-
高海拔地区最大的环境特征是大气压降低,从而使机体从空气中摄取的氧气含量减少。大脑是最耗氧的器官之一,对缺氧异常敏感,这种生理反应使机体处于缺氧环境中可能导致严重的脑损伤,如学习和记忆障碍[1-3]。近年来,治疗或改善低压低氧引起的中枢神经系统(CNS)损伤在高原医学领域引起了越来越多的关注[4-5]。有研究表明,急性暴露在缺氧环境中会加速活性氧(ROS)的积累,并在海马和皮层区域引发氧化应激,而氧化应激是高原学习记忆障碍的主要诱因[6-8],随着进入高海拔地区学习、工作和旅游的人群日益增加,寻找能够改善高原缺氧记忆损伤的药物成为高原缺氧损伤防治研究的重点。
利舒康胶囊为国药准字(Z20025932)药品,其主要成分为红景天、手掌参、甘青青兰、烈香杜鹃四味藏药辅以黄柏、甘草,经提取加工而制成,具有抗缺氧、抗疲劳、增强体力等作用,主要用于防治各种急、慢性高原病。临床实践表明,该药还可用于慢性缺血缺氧症侯群、脑梗死伴眩晕症、血管性痴呆、非痴呆性血管性认知功能障碍等缺血缺氧性相关疾病的治疗,其作用机制可能与抑制脂质过氧化,清除自由基减轻氧化应激有关[9]。本研究利用大型低压氧舱模拟高原海拔7500 m缺氧72 h,从行为学、组织形态学和Keap1/Nrf2/HO-1信号通路等方面,探讨利舒康胶囊对高原学习记忆损伤的干预作用,以期进一步阐明其可能的作用机制,为扩大利舒康胶囊的药理作用和临床适用范围提供理论依据。
-
与正常对照组相比,缺氧模型组小鼠WME、RWE、TE、TT均显著升高(P<0.01);与缺氧模型组相比,利舒康胶囊三个剂量组小鼠四项指标均有不同程度降低,其中利舒康胶囊高剂量组800 mg/(kg·d)具有显著性差异(P<0.05或P<0.01),表明高原低氧缺氧能导致小鼠空间记忆障碍,而利舒康胶囊高剂量组能够增强高原缺氧小鼠的短期记忆和长期记忆能力,改善大鼠认知,有望对高原缺氧导致的学习记忆损伤患者提供预防和治疗。结果见表1和图2。
表 1 利舒康胶囊对高原缺氧小鼠空间记忆的影响(
$ \bar x \pm s $ ,n=10)组别 剂量(mg/kg) WME(次) RME(次) TT(秒) TE(次) 正常对照组 − 0.43±0.35 0.50±0.41 63.47±9.64 1.50±0.17 缺氧模型组 − 3.00±1.58## 2.70±1.52## 119.99±43.00## 3.10±0.48## 红景天胶囊组 400 0.57±0.35** 0.79±0.49* 81.78±28.20 1.83±0.29#** 利舒康胶囊低剂量组 400 2.43±0.89#▲ 1.36±0.56 99.45±25.27## 3.06±0.58##▲▲ 利舒康胶囊中剂量组 600 2.14±1.14#▲ 1.43±1.10 98.75±30.51 3.04±0.52##▲▲ 利舒康胶囊高剂量组 800 1.07±0.61* 0.71±0.49** 69.38±31.69* 1.73±0.37** # P<0.05, ## P<0.01, 与正常对照组比较;* P<0.05, ** P<0.01,与缺氧组比较 ;▲P<0.05, ▲▲P<0.01,与红景天胶囊组比较 -
海马体在学习和记忆中发挥关键作用,尤其容易受到缺氧损伤。在本研究中,小鼠脑组织病理学切片结果显示(见图3),正常对照组小鼠海马神经元密集,细胞核大而圆,神经元呈层状有序分布,各层细胞形态饱满而数量多,细胞排列整齐且致密;缺氧模型组小鼠海马神经元细胞分布受到破坏,细胞形态不规则,部分细胞缺失或有空泡(黑色箭头),出现明显核固缩(蓝色箭头),细胞排列紊乱,CA1区有较多不规则形状的深染细胞(绿色箭头),表明脑组织出现神经元死亡;经过红景天胶囊和利舒康胶囊药物干预后,缺氧模型组脑组织损伤有所改善;神经元细胞排列整齐,核固缩现象减轻(蓝色箭头),深染细胞数量减少(绿色箭头),少有细胞缺失或空泡(黑色箭头),细胞形态较好,排列整齐,且利舒康胶囊高剂量组脑组织形态趋于正常。
-
与正常对照组比较,缺氧模型组小鼠海马组织中Keap1蛋白含量显著升高(P<0.01),Nrf2、HO-1蛋白含量显著降低(P<0.01);与缺氧模型组相比,利舒康胶囊组Keap1蛋白含量降低(P<0.01),Nrf2、HO-1蛋白含量显著升高(P<0.01),其中以利舒康胶囊高剂量组蛋白水平变化最为显著,且效果优于阳性药红景天胶囊组。结果见图4和表2。
表 2 各组小鼠脑组织蛋白表达比较(
$ \bar x \pm s $ ,n=3)分组 剂量
(mg/kg)Keap1 Nrf2 HO-1 Caspase-3 Bax BCL-2 正常对照组 − 0.36±0.08 0.76±0.08 1.03±0.14 0.03±0.01 0.09±0.02 0.79±0.09 缺氧模型组 − 0.91±0.15## 0.45±0.03## 0.31±0.05## 0.95±0.12## 0.99±0.08## 0.31±0.02## 红景天胶囊组 400 0.44±0.03** 0.92±0.15** 0.86±0.03 0.75±0.16## 0.73±0.11##** 0.65±0.02#** 利舒康胶囊低剂量组 400 0.42±0.03** 0.77±0.09** 0.67±0.02## 0.86±0.17## 0.81±0.05##** 0.62±0.02##** 利舒康胶囊中剂量组 600 0.41±0.04** 0.83±0.09** 0.98±0.13 0.64±0.06## 0.79±0.06##** 0.73±0.09** 利舒康胶囊高剂量组 800 0.33±0.02**▲▲ 1.21±0.11##**▲▲ 1.23±0.08**▲▲ 0.46±0.01##**▲▲ 0.52±0.05##**▲ 0.76±0.11** # P<0.05, ## P<0.01, 与正常对照组比较;* P<0.05, ** P<0.01,与缺氧组比较;▲P<0.05, ▲▲P<0.01,与红景天胶囊组比较 -
与正常对照组相比,缺氧模型组小鼠海马组织中Caspase-3和Bax的表达显著升高(P<0.01),Bcl-2蛋白表达显著降低(P<0.01);与缺氧模型组相比,利舒康胶囊组Caspase-3和Bax蛋白表达显著降低(P<0.01),BCL-2蛋白表达显著升高(P<0.01),其中利舒康胶囊高剂量组蛋白水平变化尤为显著,且效果优于阳性药红景天胶囊组。结果见图5和表2。
Study on the effect of Lishukang capsule on learning and memory impairment in mice with high altitude hypoxia based on Keap1/Nrf2/HO-1 signal pathway
-
摘要:
目的 基于Keap1/Nrf2/HO-1信号通路探讨利舒康胶囊对高原缺氧小鼠学习记忆障碍的改善作用。 方法 将 60只Balb/C雄性小鼠随机分为正常对照组、缺氧模型组、红景天胶囊组:400 mg/kg、利舒康胶囊低、中、高剂量组:400 mg/kg、600 mg/kg、800 mg/kg,每组10只。各组灌胃给药7 d,第4 d给药结束后,正常对照组饲养于当地海拔(1500 m),其余各组置于低压低氧动物实验舱模拟高原海拔7500 m缺氧3 d,期间每天灌胃给药一次,正常对照组和缺氧模型组给予生理盐水,末次给药后1 h,采用八臂迷宫检测小鼠在模拟高原缺氧状态下空间记忆能力;HE染色观察小鼠脑组织海马区的形态学变化;Western blot 法检测海马组织中Keap1/Nrf2/HO-1信号通路蛋白含量变化及凋亡相关蛋白含量变化。 结果 与正常对照组相比,缺氧模型组小鼠空间记忆能力明显损伤(P<0.01);HE染色观察小鼠海马神经元损伤严重;Keap1蛋白含量及凋亡相关蛋白Bax、Caspase-3含量均上升(P<0.01);Nrf2、HO-1及凋亡相关蛋白Bcl-2含量下降(P<0.01)。与缺氧模型组相比,利舒康胶囊高剂量组小鼠在八臂迷宫行为学实验中错误率显著降低(P<0.05,P<0.01);HE染色观察神经元细胞排列整齐,细胞形态较好;Keap1蛋白含量及凋亡相关蛋白Bax、Caspase-3含量均下降(P<0.01);Nrf2、HO-1及凋亡相关蛋白Bcl-2含量上升(P<0.01)。 结论 高原缺氧可以导致小鼠氧化应激损伤、诱导凋亡相关基因的表达,从而加重了小鼠认知功能的障碍;利舒康胶囊能有效改善缺氧引起的小鼠学习记忆障碍,其作用机制可能与调控Keap1/Nrf2/HO-1信号通路、降低凋亡有关。 Abstract:Objective Study on the effect of Lishukang capsule on learning and memory impairment in mice with high altitude hypoxia based on Keap1/Nrf2/HO-1 signal pathway. Methods Sixty male Balb/C mice were randomly divided into normal control group, hypoxia model group, Rhodiola capsule group: 400 mg/kg, low, medium and high dose groups of Lishukang capsule: 400 mg/kg, 600 mg/kg, 800 mg/kg, with 10 mice in each group. The normal control group was fed at the local altitude (1500m) after 7 days of intragastric administration in each group, and the rest groups were fed at the low pressure and hypoxia animal experimental cabin to simulate the altitude of 7500 m for hypoxia for 3 days. During this period, the normal control group and the hypoxia model group were given normal saline once a day, and 1 hour after the last administration, the eight arm maze was used to test the spatial memory ability of mice under simulated high altitude hypoxia; HE staining was used to observe the morphological changes of hippocampus in mice; Western blot was used to detect the changes of protein content of Keap1/Nrf2/HO-1 signal pathway and apoptosis related protein in hippocampus of mice. Results Compared with the normal control group, the spatial memory ability of mice in the hypoxia model group was significantly impaired (P<0.01); HE staining showed that hippocampal neurons in mice were seriously injured; the content of brain tissue Keap1 protein and apoptosis related protein Bax and Caspase-3 increased (P<0.01); the content of Nrf2, HO-1 and apoptosis related protein Bcl-2 decreased (P<0.01). Compared with the hypoxia model group, the error rate of mice in the high dose group of Lishukang capsule in the eight arm maze behavior experiment was significantly reduced (P<0.05, P<0.01); HE staining showed that the neurons were arranged orderly and the cell morphology was good; the content of Keap1 protein and apoptosis related protein Bax and Caspase-3 decreased (P<0.01); the content of Nrf2, HO-1 and apoptosis related protein Bcl-2 increased (P<0.01). Conclusion High altitude hypoxia can lead to oxidative stress injury in mice and induce the expression of apoptosis related genes, thus aggravating the cognitive dysfunction of mice; Lishukang capsule can effectively improve the learning and memory impairment in mice caused by hypoxia, and its mechanism may be related to regulating the Keap1/Nrf2/HO-1 signal pathway and reducing apoptosis. -
表 1 利舒康胶囊对高原缺氧小鼠空间记忆的影响(
$ \bar x \pm s $ ,n=10)组别 剂量(mg/kg) WME(次) RME(次) TT(秒) TE(次) 正常对照组 − 0.43±0.35 0.50±0.41 63.47±9.64 1.50±0.17 缺氧模型组 − 3.00±1.58## 2.70±1.52## 119.99±43.00## 3.10±0.48## 红景天胶囊组 400 0.57±0.35** 0.79±0.49* 81.78±28.20 1.83±0.29#** 利舒康胶囊低剂量组 400 2.43±0.89#▲ 1.36±0.56 99.45±25.27## 3.06±0.58##▲▲ 利舒康胶囊中剂量组 600 2.14±1.14#▲ 1.43±1.10 98.75±30.51 3.04±0.52##▲▲ 利舒康胶囊高剂量组 800 1.07±0.61* 0.71±0.49** 69.38±31.69* 1.73±0.37** # P<0.05, ## P<0.01, 与正常对照组比较;* P<0.05, ** P<0.01,与缺氧组比较 ;▲P<0.05, ▲▲P<0.01,与红景天胶囊组比较 表 2 各组小鼠脑组织蛋白表达比较(
$ \bar x \pm s $ ,n=3)分组 剂量
(mg/kg)Keap1 Nrf2 HO-1 Caspase-3 Bax BCL-2 正常对照组 − 0.36±0.08 0.76±0.08 1.03±0.14 0.03±0.01 0.09±0.02 0.79±0.09 缺氧模型组 − 0.91±0.15## 0.45±0.03## 0.31±0.05## 0.95±0.12## 0.99±0.08## 0.31±0.02## 红景天胶囊组 400 0.44±0.03** 0.92±0.15** 0.86±0.03 0.75±0.16## 0.73±0.11##** 0.65±0.02#** 利舒康胶囊低剂量组 400 0.42±0.03** 0.77±0.09** 0.67±0.02## 0.86±0.17## 0.81±0.05##** 0.62±0.02##** 利舒康胶囊中剂量组 600 0.41±0.04** 0.83±0.09** 0.98±0.13 0.64±0.06## 0.79±0.06##** 0.73±0.09** 利舒康胶囊高剂量组 800 0.33±0.02**▲▲ 1.21±0.11##**▲▲ 1.23±0.08**▲▲ 0.46±0.01##**▲▲ 0.52±0.05##**▲ 0.76±0.11** # P<0.05, ## P<0.01, 与正常对照组比较;* P<0.05, ** P<0.01,与缺氧组比较;▲P<0.05, ▲▲P<0.01,与红景天胶囊组比较 -
[1] MA J Q, WANG C Y, SUN Y B, et al. Comparative study of oral and intranasal puerarin for prevention of brain injury induced by acute high-altitude hypoxia[J]. Int J Pharm, 2020, 591:120002. doi: 10.1016/j.ijpharm.2020.120002 [2] 赵敏, 陈垚, 李文华. 高原低氧影响学习记忆功能的机制研究进展[J]. 西北国防医学杂志, 2019, 40(9):536-541. doi: 10.16021/j.cnki.1007-8622.2019.09.002 [3] LI Y, WANG Y. Effects of long-term exposure to high altitude hypoxia on cognitive function and its mechanism: a narrative review[J]. Brain Sci, 2022, 12(6):808. doi: 10.3390/brainsci12060808 [4] ZHU M X, XU M K, ZHANG K X, et al. Effect of acute exposure to hypobaric hypoxia on learning and memory in adult Sprague-Dawley rats[J]. Behav Brain Res, 2019, 367:82-90. doi: 10.1016/j.bbr.2019.03.047 [5] WANG X B, HOU Y, LI Q Y, et al. Rhodiola crenulata attenuates apoptosis and mitochondrial energy metabolism disorder in rats with hypobaric hypoxia-induced brain injury by regulating the HIF-1α/microRNA 210/ISCU1/2(COX10) signaling pathway[J]. J Ethnopharmacol, 2019, 241:111801. doi: 10.1016/j.jep.2019.03.028 [6] ZHOU F, WANG M D, JU J, et al. Schizandrin A protects against cerebral ischemia-reperfusion injury by suppressing inflammation and oxidative stress and regulating the AMPK/Nrf2 pathway regulation[J]. Am J Transl Res, 2019, 11(1):199. [7] CHEN C, LI B, CHEN H T, et al. Epigallocatechin-3-gallate ameliorated iron accumulation and apoptosis and promoted neuronal regeneration and memory/cognitive functions in the hippocampus induced by exposure to a chronic high-altitude hypoxia environment[J]. Neurochem Res, 2022, 47(8):2254-2262. doi: 10.1007/s11064-022-03611-2 [8] ZHANG X Y, ZHANG X J, DANG Z C, et al. Cognitive protective mechanism of crocin pretreatment in rat submitted to acute high-altitude hypoxia exposure[J]. BioMed Res Int, 2020, 2020:1-15. [9] 李燕. 利舒康胶囊对慢性缺血缺氧症侯群的疗效探讨[J]. 中西医结合心血管病电子杂志, 2018, 6(22): 102, 104. [10] XU H B, BARACSKAY P, O'NEILL J, et al. Assembly responses of hippocampal CA1 place cells predict learned behavior in goal-directed spatial tasks on the radial eight-arm maze[J]. Neuron, 2019, 101(1): 119-132. e4. [11] LI M X, ZHU Y T, LI J, et al. Effect and mechanism of verbascoside on hypoxic memory injury in plateau[J]. Phytother Res, 2019, 33(10):2692-2701. doi: 10.1002/ptr.6443 [12] DU X, LIU T L, TAO W D, et al. Effect of aqueous extract of Astragalus membranaceus on behavioral cognition of rats living at high altitude[J]. Chung I Tsa Chih Ying Wen Pan, 2022, 42(1):58-64. [13] ZHANG Z A, SUN Y F, YUAN Z Y, et al. Insight into the effects of high-altitude hypoxic exposure on learning and memory[J]. Oxid Med Cell Longev, 2022, 2022:4163188. [14] JING L L, WU N Z, ZHANG J, et al. Protective effect of 5, 6, 7, 8-Tetrahydroxyflavone on high altitude cerebral edema in rats[J]. Eur J Pharmacol, 2022, 928:175121. doi: 10.1016/j.ejphar.2022.175121 [15] KOESTER-HEGMANN C, BENGOETXEA H, KOSENKOV D, et al. High-altitude cognitive impairment is prevented by enriched environment including exercise via VEGF signaling[J]. Front Cell Neurosci, 2018, 12:532. doi: 10.3389/fnins.2018.00532 [16] WU C T, DENG J S, HUANG W C, et al. Salvianolic acid C against acetaminophen-induced acute liver injury by attenuating inflammation, oxidative stress, and apoptosis through inhibition of the Keap1/Nrf2/HO-1 signaling[J]. Oxid Med Cell Longev, 2019, 2019:9056845. [17] 姚娟, 吴平安, 李芸, 等. Keap1-Nrf2-ARE信号通路及其激活剂的研究进展[J]. 中国药理学通报, 2019, 35(10):1342-1346. [18] HUANG C Y, DENG J S, HUANG W C, et al. Attenuation of lipopolysaccharide-induced acute lung injury by hispolon in mice, through regulating the TLR4/PI3K/akt/mTOR and Keap1/Nrf2/HO-1 pathways, and suppressing oxidative stress-mediated ER stress-induced apoptosis and autophagy[J]. Nutrients, 2020, 12(6):1742. doi: 10.3390/nu12061742 [19] LI J C, LU K M, SUN F L, et al. Panaxydol attenuates ferroptosis against LPS-induced acute lung injury in mice by Keap1-Nrf2/HO-1 pathway[J]. J Transl Med, 2021, 19(1):96. doi: 10.1186/s12967-021-02745-1 [20] 马慧萍, 张俊, 贾正平, 等. 利舒康胶囊对模拟高原缺氧动物的保护作用研究[J]. 药学实践杂志, 2018, 36(3):255-259. [21] LIU X T, LIN X, ZHANG S Y, et al. Lycopene ameliorates oxidative stress in the aging chicken ovary via activation of Nrf2/HO-1 pathway[J]. Aging, 2018, 10(8):2016-2036. doi: 10.18632/aging.101526 [22] MOKHTARI SANGDEHI S R, HAJIZADEH MOGHADDAM A, RANJBAR M. Anti-apoptotic effect of silymarin-loaded chitosan nanoparticles on hippocampal caspase-3 and Bcl-2 expression following cerebral ischemia/reperfusion injury[J]. Int J Neurosci, 2022, 132(11):1102-1109. doi: 10.1080/00207454.2020.1860971 [23] ABOUTALEB N, SHAMSAEI N, KHAKSARI M, et al. Pre-ischemic exercise reduces apoptosis in hippocampal CA3 cells after cerebral ischemia by modulation of the Bax/Bcl-2 proteins ratio and prevention of caspase-3 activation[J]. J Physiol Sci, 2015, 65(5):435-443. doi: 10.1007/s12576-015-0382-7 [24] WANG J L, XU X X, JIA W Y, et al. Calcium-/ calmodulin-dependent protein kinase II (CaMKII) inhibition induces learning and memory impairment and apoptosis[J]. Oxid Med Cell Longev, 2021, 2021:4635054. [25] GUO Z Y, RUAN Z Z, ZHANG D D, et al. Rotenone impairs learning and memory in mice through microglia-mediated blood brain barrier disruption and neuronal apoptosis[J]. Chemosphere, 2022, 291(Pt 2): 132982.