药学院知识库

高原低氧将导致新陈代谢改变从而增加体内活性氧（reactive oxygen species，ROS）的合成。低氧诱导产生的ROS将导致机体的氧化损伤，包括蛋白羰基化，DNA损伤，脂质过氧化等。高原低氧也会导致炎症因子的大量释放。最新研究表明槟榔提取物具有抗氧化、抗炎等多种生物活性，而槟榔提取物是否具有抗高原低氧诱导的氧化损伤和炎症反应的作用未见报道。本研究将从以下几个方面进行研究：第一、为了筛选槟榔中具有抗高原缺氧作用的有效组分，采用模拟高原环境缺氧大鼠模型，考察了250 mg/kg的槟榔碱、槟榔无水乙醇提取物、50%乙醇提取物对大鼠血气指标、组织病理变化、氧化应激指标的影响。模拟高原环境急性缺氧实验结果表明，无水乙醇提取物和50%乙醇提取物改善了缺氧大鼠的低氧血症，其中无水乙醇提取物使缺氧大鼠血气指标动脉血氧分压（arterial oxygen partial pressure，PaO₂）、动脉血氧饱和度（arterial oxygen saturation，SatO₂）分别增加了12.99%、2.46%（P<0.05），50%乙醇提取物组血气指标PaO₂、SatO₂分别增加了20.00%、5.79%（P<0.01）。病理结果可见，无水乙醇提取物和50%乙醇提取物能够减轻缺氧大鼠心肌组织炎性损伤，减少炎症细胞浸润，减轻脑组织损伤，改善了神经元细胞的核固缩。氧化应激指标表明，无水乙醇提取物和50%乙醇提取物能提高组织抗氧化酶活性，减轻脂质过氧化损伤，其中无水乙醇提取物组心肌、脑组织中超氧化物歧化酶（superoxide dismutase，SOD）活力分别升高了15.15%、7.82%（P<0.05），脑组织丙二醛（maleic dialdehyde，MDA）含量下降了28.20%（P<0.05），50%乙醇提取物组心肌、脑组织SOD活力分别显著增加了16.79%、7.89%（P<0.05），脑组织一氧化氮（nitric oxide，NO）含量显著升高了29.03%（P<0.05）。槟榔碱对缺氧大鼠血气指标、心肌、脑组织病理变化、心肌、脑组织氧化应激指标均无显著影响（P>0.05）。第二、为了进一步研究槟榔无水乙醇提取物和50%乙醇提取物的抗高原缺氧药效学，采用高原实地缺氧大鼠模型，考察了250 mg/kg、500 mg/kg、750 mg/kg的槟榔无水乙醇提取物和50%乙醇提取物对缺氧大鼠血气指标、组织病理变化、心肌和脑组织氧化应激指标、血清中炎症因子的影响。急进高原实地缺氧实验结果表明，低、中剂量的无水乙醇提取物均能改善缺氧大鼠的低氧血症，其中低剂量组PaO₂、SatO₂升高了16.78%、6.29%（P<0.05），中剂量组PaO₂、SatO₂升高了13.40%、5.13%（P<0.05）。病理结果可见，无水乙醇提取物、50%乙醇提取物能减轻心肌组织胞浆深染程度，改善脑组织神经元细胞核固缩。同时，无水乙醇提取物和50%乙醇提取物均能减轻缺氧大鼠的氧化应激程度，其中无水乙醇提取物中、高剂量组大鼠心肌组织中MDA含量显著下降（P<0.05）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH）活力显著升高（P<0.05），高剂量组SOD活力、NO含量显著升高（P<0.05），中、高剂量组脑组织中SOD、GSH活力均显著升高（P<0.05或P<0.01），高剂量组NO含量显著升高（P<0.05）；50%乙醇提取物中、高剂量组大鼠心肌组织SOD活力显著升高（P<0.05），高剂量组NO含量显著升高，高剂量组脑组织中GSH活力显著升高（P<0.05），低、中、高剂量组NO含量均显著升高（P<0.05）。此外，无水乙醇提取物和50%乙醇提取物能够抑制促炎因子单核细胞趋化因子-1（monocyte chemotactic protein，MCP-1）、白介素-6（interleukin-6，IL-6）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的释放，其中给药组血清中MCP-1表达量均显著降低（差异倍数≤0.85），50%乙醇提取物中、高剂量组IL-6表达量分别下降了0.85、0.79倍，无水乙醇提取物中、高剂量组，50%乙醇提取物高剂量组TNF-α表达量分别降低了0.60、0.66、0.73倍。第三、为了进一步探究槟榔无水乙醇提取物的抗缺氧机制，通过建立H9C2心肌细胞体外缺氧模型，检测了5 μg/mL、10 μg/mL、20 μg/mL的无水乙醇提取物对H9C2 细胞存活率的影响，同时研究了20 μg/mL的无水乙醇提取物对细胞内MDA含量，SOD、GSH活力，核因子相关因子2（nuclear factor correlation factor2，Nrf2）、半胱氨酸蛋白酶3（cysteine protease 3，Caspase-3）mRNA表达量的影响。结果表明，槟榔无水乙醇提取物能明显提高H9C2细胞存活率（P<0.01），提高了细胞内抗氧酶SOD、GSH的活力（分别增加了14.90%、28.94%，P<0.05），使氧化应激标志物Nrf2 mRNA相对表达量下降了0.66倍（P<0.05）。

 综上所述，结论如下：1.槟榔无水乙醇提取物及50%乙醇提取物是具有抗高原缺氧作用的有效组分；2.槟榔提取物的抗缺氧作用与其提高机体抗氧化能力，清除过多的氧自由基，减少氧化应激损伤，抑制炎症反应有关；3.槟榔无水乙醇提取物能够增强H9C2细胞的耐缺氧能力，提高细胞的抗氧化酶活性，使缺氧细胞内Nrf2的mRNA相对表达趋于常氧水平，保护细胞免受氧化应激损伤而发挥抗缺氧作用。

High-altitude hypoxia will lead to metabolic changes and increase ROS synthesis in vivo. Hypoxia induced ROS will lead to oxidative damage to the body, including protein carbonylation, DNA damage, lipid peroxidation and so on. High-altitude hypoxia can also cause massive release of inflammatory factors. Recent studies have shown that Areca catechu L extracts have various biological activities such as anti-oxidation and anti-inflammation. However, the effects of Areca catechu L extract on the oxidative damage and inflammatory response induced by hypoxia were not reported.We will study from the following aspects:First, the Areca catechu L was screened for its anti-hypoxic potential by using the simulated hypoxic rat model. The dose of 250 mg/kg of arecoline, anhydrous ethanol extract of Areca catechu L, 50% ethanol extract were tested for blood gas, histopathological changes and oxidative stress. The result of the experiment showed that the hypoxemia can be improved by anhydrous ethanol extract of Areca catechu L and 50% ethanol extract. The PaO₂ and SatO₂ in hypoxic rats were increased respectively by 12.99%, and 2.46% (P<0.05) by anhydrous ethanol extract, and 20.00%、5.79%（P<0.01）by 50% ethanol extract. The result of pathology indicated that the inflammatory injury of myocardial tissue and the infiltration of inflammatory cells can be reduced by the anhydrous ethanol extract of Areca catechu L and 50% ethanol extract, as well as the damage of brain tissue and the nuclear condensation of neuronal cells can be improved. The oxidative stress data showed that the activity of antioxidant enzymes was increased and the lipid peroxidation injury was reduced by the anhydrous ethanol extract of Areca catechu L and 50% ethanol extract. Thereinto, in the anhydrous ethanol extract of Areca catechu L group, the activity of SOD in myocardium and brain tissue was respectively increased by 15.15 % and 7.82% (P<0.05), the content of MDA in brain tissue decreased by 28.20 % (P<0.05). In 50% ethanol extract group, the SOD activity in myocardium and brain tissue was increased significantly by 16.79% and 7.89%(P<0.05). The content of NO in brain tissue was increased significantly by 29.03% (P<0.05). At the same time, the arecoline had no effect on blood gas, histopathological changes and oxidative stress of hypoxic rat.Second, the anti-plateau anoxic pharmacodynamics of anhydrous ethanol extract and 50% alcohol extract of Areca catechu L was studied by using the plateau hypoxia-induced rat model. Three dose of anhydrous ethanol extract of Areca catechu L (250 mg/kg, 500 mg/kg, and 750 mg/kg) and 50% ethanol extract were performed for blood gas, histopathological changes, oxidative stress of myocardium and brain tissue and serum inflammatory factors. The results of acute hypoxia in plateau showed that the hypoxemia can be improved by the low and medium dosage of Areca catechu L anhydrous alcohol extract. PaO₂ and SatO₂ in low dose group were respectively increased by 16.78% and 6.29% (P<0.05), and in the middle-dose group which were increased by 13.40% and 5.13% respectively(P<0.05). The result of pathology indicated that the hyperchromatic cytoplasm of myocardial tissue and the nuclear condensation of neuronal cells can be improved by anhydrous ethanol extract of Areca catechu L and 50% ethanol extract., as well as the damage of brain tissue and the infiltration of inflammatory cells can be reduced, the nuclear condensation of neuronal cells can be improved. At the same time, both anhydrous ethanol extracts and 50% ethanol extracts could release the oxidative stress in hypoxic rats. In the medium and high dose groups of Areca catechu L anhydrous alcohol extract，the content of MDA in myocardial tissue of the rats was significantly decreased (P<0.05) and the activity of GSH was significantly increased, in the high dose groups, the activity of SOD and the content of NO were significantly increased (P<0.05).;as for the brain tissue, in the medium and high dose groups of Areca catechu L anhydrous alcohol extract, the activity of SOD and GSH was significantly increased (P<0.05 or P<0.01), in the high dose group, the content of NO was significantly increased (P<0.05). The activity of SOD in myocardial tissue was significantly increased in the medium and high dose groups of the Areca catechu L 50% ethanol extract, the content of NO was significantly increased in the high dose groups; as for the brain tissue, the activity of GSH was significantly increased in the high dose groups, the content of NO was all significantly increased (P<0.05) in the low, middle and high dose groups. In addition, the release of pro-inflammatory cytokines MCP-1, IL-6, TNF-α can be inhibited by the anhydrous alcohol extract and 50% ethanol extract. The expression of MCP-1 in the serum of each drug group was significantly decreased (the fold difference was less than or equal to 0.85). The expression of IL-6 in medium and high doses of 50% ethanol extract were decreased respectively by 0.85 and 0.79 times. The expression of TNF-α respectively decreased by 0.60, 0.66, and 0.73 fold in medium and high dose of anhydrous ethanol extracts and high dose of 50% alcohol extracts.Third, the anhydrous ethanol extract of Areca catechu L was further explored for the anti-hypoxia mechanism by establishing an in vitro hypoxia model of H9C2 cardiomyocytes. Three dose of Areca catechu L anhydrous alcohol extract (5, 10, and 20 μg/mL) was investigated for the effects on H9C2 cell survival rate. The 20 μg/mL Areca catechu L alcohol extracts was tested for the the effects on the intracellular MDA content, SOD, GSH activity, Nrf2 and Caspase-3 mRNA expression levels. The results showed that the survival rate of H9C2 cells (P<0.01)and the activities of intracellular antioxidant enzymes SOD and GSH could be significantly increased (respectively by 14.90% and 28.94%, P<0.05), the relative expression of Nrf2 mRNA was decreased by 0.66-fold (P<0.05) by the anhydrous ethanol extract of Areca catechu L.

The following conclusions can be drawn from the above study: 1) anhydrous ethanol extracts and 50% ethanol extracts of Areca catechu L are effective components to against hypoxia. 2) anti-hypoxia effect of Areca catechu L extract is related to its antioxidant capacity, excess oxygen free radicals clearance, oxidative stress damage reduction and inflammatory response inhibition. 3) Areca catechu L alcohol extract can enhance hypoxia tolerance of H9C2 cell and increase the activity of antioxidant enzyme, so that mRNA expression of Nrf2 in hypoxic cells tend to normoxia level, which protects cells from oxidative stress producing the anti-hypoxia effect.