莱菔硫烷改善肝细胞脂肪变及内质网调控机制
发布时间：2018-07-25 11:31
【摘要】：非酒精性脂肪肝是高发且严重危害人类健康的饮食相关性肝损伤，从膳食角度寻找其防治因子具有重要意义。前期研究表明莱菔硫烷（SFN）可减轻酒精性肝损伤及调控脂代谢，但对非酒精性肝损伤的作用及机制尚无深入探讨。本研究探讨SFN预保护人肝细胞HHL-5后，能否抵抗油酸/软脂酸对肝细胞脂代谢的病理性损伤，重点探究内质网应激调控通路的作用，主要研究结果如下。 油酸/软脂酸（1:2）作诱导剂，100-500μmol/L作用于HHL-5细胞，油红O染色检测脂滴，再检测肝细胞代谢酶和甘油三酯（TC）及胆固醇（TG），发现油酸/软脂酸300μmol/L时，多数细胞内出现红色圆珠状且非常饱满的脂滴，诱导剂剂量增大，脂滴增多。油酸/软脂酸（1:2）300μmol/L作用24h，TC和TG含量明显升高（P0.01），谷丙转氨酶（ALT）与谷草转氨酶（AST）活性显著升高；诱导剂浓度大于400μmol/L，细胞死亡。因此，将油酸/软脂酸（1:2）300μmol/L作用24h作为诱导肝细胞HHL-5脂代谢异常的作用方式。SFN分别以时间效应和剂量效应方式预保护细胞，再用油酸/软脂酸刺激24h，发现TG和TC含量与阳性对照组比较显著降低（P0.01），10μmol/L SFN对TG与TC含量降低最为明显，抑制AST和ALT增加，抑制后水平与阴性对照组接近。 以内质网为切入点，透射电镜检测结果表明，油酸/软脂酸作用细胞24h可引起内质网扩张，提示内质网功能受损，同时影响线粒体功能，表现为线粒体颜色加深。SFN预保护后能明显改善内质网的扩张，内质网结构基本恢复到阴性对照组水平，表现为内质网成条索状，，并对线粒体损伤明显改善。Real-timePCR检测内质网调控蛋白XBP1、GRP78转录水平的表达情况。发现油酸/软脂酸（1:2）300μmol/L诱导肝细胞24h后，XBP1与GRP78mRNA表达量显著增加。10μmol/L SFN使二者表达量比诱导组降低。SFN作用时间延长，XBP1与GRP78mRNA表达量减小，24h最显著。流式细胞术检测内质网调控网络中心调控因子Ca2+，发现油酸/软脂酸能诱导内质网中Ca2+流出，胞浆中Ca2+浓度升高明显高于阴性对照组。随着SFN作用浓度增加，胞浆中钙离子浓度下降。 本研究结果表明，SFN能明显改善油酸/软脂酸引起的肝细胞脂代谢异常，内质网调控在其中发挥重要作用。本研究将为SFN的开发应用提供理论基础。
[Abstract]:Non-alcoholic fatty liver is a kind of diet-related liver injury with high incidence and serious harm to human health. It is of great significance to look for its preventive and therapeutic factors from the point of view of diet. Previous studies have shown that sulforaphane (SFN) can attenuate alcoholic liver injury and regulate lipid metabolism, but the role and mechanism of sulforaphane (SFN) in non-alcoholic liver injury have not been thoroughly discussed. The aim of this study was to investigate whether SFN could protect human hepatocytes from the pathological damage of lipid metabolism induced by oleic acid / palmitate, and to explore the role of endoplasmic reticulum stress regulation pathway. The main results were as follows. Oleic acid / palmitic acid (1:2) acted on HHL-5 cells with 100-500 渭 mol/L, oil red O staining was used to detect lipid droplets, and liver cell metabolic enzymes, triglyceride (TC) and cholesterol (TG), were detected when oleic acid / palmitic acid was found to be 300 渭 mol/L. Red globular and very full lipid droplets were found in most cells. The dose of inducer increased and lipid droplets increased. The contents of TC and TG increased significantly (P0.01), the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased significantly after treatment with oleic acid / palmitic acid (1:2) 300 渭 mol/L for 24 h, and the concentration of inducer was more than 400 渭 mol / L, the cell died. Therefore, 渭 mol/L of oleic acid / palmitate (1:2) was used to induce abnormal lipid metabolism of HHL-5 in hepatocytes for 24 hours. SFN preprotected the cells by time effect and dose effect, respectively. After stimulation with oleic acid / palmitic acid for 24 h, it was found that TG and TC decreased significantly (P0.01) 10 渭 mol/L SFN compared with that of the positive control group, the decrease of TG and TC content was the most obvious, the increase of AST and ALT was inhibited, and the level after inhibition was close to that of the negative control group. Using endoplasmic reticulum as the starting point, the results of transmission electron microscopy showed that oleic acid / palmitic acid could induce endoplasmic reticulum dilatation for 24 hours, suggesting that endoplasmic reticulum function was damaged and mitochondria function was affected. The results showed that the extension of endoplasmic reticulum (ER) could be improved obviously after pre-protection of mitochondria. The structure of endoplasmic reticulum (ER) returned to the level of negative control group, showing that the endoplasmic reticulum (ER) was striped. The expression of endoplasmic reticulum regulatory protein XBP1 GRP78 was improved by real-time PCR. It was found that the expression of XBP1 and GRP78mRNA in hepatocytes induced by oleic acid / palmitate (1:2) at 300 渭 mol/L for 24 hours increased significantly. 10 渭 mol/L SFN significantly decreased the expression of XBP1 and GRP78mRNA in hepatocytes. Flow cytometry was used to detect the central regulator of endoplasmic reticulum network (Ca2). It was found that oleic acid / palmitate could induce Ca2 outflow from the endoplasmic reticulum and the concentration of Ca2 in the cytoplasm was significantly higher than that in the negative control group. With the increase of the concentration of SFN, the concentration of Ca ~ (2 +) in the cytoplasm decreased. The results showed that SFN could significantly improve lipid metabolism of hepatocytes induced by oleic acid / palmitate, and endoplasmic reticulum regulation played an important role in it. This study will provide a theoretical basis for the development and application of SFN.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5
本文编号：2143690
[Abstract]:Non-alcoholic fatty liver is a kind of diet-related liver injury with high incidence and serious harm to human health. It is of great significance to look for its preventive and therapeutic factors from the point of view of diet. Previous studies have shown that sulforaphane (SFN) can attenuate alcoholic liver injury and regulate lipid metabolism, but the role and mechanism of sulforaphane (SFN) in non-alcoholic liver injury have not been thoroughly discussed. The aim of this study was to investigate whether SFN could protect human hepatocytes from the pathological damage of lipid metabolism induced by oleic acid / palmitate, and to explore the role of endoplasmic reticulum stress regulation pathway. The main results were as follows. Oleic acid / palmitic acid (1:2) acted on HHL-5 cells with 100-500 渭 mol/L, oil red O staining was used to detect lipid droplets, and liver cell metabolic enzymes, triglyceride (TC) and cholesterol (TG), were detected when oleic acid / palmitic acid was found to be 300 渭 mol/L. Red globular and very full lipid droplets were found in most cells. The dose of inducer increased and lipid droplets increased. The contents of TC and TG increased significantly (P0.01), the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased significantly after treatment with oleic acid / palmitic acid (1:2) 300 渭 mol/L for 24 h, and the concentration of inducer was more than 400 渭 mol / L, the cell died. Therefore, 渭 mol/L of oleic acid / palmitate (1:2) was used to induce abnormal lipid metabolism of HHL-5 in hepatocytes for 24 hours. SFN preprotected the cells by time effect and dose effect, respectively. After stimulation with oleic acid / palmitic acid for 24 h, it was found that TG and TC decreased significantly (P0.01) 10 渭 mol/L SFN compared with that of the positive control group, the decrease of TG and TC content was the most obvious, the increase of AST and ALT was inhibited, and the level after inhibition was close to that of the negative control group. Using endoplasmic reticulum as the starting point, the results of transmission electron microscopy showed that oleic acid / palmitic acid could induce endoplasmic reticulum dilatation for 24 hours, suggesting that endoplasmic reticulum function was damaged and mitochondria function was affected. The results showed that the extension of endoplasmic reticulum (ER) could be improved obviously after pre-protection of mitochondria. The structure of endoplasmic reticulum (ER) returned to the level of negative control group, showing that the endoplasmic reticulum (ER) was striped. The expression of endoplasmic reticulum regulatory protein XBP1 GRP78 was improved by real-time PCR. It was found that the expression of XBP1 and GRP78mRNA in hepatocytes induced by oleic acid / palmitate (1:2) at 300 渭 mol/L for 24 hours increased significantly. 10 渭 mol/L SFN significantly decreased the expression of XBP1 and GRP78mRNA in hepatocytes. Flow cytometry was used to detect the central regulator of endoplasmic reticulum network (Ca2). It was found that oleic acid / palmitate could induce Ca2 outflow from the endoplasmic reticulum and the concentration of Ca2 in the cytoplasm was significantly higher than that in the negative control group. With the increase of the concentration of SFN, the concentration of Ca ~ (2 +) in the cytoplasm decreased. The results showed that SFN could significantly improve lipid metabolism of hepatocytes induced by oleic acid / palmitate, and endoplasmic reticulum regulation played an important role in it. This study will provide a theoretical basis for the development and application of SFN.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5
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相关期刊论文 前2条
1 拜明军;沈薇;洪江龙;;不同性质的脂肪酸对脂肪变性L02肝细胞凋亡和葡萄糖调节蛋白78表达的影响[J];中国生物制品学杂志;2008年10期
2 李宝龙;田思聪;谭洁;李冰;陈镜羽;单毓娟;;绿花椰菜片剂中莱菔硫烷含量测定及对急性酒精性肝损伤的保护作用[J];浙江大学学报(农业与生命科学版);2013年02期
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