安石榴苷调控内质网应激介导肝细胞脂凋亡的分子机制研究

Lipid metabolism balance plays an important role in maintaining the normal levels of sterols, triglycerides and free fatty acids in human blood and peripheral tissues. Systematically elucidating the underlying mechanism of food components in regulating lipid metabolism is of great significance for maintaining lipid homeostasis and preventing lipid metabolism disorder-related diseases. Our previous studies found that punicalagin (PU) could inhibit palmitic acid (PA)-induced hepatocyte lipoapoptosis by regulating the endoplasmic reticulum (ER) stress signaling pathway. This study will systematically clarify the mechanism of PU in regulating endoplasmic reticulum stress-mediated lipoapoptosis. We conducted the experiment by using the PA-treatment hepatocyte cells model under PU non- and pre-treatment. Sirtuin1 was silenced by RNA interference, the deacetylation status of PGC1-alpha, HSF1/HSP signaling pathways and autophagy-associated proteins were evaluated by immunoprecipitation and western blotting. RT-RCR and western blotting were performed to detect the expression of Sirtuin 1 which regulated by miR-34a. Furthermore, the effect of PU on ER stress-mediated lipoapoptosis was verified in MCD animal model. This study will provide a novel basis for the mechanism of PU regulating lipid metabolism and the development of pomegranate polyphenol dietary supplements.

脂质代谢平衡对于维持人体固醇、甘油三酯、游离脂肪酸的正常水平起着至关重要的作用。石榴多酚能有效调节脂质代谢，食源性地预防脂质代谢紊乱导致的脂肪肝、心血管等慢性疾病。项目申请人前期研究发现，石榴皮中特异性成分安石榴苷（PU）可通过调控内质网应激信号通路蛋白的表达而抑制棕榈酸（PA）诱导的肝细胞脂凋亡。本课题系统地解析PU调控内质网应激介导脂凋亡的作用机理。拟利用PA诱导的肝细胞模型，采用小干扰RNA基因沉默、免疫共沉淀、RT-RCR、蛋白免疫印迹等手段，通过探究miR-34a调控Sirtuin1的作用，以及Sirtuin1对下游PGC1α、HSF1/HSP信号通路和自噬相关蛋白去乙酰化水平的影响，阐明Sirtuin1介导PU调控内质网应激的分子机制。利用MCD小鼠模型进一步确证PU抑制内质网应激介导脂凋亡的现象与机理，为开发功效确切、成分和机制明确的石榴多酚功能因子提供可靠的理论依据。

饱和脂肪酸和高热量饮食等因素都会引起脂代谢的异常，导致脂质的蓄积、脂源性的细胞凋亡等生理病理现象，进而引发脂肪肝、肥胖、心血管等疾病的发生。深入探究脂质代谢紊乱的机制，对于开发功效确切、机制明确的食品功能因子提供理论依据，同时也为慢性疾病的预防及干预提供重要参考。.本项目利用饱和脂肪酸棕榈酸（PA）诱导的HepG2肝细胞脂凋亡模型，证实了SIRT1在安石榴苷（PU）抑制内质网应激介导脂凋亡的作用，通过对SIRT1调控细胞自噬、氧化应激以及HSF1/HSP信号通路的探究，阐明了SIRT1抑制PA诱导内质网应激的分子机制。然而，进一步对饱和脂肪酸PA的研究发现，PA能够增强甲基硒酸（MSeA）抗癌的敏感性。在HepG2肝癌细胞中，二者联合处理激活了内质网应激反应，而PERK和IRE1信号通路在PA+MSeA诱导的凋亡中发挥着相反的作用。此外，PA的加入增加了MSeA对HepG2细胞葡萄糖代谢、线粒体功能的抑制。在L02人正常肝细胞中，MSeA却能够保护PA诱导的细胞毒性。该研究对饱和脂肪酸PA在抗癌方面的新用途提供了重要的理论依据。.此外，本项目还深入探究了多不饱和脂肪酸中ω-3家族的DHA通过其免疫调节功能发挥抗肿瘤的活性机制。在体外模型中，验证了DHA能够恢复T细胞杀伤敏感性，增强肿瘤浸润性T细胞的免疫功能；DHA通过抑制PD-L1的棕榈酰化和CSN5活性来加速PD-L1泛素依赖性降解。在T细胞缺陷的裸鼠中，DHA对LLC肿瘤的抑制作用显著减弱。

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