Ren Wei, Yang Yushan, Duan Huiming, Nong Wei, Tang Aihua, Lin Hongsheng, Li Lu, Wang Chunling, Feng Xiaotao
Institute of Traditional Chinese and Zhuang-Yao Ethnic Medicine, Guangxi University of Chinese Medicine, 13 Wuhe Road, Qingxiu District, Nanning, 530200, China.
The First Affiliated Hospital, Guangxi University of Chinese Medicine, 89-9 Dongge Road, Qingxiu District, Nanning, 530023, China.
BMC Complement Med Ther. 2025 Aug 28;25(1):315. doi: 10.1186/s12906-025-05024-4.
Pollen Typhae (PT), a traditional Chinese medicine herb utilized in diabetes management, exerts anti-inflammatory effects through its flavonoids, yet the active constituents and mechanisms remain unclear.
PT total flavone (PTF) was extracted from PT and identified the compounds by UHPLC-MS. Network pharmacology and molecular docking were used to predict the underlying targets and anti-inflammatory mechanisms of PTF. The prediction was validated in RAW264.7 macrophages. IL-1β and IL-18 in culture supernatants were analyzed by ELISA. The protein and gene expression were checked by western blotting and Real-time PCR, respectively. Intracellular ROS production was detected by DCFH-DA method. Intracellular lipids were analyzed by ELISA and Enzyme assay. The Caspase-1 activity was evaluated by bioluminescent method.
PTF was identified 47 flavonoid compounds, including typhaneoside (TYP). Network pharmacology and molecular docking indicated that the flavonoid compounds might regulate inflammatory response, fatty acid metabolism, and the NOD-like receptor, AMPK pathways. PTF and TYP inhibited palmitic acid (PA)-induced NLRP3 inflammasome activation in lipopolysaccharide-primed RAW264.7 macrophages, leading to decreased secretion of IL-1β and IL-18. Furthermore, PTF and TYP improved intracellular lipid metabolism in PA-induced macrophages, indicating decreased free fatty acid and triglyceride contents, reduced protein expression of CD36, PPARγ, FAS, DGAT1, and CPT-1, as well as declined ROS with increased ATP production. Additionally, PTF and TYP increased the p-AMPK/AMPK ratio and upstream p-LKB1/LKB ratio. Activated AMPK, in turn, ameliorated lipid metabolism dysfunction, thus abolishing PA-induced ROS production and NLRP3 inflammasome activation. Antioxidant and improving lipid metabolism by suppressing ACC also inhibited NLRP3 inflammasome activation, respectively. Importantly, AMPK inhibition attenuated or abolished the inhibitory effects of PTF or TYP on ROS production, IL-1β and IL-18 secretion, and Caspase-1 activity.
The findings highlight the ability of PTF and its active component TYP to inhibit PA-induced NLRP3 inflammasome activation in macrophages involving AMPK-mediated lipid metabolism, implying the potential use of PT flavonoid compounds as anti-diabetic inflammation lead compounds.
蒲黄是一种用于糖尿病治疗的传统中药，其黄酮类成分具有抗炎作用，但其活性成分和作用机制尚不清楚。
从蒲黄中提取蒲黄总黄酮（PTF），采用超高效液相色谱-质谱联用仪（UHPLC-MS）鉴定化合物。运用网络药理学和分子对接技术预测PTF的潜在靶点和抗炎机制。在RAW264.7巨噬细胞中验证该预测结果。采用酶联免疫吸附测定法（ELISA）分析培养上清液中的白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）。分别通过蛋白质印迹法和实时荧光定量聚合酶链反应（Real-time PCR）检测蛋白质和基因表达。采用2′,7′-二氯二氢荧光素二乙酸酯（DCFH-DA）法检测细胞内活性氧（ROS）的产生。通过ELISA和酶法分析细胞内脂质。采用生物发光法评估半胱天冬酶-1（Caspase-1）的活性。
鉴定出PTF中的47种黄酮类化合物，包括异鼠李素-3-O-新橙皮糖苷（TYP）。网络药理学和分子对接表明，这些黄酮类化合物可能调节炎症反应、脂肪酸代谢以及核苷酸结合寡聚化结构域样受体（NOD样受体）、腺苷酸活化蛋白激酶（AMPK）信号通路。PTF和TYP抑制脂多糖预处理的RAW264.7巨噬细胞中棕榈酸（PA）诱导的NLRP3炎性小体激活，导致IL-1β和IL-18分泌减少。此外，PTF和TYP改善PA诱导的巨噬细胞内脂质代谢，表现为游离脂肪酸和甘油三酯含量降低，脂肪酸转运蛋白CD36、过氧化物酶体增殖物激活受体γ（PPARγ）、脂肪酸合成酶（FAS）、二酰甘油酰基转移酶1（DGAT1）和肉碱棕榈酰转移酶1（CPT-1）的蛋白质表达降低，同时ROS水平下降，三磷酸腺苷（ATP）生成增加。此外，PTF和TYP增加磷酸化AMPK（p-AMPK）与AMPK的比值以及上游磷酸化肝脏激酶B1（p-LKB1）与LKB1的比值。激活的AMPK进而改善脂质代谢功能障碍，从而消除PA诱导的ROS产生和NLRP3炎性小体激活。抗氧化以及通过抑制乙酰辅酶A羧化酶（ACC）改善脂质代谢也分别抑制了NLRP3炎性小体激活。重要的是，抑制AMPK减弱或消除了PTF或TYP对ROS产生、IL-1β和IL-18分泌以及Caspase-1活性的抑制作用。
这些发现突出了PTF及其活性成分TYP抑制巨噬细胞中PA诱导的NLRP3炎性小体激活的能力，这一过程涉及AMPK介导的脂质代谢，提示蒲黄黄酮类化合物作为抗糖尿病炎症先导化合物的潜在用途。
