鱼腥草提取物去甲头花千金藤二酮B对H2O2诱导的海马神经元损伤的作用及可能机制

Effects of norcepharadion B extracted from Houttuynia cordata on hippocampal neurons injury induced by H2O2 and its possible mechanism

摘要:

目的 探讨鱼腥草提取物去甲头花千金藤二酮B(NB)对H₂O₂诱导的海马神经元细胞损伤的作用及可能机制。方法 将对数生长期的HT22海马神经细胞分为正常对照组、H₂O₂处理组、NB干预组(H₂O₂₊NB)、单独NB组和阳性药物对照组(H₂O₂+NAC)。其中H₂O₂组为加入终浓度为300μmol/L的H₂O₂与细胞共孵育24h；H₂O₂+NB组和H₂O₂+NAC组是于H₂O₂处理前2h分别给予100μmol/L的NB或5mmol/L的NAC共孵育24h。为明确磷脂酰肌醇-3激酶/蛋白激酶B/血红素氧合酶1(PI3K/Akt/HO-1)途径是否参与了NB的神经保护作用,对细胞给予PI3K抑制剂Ly294002预处理30min,再加入NB及H₂O₂共孵育24h。CCK-8法检测细胞存活率,流式细胞仪检测细胞凋亡率,生化试剂盒检测细胞超氧化物歧化酶(SOD)、丙二醛(MDA)及谷胱甘肽(GSH)及细胞上清液乳酸脱氢酶(LDH)水平,Western blotting检测凋亡蛋白Bcl-2和Bax,及相关信号通路蛋白p-Akt和HO-1水平。采用SPSS 19.0软件进行统计分析,组间比较采用方差分析或Tukey′s检验。结果 (1)细胞活性。与空白对照组比较,H₂O₂组细胞存活率明显下降,细胞上清LDH含量显著增加,细胞形态出现明显皱缩、空泡变性及细胞减少。给予NB或NAC干预可显著增加细胞活性、减少LDH释放,细胞形态的破坏得到明显改善。(2)氧化损伤指标。与正常对照组比较,H₂O₂组细胞MDA水平显著增加,SOD及GSH活性显著降低；与H₂O₂组比较,加入NB或NAC的细胞MDA漏出量显著降低,SOD、GSH活性显著升高。(3)细胞凋亡情况。流式细胞术结果显示,与正常对照组比较,H₂O₂组细胞凋亡率明显增高,加入NB及NAC处理后,与H₂O₂组比较,细胞凋亡率显著降低(P<0.05)。Western blotting结果显示,与正常对照组比较,H₂O₂处理组Bcl-2表达显著下降,Bax水平显著升高；与H₂O₂处理组比较,给予NB或阳性药物NAC处理的细胞Bcl-2显著升高,Bax水平显著下降。(4)相关信号通路蛋白表达。Western blotting显示,与正常对照组比较,H₂O₂组p-Akt、HO-1水平升高；与H₂O₂组相比,NB+H₂O₂组p-Akt、HO-1水平显著升高 (P＜0.05)。上述所有指标在正常对照组与NB组细胞间比较差异均无统计学意义(P＞0.05)。另外,加入Ly294002组的细胞p-AKT及HO-1表达水平均较H₂O₂+NB组显著降低。结论 NB可通过抗氧化、抗凋亡减轻H₂O₂诱导的神经元损伤,发挥神经保护作用,其机制可能是激活PI3K/Akt/HO-1信号通路。

Abstract:

Objective To investigate the effect of norcepharadion B (NB) extracted from Houttuynia cordata on the injury of hippocampal neurons induced by H₂O₂ and its possible mechanism. Methods Hippocampal nerve cells (HT22) in logarithmic growth phase were divided into control group, H₂O₂ group, H₂O₂+NB group, NB group and H₂O₂+NAC group. H₂O₂ group were incubated with H₂O₂ at a final concentration of 300μmol/L for 24h; the H₂O₂+NB group were treated with 100μmol/L NB and H₂O₂+NAC group with 5mmol/L NAC for 2 hours before incubation with H₂O₂ for 24h. To determine whether phosphatidylinositol-3 kinase/protein kinase B/heme oxygenase-1 (PI3K/Akt/HO-1) pathway is involved in the neuroprotection of NB, the neurons were pretreated with Ly294002, a PI3K inhibitor, for 30min before incubation with NB and H₂O₂ for 24h. Cell survival rate was detected by CCK-8 test and apoptosis by flow cytometry. Lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione (GSH) and malondialdehydemalonic dialdehyde (MDA) were measured by biochemical kit. Western blotting was performed to detect the expression of B-celllymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein(Bax), and phosphorylated Akt (p-Akt) and HO-1. SPSS statistics 19.0 was used for analysis. ANOVA or Tukey′s test was used for intergroup comparison.Results (1) Cell activity. Compared with control group, the survival rate of H₂O₂ group decreased significantly, LDH in the cell supernatant increased significantly, cell shrinkage, vacuole degeneration and cell reduction were observed. However, intervention with NB or NAC significantly increased cell activity, reduced LDH release, and significantly improved cell morphological damage. (2) Oxidative damage indicators. Compared with control group, MDA in H₂O₂ group increased significantly, and SOD and GSH decreased significantly; compared with H₂O₂ group, MDA leakage decreased significantly after the intervention with NB or NAC, and the SOD and GSH activities increased significantly. (3) Apoptosis. Flow cytometry showed that the apoptosis rate in the H₂O₂ group was significantly higher than that in the control group, and the apoptosis rate of H₂O₂ group was significantly lower than that in H₂O₂＋NB group and H₂O₂＋NAC group (P<0.05). Western blotting showed that compared with control group, Bcl-2 in H₂O₂ group decreased significantly, and the Bax level increased significantly; compared with H₂O₂ group, Bcl-2 in the H₂O₂+NB group or H₂O₂+NAC group increased significantly, and the Bax level decreased significantly. (4) Western blotting showed that p-Akt and HO-1 in H₂O₂ group were higher than those in control group, and p-Akt and HO-1 in the NB+H₂O₂ group were significantly higher than those in H₂O₂ group (P<0.05). There was no significant differences between control group and NB group in the indicators all above (P>0.05). In addition, the expression level of p-Akt and HO-1 in Ly294002+H₂O₂+NB group was significantly lower than that in H₂O₂₊NB group. Conclusion NB provides neuroprotection by mitigating the neural injuries induced by H₂O₂ through anti-oxidation and anti-apoptosis, whose underlying mechanism may be attributed to signaling pathway of the activation of the PI3K/Akt/HO-1.