摘要
目的探讨Adropin对脂肪细胞自噬及磷脂酰肌醇-3激酶/丝氨酸苏氨酸蛋白激酶(PI3K/AKT)通路的影响。方法将小鼠胚胎成纤维前脂肪细胞3T3-L1诱导分化为成熟脂肪细胞,随机分为对照组、Adropin组、自噬激动剂组、Adropin+自噬激动剂组。对照组细胞不予特殊处理,Adropin组细胞加入Adropin(终浓度为1000 nmol·L^-1),自噬激动剂组加入西罗莫司(终浓度为100 nmol·L^-1),Adropin+自噬激动剂组细胞加入Adropin(终浓度为1000 nmol·L^-1)和西罗莫司(终浓度为100 nmol·L^-1),继续培养2 h;采用单丹磺酰尸胺染色检测各组细胞中自噬空泡情况,细胞免疫荧光染色法检测各组细胞微管相关蛋白轻链3(LC3)表达,采用Western blot法检测各组细胞自噬相关蛋白Beclin-1、LC3-Ⅱ、p62及PI3K/AKT通路蛋白PI3K、磷酸化PI3K(p-PI3K)、AKT、磷酸化AKT(p-AKT)的表达。结果与对照组比较,Adropin组细胞中自噬空泡相对含量、LC3阳性细胞比例及LC3-Ⅱ、Beclin-1蛋白相对表达量显著降低(P<0.05),p62、p-PI3K、p-AKT蛋白相对表达量显著升高(P<0.05)。与对照组比较,自噬激动剂组细胞中自噬空泡相对含量、LC3阳性细胞比例及LC3-Ⅱ、Beclin-1蛋白相对表达量显著升高(P<0.05),p62、p-PI3K、p-AKT蛋白相对表达量显著降低(P<0.05)。Adropin+自噬激动剂组与对照组细胞中自噬空泡相对含量、LC3阳性细胞比例及LC3-Ⅱ、Beclin-1、p62、p-PI3K、p-AKT蛋白相对表达量比较差异无统计学意义(P>0.05)。与Adropin组比较,自噬激动剂组、Adropin+自噬激动剂组细胞中自噬空泡相对含量、LC3阳性细胞比例及LC3-Ⅱ、Beclin-1蛋白相对表达量显著升高(P<0.05),p62、p-PI3K、p-AKT蛋白相对表达量显著降低(P<0.05)。与自噬激动剂组比较,Adropin+自噬激动剂组细胞中自噬空泡相对含量、LC3阳性细胞比例及LC3-Ⅱ、Beclin-1蛋白相对表达量显著降低(P<0.05),p62、p-PI3K、p-AKT蛋白相对表达量显著升高(P<0.05)。4组�
Objective To investigate the effects of Adropin on adipocyte autophagy and phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)pathway.Methods Mouse embryonic fibroblast-adipose like cell line(3T3-L1)was induced to differentiate into mature adipocytes,then the cells were randomly divided into control group,Adropin group,autophagy agonist group and Adropin+autophagy agonist group.The cells in the control group were not given special treatment.The cells in the Adropin group were given Adropin(final concentration was 1000 nmol·L^-1).The cells in the autophagy agonist group was given sirolimus(final concentration was 100 nmol·L^-1).The cells in the Adropin+autophagy agonist group were given Adropin(final concentration was 1000 nmol·L^-1)and sirolimus(final concentration was 100 nmol·L^-1).Then the cells in were cultured for 2 hours.The autophagic vacuoles in cells of each group were detected by monodansylcadaverine staining.The expression of microtubule-associated protein 1 light chain 3(LC3)in cells of each group was detected by immunofluorescence staining.The expressions of autophagy-related proteins(Beclin-1,LC3-Ⅱ,p62)and PI3K/AKT pathway-related proteins such as PI3K,phosphorylated-PI3K(p-PI3K),AKT,phosphorylated-AKT(p-AKT)in cells of each group were detected by Western blot.Results Compared with the control group,the relative content of autophagic vacuoles,the proportion of LC3 positive cells,the relative expressions of LC3-Ⅱand Beclin-1 proteins was lower(P<0.05),while the relative expressions of p62,p-PI3K and p-AKT proteins were higher in the Adropin group(P<0.05);the relative content of autophagic vacuoles,the proportion of LC3 positive cells,the relative expressions of LC3-Ⅱand Beclin-1 proteins in the autophagy agonist group were higher(P<0.05),while the relative expressions of p62,p-PI3K and p-AKT proteins were lower(P<0.05).There was no significant difference in the relative content of autophagic vacuoles,the proportion of LC3 positive cells and the relative expressions of LC3-Ⅱ,Beclin-1,
作者
李凤丽
李茂山
何丽
罗方
康志强
LI Feng-li;LI Mao-shan;HE Li;LUO Fang;KANG Zhi-qiang(Department of Endocrinology,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450007,Henan Province,China;Department of Orthopaedics,the Third People′s Hospital of Henan Province,Zhengzhou 450006,Henan Province,China)
出处
《新乡医学院学报》
CAS
2019年第11期1018-1023,1029,共7页
Journal of Xinxiang Medical University
基金
河南省科技发展计划项目资助(编号:182102310089)
