背景:急性胰腺炎(AP)是一种外分泌胰腺的炎症性疾病,尤其是高脂血症性急性胰腺炎(HLAP)是急性胰腺炎的第三大病因,急性胰腺炎更严重,持续性多器官功能衰竭的发生率更高。HLAP对腺泡细胞内的细胞器造成损伤,特别是线粒体,内溶酶体-自噬系统,并伴有衰老相关分泌表型(SASP)。RAD,只有两个由泽泻和白术组成,这是最著名的抗炎和降脂的能力。然而,RAD缓解HLAP的机制仍然模糊,需要进一步调查。
目的:该研究旨在评估RAD对HLAP的影响,并阐明体内和体外的潜在机制,为HLAP的临床治疗提供了潜在的药物。
方法:C57BL/6小鼠HFD和CER诱导高脂血症性急性胰腺炎。然后用RAD管理。AR42J用cerulein或条件培养基刺激,然后用RAD培养。分析血清以评估潜在的胰腺和肝脏损伤。此外,组织样本用于组织学,和H&E的蛋白质研究,油红染色,和Westernblot。此外,蛋白质印迹和免疫荧光染色用于评估RAD对线粒体功能的影响,自噬通量,和SASP。
结果:体内,RAD显著缓解全身炎症,同时减弱TC,TG,艾米,LPS,炎性细胞因子,组织病理学改变,氧化损伤,线粒体裂变,和HLAP小鼠的自噬标记。自噬通量受损和线粒体功能障碍导致胰腺中NLRP3和IL-1β的显着增强。RAD可以逆转这些变化。体外,RAD显著恢复线粒体膜电位和氧化磷酸化水平。RAD降低Beclin-1和LC3-II表达,增加LAMP-1和Parkin-Pink表达,结果表明,RAD通过抑制线粒体氧化损伤,增强自噬通量和线粒体自噬清除受损线粒体,显著改善HLAP诱导的线粒体功能损伤。此外,我们发现RAD可以上调BAX的表达,并下调p16和p21的表达,表明RAD可以促进受损细胞凋亡和减轻SASP。
结论:这项研究表明,RAD通过增强自噬通量改善线粒体功能以减轻SASP,线粒体自噬,和细胞凋亡,为抗HLAP保护策略的发展和发展提供了分子基础。
BACKGROUND: Acute pancreatitis (AP) is an inflammatory disorder of the exocrine pancreas, especially hyperlipidemia acute pancreatitis (HLAP) is the third leading cause of acute pancreatitis which is more severe with a greater incidence of persistent multiorgan failure. HLAP inflicts injury upon the organelles within the acinar cell, particularly mitochondria, the endolysosomal-autophagy system, and is accompanied by senescence-associated secretory phenotype (SASP). RAD, only two consists of Rhizoma Alismatis and Atractylodes macrocephala Rhizoma, which is best known for its ability to anti-inflammatory and lipid-lowering. Nevertheless, the mechanism by which RAD alleviates HLAP remains obscure, necessitating further investigation.
OBJECTIVE: The study aimed to assess the effects of the RAD on HLAP and to elucidate the underlying mechanism in vivo and in vitro, offering a potential medicine for clinical treatment for HLAP.
METHODS: C57BL/6 mice with hyperlipidemia acute pancreatitis were induced by HFD and CER, then administrated with RAD. AR42J were stimulated by cerulein or conditioned medium and then cultured with RAD. Serums were analyzed to evaluate potential pancreas and liver damage. Furthermore, tissue samples were obtained for histological, and protein investigations by H&E, Oil red staining, and Western blot. In addition, western blot and immunofluorescent staining were utilized to estimate the effect of RAD on mitochondrial function, autophagy flux, and SASP.
RESULTS: In vivo, RAD considerably alleviated systemic inflammation while attenuating TC, TG, AMY, LPS, inflammatory cytokines, histopathology changes, oxidative damage, mitochondrial fission, and autophagy markers in HLAP mice. Impaired autophagy flux and mitochondrial dysfunction resulted in a significant enhancement of NLRP3 and IL-1β in the pancreas. RAD could reverse these changes. In vitro, RAD significantly restored mitochondrial membrane potential and oxidative phosphorylation levels. RAD decreased Beclin-1 and LC3-II expression and increased LAMP-1 and Parkin-Pink expression, which showed that RAD significantly ameliorated HLAP-induced damage to the mitochondria function by suppressing mitochondrial oxidative damage and enhancing autophagy flux and mitophagy to remove the damaged mitochondria. In addition, we found that RAD could up-regulate the expression of BAX, and Bad and down-regulate the expression of p16, and p21, indicating that RAD could promote damaged cell apoptosis and alleviate SASP.
CONCLUSIONS: This study revealed that RAD ameliorates mitochondrial function to alleviate SASP through enhancing autophagy flux, mitophagy, and apoptosis which provided a molecular basis for the advancement and development of protection strategies against HLAP.