Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China; MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China; MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
Int Immunopharmacol. 2024 Dec 25;143(Pt 2):113432. doi: 10.1016/j.intimp.2024.113432. Epub 2024 Oct 23.
Matrine is a tetracyclic quinolizidine alkaloid with diverse bioactive properties, including anti-inflammatory and neuroprotective properties. However, the underlying anti-inflammatory mechanisms remain unclear.
This study aimed to explore how matrine reduces Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and to assess its protective effects against LPS-induced intestinal damage.
The effect of matrine on cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Additionally, its impact on inflammatory cytokines and macrophage polarization was assessed using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The effects on intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nitric oxide (NO) production, and oxidative stress were evaluated using 2',7'-dichlorodihydrofluorescein diacetate staining and JC-1 and Griess assays. Immunofluorescence staining was used to observe the translocation of the NF-κB p65 subunit. Western blotting (WB) and qRT-PCR were employed to analyze the expression levels of proteins related to the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. An LPS-induced mouse model was established to study the intestinal inflammation and barrier injury. Mouse feces characteristics, colon length, and disease activity index (DAI) were recorded. Hematoxylin-eosin (H&E) and alcian blue/periodic acid schiff (AB/PAS) staining were used to observe morphological changes and barrier damage in the duodenum, jejunum, ileum, and colon and to measure villus length, crypt depth, goblet cell count, and positive areas in the duodenum, jejunum, and ileum. The content of short-chain fatty acids (SCFAs) in the colon was determined using gas chromatography (GC).
Matrine inhibited LPS-induced inflammatory cytokine levels, suppressed macrophage M1 polarization, and promoted M2 macrophage polarization. Matrine reduced LPS-induced increases in ROS and NO levels and regulates oxidative stress. Additionally, matrine inhibited the nuclear translocation of the NF-κB p65 subunit and exerted anti-inflammatory effects by suppressing the activation of the TLR4/NF-κB/MAPK pathway. In vivo experiments indicated that matrine significantly alleviated LPS-induced diarrhea, increased DAI, and shortened the colon. Matrine reduced the production of the pro-inflammatory cytokine interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α and the pro-inflammatory mediator NO in mouse intestinal tissues while promoting the content of the anti-inflammatory cytokine IL-10. Furthermore, it improved intestinal tissue structure and alleviated LPS-induced intestinal barrier damage. Finally, matrine increased the SCFA levels in the intestine.
Matrine exerted its anti-inflammatory effects and protects against intestinal injury through the TLR4/NF-κB/MAPK signaling pathway.
苦参碱是一种四环喹诺里西啶生物碱，具有多种生物活性，包括抗炎和神经保护作用。然而，其抗炎的潜在机制尚不清楚。
本研究旨在探讨苦参碱如何降低脂多糖（LPS）诱导的 RAW 264.7 细胞炎症，并评估其对 LPS 诱导的肠道损伤的保护作用。
使用细胞计数试剂盒-8（CCK-8）测定苦参碱对细胞活力的影响。此外，通过酶联免疫吸附测定（ELISA）、流式细胞术和实时定量聚合酶链反应（qRT-PCR）分析评估苦参碱对炎性细胞因子和巨噬细胞极化的影响。通过 2'，7'-二氯二氢荧光素二乙酸酯染色和 JC-1 和格里斯测定评估细胞内活性氧（ROS）、线粒体膜电位（MMP）、一氧化氮（NO）产生和氧化应激的影响。免疫荧光染色用于观察核因子-κB（NF-κB）p65 亚基的易位。采用 Western blot（WB）和 qRT-PCR 分析 Toll 样受体 4（TLR4）/核因子-κB（NF-κB）/丝裂原活化蛋白激酶（MAPK）通路相关蛋白的表达水平。建立 LPS 诱导的小鼠模型研究肠道炎症和屏障损伤。记录小鼠粪便特征、结肠长度和疾病活动指数（DAI）。苏木精-伊红（H&E）和阿尔辛蓝/过碘酸希夫（AB/PAS）染色观察十二指肠、空肠和回肠及结肠的形态变化和屏障损伤，并测量十二指肠、空肠和回肠的绒毛长度、隐窝深度、杯状细胞计数和阳性面积。采用气相色谱（GC）法测定结肠中短链脂肪酸（SCFA）的含量。
苦参碱抑制 LPS 诱导的炎性细胞因子水平，抑制巨噬细胞 M1 极化，促进 M2 巨噬细胞极化。苦参碱降低 LPS 诱导的 ROS 和 NO 水平升高，并调节氧化应激。此外，苦参碱抑制 NF-κB p65 亚基的核易位，通过抑制 TLR4/NF-κB/MAPK 通路的激活发挥抗炎作用。体内实验表明，苦参碱可显著减轻 LPS 诱导的腹泻，增加 DAI，并缩短结肠。苦参碱降低了小鼠肠道组织中促炎细胞因子白细胞介素（IL）-6、IL-1β和肿瘤坏死因子（TNF）-α以及促炎介质 NO 的产生，同时促进抗炎细胞因子 IL-10 的产生。此外，它改善了肠道组织结构，减轻了 LPS 诱导的肠道屏障损伤。最后，苦参碱增加了肠道中的 SCFA 含量。
苦参碱通过 TLR4/NF-κB/MAPK 信号通路发挥抗炎作用并保护肠道免受损伤。
