背景:柚皮素是从具有许多药理活性的柑橘类水果中天然分离的。然而,在代谢综合征模型中,柚皮素对非酒精性脂肪性肝炎(NASH)的影响知之甚少。
目的:本研究旨在探讨柚皮素对12月龄雄性ApoE-/-小鼠NASH的影响及其可能的作用机制。
方法:体内,12月龄雄性ApoE-/-小鼠通过胃内灌胃施用柚皮素12周。实验结束时,收集血液样本和肝脏组织。血清代谢参数,甘油三酯的水平,胆固醇和羟脯氨酸,抗氧化酶的活性,用相应的试剂盒检测肝脏中炎性细胞因子(TNF-α和IL-6)的含量。苏木精-伊红观察肝脏病理变化,油红O,马斯顿的三色,小天狼星红和衰老β-半乳糖苷酶染色。二氢乙锭用于检测活性氧(ROS)。体外,在存在或不存在柚皮素的情况下用油酸处理AML-12细胞24小时。还在体外进行SIRTlsiRNA的转染。脂质积累,细胞ROS的产生,丙二醛含量,检测了抗氧化酶活性和TNF-α和IL-6的分泌水平。在体内和体外,通过实时PCR或Westernblot检测基因表达。
结果:柚皮素给药改善了代谢参数,抑制肝脏脂肪变性,脂质代谢相关基因的表达调控(FASN,SCD1、PPARα和CPT1α),减少肝纤维化和细胞衰老,通过减少巨噬细胞募集和TNF-α和IL-6的含量来抑制肝脏炎症,并通过抑制ROS的产生和使抗氧化酶的活性正常化来减少肝脏氧化应激。值得注意的是,柚皮素给药增加肝脏SIRT1蛋白的表达和活性,同时增加肝脏激酶B1(LKB1)的脱乙酰作用,PGC1α和NF-κB。体外研究,柚皮素对脂质积累的好处,SIRT1siRNA转染可减少氧化应激和炎症反应。
结论:这些结果表明,柚皮素可能是NASH治疗的潜在治愈性疗法,肝脏SIRT1介导的信号传导级联的激活与其有益作用有关。
BACKGROUND: Naringenin is naturally isolated from citrus fruits possessing many pharmacological activities. However, little is known about the effect of naringenin on nonalcoholic steatohepatitis (NASH) in the model of metabolic syndrome.
OBJECTIVE: The present study is aimed to investigate the effect of naringenin on NASH in 12-mo-old male ApoE-/- mice and its possible underlying mechanism.
METHODS: In vivo, 12-mo-old male ApoE-/- mice were administrated with naringenin by intragastric gavage for 12 weeks. At the end of experiment, the blood samples and liver tissues were collected. Metabolic parameters in serum, levels of triglyceride, cholesterol and hydroxyproline, activities of antioxidant enzymes, and content of inflammatory cytokines (TNF-α and IL-6) in liver were examined by corresponding assay kits. Pathological changes in liver were observed by hematoxylin-eosin, oil red O, masson\'s trichrome, picro-sirius red and senescence β-galactosidase staining. Dihydroethidium was used for detection of reactive oxygen species (ROS). In vitro, AML-12 cells were treated with oleic acid in the presence or absence of naringenin for 24 h. Transfection of SIRT1 siRNA was also conducted in vitro. Lipid accumulation, cellular ROS generation, malondialdehyde content, antioxidant enzyme activities and secretion levels of TNF-α and IL-6 were examined. Both in vivo and in vitro, gene expressions were detected by real-time PCR or western blot.
RESULTS: Naringenin administration improved metabolic parameters, suppressed hepatic steatosis, regulated expression of genes involved in lipid metabolism (FASN, SCD1, PPARα and CPT1α), reduced hepatic fibrosis and cell senescence, inhibited hepatic inflammation as evidenced by the decreased macrophage recruitment and content of TNF-α and IL-6, and reduced hepatic oxidative stress by suppressing ROS generation and normalizing activities of antioxidant enzymes. Notably, naringenin administration increased hepatic SIRT1 protein expression and activity along with the increased deacetylation of liver kinase B1 (LKB1), PGC1α and NF-κB. In vitro study, the benefits of naringenin on lipid accumulation, oxidative stress and inflammation were diminished by SIRT1 siRNA transfection.
CONCLUSIONS: These results indicate that naringenin administration may be a potential curative therapy for NASH treatment and the activation of hepatic SIRT1-mediated signaling cascades is involved in its beneficial effects.