63±0.03 mmol/L vs 0.71±0.00 mmol/L, p<0.01) was elevated and the level of FBG (CDVDG5 vs CDG:7.70±0.03 mmol/L vs 8.44±0.11 mmol/L,p<0.05) was decreased in CDVDG5 group; the NAS (CDVDG5 vs CDG:5.20±0.20 vs 7.40±0.40,p<0.01) was significantly decreased in CDVDG5 group, fibrosis scores were 0 in CDVDG1,5 and 10 group, was 0.60±0.25 in CDG group (p<0.01); the hepatic levels of FFA (CDVDG1,5 vs CDG: 432.54±3.70 mmol/L/mg Liver,403.66±0.59 mmol/L/mg Liver, vs 456.61±3.70 mmol/L/mg Liver, p<0.05, p<0.01) were significantly decreased in CDVDG1 and 5 group, the hepatic level of TG (CDVDG5 vs CDG:0.37±0.03 mmol/L/mg Liver vs 1.29±0.00 mmol/L/mg Liver,p<0.01) was significantly decreased in CDVDG5 group; the serum levels of FINS (CDVDG1,5,10 vs CDG:27.20±0.91 μIU/mL,22.94±0.90 μIU/mL,21.47±0.77 μIU/mL vs 29.50±0.34 μIU/mL,p<0.05, p<0.01,p<0.01) were significantly decreased in CDVDGl,5 and 10 group, the serum levels of HOMA-IR (CDVDG5,10 vs CDG:7.85±0.32,7.56±0.59 vs 11.07±0.20,p<0.01,p<0.05) were significantly decreased in CDVDG5 and 10 group; the serum level of LEP (CDVDG5 vs CDG:445.87±14.98 ng/L vs 569.71±38.97 ng/L,p<0.05) was decreased in CDVDG5 group; the hepatic levels of TBARS (CDVDG1,5,10 vs CDG: 206.21±1.15 pg/mL/mg Liver,164.19±6.14 pg/mL/mg Liver,188.85±2.61 pg/mL/mg Liver vs 233.46±1.03 pg/mL/mg Liver, all p<0.01) were significantly decreased in CDVDG1,5 and 10 group; the TUNEL positive cells (CDVDGl,5,10 vs CDG: 27.60±2.80,11.60±1.33,29.00±2.05 vs 36.80±1.83, all p<0.01) were significantly decreased in CDVDG1,5 and 10 group; the serum levels of CK18-M30 (CDVDG1,5, 10 vs CDG:144.66±9.88 ng/L,105.59±8.48 ng/L,141.64±12.80 ng/L vs 179.45±15.01 ng/L, p<0.05,p<0.01,p<0.05) were significantly down-regulated in CDVDGl,5 and 10 group; the hepatic levels of CK18-M30 (CDVDG1,5,10 vs CDG: 11.09±0.24 μg/L/mg Liver,12.90±0.08 μg/L/mg Liver,11.38±0.06 μg/L/mg Liver vs 9.05±0.43 μg/L/mg Liver, all p<0.01) were significantly elevated in CDVDG1,5 and 10 group; the hepatic expression of levels of a-SMA (CDVDG5 vs CDG:21.60±1.86 vs 45.40±2.42,p<0.01) and TGF-β1 (CDVDG5 vs CDG:53.60±1.57 vs 62.80±2.92, p<0.05) were significantly decreased in CDVDG5 group; the hepatic level of TIMP-1 (CDVDG1,5 vs CDG:2.04±0.05,1.56±0.05 vs 2.69±0.02, both;p<0.01) was significantly decreased in CDVDG1 and 5 group; the serum levels of 25(OH)D3 (CDVDG1,5,10 vs CDG:293.43±4.68 μg/L,342.71±0.51 μg/L,348.52±4.37 μg/L vs 253.72±4.10 μg/L, all p<0.01) and the hepatic expression of levels of VDR (CDVDG1,5,10 vs CDG:2.62±0.02,3.36±0.05,2.28±0.00 vs 2.07±0.01, all p<0.01) were significantly increased in CDVDG1,5 and 10 group.Compared to the CDVDG5 group, in the CDVDG10 group the NAS (CDVDG10 vs CDVDG5:6.40±0.51 vs 5.20±0.20,p<0.05), the hepatic levels of TBARS (CDVDG10 vs CDVDG5:188.85±2.61 pg/mL/mg Liver vs 164.19±6.14 pg/mL/mg Liver, p<0.01) and TUNEL positive cells (CDVDG10 vs CDVDG5:29.00±2.05 vs 11.6±1.33,p<0.01) were increased, serum level of CK18-M30 (CDVDG10 vs CDVDG5:141.64±12.80 ng/L vs 105.59±8.48 ng/L, p<0.05) was elevated, hepatic level of CK18-M30 (CDVDG10 vs CDVDG5:11.38±0.06 μg/L/mg Liver vs 12.90Π0.08 μg/L/mg Liver, p<0.01) was down-regulated, the hepatic levels of a-SMA (CDVDG10 vs CDVDG5:38.20±3.71 vs 21.60±1.86,p<0.05) and TIMP-1 (CDVDG10 vs CDVDG5:2.44±0.08 vs 1.56±0.05,p<0.01) were increased; the hepatic level of VDR (CDVDG10 vs CDVDG5:2.28±0.00 vs 3.36±0.05,p<0.01) was decreased.1,25-VD3 produced preventive effects and there was an obvious dose-effect relationship. The dose-effect curves were either "U-shaped" or "inverted U-shaped" for the serum level of CK18-M30 and liver lipid peroxidation level, hepatocyte apoptosis, hepatic stellate cell (HSC) activation and hepatic VDR, indicating that higher dosage may lead to adverse effects. Conclusion:These results suggest that the preventing effect of 1,25-VD3 supplementation against CDAA-diet induced NASH is related to the inhibition of hepatic accumulation of FFA and TG, the amelioration of IR, LEP resistance and lipid peroxidation, the inhibition of hepatocyte apoptosis and activation of HSC in rat liver with alteration of the serum level of 25(OH)D3 and the hepatic expression of level of VDR. However, we observed adverse affects of supplementation of higher dosage in the serum CK18-M30 and liver NAS, lipid peroxidation, hepatocyte apoptosis, HSC activation and hepatic VDR. |
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