摘要
目的研究重组糖蛋白激素β5/α2(rCGH)对3T3-L1脂肪细胞脂解的影响,并探究其潜在机制。方法体外培养3T3-L1前脂肪细胞,并诱导分化为成熟脂肪细胞,给予不同浓度rCGH干预24 h。以CCK-8法检测3T3-L1脂肪细胞的细胞活力,酶学方法测定细胞内甘油三酯(TG)及培养上清中甘油的水平,油红O染色观察脂滴变化。Western blot检测各组脂肪细胞中HSL和ATGL脂解蛋白的表达水平。不同浓度rCGH加或不加PKA抑制剂H89对已分化成熟的3T3-L1脂肪细胞进行联合干预实验,将培养细胞分为对照组、H89预处理组、1μmol·L^(-1)rCGH组和(1μmol·L^(-1)rCGH+H89)联合干预组。酶法测定胞内TG及游离甘油的含量,Western blot检测CREB及脂解相关蛋白的表达。结果不同浓度rCGH(0.25、0.5、1、2μmol·L^(-1))对脂肪细胞细胞活力无显著影响(P>0.05);与对照组相比,rCGH处理显著降低了脂滴大小和细胞内TG含量,同时显著升高了细胞上清液中的甘油浓度。rCGH处理还刺激了p-HSL、ATGL和p-PKA的蛋白表达。此外,加入PKA抑制剂H89,减弱了rCGH对游离甘油水平、细胞内TG含量以及p-HSL、p-PLIN1和p-CREB表达的影响。结论rCGH通过上调HSL、ATGL和PKA活性促进3T3-L1脂肪细胞的脂质分解,促进甘油释放,抑制TG合成和脂质积累,其作用机制与cAMP/PKA/CREB信号通路激活有关。
Aim To investigate the effect of recombinant glycoprotein hormoneβ5/α2(rCGH)on lipolysis in 3T3-L1 adipocytes,and explore the underlying mechanism.Methods 3T3-L1 preadipocytes were cultured and induced to differentiate into mature adipocytes,then treated with different concentrations of rCGH for 24 h in vitro.Cell viability of 3T3-L1 adipocytes was evaluated by CCK-8 assay,the levels of intracellular triglyceride(TG)and glycerol in the culture supernatant were measured by enzymatic method,and the changes of lipid droplets were observed by oil red O staining.The expression levels of HSL and ATGL lipolytic proteins in adipocytes were detected by Western blot.To carry out the intervention experiment with different concentrations of rCGH with or without the PKA inhibitor,H89,on the mature 3T3-L1 adipocytes,the cultured cells were divided into the control group,H89 pretreatment group,1μmol·L^(-1)rCGH group,and(1μmol·L^(-1)rCGH+H89)combined intervention group.The contents of intracellular TG and free glycerol were measured by enzymatic method,and the expression of CREB and lipolysis-related proteins was detected using Western blot.Results Different concentrations of rCGH(0.25,0.5,1,and 2μmol·L^(-1))had no significant effect on the cell viability of adipocytes(P>0.05).Compared with the control group,the treatment with rCGH significantly decreased the size of lipid droplets and intracellular TG content,while significantly elevated glycerol concentration in cell supernatant.rCGH treatment also stimulated the protein expression of p-HSL,ATGL,and p-PKA.In addition,the addition of a PKA inhibitor,H89,attenuated the effects of rCGH on free glycerol level,intracellular TG content,and the expression of p-HSL,p-PLIN1,and p-CREB.Conclusions rCGH enhances the lipolysis of 3T3-L1 adipocytes by up-regulating the activities of HSL,ATGL and PKA,promoting glycerol release,inhibiting TG synthesis and lipid accumulation,and its mechanism of action is related to the activation of cAMP/PKA/CREB signaling pathway.
作者
千爱君
萧耿苗
李壮
田雪
刘晓红
宋玉萍
赵正刚
赵子建
李芳红
QIAN Ai-jun;XIAO Geng-miao;LI Zhuang;TIAN Xue;LIU Xiao-hong;SONG Yu-ping;ZHAO Zheng-gang;ZHAO Zi-jian;LI Fang-hong(School of Biomedical and Pharmaceutical Science,Guangdong University of Technology,Guangzhou 510006,China;Shunde Hospital of Southern Medical University,Foshan Guangdong 528300,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2024年第7期1272-1278,共7页
Chinese Pharmacological Bulletin
基金
国家重点研发计划(No 2018YFA0800603)
广州市科技计划项目(No 2023B03J1291)
广东省创新团队(No 2016ZT06Y432)
广东省重点领域研发计划“新药创制”专项(No 2019B020201015)。
