摘要
运用CCK-8法检测不同浓度CoCl2(0、50、100、200、300、400 μmol/L)及其不同处理时间(0、6、12、24、48 h)对脂肪细胞活力的影响,并选择最适宜的处理条件;采用 Western blot方法检测不同浓度CoCl2(0、50、100、200、400 μmol/L)对脂肪细胞低氧及其下游关键分子蛋白表达的影响.结果显示,CoCl2处理组浓度越高,脂肪细胞活力越低,其中300、400 μmol/L处理组细胞活力显著下降(P＜0.01),200 μmol/L处理组细胞活力最高.与对照组相比,200 μmol/L CoCl2处理组低氧及其下游信号通路关键分子:缺氧诱导因子1-α(hypoxia inducible factor 1-alpha,HIF-1α)、葡萄糖转运蛋白 4(recombinant glucose transporter 4,GLUT4)、血管内皮生长因子受体 1(vascular endo-thelial growth factor receptor 1,FLT-1)、脯氨酰羟化酶 2(proline hydroxylase2,PHD2)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的蛋白表达水平显著上调(P＜0.01).与对照组相比,200 μmol/L CoCl2处理组中脂解代谢关键酶脂肪甘油三酯脂肪酶(adipose triglyceride lipase,ATGL)、围脂滴蛋白1(perilipin 1,PLIN1)、蛋白激酶 A(protein kinase A,PKA)和 300、400 μmol/L 处理组中激素敏感性甘油三酯脂肪酶(hormone-sensitive lipase,HSL)磷酸化的水平高于其他各组(P＜0.01).200 μmol/L处理组CoCl2介导的缺氧上调了胞内磷脂酰肌醇激酶(phosphatidylinositol 3-kinase,PI3K)的蛋白表达和蛋白激酶B(protein kinase B,Akt)的磷酸化水平.结果表明,添加200 μmol/LCoC12能够促进原代牛脂肪细胞缺氧相关蛋白和脂解代谢酶及胰岛素相关信号蛋白的表达.
Abstract
This study utilized the CCK-8 assay to examine the effects of various concentrations of CoCl2(0,50,100,200,300,400 μmol/L)and different treatment durations(0,6,12,24,48 h)on the viability of adipocytes,in order to determine the most suitable treatment conditions.Western blot analysis was employed to investigate the impact of different concentrations of CoCl2(0,50,100,200,400 μmol/L)on the expression of hypoxia and its downstream key proteins in adipocytes.The results indicated that higher concentrations of CoCl2 led to lower adipocyte viability,with sig-nificant decreases in cell viability observed in the 300,400 μmol/L treatment groups(P＜0.01),while the 200 μmol/L group exhibited the highest cell viability.Compared to the control group,the 200 μmol/L CoCl2 treatment group showed a significant upregulation in the expression of hypoxia and its downstream signaling pathway key molecules:hypoxia-inducible factor 1-alpha(HIF-1α),glucose transporter type 4(GLUT4),vascular endothelial growth factor receptor 1(FLT-1),prolyl hydroxylase 2(PHD2),and vascular endothelial growth factor(VEGF)(P＜0.01).Addi-tionally,the 200 μmol/L CoCl2 treatment group exhibited higher levels of key lipolytic enzymes,including adipose triglyceride lipase(ATGL),perilipin 1(PLIN1),protein kinase A(PKA),and increased phosphorylation levels of hormone-sensitive lipase(HSL)in the 300 and 400 μmol/L groui ps(P＜0.01).CoCl2-mediated hypoxia in the 200 μmol/L treatment group also in-creased the protein expression of phosphatidylinositol 3-kinase(PI3K)and the phosphorylation level of protein kinase B(Akt).These findings suggest that adding 200 μmol/L CoCl2 can enhance the expression of hypoxia-related proteins,lipolytic enzymes,and insulin-related signaling proteins in primary bovine adipocytes.
基金项目
国家自然科学基金资助项目(32102743)
国家自然科学基金资助项目(32125038)
黑龙江八一农垦大学引进人才科研启动基金资助项目(XYB202105)
维普
万方数据