目的:探讨缺氧对脑内胆固醇代谢稳态及神经元衰老的影响。方法:8~12周龄的C57BL/6野生型雄性小鼠随机分为常氧(Normoxia)组和缺氧(Hypoxia)组;以缺氧处理小鼠海马神经元HT22细胞并分为对照(Control)组和缺氧(Hypoxia)组;以10µmol/...目的:探讨缺氧对脑内胆固醇代谢稳态及神经元衰老的影响。方法:8~12周龄的C57BL/6野生型雄性小鼠随机分为常氧(Normoxia)组和缺氧(Hypoxia)组;以缺氧处理小鼠海马神经元HT22细胞并分为对照(Control)组和缺氧(Hypoxia)组;以10µmol/L剂量的24-羟基胆固醇(24S-Hydroxycholesterol,24S-OHC)处理HT22细胞并分为Control组和24S-OHC组。苏木精-伊红染色(hematoxylin-ensin,HE)和尼氏(nissl)染色观察脑组织结构;酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测血清、脑组织和细胞24S-OHC水平;微量法检测脑组织和细胞总胆固醇(total cholesterol,TC)与甘油三酯(triglyceride,TG)水平;蛋白质免疫印迹(western blot,WB)法检测脑组织和细胞胆固醇代谢相关酶3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-coenzyme A reductase,HMGCR)、细胞色素P450家族46亚家族A成员1(cytochrome P450 family 46 subfamily A member 1,CYP46A1)、衰老相关蛋白细胞周期蛋白依赖性激酶抑制因子2A(cyclin-dependent kinase inhibitor 2A,CDKN2A/p16)、细胞周期蛋白依赖性激酶抑制因子1A(cyclin-dependent kinase inhibitor 1A,CDKN1A/p21)、磷酸化组蛋白(phospho-H2AX,γH2AX)和脂质合成相关分子抗硬脂酰辅酶A去饱和酶1(stearoyl-Coenzyme A desaturase 1,SCD1)、脂肪酸合酶(fatty acid synthase,FASN)、固醇调节元件结合蛋白1c(sterol regulatory element-binding protein 1c,SREBP1c)的表达水平;免疫组织化学染色检测脑内CYP46A1和γH2AX的表达;检测各组小鼠大脑组织中基因表达谱,筛选目标差异表达基因(differentially expressed genes,DEGs),对DEGs进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析;衰老相关β半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-Gal)染色评估细胞衰老情况;BODIPY染色观察细胞脂滴蓄积情况。结果:与常氧组比较,缺氧组脑湿重差异无统计学意义(P=0.573);Nissl染色显示存活神经元数目减少;血清24S-OHC水平升高,脑内24S-OHC和TG含量增多,TC含量减少(均P<0.05);胆固醇合成酶HMGCR表达降低,胆固醇分解酶CYP46A1表达升高,p16、p21和γH2AX表达增加(均P<0.05);转录组学结果显示,在GO分析中炎症通路与脂质相关通路富集,在KEGG分析中磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K-Akt)信号通路和肿瘤蛋白p53(tumor protein 53,p53)信号通路等与衰老相关通路富集。缺氧处理HT22细胞实验中,与Control组相比,Hypoxia组细胞内TC含量减少,24S-OHC含量增多(均P<0.05);HMGCR表达降低,CYP46A1表达升高,p16、p21、γH2AX表达增加(均P<0.05);SA-β-gal阳性细胞明显增多。24S-OHC处理HT22细胞实验中,与Control组相比,24S-OHC组细胞内TC和TG含量均增多(均P<0.05);细胞内脂滴含量明显增多(P<0.05);HMGCR和CYP46A1表达降低,SCD1、FASN和SREBP1c表达升高,p16、p21和γH2AX表达升高(均P<0.05)。结论:慢性缺氧通过下调HMGCR和上调CYP46A1表达,诱导24S-OHC异常蓄积并触发SREBP1c/SCD1信号通路介导的脂毒性衰老。展开更多
Objective Age-related diseases, including neurodegenerative diseases, are associated with oxidative stress and lipid peroxidation, and increase the levels of cholesterol auto-oxidation products such as 7β-hydroxychol...Objective Age-related diseases, including neurodegenerative diseases, are associated with oxidative stress and lipid peroxidation, and increase the levels of cholesterol auto-oxidation products such as 7β-hydroxycholesterol(7β-OHC). Thus, it is imperative to identify agents that can prevent 7β-OHC-induced side-effects. Methods We evaluated the potential protective effects of Carpobrotus edulis ethanol-water extract(EWe) on murine oligodendrocytes(158 N) cultured in the absence or presence of 7β-OHC(20 μg/mL, 24 h). The cells were incubated with EWe(20-200 μg/mL) 2 h before 7β-OHC treatment. Mitochondrial activity and cell growth were evaluated with the MTT assay. Photometric methods were used to analyze antioxidant enzyme [catalase(CAT) and glutathione peroxidase(GPx)] activities and the generation of lipid and protein oxidation products [malondialdehyde(MDA), conjugated diene(CD), and carbonylated proteins(CPs)]. Results Treatment with 7β-OHC induced cell death and oxidative stress(reflected by alteration in CAT and SOD activities). Overproduction of lipid peroxidation products(MDA and CDs) and CPs was also reported. The cytotoxic effects associated with 7β-OHC were attenuated by 160 μg/mL of EWe of C. edulis. Cell death induced by 7β-OHC treatment was ameliorated, GPx and CAT activities were restored to normal, and MDA, CD, and CP levels were reduced following C. edulis extract treatment. Conclusion These data demonstrate the protective activities of C. edulis EWe against 7β-OHC-induced disequilibrium in the redox status of 158 N cells, indicative of the potential role of this plant extract in the prevention of neurodegenerative diseases.展开更多
文摘目的:探讨缺氧对脑内胆固醇代谢稳态及神经元衰老的影响。方法:8~12周龄的C57BL/6野生型雄性小鼠随机分为常氧(Normoxia)组和缺氧(Hypoxia)组;以缺氧处理小鼠海马神经元HT22细胞并分为对照(Control)组和缺氧(Hypoxia)组;以10µmol/L剂量的24-羟基胆固醇(24S-Hydroxycholesterol,24S-OHC)处理HT22细胞并分为Control组和24S-OHC组。苏木精-伊红染色(hematoxylin-ensin,HE)和尼氏(nissl)染色观察脑组织结构;酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测血清、脑组织和细胞24S-OHC水平;微量法检测脑组织和细胞总胆固醇(total cholesterol,TC)与甘油三酯(triglyceride,TG)水平;蛋白质免疫印迹(western blot,WB)法检测脑组织和细胞胆固醇代谢相关酶3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-coenzyme A reductase,HMGCR)、细胞色素P450家族46亚家族A成员1(cytochrome P450 family 46 subfamily A member 1,CYP46A1)、衰老相关蛋白细胞周期蛋白依赖性激酶抑制因子2A(cyclin-dependent kinase inhibitor 2A,CDKN2A/p16)、细胞周期蛋白依赖性激酶抑制因子1A(cyclin-dependent kinase inhibitor 1A,CDKN1A/p21)、磷酸化组蛋白(phospho-H2AX,γH2AX)和脂质合成相关分子抗硬脂酰辅酶A去饱和酶1(stearoyl-Coenzyme A desaturase 1,SCD1)、脂肪酸合酶(fatty acid synthase,FASN)、固醇调节元件结合蛋白1c(sterol regulatory element-binding protein 1c,SREBP1c)的表达水平;免疫组织化学染色检测脑内CYP46A1和γH2AX的表达;检测各组小鼠大脑组织中基因表达谱,筛选目标差异表达基因(differentially expressed genes,DEGs),对DEGs进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析;衰老相关β半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-Gal)染色评估细胞衰老情况;BODIPY染色观察细胞脂滴蓄积情况。结果:与常氧组比较,缺氧组脑湿重差异无统计学意义(P=0.573);Nissl染色显示存活神经元数目减少;血清24S-OHC水平升高,脑内24S-OHC和TG含量增多,TC含量减少(均P<0.05);胆固醇合成酶HMGCR表达降低,胆固醇分解酶CYP46A1表达升高,p16、p21和γH2AX表达增加(均P<0.05);转录组学结果显示,在GO分析中炎症通路与脂质相关通路富集,在KEGG分析中磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K-Akt)信号通路和肿瘤蛋白p53(tumor protein 53,p53)信号通路等与衰老相关通路富集。缺氧处理HT22细胞实验中,与Control组相比,Hypoxia组细胞内TC含量减少,24S-OHC含量增多(均P<0.05);HMGCR表达降低,CYP46A1表达升高,p16、p21、γH2AX表达增加(均P<0.05);SA-β-gal阳性细胞明显增多。24S-OHC处理HT22细胞实验中,与Control组相比,24S-OHC组细胞内TC和TG含量均增多(均P<0.05);细胞内脂滴含量明显增多(P<0.05);HMGCR和CYP46A1表达降低,SCD1、FASN和SREBP1c表达升高,p16、p21和γH2AX表达升高(均P<0.05)。结论:慢性缺氧通过下调HMGCR和上调CYP46A1表达,诱导24S-OHC异常蓄积并触发SREBP1c/SCD1信号通路介导的脂毒性衰老。
文摘Objective Age-related diseases, including neurodegenerative diseases, are associated with oxidative stress and lipid peroxidation, and increase the levels of cholesterol auto-oxidation products such as 7β-hydroxycholesterol(7β-OHC). Thus, it is imperative to identify agents that can prevent 7β-OHC-induced side-effects. Methods We evaluated the potential protective effects of Carpobrotus edulis ethanol-water extract(EWe) on murine oligodendrocytes(158 N) cultured in the absence or presence of 7β-OHC(20 μg/mL, 24 h). The cells were incubated with EWe(20-200 μg/mL) 2 h before 7β-OHC treatment. Mitochondrial activity and cell growth were evaluated with the MTT assay. Photometric methods were used to analyze antioxidant enzyme [catalase(CAT) and glutathione peroxidase(GPx)] activities and the generation of lipid and protein oxidation products [malondialdehyde(MDA), conjugated diene(CD), and carbonylated proteins(CPs)]. Results Treatment with 7β-OHC induced cell death and oxidative stress(reflected by alteration in CAT and SOD activities). Overproduction of lipid peroxidation products(MDA and CDs) and CPs was also reported. The cytotoxic effects associated with 7β-OHC were attenuated by 160 μg/mL of EWe of C. edulis. Cell death induced by 7β-OHC treatment was ameliorated, GPx and CAT activities were restored to normal, and MDA, CD, and CP levels were reduced following C. edulis extract treatment. Conclusion These data demonstrate the protective activities of C. edulis EWe against 7β-OHC-induced disequilibrium in the redox status of 158 N cells, indicative of the potential role of this plant extract in the prevention of neurodegenerative diseases.
