Eupatilin Suppresses OVA-Induced Asthma by Inhibiting NF-κB and MAPK and Activating Nrf2 Signaling Pathways in Mice

To investigate the effect of eupatilin in asthma treatment, we evaluated its therapeutic effect and related signal transduction in OVA-induced asthmatic mice and LPS-stimulated RAW264.7 cells. The BALF was tested for changes in lung inflammatory cells. Th2 cytokines in the BALF and OVA-IgE in the serum were measured by ELISA. H&E and PAS staining were used to evaluate histopathological changes in mouse lungs. The key proteins NF-κB, MAPK, and Nrf2 in lung tissues were quantitatively analyzed by Western blotting. Finally, we evaluated the effect of eupatilin on cytokines and related protein expression in LPS-stimulated RAW 264.7 cells in vitro. In OVA-induced asthmatic mice, eupatilin reduced the numbers of inflammatory cells, especially neutrophils and eosinophils. Eupatilin also decreased the levels of IL-5, IL-13 in the BALF and OVA-IgE in the serum. Furthermore, eupatilin inhibited the activation of NF-κB and MAPK pathways and increased the expression of Nrf2 in OVA-induced asthmatic mice. In vitro, eupatilin significantly reduced LPS-stimulated NO, IL-6, and ROS production. Additionally, the NF-κB, MAPK, and Nrf2 protein expression in LPS-stimulated RAW264.7 cells was consistent with that in OVA-induced asthmatic lung tissues. In summary, eupatilin attenuated OVA-induced asthma by regulating NF-κB, MAPK, and Nrf2 signaling pathways. These results suggest the utility of eupatilin as an anti-inflammatory drug for asthma treatment.     

艾叶黄酮异泽兰黄素对运动损伤大鼠炎症和抗氧化系统的影响

目的：研究异泽兰黄素（Eupatilin,Eptl）对大鼠酒精戒断（Ethanol withdrawal,EtOHWI）焦虑样行为的改善作用及其与腹侧海马（Ventral hippocampus,vHippo）相关机制。方法：将32只成年雄性Sprague-Dawley大鼠随机分为4组,分别为生理盐水对照组、EtOHWI模型组、Eptl低剂量治疗组和Eptl高剂量治疗组,每组各8只。EtOHWI模型组、Eptl低、高剂量治疗组每天1次腹腔注射3 g/kg乙醇（Ethanol,EtOH）,连续28 d,戒断3 d制备大鼠EtOHWI模型,生理盐水对照组腹腔注射等容积生理盐水。在3 d戒断期间,Eptl低、高剂量治疗组分别每天1次灌胃10和30 mg/kg Eptl,第3次给药30 min后,利用旷场（Open filed,OF）和高架十字迷宫（Elevated plus maze,EPM）实验对各组大鼠进行焦虑样行为学检测。利用ELISA检测血清皮质酮（Coritosterone,CORT）浓度、vHippo组织中GABA水平;利用实时荧光定量PCR检测vHippo组织中谷氨酸脱羧酶67（GAD 67）mRNA相对表达量;利用Western blot（WB）检测vHippo组织中GABAaRα1（GABAa receptor α1,GABAaRα1）、GABAaRα2、核因子E2相关因子2（Nrf2）、血红素加氧酶1（HO-1）蛋白表达;利用试剂盒检测vHippo组织中MDA、T-SOD、CAT、GSH以及IL-6和TNF-α的含量。利用免疫荧光检测技术观察HT22细胞的核中Nrf2蛋白水平。结果：与EtOHWI模型组相比,Eptl低、高剂量治疗组大鼠在OF中央区活动距离极显著升高（P<0.01）,分别为70.62%和124.21%,活动时间显著升高（P<0.05或P<0.01）,分别为251.75%和371.62%,EPM的开放臂进入次数百分比显著升高（P<0.05或P<0.01）,分别为110.33%和207.32%,滞留时间百分比显著升高（P<0.05或P<0.01）,分别为99.56%和184.18%;血清CORT含量极显著降低（P<0.01）;vHippo组织中GABA含量和GAD67mRNA表达显著升高（P<0.05或P<0.01）;GABAaRα1、GABAaRα2、Nrf2和HO-1蛋白表达显著增多（P<0.05或P<0.01）;MDA水平极显著降低（P<0.01）,而T-SOD、CAT和GSH的活性或水平显著升高（P<0.05或P<0.01）;IL-6、TNF-α含量显著降低（P<0.05或P<0.01）。体外实验结果显示,与空白对照组相比,200 μmol/L H₂O₂刺激的HT22细胞核中Nrf2的水平极显著增多（P<0.01）,但30 μmol/L Eptl预处理显著抑制了Nrf2水平的增多（P<0.05）。结论：Eptl具有改善大鼠EtOHWI焦虑样行为的作用,其机制可能通过Eptl的抗氧化和抗炎作用而调节EtOHWI大鼠vHippo的GABAaR传递紊乱所介导。

     

Optimized Conditions for the Extraction of Eupatilin in Artemisia asiatica by Pressurized Liquid Extraction

Abstract

The effect of temperature and solvent on the extraction of Artemisia asiatica using pressurized liquid extraction was examined. These two parameters were operated according to central composite design followed by response surface analysis to obtain the highest extraction efficiency. Determination of eupatilin was carried out by high performance liquid chromatography coupled with a diode array detector. While the rise in temperature could increase the yield of the total extract, the content of eupatilin was decreased in high temperature. In addition, high ethanol proportion maximized the contents of eupatilin in the total extract.     

Inhibition of Synaptic Glutamate Exocytosis and Prevention of Glutamate Neurotoxicity by Eupatilin from Artemisia argyi in the Rat Cortex

The inhibition of synaptic glutamate release to maintain glutamate homeostasis contributes to the alleviation of neuronal cell injury, and accumulating evidence suggests that natural products can repress glutamate levels and associated excitotoxicity. In this study, we investigated whether eupatilin, a constituent of Artemisia argyi, affected glutamate release in rat cortical nerve terminals (synaptosomes). Additionally, we evaluated the effect of eupatilin in an animal model of kainic acid (KA) excitotoxicity, particularly on the levels of glutamate and N-methyl-D-aspartate (NMDA) receptor subunits (GluN2A and GluN2B). We found that eupatilin decreased depolarization-evoked glutamate release from rat cortical synaptosomes and that this effect was accompanied by a reduction in cytosolic Ca²⁺ elevation, inhibition of P/Q-type Ca²⁺ channels, decreased synapsin I Ca²⁺-dependent phosphorylation and no detectable effect on the membrane potential. In a KA-induced glutamate excitotoxicity rat model, the administration of eupatilin before KA administration prevented neuronal cell degeneration, glutamate elevation, glutamate-generating enzyme glutaminase increase, excitatory amino acid transporter (EAAT) decrease, GluN2A protein decrease and GluN2B protein increase in the rat cortex. Taken together, the results suggest that eupatilin depresses glutamate exocytosis from cerebrocortical synaptosomes by decreasing P/Q-type Ca²⁺ channels and synapsin I phosphorylation and alleviates glutamate excitotoxicity caused by KA by preventing glutamatergic alterations in the rat cortex. Thus, this study suggests that eupatilin can be considered a potential therapeutic agent in the treatment of brain impairment associated with glutamate excitotoxicity.