欧前胡素通过AMPK/SIRT1/PGC-1α通路促白色脂肪棕色化

Imperatorin promotes the browning of white fat through the AMPK/SIRT1/PGC-1α pathway

关键词:  网络药理学  欧前胡素  产热  白色脂肪棕色化  生存时间

Key words:  network pharmacology  imperatorin  thermogenesis  white adipose tissue browning  survival time

摘要:

[目的] 通过网络药理学筛选促白色脂肪棕色化的中药单体，评价活性并探究其作用机制。[方法] 1）中药单体的筛选：用GEO2R分析处理一周与五周冷暴露下C57BL/6小鼠皮下白色脂肪的差异基因，筛选出交集差异基因（DEGs）；建立蛋白相互作用图谱，筛选核心（Hub）基因；ITCM平台预测可调控hub基因的中药单体，给予10 μmol/L不同药物诱导3T3-L1前脂肪细胞分化，实时荧光定量PCR法（RT-PCR）筛选对产热基因表达上调效果最强的中药单体。2）中药单体的生物学活性评价与机制研究：最优单体干预细胞后，CCK8法检测细胞活力，油红O染色观察细胞脂肪蓄积情况；免疫荧光定量解偶联蛋白1（Ucp1）表达；线粒体红色荧光（Mito-tracker-Red）探针检测线粒体数量；RT-PCR法和免疫蛋白印迹（Western blot）法检测棕色化基因蛋白水平变化；加入AMP活化蛋白激酶（AMPK）抑制剂多索吗啡验证单体是否通过激活AMP活化蛋白激酶/沉默信息调节因子2同源蛋白1/过氧化物酶体增殖物激活受体γ共激活因子1α（AMPK/SIRT1/PGC-1α）途径发挥产热作用。将8周龄雄性C57BL/6小鼠分为对照组、低剂量组，高剂量组、2周冷暴露组，给药28天后眼眶取血，检测血清中总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-C）和高密度脂蛋白胆固醇（HDL-C）含量。分离腹股沟白色脂肪（iWAT），苏木精-伊红（HE）染色与UCP1免疫组化观察组织形态学，RT-PCR实验、Western blot实验测定相关通路基因和蛋白的表达变化；平行一组-15℃暴露测定其生存时间。[结果] 网络药理学筛选出的五个中药单体干预细胞后，对产热基因表达上调效果最强的是欧前胡素（IMP）。CCK8法筛选出IMP最优浓度10 μmol/L，IMP干预后脂肪细胞内脂滴含量减少，UCP1表达量与线粒体数量显著上调（P<0.01）；线粒体生物合成基因核呼吸因子1（Nrf1）、线粒体转录因子A（Tfam）与米色特异性基因Cbp/P300相互作用转录激活因子1（Cited1）、肿瘤坏死因子受体超家族成员9（Cd137）、跨膜蛋白26（Tmem26）、Ⅱ型脱碘酶（Dio2）（P<0.05）表达显著上调；在1、3、10μmol/L浓度下，IMP呈剂量依赖性促进产热基因UCP1、PR结构域蛋白16（PRDM16）、PGC-1α（P<0.05）和产热、脂肪酸氧化过氧化物酶体增殖物激活受体α（PPARα）蛋白以及AMPK通路蛋白P-AMPK、SIRT1的表达，抑制脂质合成基因过氧化物酶体增殖物激活受体γ（PPARγ）、CCAAT增强子结合蛋白（C/EBP）的表达；AMPK抑制剂可阻断IMP对棕色化脂肪的作用。IMP干预后小鼠TG、TC、LDL-C低于对照组，HDL-C高于对照组，iWAT中脂滴形态相对变小，高剂量组UCP1阳性细胞数量显著增多（P<0.01），高剂量IMP显著上调UCP1、PGC-1α基因和蛋白表达、AMPK通路蛋白表达，下调PPARγ蛋白表达（P<0.05）。低剂量IMP可显著延长-15℃小鼠的生存时间（P<0.01）。[结论] 1）通过网络药理学可预测促进脂肪细胞棕色化的中药单体。2）IMP可促进白色脂肪棕色化，有效延长了小鼠在极寒温度下的存活时间。3）IMP促白色脂肪棕色化通过调控AMPK/SIRT1/PGC-1α信号通路实现。

Abstract:

[Objective] To screen traditional Chinese medicine（TCM）monomers that promote white adipose tissue browning using network pharmacology，evaluate their activity，and investigate their mechanism of action. [Methods] 1）Screening of TCM monomers：GEO2R was used to analyze differential genes in subcutaneous white adipose tissue of C57BL / 6 mice after one week and five weeks of cold exposure，and intersecting differentially expressed genes（DEGs）were screened. A protein-protein interaction network was constructed to identify core （Hub）genes. The ITCM platform predicted TCM monomers capable of regulating hub genes. 3T3-L1 preadipocytes were induced to differentiate with 10 μmol/L of different drugs，and real-time quantitative PCR（RT-PCR）was used to screen for the TCM monomer with the strongest up-regulatory effect on thermogenic gene expression. 2）Biological activity evaluation and mechanism study of the TCM monomer：After intervention with the optimal monomer，cell viability was detected by CCK-8 assay；lipid accumulation was observed by Oil Red O staining；uncoupling protein 1 (UCP1) expression was quantified by immunofluorescence；mitochondrial number was detected using MitoTracker Red probe；changes in browning gene and protein levels were measured by RT-PCR and Western blot；the AMP- activated protein kinase（AMPK） inhibitor dorsomorphin was added to verify whether the monomer exerts thermogenic effects by activating the AMPK/SIRT1/PGC-1α pathway. Eight-week-old male C57BL/6 mice were divided into control，low-dose，high-dose，and 2-week cold exposure groups. After 28 days of administration，orbital blood was collected to measure serum total cholesterol（TC），triacylglycerol（TG），low-density lipoprotein cholesterol (LDL-C)，and high-density lipoprotein cholesterol（HDL-C）levels. Inguinal white adipose tissue(iWAT) was isolated；histomorphology was observed by hematoxylin-eosin（HE）staining and UCP1 immunohistochemistry；gene and protein expression of related pathways was determined by RT-PCR and Western blot；a parallel group was exposed to-15 ℃ to determine survival time. [Results] Among the five TCM monomers screened by network pharmacology，imperatorin（IMP）showed the strongest up-regulatory effect on thermogenic gene expression. The optimal concentration of IMP was 10 μmol/L as determined by CCK-8 assay. After IMP intervention，lipid droplet content in adipocytes decreased，while UCP1 expression and mitochondrial number were significantly up-regulated （P<0.01）. Mitochondrial biogenesis genes nuclear respiratory factor 1（Nrf1），mitochondrial transcription factor A （Tfam），and beige-specific genes Cited1，Cd137，Tmem26，and Dio2 were significantly up-regulated（P<0.05）. At concentrations of 1，3，and 10 μmol/L，IMP dose-dependently promoted the expression of thermogenic genes UCP1， PRDM16，PGC-1α（P<0.05），thermogenic and fatty acid oxidation protein PPARα，and AMPK pathway proteins p- AMPK and SIRT1，while inhibiting the expression of lipid synthesis genes PPARγ and C/EBP. The AMPK inhibitor blocked IMP’s effects on adipose browning. After IMP intervention，mice had lower TG，TC，and LDL-C，and higher HDL-C than the control group. Lipid droplet morphology in iWAT was relatively smaller；UCP1-positive cell number significantly increased in the high-dose group（P<0.01）；high-dose IMP significantly up-regulated UCP1，PGC-1α gene and protein expression，and AMPK pathway protein expression，while down-regulating PPARγ protein expression（P<0.05）. Low-dose IMP significantly extended mouse survival time at -15 ℃（P<0.01）. [Conclusion] 1）Network pharmacology can predict TCM monomers that promote adipocyte browning. 2）IMP can promote white adipose tissue browning and effectively extend mouse survival time under extreme cold. 3）IMP promotes white adipose tissue browning by regulating the AMPK/SIRT1/PGC-1α signaling pathway.