本研究旨在对水牛固醇携带蛋白2(sterol carrier protein 2,SCP2)基因进行克隆及生物信息学分析,并检测其在水牛不同组织中的表达。以黄牛SCP2基因(登录号:NM_001033990.3)为种子序列成功克隆了水牛SCP2基因完整CDS区,该序列长1 632bp,...本研究旨在对水牛固醇携带蛋白2(sterol carrier protein 2,SCP2)基因进行克隆及生物信息学分析,并检测其在水牛不同组织中的表达。以黄牛SCP2基因(登录号:NM_001033990.3)为种子序列成功克隆了水牛SCP2基因完整CDS区,该序列长1 632bp,可编码543个氨基酸;其与黄牛、绵羊、山羊、白鲸、人、家犬和家猫的同源性分别为95.9%、93.4%、92.4%、89.4%、88.3%、86.3%和86.9%,说明SCP2基因CDS区在不同物种间具有较高的保守性。聚类分析则表明水牛与黄牛的分子进化关系最近;氨基酸序列分析表明,SCP2蛋白的分子式为C_(2602)H_(4131)N_(709)O_(774)S_(298),分子质量为58.66ku,理论等电点(pI)为8.59,不稳定系数为27.94,平均亲水性为-0.215,属于碱性、稳定、亲水蛋白质;二级结构分析表明水牛SCP2蛋白由α-螺旋、无规则卷曲和延伸链构成,其中α-螺旋占35.54%,无规则卷曲占48.99%,延伸链占15.47%,与三级结构预测结果一致;亚细胞定位分析表明,水牛SCP2蛋白分布在细胞质(43.5%)、过氧化物酶体(21.7%)、线粒体(17.4%)、细胞核(13.0%)和细胞骨架(4.4%);跨膜结构和信号肽预测分析表明,水牛SCP2蛋白不含跨膜结构和信号肽;磷酸化位点分析发现,水牛SCP2蛋白有13个Ser、3个Thr和2个Tyr可能成为蛋白激酶磷酸化位点;蛋白质结合位点预测结果显示,水牛SCP2蛋白含有12个蛋白质结合位点和1个多核苷酸结合位点;实时荧光定量PCR结果表明,水牛SCP2基因在肝脏中表达量最高,其他组织中表达量从高到低依次为乳腺、淋巴、肾脏、大肠、胃、肺脏、脾脏、卵巢、垂体、大脑和心脏。本试验为今后进一步探讨SCP2基因的功能奠定了基础。展开更多
Background Sterol regulatory element binding protein （SREBP）-2 plays a key role in lipid homeostasis by stimulating gene expression of cholesterol biosynthetic pathways. The insulin-like growth factor binding prote...Background Sterol regulatory element binding protein （SREBP）-2 plays a key role in lipid homeostasis by stimulating gene expression of cholesterol biosynthetic pathways. The insulin-like growth factor binding protein （IGFBP） family regulates growth and metabolism, especially bone cell metabolism, and correlates with osteonecrosis. However, association of their gene polymorphisms with risk of avascular necrosis of the femoral head （ANFH） has rarely been reported. We determined whether SREBP-2 and IGFBP-3 gene polymorphisms were associated with increased ANFH risk in the Chinese population. Methods Two single nucleotide polymorphisms of SREBP2 gene, rs2267439 and rs2267443, and one of IGFBP-3 gene, rs2453839, were selected and genotyped in 49 ANFH patients and 42 control individuals by direct sequencing assay. Results The frequencies of rs2267439 TT and rs2267443 GA of SREBP2 and rs2453839 TT and CT of IGFBP-3 in the ANFH group showed increased and decreased tendencies （against normal control group）, respectively. Interaction analysis of genes revealed that the frequency of carrying rs2267439 TT and rs2267443 GA genotypes of SREBF-2 in ANFH patients was significantly higher than in the control group （P 〈0.05）. Association analysis between polymorphisms and clinical phenotype demonstrated that the disease course in ANFH patients with the rs2453839 TT genotype of IGFBP-3 was significantly shorter than that of CT＋CC carriers （P 〈0.01）. CT＋CC genotype frequency in patients with stage Ill/IV bilateral hip lesions was significantly higher than in those with stage Ill/IV unilateral lesions and stage II/111 bilateral lesions （P 〈0.05-0.02）. Conclusions Our results suggested that interaction of SREBP-2 gene polymorphisms and the relationship between the polymorphisms and clinical phenotype of IGFBP-3 were closely related to increased ANFH risk in the Chinese population. The most significant finding was that the CT＋CC genotype carriers of IGFBP-3 rs2453839 were highly associated with the development of ANFH.展开更多
目的:探讨升清胶囊对人肝细胞氧化损伤模型中胆固醇7α-羟化酶(CYP7A1)和固醇携带蛋白2(SCP2)m RNA及蛋白表达的影响。方法:选用人L-02肝细胞,分为正常组、模型组、空白血清组及升清胶囊组。采用H2O2诱导建立氧化损伤模型,空白血清组和...目的:探讨升清胶囊对人肝细胞氧化损伤模型中胆固醇7α-羟化酶(CYP7A1)和固醇携带蛋白2(SCP2)m RNA及蛋白表达的影响。方法:选用人L-02肝细胞,分为正常组、模型组、空白血清组及升清胶囊组。采用H2O2诱导建立氧化损伤模型,空白血清组和升清胶囊组每孔分别加入2 m L含10%血清或含药血清的RPMI-1640培养液,培养24 h后采用real-time PCR法和western-blot法分别检测各组肝细胞CYP7A1和SCP2 m RNA及蛋白表达。结果:与正常组比较,模型组CYP7A1 m RNA及蛋白表达降低,SCP2 m RNA及蛋白表达升高(P<0.01);与模型组比较,空白血清和升清胶囊组CYP7A1m RNA及蛋白表达升高,SCP2 m RNA及蛋白表达降低(P<0.01);与空白血清组比较,升清胶囊组CYP7A1 m RNA及蛋白表达增强,SCP2 m RNA及蛋白表达降低(P<0.05)。结论:升清胶囊可以通过下调SCP2 m RNA及蛋白表达来减少肝细胞分泌胆固醇入胆汁,同时上调CYP7A1 m RNA及蛋白表达来加速胆汁酸生成,从而防止胆固醇结晶析出,达到降低胆结石形成的作用。展开更多
Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated L...Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line （HepG2） cells under physiological and inflammatory conditions. Methods Intracellular total cholesterol （TC）, free cholesterol （FC） and cholesterol ester （CE） were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein （SREBP）-2 and SREBP cleavage-activating protein （SCAP） mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum （ER） to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies. Results LDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1β （IL-1β） further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1β caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium. Conclusions IL-1β disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.展开更多
AIM: To evaluate the effect of resveratrol, alone and in combination with fenofibrate, on fructose-induced metabolic genes abnormalities in rats.METHODS: Giving a fructose-enriched diet (FED) to rats for 12 wk was use...AIM: To evaluate the effect of resveratrol, alone and in combination with fenofibrate, on fructose-induced metabolic genes abnormalities in rats.METHODS: Giving a fructose-enriched diet (FED) to rats for 12 wk was used as a model for inducing hepatic dyslipidemia and insulin resistance. Adult male albino rats (150-200 g) were divided into a control group and a FED group which was subdivided into 4 groups, a control FED, fenofibrate (FENO) (100 mg/kg), resveratrol (RES) (70 mg/kg) and combined treatment (FENO + RES) (half the doses). All treatments were given orally from the 9<sup>th</sup> week till the end of experimental period. Body weight, oral glucose tolerance test (OGTT), liver index, glucose, insulin, insulin resistance (HOMA), serum and liver triglycerides (TGs), oxidative stress (liver MDA, GSH and SOD), serum AST, ALT, AST/ALT ratio and tumor necrosis factor-α (TNF-α) were measured. Additionally, hepatic gene expression of suppressor of cytokine signaling-3 (SOCS-3), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), malonyl CoA decarboxylase (MCD), transforming growth factor-β1 (TGF-β1) and adipose tissue genes expression of leptin and adiponectin were investigated. Liver sections were taken for histopathological examination and steatosis area were determined.RESULTS: Rats fed FED showed damaged liver, impairment of glucose tolerance, insulin resistance, oxidative stress and dyslipidemia. As for gene expression, there was a change in favor of dyslipidemia and nonalcoholic steatohepatitis (NASH) development. All treatment regimens showed some benefit in reversing the described deviations. Fructose caused deterioration in hepatic gene expression of SOCS-3, SREBP-1c, FAS, MDA and TGF-β1 and in adipose tissue gene expression of leptin and adiponectin. Fructose showed also an increase in body weight, insulin resistance (OGTT, HOMA), serum and liver TGs, hepatic MDA, serum AST, AST/ALT ratio and TNF-α compared to control. All treatments improved SOCS-3, FAS, MCD, TGF-β1 and leptin genes expression while only RES and FENO + RES groups showed an improvement in SREBP-1c expression. Adiponectin gene expression was improved only by RES. A decrease in body weight, HOMA, liver TGs, AST/ALT ratio and TNF-α were observed in all treatment groups. Liver index was increased in FENO and FENO + RES groups. Serum TGs was improved only by FENO treatment. Liver MDA was improved by RES and FENO + RES treatments. FENO + RES group showed an increase in liver GSH content.CONCLUSION: When resveratrol was given with half the dose of fenofibrate it improved NASH-related fructose-induced disturbances in gene expression similar to a full dose of fenofibrate.展开更多
文摘本研究旨在对水牛固醇携带蛋白2(sterol carrier protein 2,SCP2)基因进行克隆及生物信息学分析,并检测其在水牛不同组织中的表达。以黄牛SCP2基因(登录号:NM_001033990.3)为种子序列成功克隆了水牛SCP2基因完整CDS区,该序列长1 632bp,可编码543个氨基酸;其与黄牛、绵羊、山羊、白鲸、人、家犬和家猫的同源性分别为95.9%、93.4%、92.4%、89.4%、88.3%、86.3%和86.9%,说明SCP2基因CDS区在不同物种间具有较高的保守性。聚类分析则表明水牛与黄牛的分子进化关系最近;氨基酸序列分析表明,SCP2蛋白的分子式为C_(2602)H_(4131)N_(709)O_(774)S_(298),分子质量为58.66ku,理论等电点(pI)为8.59,不稳定系数为27.94,平均亲水性为-0.215,属于碱性、稳定、亲水蛋白质;二级结构分析表明水牛SCP2蛋白由α-螺旋、无规则卷曲和延伸链构成,其中α-螺旋占35.54%,无规则卷曲占48.99%,延伸链占15.47%,与三级结构预测结果一致;亚细胞定位分析表明,水牛SCP2蛋白分布在细胞质(43.5%)、过氧化物酶体(21.7%)、线粒体(17.4%)、细胞核(13.0%)和细胞骨架(4.4%);跨膜结构和信号肽预测分析表明,水牛SCP2蛋白不含跨膜结构和信号肽;磷酸化位点分析发现,水牛SCP2蛋白有13个Ser、3个Thr和2个Tyr可能成为蛋白激酶磷酸化位点;蛋白质结合位点预测结果显示,水牛SCP2蛋白含有12个蛋白质结合位点和1个多核苷酸结合位点;实时荧光定量PCR结果表明,水牛SCP2基因在肝脏中表达量最高,其他组织中表达量从高到低依次为乳腺、淋巴、肾脏、大肠、胃、肺脏、脾脏、卵巢、垂体、大脑和心脏。本试验为今后进一步探讨SCP2基因的功能奠定了基础。
文摘Background Sterol regulatory element binding protein （SREBP）-2 plays a key role in lipid homeostasis by stimulating gene expression of cholesterol biosynthetic pathways. The insulin-like growth factor binding protein （IGFBP） family regulates growth and metabolism, especially bone cell metabolism, and correlates with osteonecrosis. However, association of their gene polymorphisms with risk of avascular necrosis of the femoral head （ANFH） has rarely been reported. We determined whether SREBP-2 and IGFBP-3 gene polymorphisms were associated with increased ANFH risk in the Chinese population. Methods Two single nucleotide polymorphisms of SREBP2 gene, rs2267439 and rs2267443, and one of IGFBP-3 gene, rs2453839, were selected and genotyped in 49 ANFH patients and 42 control individuals by direct sequencing assay. Results The frequencies of rs2267439 TT and rs2267443 GA of SREBP2 and rs2453839 TT and CT of IGFBP-3 in the ANFH group showed increased and decreased tendencies （against normal control group）, respectively. Interaction analysis of genes revealed that the frequency of carrying rs2267439 TT and rs2267443 GA genotypes of SREBF-2 in ANFH patients was significantly higher than in the control group （P 〈0.05）. Association analysis between polymorphisms and clinical phenotype demonstrated that the disease course in ANFH patients with the rs2453839 TT genotype of IGFBP-3 was significantly shorter than that of CT＋CC carriers （P 〈0.01）. CT＋CC genotype frequency in patients with stage Ill/IV bilateral hip lesions was significantly higher than in those with stage Ill/IV unilateral lesions and stage II/111 bilateral lesions （P 〈0.05-0.02）. Conclusions Our results suggested that interaction of SREBP-2 gene polymorphisms and the relationship between the polymorphisms and clinical phenotype of IGFBP-3 were closely related to increased ANFH risk in the Chinese population. The most significant finding was that the CT＋CC genotype carriers of IGFBP-3 rs2453839 were highly associated with the development of ANFH.
文摘目的:探讨升清胶囊对人肝细胞氧化损伤模型中胆固醇7α-羟化酶(CYP7A1)和固醇携带蛋白2(SCP2)m RNA及蛋白表达的影响。方法:选用人L-02肝细胞,分为正常组、模型组、空白血清组及升清胶囊组。采用H2O2诱导建立氧化损伤模型,空白血清组和升清胶囊组每孔分别加入2 m L含10%血清或含药血清的RPMI-1640培养液,培养24 h后采用real-time PCR法和western-blot法分别检测各组肝细胞CYP7A1和SCP2 m RNA及蛋白表达。结果:与正常组比较,模型组CYP7A1 m RNA及蛋白表达降低,SCP2 m RNA及蛋白表达升高(P<0.01);与模型组比较,空白血清和升清胶囊组CYP7A1m RNA及蛋白表达升高,SCP2 m RNA及蛋白表达降低(P<0.01);与空白血清组比较,升清胶囊组CYP7A1 m RNA及蛋白表达增强,SCP2 m RNA及蛋白表达降低(P<0.05)。结论:升清胶囊可以通过下调SCP2 m RNA及蛋白表达来减少肝细胞分泌胆固醇入胆汁,同时上调CYP7A1 m RNA及蛋白表达来加速胆汁酸生成,从而防止胆固醇结晶析出,达到降低胆结石形成的作用。
基金This study was supported by grants from the National Natural Science Foundation of China(Key Program,No.30530360)the National Basic Research Program of China(No.2006CB503907)Royal Free Hospital Special Trustees grant
文摘Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line （HepG2） cells under physiological and inflammatory conditions. Methods Intracellular total cholesterol （TC）, free cholesterol （FC） and cholesterol ester （CE） were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein （SREBP）-2 and SREBP cleavage-activating protein （SCAP） mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum （ER） to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies. Results LDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1β （IL-1β） further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1β caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium. Conclusions IL-1β disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.
文摘AIM: To evaluate the effect of resveratrol, alone and in combination with fenofibrate, on fructose-induced metabolic genes abnormalities in rats.METHODS: Giving a fructose-enriched diet (FED) to rats for 12 wk was used as a model for inducing hepatic dyslipidemia and insulin resistance. Adult male albino rats (150-200 g) were divided into a control group and a FED group which was subdivided into 4 groups, a control FED, fenofibrate (FENO) (100 mg/kg), resveratrol (RES) (70 mg/kg) and combined treatment (FENO + RES) (half the doses). All treatments were given orally from the 9<sup>th</sup> week till the end of experimental period. Body weight, oral glucose tolerance test (OGTT), liver index, glucose, insulin, insulin resistance (HOMA), serum and liver triglycerides (TGs), oxidative stress (liver MDA, GSH and SOD), serum AST, ALT, AST/ALT ratio and tumor necrosis factor-α (TNF-α) were measured. Additionally, hepatic gene expression of suppressor of cytokine signaling-3 (SOCS-3), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), malonyl CoA decarboxylase (MCD), transforming growth factor-β1 (TGF-β1) and adipose tissue genes expression of leptin and adiponectin were investigated. Liver sections were taken for histopathological examination and steatosis area were determined.RESULTS: Rats fed FED showed damaged liver, impairment of glucose tolerance, insulin resistance, oxidative stress and dyslipidemia. As for gene expression, there was a change in favor of dyslipidemia and nonalcoholic steatohepatitis (NASH) development. All treatment regimens showed some benefit in reversing the described deviations. Fructose caused deterioration in hepatic gene expression of SOCS-3, SREBP-1c, FAS, MDA and TGF-β1 and in adipose tissue gene expression of leptin and adiponectin. Fructose showed also an increase in body weight, insulin resistance (OGTT, HOMA), serum and liver TGs, hepatic MDA, serum AST, AST/ALT ratio and TNF-α compared to control. All treatments improved SOCS-3, FAS, MCD, TGF-β1 and leptin genes expression while only RES and FENO + RES groups showed an improvement in SREBP-1c expression. Adiponectin gene expression was improved only by RES. A decrease in body weight, HOMA, liver TGs, AST/ALT ratio and TNF-α were observed in all treatment groups. Liver index was increased in FENO and FENO + RES groups. Serum TGs was improved only by FENO treatment. Liver MDA was improved by RES and FENO + RES treatments. FENO + RES group showed an increase in liver GSH content.CONCLUSION: When resveratrol was given with half the dose of fenofibrate it improved NASH-related fructose-induced disturbances in gene expression similar to a full dose of fenofibrate.
