火龙果皮发酵物对脂多糖诱导RAW264.7细胞炎症的缓解作用

Attenuation of Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells by Fermented Hylocereus undulatus Peels

摘要:

本文研究了火龙果皮发酵物（Fermented Hylocereus undulatus peel, FHP）对脂多糖（LPS）诱导RAW264.7细胞炎症的缓解作用及机制。通过干酪乳杆菌CICC20280发酵火龙果皮，制备FHP。采用50、100、200、400、800 μg/mL FHP处理RAW264.7细胞，噻唑蓝比色法确定FHP细胞毒性。在此基础上，将细胞分为对照组、LPS组及FHP处理组，格里斯试剂法、DCFH-DA荧光法和ELISA分别检测各组细胞培养上清中一氧化氮（NO）、活性氧（ROS）和细胞因子（TNF-α、IL-1β、IL-6、IL-10）含量；RT-PCR检测NF-κB通路的信号转导元件TLR4/MyD88/NF-κB及下游炎性因子的表达。结果显示：FHP作用RAW264.7细胞的安全浓度≤400 μg/mL。与LPS组相比，FHP呈剂量依赖性地显著（p<0.05）抑制细胞分泌NO、ROS、TNF-α、IL-1β、IL-6，平均抑制率76.40%、提高IL-10浓度，提高率达173.72%，同时极显著（p<0.01）下调TLR4、MyD88、NF-κB及TNF-α、IL-1β、IL-6的表达。综上可知：FHP对LPS诱导RAW264.7细胞炎症具有缓解作用，其抗炎活性通过抑制促炎介质并提高抑炎因子的水平实现，机制与沉默NF-κB通路的信号转导功能有关。

Abstract:

To investigate the attenuating effects of fermented Hylocereus undulatus peels (FHP) on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells, and the mechanisms of attenuation, Hylocereus undulatus peels were fermented using Lactobacillus casei CICC20280. A methyl-thiazolyl-tetrazolium reduction assay was used to determine FHP cytotoxicity at different concentrations (50, 100, 200, 400 and 800 μg/mL). The following groups were included: a control group, an LPS-induced inflammation model group, and five groups treated with LPS and different concentrations of FHP. Nitric oxide (NO), reactive oxygen species (ROS), and cytokines (TNF-α, IL-1β, IL-6, and IL-10) in cell culture supernatants were quantitated using various molecular analysis techniques, including Griess, DCFH-DA fluorescence, and ELISA assays. RT-PCR was used to detect expression of NF-κB pathway components (TLR4/MyD88/NF-κB) and pro-inflammatory factors. FHP at concentrations of ≤400 μg/mL is non-toxic to RAW264.7 cells. FHP significantly decreases (p<0.05) concentrations of pro-inflammatory factors including NO, ROS, TNF-α, IL-1β and IL-6, by an average of 76.40%, and increases the concentration of the anti-inflammatory factor IL-10, by an average of 173.72%, in a dose-dependent manner, compared to the LPS-induced inflammation model group. Expression levels of TLR4, MyD88, NF-κB and TNF-α, IL-1β, IL-6 were significantly down-regulated (p<0.01) by FHP. In summary, FHP displays readily detectable attenuating effects on LPS-induced inflammation in RAW264.7 cells, probably by inhibiting secretion of pro-inflammatory mediators and increasing levels of anti-inflammatory factors, via a mechanism possibly associated with an NF-κB pathway.