异泽兰黄素通过改善腹侧海马GABAa受体传递缓解大鼠酒精戒断焦虑样行为

李露露; 焦宇; 姜敏; 李秋月; 吴朋烊; 马淑丽; 郑晓宇; 赵容杰; 赵正林

doi:10.13386/j.issn1002-0306.2023060034

异泽兰黄素通过改善腹侧海马GABAa受体传递缓解大鼠酒精戒断焦虑样行为

Eupatilin Attenuates Ethanol Withdrawal-Induced Anxiety-like Behavior in Rats by Improving Ventral Hippocampus GABAa Transmission

摘要

摘要: 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。

Abstract: Objective: To study the improving effect of Eupatilin (Eptl) on ethanol withdrawal (EtOHWI)-induced anxiety-like behavior in rats and probe the mechanisms related to ventral hippocampus (vHippo). Methods: Thirty-two adult male Sprague-Dawley rats were randomly divided into four groups, 8 rats per group: Saline-treated control group, EtOHWI model group, low-dose Eptl treatment group and high-dose Eptl treatment group. The EtOHWI was established by intraperitoneal injection of 3 g/kg of ethanol (20% volume/volume, dissolved in saline) once a day for 28 days followed by 3 days of withdrawal, during the withdrawal period, the low-and the high-dose Eptl treatment groups were respectively given 10 and 30 mg/kg Eptl through oral route once a day, while the saline control group was administered with an equal volume of saline. Thirty minutes after the third Eptl, all the rats were subjected to open filed (OF) and elevated plus maze (EPM) tests to detect anxiety-like behaviors. The serum coritosterone (CORT) concentration and vHippo γ-aminobutyric acid (GABA) secretion were measured by enzyme linked immunosorbent assay (ELISA), and vHippo glutamic acid decarboxylase 67 (GAD 67) mRNA relative expression was assayed by real-time quantitative polymerase chain reaction. The protein expression of GABAa receptor α1 (GABAaRα1), GABAaRα2, nuclear factor E2-related factor 2 (Nrf2), heme oxygense-1 (HO-1) in the vHippo were analyzed by Western blot. The levels of MDA, T-SOD, CAT and GSH, IL-6 and TNF-α were measured by commercial kits. Meanwhile, in the in vitro experiment, the nuclear levels of Nrf2 in HT22 cells were detected via immunofluorescent technique. Results: Compared with the rats in the EtOHWI group, the rats in low and high-dose Eptl treatment groups moving distance increased significantly (P<0.01) in the central region of OF which was 70.62% and 124.21% respectively, and moving time increased significantly (P<0.05 or P<0.01) which was 251.75% and 371.62% respectively in the central zone of the OF. Visited more frequently (P<0.05 or P<0.01) which was 110.33% and 207.32% respectively, and stayed time increased significantly (P<0.05 or P<0.01) which was 99.56% and 184.18% respectively in the open arms of the EPM1. In biochemical assays, compared with those in EtOHWI rats, in the rats of low-dose and high-dose Eptl treatment groups, the serum CORT concentrations decreased significantly (P<0.01). The vHippo GABA and GAD67 mRNA levels increased significantly (P<0.05 or P<0.01). The protein expression of GABAaRα1, GABAaRα2, Nrf2, HO-1 in the vHippo increased significantly (P<0.05 or P<0.01). The level of MDA decreased significantly (P<0.01), while the activities of T-SOD and CAT, as well as the level of GSH increased respectively significantly (P<0.05 or P<0.01). The levels of IL-6 and TNF-α decreased significantly (P<0.05 or P<0.01). In the in vitro experiment, the immunofluorescent assay showed that compared with blank control group, Nrf2 level in nucleus of HT22 stimulated by 200 μmol/L H₂O₂ increased significantly (P<0.01), whereas pretreatment with 30 μmol/L Eptl inhibited the increase of Nrf2 level (P<0.05). Conclusion: Eptl attenuates EtOHWI-induced anxiety-like behavior in rats, which may be mediated by regulating the vHippo GABAaR transmissional disorder of EtOHWI rats via its antioxidant and anti-inflammatory activities.