目的 研究白藜芦醇通过诱导自噬促进肥胖小鼠骨骼肌组织中纤连蛋白Ⅲ型结构域包含5(FNDC5)降解的机制.方法 将6周龄雄性C57BL/6小鼠随机分为标准饮食(SCD)组、高脂(HFD)组及高脂+白藜芦醇(HFD+RES)组,HFD+RES组在HFD的同时胃饲白藜芦醇(400 mg/kg·d),共持续20周.测定体质量、血清甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白,HE染色检测骨骼肌的病理学变化.免疫组化、RT-PCR和Western blot分析和纤连蛋白Ⅲ型结构域包含5(FNDC5)、沉默信息调节因子(SIRT)-1、SIRT2、微管相关蛋白1轻链3α(LC3)、选择性自噬接头蛋白(p62)、Bcl-2同源结构域蛋白抗体(Beclin-1)、噬相关5同源物(ATG5)、噬相关7同源物(ATG7)的表达.结果 HFD组小鼠体质量增高,血清TG、TC和LDL-C水平显著升高,HDL-C水平下降;骨骼肌纤维间出现脂肪沉积;SIRT1、SIRT2和LC3的蛋白表达水平降低,而FNDC5和p62的蛋白表达升高.FNDC5的表达水平升高,SIRT1、SIRT2、LC3、Atg7和Beclin-1的基因表达水平降低.RES组可逆转HFD的效应,增加SIRT1、SIRT2和自噬相关基因的表达.结论 白藜芦醇可减少骨骼肌FNDC5表达的效应,其机制可能与其增加SIRT1和SIRT2的表达,进而促进自噬和FNDC5的降解有关.

Liraglutide (LRG), one agonist of glucagon-like peptide-1 receptor (GLP1R), has multiple lipid-lowering effects in type 2 diabetes mellitus, however, studies on the role of LRG in saturated fatty acid-induced bone loss are limited. Therefore, our aim was to investigate whether LRG reduces palmitate (PA)-induced apoptosis and whether the mechanism involves PKA/β-catenin/Bcl-2/Bax in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were treated with different concentrations of PA, LRG, or pretreated with Exendin 9-39 and H89, cell viability, intracellular reactive oxygen species (ROS), cAMP levels, apoptosis and the expression of protein kinase A (PKA) and phosphorylation of PKA (p-PKA), β-catenin and phosphorylation of β-catenin (Ser675)(p-β-catenin), GLP1R, cleaved-capase 3, Bcl2-Associated X Protein (Bax) and B-cell lymphoma-2 (Bcl-2) along with expression of Osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were evaluated. PA treatment inhibited cell proliferation and cAMP levels, elevated intracellular ROS levels and promoted apoptosis, increased protein expressions of RANKL, Bax and cleaved-caspase3, meanwhile decreased protein expression of OPG and Bcl-2 in a dose-dependent manner. LRG inverted PA-induced apoptosis, increased cAMP levels, promoted expression of p-PKA, p-β-catenin (Ser675) and reversed these gene expressions via increasing GLP1R expression. Pretreatment of the cells with Exendin 9-39 and H89 partially eradicated the protective effect of LRG on PA-induced apoptosis and gene expressions. Therefore, these findings indicated that LRG attenuates PA-induced apoptosis possibly by GLP1R-mediated PKA/β-catenin/Bcl-2/Bax pathway in MC3T3-E1 cells. Our results point to LRG as a new strategy to attenuate bone loss associated with high fat diet beyond its lipid-lowering actions.

目的 研究白藜芦醇(resveratrol,RES)减少肥胖小鼠细胞焦亡致肝损伤的机制.方法 将雄性C57BL/6小鼠随机分为标准(SCD)、高脂(HFD)及高脂+白藜芦醇(HFD+RES)饮食3组,标准或高脂饮食喂养,HFD+RES小鼠在高脂饮食的同时胃饲白藜芦醇(400 mg/(kg?d)),持续22 w.测定体重,分析血脂和interleukin-1 beta(IL-1β)、interleukin-18(IL-18)水平,HE染色检测肝脏组织的形态学变化,免疫组化检测去乙酰化酶sirtuin-1(SIRT1)、P66SHC、NLR family pyrin domain containing 3(NLRP3)的定位和表达,实时荧光定量RT-PCR和Western blot分析SIRT1、P66SHC(66ku Src homology 2 domain-containing protein)、NLRP3、cysteinyl aspartate specific proteinase-1(caspase-1)、gasdermin-D(GSDMD)、interleukin-1 beta(IL-1β)、interleukin-18(IL-18)等的表达.结果 体重、血液指标和HE染色结果均显示饮食诱导肥胖小鼠的炎症损伤模型构建成功;SIRT1表达量明显减少,P66SHC、NLRP3、Caspase-1、GSDMD、IL-1β、IL-18表达均显著增加.与HFD小鼠相比,HFD+RES小鼠的体重、血脂指标和肝脏脂质沉积均显著减少,SIRT1表达量明显增加,P66SHC、NLRP3、Caspase-1、GSDMD、IL-1β、IL-18表达均显著降低,且具有统计学意义.结论 白藜芦醇有明显的抑制P66SHC-NLRP3炎性小体介导的GSDMD活化和细胞焦亡的效应,其机制可能与增加肥胖小鼠肝脏组织SIRT1的表达有关.

Macrovascular disease is tightly associated with obesity-induced metabolic syndrome. Sitagliptin (SIT), an orally stable selective inhibitor of Dipeptidyl peptidase-4 (DPP-4), has protective effects on endothelium. However, the mechanisms enabling SIT to exhibit resistance to diet-induced obesity (DIO) related with reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress in the aorta and endothelial cells have not been reported yet. Therefore, the present study was conducted to determine if SIT exerts protective role in the thoracic aortas isolated from the high-fat diet (HFD)-treated rats and palmitate (PA)-treated endothelial cells by alleviating ROS and ER stress. Male Sprague Dawley rats were randomly divided into standard chow diet (SCD), HFD and HFD plus sitagliptin administration (HFD + SIT) groups. The rats of latter two groups were given HFD fodder for 12 weeks, then the HFD + SIT rats were treated with SIT (10 mg/kg/d) by intragastric administration for another 8 weeks. The body mass, vascular tension, serum oxidative stress indices and inflammatory parameters, pathological changes, protein expression of endothelial nitric oxide synthase (eNOS), the genes associated with ER stress and apoptosis in the thoracic aorta were measured. Furthermore, cell proliferation, ROS and the protein expression associated with ER stress (especially CHOP) and apoptosis were assessed in human umbilical vein endothelial cells (HUVECs) incubated with SIT and PA. Compared to the SCD rats, the HFD rats had higher serum lipid levels, decreased vascular tension, increased inflammation, oxidative and ER stress, and apoptosis of endothelial cells. PA promoted ROS generation, ER stress and apoptosis, inhibited cell proliferation in HUVECs. SIT treatment obviously ameliorated apoptosis via alleviating ROS and ER stress in the thoracic aortas isolated from HFD-fed rats and PA-treated HUVECs. The results suggest that SIT improved endothelial function via promoting cell proliferation and alleviating ROS-ER stress-CHOP pathway both in vivo and in vitro.