体内外研究羟基胆固醇硫酸基转移酶（SULT2B1b）在脂类代谢中的功能及机制
发布时间：2018-08-17 11:06
【摘要】：胞浆硫酸基转移酶(SULT) 2B1b催化25-羟化胆固醇(25HC)硫酸化,反应主要产物为3-硫酸-25-羟化胆固醇(25HC3S)。研究发现该硫酸化产物可通过抑制LXR/SREBPs信号转导通路降低THP-1来源的巨噬细胞内脂质水平。因此,合成25HC3S的关键限速酶SULT2B1b可能在调节脂质代谢中起重要作用。本实验中,我们在原代人主动脉内皮细胞内研究SULT2B1b对脂质代谢的作用及机制,并在小鼠非酒精性脂肪性肝病模型中检测SULT2B1b的表达及其重要的生理意义。 本研究共包括三部分内容,详细如下： 第一部分：预实验及其它文献报道,羟基类固醇硫酸基转移酶(SULT2B1b)同样表达于原代人主动脉内皮细胞及小鼠肝脏组织内,但表达量较低。为了更好的研究SULT2B1b及其所产生的内源性硫化固醇对脂质代谢的作用及机制,在这些细胞或组织内过表达SULT2B1b是一种比较合理的方法,所以我们构建含SULT2B1b基因的腺病毒载体。 第二部分：利用我们所构建的含SULT2B1b基因的腺病毒载体转染原代人主动脉内皮细胞,研究SULT2B1b对脂质代谢的影响。选用该细胞系作为我们的研究对象,是因为血管内皮细胞在动脉粥样硬化等脂质代谢紊乱相关疾病中发挥重要作用。结果显示,腺病毒(Ad-SULT2B1b)感染后,SULT2B1b基因在原代人主动脉内皮细胞内成功过表达；加入[H3]标记的25-羟化胆固醇(25HC)追踪其代谢变化,2小时后约40%25HC被硫酸化,24小时后大于50%25HC被硫酸化,HPLC鉴定硫酸化产物发现主要为3-硫酸-25-羟化胆固醇(25HC3S)；进一步检测细胞内脂质水平的变化,发现在25HC存在的情况下,过表达SULT2B1b显著降低细胞内甘油三酯、总胆固醇、游离胆固醇及游离脂肪酸,并降低脂质合成相关基因,包括LXRα、SREBP-1、ACC1、FAS和HMGR等蛋白及mRNA水平；然而在没有25-羟化胆固醇存在,或在T0901317 (化学合成的LXR激动剂)存在的情况下,过表达SULT2B1b降脂作用不明显。表明SULT2B1b的降脂作用主要是通过其形成的内源性25HC3S而产生的,并非其自身直接作用。另外,我们还在相对高表达SULT2B1b的HepG2细胞内,采用干扰RNA技术敲除SULT2B1b基因的表达,反面验证氧化固醇硫酸化的降脂作用。 第三部分：高脂或高胆固醇饲养C57BL/6及LDLR-/-敲基因小鼠建立非酒精性脂肪性肝病模型,通过尾静脉注射含SULT2B1b基因的腺病毒,研究其在小鼠体内对脂质代谢的作用。小鼠被随机分为三组,即C57BL/6小鼠高胆固醇饲养组,C57BL/6小鼠高胆固醇饲养并腹腔注射25HC组,LDLR-/-敲基因小鼠高脂饲养组。每组又分为感染SULT2B1b和对照病毒β-Gal组。每小组5-6只小鼠。结果显示,过表达SULT2B1b的小鼠血清及肝脏组织内脂质水平(包括甘油三酯、胆固醇及游离脂肪酸)与相应组别内的对照病毒组明显下降,其中C57BL/6小鼠高胆固醇饲养并腹腔注射25HC组及LDLR-/-敲基因小鼠高脂饲养组更为明显。HPLC分析肝脏内氧化固醇及硫酸化的氧化固醇发现,C57BL/6小鼠高胆固醇饲养并腹腔注射25HC组过表达SULT2B1b后,氧化固醇明显下降而硫酸化的氧化固醇则明显升高,而没有腹腔注射25HC的小鼠变化不明显。脂质相关基因检测发现过表达SULT2B1b后,LXRα、SREBP-1、ACC1和FAS蛋白及mRNA水平均明显下降。另外,我们也分离了小鼠主动脉,正常C57BL/6小鼠饲养高胆固醇10周后均未检测到动脉粥样硬化,而在LDLR-/-敲基因小鼠内转染对照病毒组可见脂质堆积和动脉粥样硬化,过表达SULT2Blb后,动脉粥样硬化现象消失。 综上所述,羟基类固醇硫酸基转移酶(SULT2Blb)通过硫酸化氧化固醇抑制LXR/SREBPs脂质合成途径,从而有效降低细胞内、小鼠血清及肝脏组织内脂质水平,在脂质代谢中发挥重要作用。这些发现也为脂质代谢紊乱相关疾病如动脉粥样硬化及非酒精性脂肪性肝病等的防治提供了新的理论依据及途径。
[Abstract]:Cytoplasmic sulfate transferase (SULT) 2B1b catalyzes the sulfation of 25-hydroxycholesterol (25HC) with the main product of 3-sulfuric acid-25-hydroxycholesterol (25HC3S). It has been found that the sulfated product can reduce lipid levels in THP-1-derived macrophages by inhibiting LXR/SREBPs signal transduction pathway. Therefore, the key rate-limiting enzyme for the synthesis of 25HC3S, SULT2B1b In this study, we studied the effect and mechanism of SULT2B1b on lipid metabolism in primary human aortic endothelial cells, and detected the expression of SULT2B1b in non-alcoholic fatty liver disease model in mice.
This study consists of three parts, as follows:
Part I: Preliminary studies and other literature have reported that hydroxysteroid sulfate transferase (SULT2B1b) is also expressed in primary human aortic endothelial cells and mouse liver tissues, but the expression is low. Overexpression of SULT2B1b in tissues is a reasonable method, so we constructed adenovirus vector containing SULT2B1b gene.
The second part is to study the effect of SULT2B1b on lipid metabolism by transfecting primary human aortic endothelial cells with adenovirus vector containing SULT2B1b gene. The results showed that SULT2B1b gene was overexpressed in primary human aortic endothelial cells after adenovirus (Ad-SULT2B1b) infection, and 25-hydroxycholesterol (25HC) labeled with [H3] was added to track its metabolic changes. About 40% of 25HC was sulfated 2 hours later, and more than 50% of 25HC was sulfated 24 hours later. - Hydroxycholesterol (25HC3S) was further detected, and the expression of SULT2B1b significantly decreased triglycerides, total cholesterol, free cholesterol and free fatty acids in the cells in the presence of 25HC, and reduced the levels of protein and mRNA related to lipid synthesis, including LXR alpha, SREBP-1, ACC1, FAS and HMGR. However, in the absence of 25-hydroxycholesterol, or in the presence of T0901317 (a chemically synthesized LXR agonist), the lipid-lowering effect of SULT2B1b is not obvious. This suggests that the lipid-lowering effect of SULT2B1b is mainly produced by the endogenous 25HC3S formed by SULT2B1b, but not directly by itself. In HepG2 cells, the expression of SULT2B1b gene was knocked out by interfering RNA technology, and the lipid-lowering effect of oxidized sterol sulfonation was tested.
Part III: Non-alcoholic fatty liver disease models were established in C57BL/6 and LDLR-/-knocking mice fed with high-fat or high-cholesterol diet. Adenoviruses containing SULT2B1b gene were injected into tail vein to study their effects on lipid metabolism in mice. Mice were randomly divided into three groups: C57BL/6 mice fed with high-cholesterol diet and C57BL/6 mice fed with high-cholesterol diet. The serum and liver lipid levels (including triglycerides, cholesterol and free fatty acids) in mice overexpressing SULT2B1b were compared with those in the corresponding groups. The levels of serum oxysterol in C57BL/6 mice were significantly lower than those in C57BL/6 mice fed with high cholesterol and intraperitoneally injected with 25HC and LDLR-/-knocking mice fed with high cholesterol. The levels of LXR-alpha, SREBP-1, ACC 1 and FAS protein and mRNA were significantly decreased after the expression of SULT2B1b. In addition, the aorta of normal C57BL/6 mice were isolated and the cholesterol levels of normal C57BL/6 mice were not detected after 10 weeks. Atherosclerosis was detected, and lipid accumulation and atherosclerosis were observed in the LDLR-/-knocking mice transfected with the control virus. After overexpression of SULT2Blb, atherosclerosis disappeared.
To sum up, hydroxysteroid sulfate transferase (SULT2Blb) inhibits LXR/SREBPs lipid biosynthesis via sulfated oxidative steroids, thereby effectively reducing lipid levels in cells, mouse serum and liver tissues, and plays an important role in lipid metabolism. These findings also play an important role in lipid metabolism disorders related diseases such as atherosclerosis and atherosclerosis. The prevention and treatment of nonalcoholic fatty liver disease provide a new theoretical basis and approach.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
[Abstract]:Cytoplasmic sulfate transferase (SULT) 2B1b catalyzes the sulfation of 25-hydroxycholesterol (25HC) with the main product of 3-sulfuric acid-25-hydroxycholesterol (25HC3S). It has been found that the sulfated product can reduce lipid levels in THP-1-derived macrophages by inhibiting LXR/SREBPs signal transduction pathway. Therefore, the key rate-limiting enzyme for the synthesis of 25HC3S, SULT2B1b In this study, we studied the effect and mechanism of SULT2B1b on lipid metabolism in primary human aortic endothelial cells, and detected the expression of SULT2B1b in non-alcoholic fatty liver disease model in mice.
This study consists of three parts, as follows:
Part I: Preliminary studies and other literature have reported that hydroxysteroid sulfate transferase (SULT2B1b) is also expressed in primary human aortic endothelial cells and mouse liver tissues, but the expression is low. Overexpression of SULT2B1b in tissues is a reasonable method, so we constructed adenovirus vector containing SULT2B1b gene.
The second part is to study the effect of SULT2B1b on lipid metabolism by transfecting primary human aortic endothelial cells with adenovirus vector containing SULT2B1b gene. The results showed that SULT2B1b gene was overexpressed in primary human aortic endothelial cells after adenovirus (Ad-SULT2B1b) infection, and 25-hydroxycholesterol (25HC) labeled with [H3] was added to track its metabolic changes. About 40% of 25HC was sulfated 2 hours later, and more than 50% of 25HC was sulfated 24 hours later. - Hydroxycholesterol (25HC3S) was further detected, and the expression of SULT2B1b significantly decreased triglycerides, total cholesterol, free cholesterol and free fatty acids in the cells in the presence of 25HC, and reduced the levels of protein and mRNA related to lipid synthesis, including LXR alpha, SREBP-1, ACC1, FAS and HMGR. However, in the absence of 25-hydroxycholesterol, or in the presence of T0901317 (a chemically synthesized LXR agonist), the lipid-lowering effect of SULT2B1b is not obvious. This suggests that the lipid-lowering effect of SULT2B1b is mainly produced by the endogenous 25HC3S formed by SULT2B1b, but not directly by itself. In HepG2 cells, the expression of SULT2B1b gene was knocked out by interfering RNA technology, and the lipid-lowering effect of oxidized sterol sulfonation was tested.
Part III: Non-alcoholic fatty liver disease models were established in C57BL/6 and LDLR-/-knocking mice fed with high-fat or high-cholesterol diet. Adenoviruses containing SULT2B1b gene were injected into tail vein to study their effects on lipid metabolism in mice. Mice were randomly divided into three groups: C57BL/6 mice fed with high-cholesterol diet and C57BL/6 mice fed with high-cholesterol diet. The serum and liver lipid levels (including triglycerides, cholesterol and free fatty acids) in mice overexpressing SULT2B1b were compared with those in the corresponding groups. The levels of serum oxysterol in C57BL/6 mice were significantly lower than those in C57BL/6 mice fed with high cholesterol and intraperitoneally injected with 25HC and LDLR-/-knocking mice fed with high cholesterol. The levels of LXR-alpha, SREBP-1, ACC 1 and FAS protein and mRNA were significantly decreased after the expression of SULT2B1b. In addition, the aorta of normal C57BL/6 mice were isolated and the cholesterol levels of normal C57BL/6 mice were not detected after 10 weeks. Atherosclerosis was detected, and lipid accumulation and atherosclerosis were observed in the LDLR-/-knocking mice transfected with the control virus. After overexpression of SULT2Blb, atherosclerosis disappeared.
To sum up, hydroxysteroid sulfate transferase (SULT2Blb) inhibits LXR/SREBPs lipid biosynthesis via sulfated oxidative steroids, thereby effectively reducing lipid levels in cells, mouse serum and liver tissues, and plays an important role in lipid metabolism. These findings also play an important role in lipid metabolism disorders related diseases such as atherosclerosis and atherosclerosis. The prevention and treatment of nonalcoholic fatty liver disease provide a new theoretical basis and approach.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
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