葛根调控PPARs信号通路改善脂肪细胞胰岛素抵抗的体外研究  

Study on the Pueraria lobata(Willd.) Regulating PPARs Signaling Pathway to Improve Insulin Resistance in Adipocytes In Vitro

作　　者：朱水兰 许文华 李冰涛[1,2] 涂珺 ZHU Shuilan;XU Wenhua;LI Bingtao;TU Jun(Research Center for Differentiation and Development of Chinese Medicine Basic Theory&Jiangxi Province Key Laboratory of Chinese Medicine Etiopathogenisis,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China;Key Laboratory of Traditional Chinese Medicine Pharmacology of Jiangxi Province,Nanchang 330004 Jiangxi,China)

机构地区：[1]江西中医药大学中医基础理论分化发展研究中心/江西省中医病因学重点实验室,江西南昌330004 [2]江西省中药药理重点实验室,江西南昌330004

出　　处：《中药新药与临床药理》2023年第3期287-295,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(82160838,81960809);江西中医药大学校级科技创新团队发展计划项目(CXTD22007)。

摘　　要：目的通过体外研究探讨葛根改善脂肪细胞胰岛素抵抗的关键分子机制。方法优化建立胰岛素抵抗(IR)-3T3-L1细胞模型,以5%、10%和15%葛根含药血清干预24 h后,采用ELISA法检测脂肪细胞上清液中脂联素(ADPN)含量;油红O染色法观察脂肪细胞的形态变化。分子对接预测葛根化学成分与过氧化物酶体增殖物激活受体家族(PPARs)的结合活性。采用Western Blot法检测脂肪细胞PPARγ/α蛋白及其磷酸化表达水平,并采用试剂盒检测其PPARγ/α活性;以PPARγ/α特异拮抗剂GW9662/GW6471分别干预后,采用qPCR法检测PPARγ/α及其调控基因的转录水平。结果与IR模型组比较,5%、10%和15%葛根含药血清组细胞外泌ADPN含量显著增加(P<0.05,P<0.01),小脂肪细胞数量增多,胞内脂滴变小。分子对接显示葛根中多个活性成分与PPARγ和PPARα有较强结合活性,而与PPARδ结合相对较弱。与IR模型组比较,5%、10%和15%葛根含药血清能显著上调PPARγ和PPARα蛋白表达(P<0.001),下调p-PPARγ(Ser112)蛋白表达(P<0.001),而p-PPARα(Ser12)蛋白条带检测不到,提示非磷酸化活性状态PPARγ和PPARα表达上调,与检测到的PPARγ/α活性显著增强(P<0.001)相吻合。GW9662/GW6471分别拮抗后,与非拮抗对照组比较,10%葛根含药血清组细胞PPARγ及其靶基因ADPN、葡萄糖转运蛋白4(GLUT4)mRNA表达均显著下调(P<0.001);PPARα及其靶基因中链脂酰辅酶A脱氢酶(MCAD)、长链脂酰辅酶A脱氢酶(LCAD)、酰基辅酶A氧化酶1(ACOX1)mRNA表达均显著下调(P<0.05,P<0.001)。此外,与IR模型组比较,10%葛根含药血清组细胞的胰岛素受体(InsR)、胰岛素受体底物1(IRS1)、葡萄糖转运蛋白1(GLUT1)、PPARδ和诱导细胞死亡的DFF45样效应因子B(CIDEB)mRNA表达显著上调(P<0.01,P<0.001)。结论葛根可能通过激活PPARγ/α表达及活性功能,协调调控脂肪细胞糖脂代谢,改善其IR。Objective To explore the key molecular mechanism of Pueraria lobata(Willd.)(Gegen)ameliorating adipocytic insulin resistance(IR)in vitro.Methods The IR-3T3-L1 cell model was optimized,and 5%,10%and 15% Gegen-containing serum were administered for 24 hours. ELISA were used to detect adiponectin(ADPN)content in adipocyte supernatant;oil red O staining was used to detect morphological changes in adipocytes.Molecular docking predicted the binding activity of Gegen chemical components to the family of peroxisome proliferators-activated receptors(PPARs). Western Blot was used to detect the expression levels of PPARγ/α and phosphorylation in adipocytes,PPARγ/α activity was measured using a kit,PPARγ/α-specific antagonists GW9662/GW6471 were intervened separately and qPCR detected the transcript levels of PPARγ/α and regulatory genes.Results Compared to the IR model group,the exocrine ADPN content was significantly increased in the 5%,10%and 15% of Gegen-containing serum groups(P<0.05,P<0.01). The 5%,10% and 15% Gegen-containing serum group showed an increase in the number of small adipocytes and smaller intracellular lipid droplets. Molecular docking showed that several active components of Gegen had strong binding activity to PPARγ and PPARα and relatively weak binding to PPARδ. Compared to the IR model group,the 5%,10% and 15% GG-containing serum groups up-regulated protein expressions of PPARγ and PPARα(P<0.001) and down-regulated p-PPARγ(Ser112)protein expression(P<0.001),while the p-PPARα(Ser12)protein band was undetectable,suggesting an up-regulation of the non-phosphorylated active state of PPARγ and PPARα, which was consistant with the significant increase of PPARγ/α activity(P<0.001). After antagonism by GW9662/GW6471 respectively, the mRNA expressions of PPARγ and its target genes ADPN and glucose transporter protein(GLUT) 4 were significantly reduced in the 10% Gegen-containing serum group compared with the non-antagonistic control group(P<0.001);the expressions of PPARα and its target ge

关 键 词：葛根 脂肪胰岛素抵抗 过氧化物酶体增殖物激活受体Γ 过氧化物酶体增殖物激活受体Α 糖脂代谢 3T3-L1细胞 

分 类 号：R285.5[医药卫生—中药学]