基于PI3K/AKT/mTOR通路的三百棒促进脂多糖诱导的RAW 264.7细胞自噬并抑制炎症

Toddalia asiatica promotes lipopolysaccharide-induced autophagy and inhibits inflammation in RAW 264.7 cells based on the PI3K/AKT/mTOR pathway

三百棒来源于芸香科植物飞龙掌血Toddalia asiatica(L.)Lam的根,是一种天然土家族中草药,具有抗炎、抗风湿、抗肿瘤、抗微生物等药理活性。其毒副作用小,疗效显著的特点使之成为当前研究热点。许多天然产物已被证明可通过靶向PI3K/AKT/mTOR介导的自噬来抑制炎症及自身免疫性疾病,本项研究通过调节PI3K/AKT/mTOR信号通路来研究三百棒醇提物(Toddalia asiatica alcohol extract,TAAE)对自噬的影响,用脂多糖(LPS)诱导单核巨噬细胞(RAW 264.7)建立炎症模型,通过细胞毒性检测试剂盒检测TAAE对细胞活力的影响,并筛选出药物的浓度及干预时间,透射电镜和单丹磺酰尸胺染色检测巨噬细胞的生物学功能,酶联免疫吸附法检测上清液中相关炎症因子水平,Western blot检测自噬和通路相关蛋白的表达水平;并采用自噬早期抑制剂(3-MA)和通路PI3K激动剂(740Y-P)进一步验证自噬对炎症和信号通路的影响。实验结果表明TAAE可能通过抑制PI3K/AKT/mTOR信号通路,促进自噬泡的形成、自噬体溶酶体融合和降解,降低LPS处理的RAW 264.7细胞中炎性细胞因子的表达和分泌。总体而言,本研究结果为三百棒的抗炎机制的研究提供了新的线索,并为临床更好的应用三百棒治疗炎症性疾病提供理论依据。

The root of the Rutaxaceae plant Toddalia asiatica(L.)Lam is a natural Tujia Chinese herbal medicine with anti-inflammatory,anti-rheumatic,anti-tumor,anti-microbial and other pharmacological activities.Its characteristics of small poisonous side effects and remarkable curative effect make it a hot spot in current research.Many natural products had been shown to inhibit inflammation and autoimmune diseases by targeting PI3K/AKT/mTOR mediated autophagy.In this study,the effect of TAAE on autophagy was studied by regulating PI3K/AKT/mTOR signaling pathway,and RAW 264.7 cells were induced by LPS to establish an inflammatory model.Cytotoxicity detection kit was used to detect the effect of TAAE on cell viability,the concentration and intervention time of the drug were screened out.Transmission electron microscopy and MDC staining were used to detect the biological function of macrophages.The levels of inflammatory factors in the supernatant were detected by ELISA,and the expression levels of autophagy and pathway-related proteins were detected by Western blot.The effects of autophagy on inflammation and signaling pathways were further verified by using an early autophagy inhibitor(3-MA)and pathway PI3K agonist(740Y-P).The results indicated that TAAE may inhibit the PI3K/AKT/mTOR signaling pathway,promote the formation of autophagic vesicles,fusion and degradation of autophagosomal lysosomes,and reduce the expression and secretion of inflammatory cytokines in RAW 264.7 cells treated with LPS.Overall,the results of this study provide a new clue for the research of the anti-inflammatory mechanism of Toddalia asiatica,and provide a theoretical basis for better clinical application of Toddalia asiatica in the treatment of inflammatory diseases.