背景:干细胞和微生物稳态的破坏加速了衰老过程。因此,保持这些平衡有效地延缓衰老并减轻与年龄有关的疾病的症状。最近的研究表明,靶向内质网(ER)应激和免疫缺陷(IMD)信号可能在维持衰老的肠干细胞(ISC)和微生物平衡的稳态中起积极作用。先前的研究表明,来自人参C.A.Meyer的总人参皂苷(TG)可能通过减轻ER应激和介导IMD途径而表现出潜在的抗衰老特性。然而,目前尚不清楚TG是否通过调节ER应激和IMD途径促进健康衰老来改善ISC和微生物稳态.
目的:为了阐明TG是否促进果蝇的健康及其潜在的分子机制,重点关注其在调节ER应激和IMD途径中的作用,以维持ISC和肠道菌群稳态。
方法:采用高效液相色谱法检测TG中的主要皂苷单体。存活率,肠长,屏障功能,和摄食/排泄行为测定用于评估TG对果蝇寿命和肠道健康的影响。在干细胞水平,“esg-荧光素酶”报告系统,esg-GFP/delta干细胞荧光标记,和磷酸组蛋白H3有丝分裂活性测定用于确定TG是否可以预防果蝇的自然衰老或与氧化应激相关的ISC过度增殖。免疫荧光染色检测TG对衰老过程中内质网应激的影响。利用基因编辑技术操纵ER应激靶基因及其相关c-JunN末端激酶(JNK)基因的过表达或干扰,以验证TG维持与年龄相关的ISC增殖稳态的分子机制。分子对接和等温滴定量热法验证了TG和ER应激靶基因之间的直接相互作用。此外,在肠道菌群水平,16SrDNA测序用于分析TG对果蝇肠道菌群多样性和丰度的影响以及可能涉及的功能途径。进行RT-qPCR以确定TG是否介导IMD途径中靶基因的表达。进行了显性细菌物种特异性单关联分析,以验证TG对IMD靶基因和ISC增殖的影响是否取决于显性细菌物种的直接控制。
结果:我们的结果表明,服用TG可以延缓衰老果蝇的肠道形态和功能下降。TG通过抑制ER应激IRE1介导的JNK信号传导来防止年龄相关的ISC过度增殖。此外,口服TG通过重塑肠道微生物群和抑制醋杆菌介导的IMD靶基因激活来预防衰老相关的ISC和肠道微生物群菌群失调。
结论:TG通过抑制ISC的过度增殖和减轻肠道微生物失衡促进健康衰老,从而为抗衰老TG产品的研发提供新的见解。
BACKGROUND: Disruption of stem cell and microbial homeostasis accelerates the aging process. Hence, maintaining these balances effectively delays aging and alleviates the symptoms of age-related diseases. Recent research indicates that targeting endoplasmic reticulum (ER) stress and immune deficiency (IMD) signalling may play a positive role in maintaining homeostasis in aging intestinal stem cells (ISC) and microbial equilibrium. Previous research has suggested that total ginsenosides (TG) derived from Panax ginseng C. A. Meyer may exhibit potential anti-aging properties by mitigating ER stress and mediating the IMD pathway. Nevertheless, it remains unclear whether TG improve ISC and microbial homeostasis by modulating ER stress and the IMD pathway to promote healthy aging.
OBJECTIVE: To elucidate whether TG promotes healthspan in Drosophila and its underlying molecular mechanisms, focusing on its role in regulating ER stress and the IMD pathway to maintain ISC and intestinal microbiota homeostasis.
METHODS: High performance liquid chromatography was performed to detect the main saponin monomer in TG. Survival rate, gut length, barrier function, and feeding/excretion behaviour assays were used to evaluate the effects of TG on the lifespan and gut health of Drosophila. At the stem cell level, \"esg-luciferase\" reporter system, esg-GFP/delta stem cell fluorescent labelling, and phospho-histone H3+ mitotic activity assays were employed to determine whether TG prevented natural aging or oxidative stress-associated ISC over-proliferation in Drosophila. Immunofluorescence staining was used to detect the effects of TG on ER stress during aging. Overexpression or interference of ER stress target genes and their related c-Jun N-terminal kinase (JNK) gene was manipulated using gene editing technology to verify the molecular mechanism by which TG maintains age-related ISC proliferation homeostasis. Molecular docking and isothermal titration calorimetry were used to verify the direct interactions between TG and ER stress target genes. In addition, at the intestinal flora level, 16S rDNA sequencing was used to analyse the effect of TG on the diversity and abundance of Drosophila intestinal flora and the possible functional pathways involved. RT-qPCR was performed to determine whether TG mediated the expression of target genes in the IMD pathway. A dominant bacterial species-specific mono-association analysis were performed to verify whether the effects of TG on IMD target genes and ISC proliferation depended on the direct control of the dominant bacterial species.
RESULTS: Our results suggest that administration of TG delays the decline in gut morphology and function in aging Drosophila. TG prevents age-associated ISC hyperproliferation by inhibiting ER stress IRE1-mediated JNK signaling. Furthermore, oral TG prevented aging-associated ISC and gut microbiota dysbiosis by remodelling the gut microbiota and inhibiting Acetobacter-mediated activation of IMD target genes.
CONCLUSIONS: TG promotes healthy aging by inhibiting the excessive proliferation of ISC and alleviating intestinal microbial imbalance, thereby providing new insights for the research and development of anti-aging TG products.