| Cytosolic sulfotransferase (SULT)2B1b catalyzes sulfation of 25-hydroxycholesterol (25HC). The main product of this reaction is 25-hydroxycholesterol-3-sulfate (25HC3S), which was found to decrease cellular lipids via suppressing LXR/SREBPs signaling in THP-1 derived macrophages. However, its precursor,25HC, regulates lipid metabolism in opposite ways, with increasing cellular lipid levels via activating LXR/SREBPs signaling. Therefore, the key enzyme for the biosynthesis of 25HC3S, oxysterol sulfotransferase (SULT2B1b), may play a crucial role in regulating lipid metabolism. The effect and mechanism of SULT2B1b on lipid metabolism were investagted in the cultured primary human aortic endothelial cells in vitro and the expression of SULT2Blb and its significance were also evaluated in the animal model of nonalcoholic fat liver disease (NAFLD) in vivo.The present study is composed of three parts as following.Part?. Previous studies showed that SULT2B1b was expressed in primary human oartic endothelial cells and liver tissues, but at low level. In order to evaluate the effect of SULT2Blb on lipid metabolism in these cells and tissues, it is a good idea to overexpress it for clearer demonstration. Therefore, we made an adenovirus encoding SULT2B1b gene.Part?. We evaluate the effect of SULT2Blb on lipid metabolism in primary human aortic endothelial cells (HAECs) with overexpression of SULT2Blb. Vascular endothelial cells (VECs) dysfunction is fundamental to the pathogenesis of atherosclerosis and related cardiovascular diseases, that's why we choose HAECs in our experiments. The results showed that SULT2B1b successfully overexpressed in HAECs following infection using a recombinant adenovirus encoding SULT2B1b gene (Ad-SULT2B1b). Addition of [H3]-25HC into the culture HAECs, about 40% 25HC can be sulfated in 2h, and more than 50% can be sulfated in 24h after SULT2B1b overexpression, HPLC analysis showed the main sulfated product was 25HC3S. Overexpression of SULT2Blb in the presence of 25HC decreased gene expressions related to lipid metabolism, including LXR??SREBP-1?ACC1?FAS and HMGR at protein and mRNA levels, subsequently decreased lipid levels in HAECs, including triglycerides, total cholesterol, free cholesterol and free fatty acids, as compared to control. These effects on lipid metabolism are similar to those caused by direct addition of 25HC3S. However, in the absence of 25HC or in the presence of T0901317 (synthetic liver oxysterol receptor (LXR) agonist), SULT2B1b overexpression had no significant effect on the regulation of key genes involved in lipid metabolism. Furthermore, we performed the "loss of fuction" studies by using siRNA-SULT2B1b in HepG2 cells, which has high level of SULT2B1b, the results confirmed that 25HC sulfation by SULT2Blb decreased lipid biosynthesis via LXR/SREBPs signaling pathway.Part III. The effect of SULT2Blb on lipid metabolism in vivo in C57BL/6 and LDLR-/-mice fed with high cholesterol diet (HCD) or high fat diet (HFD) for 10 weeks was evaluated by infection with Ad-SULT2B1b through tail vein injection. The mice were separated into 3 groups randomly (C57BL/6 mice fed with HCD, C57BL/6 mice fed with HCD and 25HC injection, and LDLR-'- mice fed with HFD), and each group contained 5-6 mice. The results showed that SULT2Blb overexpression decreased serum and hepatic lipid levels in mice from the C57BL/6 mice fed with HCD and 25HC injection and LDLR-/- mice fed with HFD groups, as compared to the same group mice infected with control virus, while no significant changes were detected in C57BL/6 mice fed with HCD without 25HC groups; HPLC analysis of oxysterols and sulfated oxysterols in liver tissue showed that overexpression of SULT2B1b significantly decreased oxysterols and increased sulfated oxysterols in mice in the presence of 25HC; Gene expressions involved in lipid metabolism was detected, SULT2B1b overexpression dramatically decreased LXR??SREBP-1?ACC1 and FAS protein and mRNA level in liver tissue; Furthermore, the aorta were isolated and analyzed by H&E staining, excess lipid accumulation and atherosclerosis plaque were inhibited by overexpression of SULT2Blb in LDLR-/- mice, while no lipid accumulation and atherosclerosis were detected in C57BL/6 mice with HCD or HFD for 10 weeks.In summary,25HC sulfation by SULT2B1b decreased lipid biosynthesis in primary human aortic endothelial cells and decreased serum and hepatic lipid levels in C57BL/6 and LDLR-/- mice via inhibiting LXR/SREBPs signaling pathway. These findings support the hypothesis that 25HC3S is an important endogenous regulator of lipid homeostatis. This pathway may represent a novel target for pharmacological intervention in atherosclerosis and nonalcoholic fatty liver diseases (NAFLD). |
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