人参皂苷Rg1对小鼠慢性间歇性缺氧诱导脑损伤的减轻作用及其机制

doi:10.13481/j.1671-587X.20240502

摘要/Abstract

摘要：

目的探讨人参皂苷Rg1对小鼠慢性间歇性缺氧（CIH）诱导脑损伤的减轻作用，并阐明其可能的作用机制。方法 40只C57BL/6雄性小鼠随机分为对照组、模型组、抑制剂组（给予钙蛋白酶1抑制剂）、低剂量人参皂苷Rg1组（给予10 mg·kg^-1 人参皂苷Rg1）和高剂量人参皂苷Rg1组（给予20 mg·kg^-1 人参皂苷Rg1）。除对照组外，其余各组小鼠均置于自动调节氧浓度的低氧舱中以诱导小鼠缺氧性脑损伤。检测各组小鼠尾外周血氧饱和度（SpO₂），采用Morris水迷宫实验检测各组小鼠逃逸潜伏期、路径长度和游泳路线穿过目标象限频次，采用试剂盒检测血清中血尿素氮（BUN）、乳酸（LA）、丙二醛（MDA）、白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）水平以及血清中超氧化物歧化酶（SOD）和乳酸脱氢酶（LDH）活性，采用HE染色观察各组小鼠脑组织损伤程度，采用二氢乙啶（DHE）探针检测各组小鼠海马组织CA1区中活性氧（ROS）表达水平，采用Western blotting法检测各组小鼠脑组织中钙蛋白酶1、IL-6、和TNF-α蛋白表达水平。结果与对照组比较，模型组小鼠SpO₂明显降低（P<0.01），表明模型建立成功。与模型组比较，抑制剂组和低及高剂量人参皂苷Rg1组小鼠SpO₂明显升高（P<0.01）。Morris水迷宫实验，与对照组比较，模型组小鼠逃逸潜伏期和路径长度明显延长（P<0.01），游泳路线穿过目标象限频次明显减少；与模型组比较，抑制剂组、低和高剂量人参皂苷Rg1组小鼠逃逸潜伏期和路径长度明显缩短，游泳路线穿过目标象限频次明显增多。与对照组比较，模型组小鼠血清中BUN、LA、MDA、IL-6和TNF-α水平明显升高（P<0.01），LDH活性明显升高（P<0.01），SOD活性明显降低（P<0.01）；与模型组比较，抑制剂组、低和高剂量人参皂苷Rg1组小鼠血清中BUN、LA、MDA、IL-6和TNF-α水平明显降低（P<0.01），LDH活性明显降低（P<0.01），SOD活性明显升高（P<0.01）。HE染色，与对照组比较，模型组小鼠海马组织CA1区中锥体神经元排列疏松，部分神经元呈三角形，核固缩，胞质浓染，少数神经元脱失，呈明显缺氧性神经元损伤形态；与模型组比较，抑制剂组、低和高剂量人参皂苷Rg1组小鼠海马组织CA1区中缺氧性神经元损伤表现有效改善。DHE探针检测，与对照组比较，模型组小鼠海马组织CA1区中ROS水平明显升高（P<0.01）；与模型组比较，抑制剂组、低和高剂量人参皂苷Rg1组小鼠海马组织CA1区中ROS水平明显降低（P<0.01）。Western blotting法检测，与对照组比较，模型组小鼠海马组织中钙蛋白酶1、TNF-α和IL-6蛋白表达水平明显升高（P<0.01）；与模型组比较，抑制剂组、低和高剂量人参皂苷Rg1组小鼠海马组织中钙蛋白酶1、TNF-α和IL-6蛋白表达水平明显降低（P<0.01）。结论人参皂苷Rg1可减轻小鼠CIH诱导的脑组织损伤，其机制可能与抑制脑组织炎症反应和氧化应激并下调钙蛋白酶1表达有关。

关键词: 人参皂苷Rg1, 钙蛋白酶1, 钙蛋白酶1抑制剂, 慢性间歇性缺氧, 脑损伤

Abstract:

Objective To discuss the alleviative effect of ginsenoside Rg1 on chronic intermittent hypoxia （CIH）-induced brain injury in the mice，and to clarify its possible mechanism. Methods Forty male C57BL/6 mice were randomly divided into control group， model group， inhibitor group （treated with calpain-1 inhibitor）， low dose of ginsenoside Rg1 group （treated with 10 mg·kg^-1 ginsenoside Rg1）， and high dose of ginsenoside Rg1 group （treated with 20 mg·kg^-1 ginsenoside Rg1）. Except for the control group， the mice in all other groups were placed in a hypoxic chamber with automatically regulated oxygen concentration to induce hypoxic brain injury.The peripheral blood oxygen saturation （SpO₂） of tail of the mice in various groups was detected； the escape latencies and path lengths and the frequency of swimming route crossing the target quadrant of the mice in various groups were determined by Morris water maze test； the levels of blood urea nitrogen （BUN）， lactate （LA）， malondialdehyde （MDA）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） and the activities of superoxide dismutase （SOD） and lactate dehydrogenase （LDH） in serum of the mice in various groups were detected by kits； the degrees of brain tissue injury of the mice in various groups were observed by HE staining. The levels of reactive oxygen species （ROS） in CA1 region of hippocampus tissue of the mice in various groups were detected by dihydroethidium（DHE） probe； the expression levels of calpain-1， IL-6， and TNF-α proteins in brain tissue of the mice in various groups were detected by Western blotting method. Results Compared with control group， the SpO₂ of the mice in model group was significantly decreased （P<0.01）， indicating that the model was successfully established. Compared with model group， the SpO₂ of the mice in inhibitor group， low dose of ginsenoside Rg1 group and high dose of ginsenoside Rg1 group were significantly increased （P<0.01）. The Morris water maze test results showed that compared with control group， the escape latency and path length of the mice in model group were significantly prolonged （P<0.01）， and the frequency of swimming route of crossing the target quadrant was significantly decreased； compared with model group， the escape latencies and path lengths of the mice in inhibitor group， low dose of ginsenoside Rg1 group and high dose of ginsenoside Rg1 group were significantly shortened（P<0.01）， and the frequency of swimming route of crossing the target quadrant was significantly increased. Compared with control group， the levels of BUN， LA， MDA， IL-6， and TNF-α in serum of the mice in model group were significantly increased （P<0.01）， while the activity of LDH was significantly increased （P<0.01）， and the activity of SOD was significantly decreased （P<0.01）； compared with model group， the levels of BUN， LA， MDA， IL-6， and TNF-α in serum of the mice in inhibitor group， low dose of ginsenoside Rg1 group and high dose of ginsenoside Rg1 group were significantly decreased （P<0.01）， while the activities of LDH were significantly decreased（P<0.01）， and the activities of SOD were significantly increased （P<0.01）. The HE staining results showed that compared with control group， the pyramidal neurons in CA1 region of hippocampus tissue of the mice in model group were loosely arranged， while some neurons were triangular， with nuclear pyknosis， cytoplasmic hyperchromasia， and a few neurons were lost， indicating obvious hypoxic neuronal injury； compared with model group， the hypoxic neuronal injury in CA1 region in hippocampus tissue of the mice in inhibitor group， low dose of ginsenoside Rg1 group and high dose of ginsenoside Rg1 group was effectively alleviated. The DHE probe detection showed that compared with control group， the level of ROS in CA1 region in hippocampus tissue of the mice in model group was significantly increased （P<0.01）； compared with model group， the levels of ROS in CA1 region in hippocampus tissue of the mice in inhibitor group， low dose of ginsenoside Rg1 group and high dose of ginsenoside Rg1 group were significantly decreased （P<0.01）. The Western blotting results showed that compared with control group， the expression levels of calpain-1， TNF-α， and IL-6 proteins in hippocampus tissue of the mice in model group were significantly increased （P<0.01）； compared with model group， the expression levels of calpain-1， TNF-α， and IL-6 proteins in hippocampus tissue of the mice in inhibitor group， low dose of GRg1 group and high dose of GRg1 group were significantly decreased （P<0.01）. Conclusion Ginsenoside Rg1 can alleviate brain tissue injury of the mice induced by CIH； its mechanism may be related to the inhibition of brain tissue inflammatory response and oxidative stress， and the downregulation of calpain-1 expression.

Key words: Ginsenoside Rg1, Calpain-1, Calpain-1 inhibitor, Chronic intermittent hypoxia, Brain injury

中图分类号:

R965

孟岩,王洪新,杨育红. 人参皂苷Rg1对小鼠慢性间歇性缺氧诱导脑损伤的减轻作用及其机制[J]. 吉林大学学报(医学版), 2024, 50(5): 1196-1204.

Yan MENG,Hongxin WANG,Yuhong YANG. Alleviative effect of ginsenoside Rg1 on brain injury induced by chronic intermittent hypoxia in mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2024, 50(5): 1196-1204.

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参考文献 23

1	LUO B Y， LI Y W， ZHU M M， et al. Intermittent hypoxia and atherosclerosis： from molecular mechanisms to the therapeutic treatment［J］. Oxid Med Cell Longev， 2022， 2022： 1438470.
2	SHE N N， SHI Y W， FENG Y N， et al. NLRP3 inflammasome regulates astrocyte transformation in brain injury induced by chronic intermittent hypoxia［J］. BMC Neurosci， 2022， 23（1）： 70.
3	FENG X M， VALDEARCOS M， UCHIDA Y， et al. Microglia mediate postoperative hippocampal inflammation and cognitive decline in mice［J］. JCI Insight， 2017， 2（7）： e91229.
4	MEI M， TANG F T， LU M L， et al. Astragaloside Ⅳ attenuates apoptosis of hypertrophic cardiomyocyte through inhibiting oxidative stress and calpain-1 activation［J］. Environ Toxicol Pharmacol， 2015， 40（3）： 764-773.
5	DWIVEDI D K， KUMAR D， KWATRA M， et al. Voluntary alcohol consumption exacerbated high fat diet-induced cognitive deficits by NF-κB-calpain dependent apoptotic cell death in rat hippocampus： Ameliorative effect of melatonin［J］. Biomedecine Pharmacother， 2018， 108： 1393-1403.
6	MENG Y， YU S X， ZHAO F， et al. Astragaloside Ⅳalleviates brain injury induced by hypoxia via the calpain-1 signaling pathway［J］. Neural Plast， 2022， 2022： 6509981.
7	杨远园，肖叶青，袁雪，等. 人参皂苷Rg1通过calpain-1通路减轻肥大心肌细胞凋亡［J］. 中药药理与临床， 2017， 33（4）： 17-20.
8	GAO Y， CHU S F， SHAO Q H， et al. Antioxidant activities of ginsenoside Rg1 against cisplatin-induced hepatic injury through Nrf2 signaling pathway in mice［J］. Free Radic Res， 2017， 51（1）： 1-13.
9	XU T Z， SHEN X Y， SUN L L， et al. Ginsenoside Rg1 protects against H₂O₂-induced neuronal damage due to inhibition of the NLRP1 inflammasome signalling pathway in hippocampal neurons in vitro ［J］. Int J Mol Med， 2019， 43（2）： 717-726.
10	李光才. 慢性间歇性缺氧诱导大鼠心脑损伤的机制及药物干预的研究［D］. 武汉：华中科技大学， 2018.
11	王波. 姜黄素在慢性间歇低氧诱导小鼠脑损伤中的作用及机制研究［D］. 沈阳：中国医科大学， 2019.
12	SNYDER B， SHELL B， CUNNINGHAM J T， et al. Chronic intermittent hypoxia induces oxidative stress and inflammation in brain regions associated with early-stage neurodegeneration［J］. Physiol Rep， 2017， 5（9）： e13258.
13	WU X， GONG L J， XIE L， et al. NLRP3 deficiency protects against intermittent hypoxia-induced neuroinflammation and mitochondrial ROS by promoting the PINK1-parkin pathway of mitophagy in a murine model of sleep apnea［J］. Front Immunol， 2021， 12： 628168.
14	LIU F， LIU T W， KANG J. The role of NF-κB-mediated JNK pathway in cognitive impairment in a rat model of sleep apnea［J］. J Thorac Dis， 2018， 10（12）： 6921-6931.
15	COIMBRA-COSTA D， ALVA N， DURAN M， et al. Oxidative stress and apoptosis after acute respiratory hypoxia and reoxygenation in rat brain［J］. Redox Biol， 2017， 12： 216-225.
16	ZHAO F， MENG Y， WANG Y， et al. Protective effect of Astragaloside IV on chronic intermittent hypoxia-induced vascular endothelial dysfunction through the calpain-1/SIRT1/AMPK signaling pathway［J］. Front Pharmacol， 2022， 13： 920977.
17	王永伟. Urocortin基于AMPK-PGC-1α通路对慢性缺氧大鼠神经功能改善作用研究［D］. 锦州：锦州医科大学， 2021.
18	DENG H Y， TIAN X X， SUN H N， et al. Calpain-1 mediates vascular remodelling and fibrosis via HIF-1α in hypoxia-induced pulmonary hypertension［J］. J Cell Mol Med， 2022， 26（10）： 2819-2830.
19	BAUDRY M， BI X N. Calpain-1 and calpain-2： the Yin and Yang of synaptic plasticity and neurodegeneration［J］. Trends Neurosci， 2016， 39（4）： 235-245.
20	ETEHADI MOGHADAM S， AZAMI TAMEH A， VAHIDINIA Z， et al. Neuroprotective effects of oxytocin hormone after an experimental stroke model and the possible role of calpain-1［J］. J Stroke Cerebrovasc Dis， 2018， 27（3）： 724-732.
21	YIN M H， LIU Q Q， YU L， et al. Downregulations of CD36 and calpain-1， inflammation， and atherosclerosis by simvastatin in apolipoprotein E knockout mice［J］. J Vasc Res， 2017， 54（3）： 123-130.
22	CHENG H， LIU J， ZHANG D D， et al. Ginsenoside Rg1 alleviates acute ulcerative colitis by modulating gut microbiota and microbial tryptophan metabolism［J］. Front Immunol， 2022， 13： 817600.
23	YANG S J， WANG J J， CHENG P， et al. Ginsenoside Rg1 in neurological diseases： From bench to bedside［J］. Acta Pharmacol Sin， 2023， 44（5）： 913-930.

Group	BUN [c_B/（mmol·L^-1）]	LA [c_B/（mmol·L^-1）]	LDH [λ_B/（U·L^-1）]
Control	6.21±0.55	5.36±0.71	535.66±50.43
Model	9.86±0.79^*	8.86±0.85^*	911.48±76.78^*
Inhibitor	6.92±0.72^△	7.04±0.59^△	630.56±59.57^△
Ginsenoside Rg1
Low dose	6.75±0.73^△	6.70±0.65^△	599.12±54.81^△
High dose	6.42±0.71^△	5.78±0.68^△	561.49±50.21^△

Group	MDA [c_B/（mmol·L^-1）]	TNF-α [ρ_B/（ng·L^-1）]	IL-6 [ρ_B/（ng·L^-1）]	SOD [λ_B/（U·L^-1）]
Control	30.61±1.82	103.54±7.43	165.66±12.43	110.61±8.82
Model	64.47±3.04^*	251.78±16.84^*	336.74±22.84^*	54.47±5.04^*
Inhibitor	41.27±2.40^△	164.12±10.04^△	221.41±14.04^△	90.27±7.40^△
Ginsenoside Rg1
Low dose	37.17±2.24^△	133.21±9.21^△	199.45±13.21^△	94.17±8.24^△
High dose	33.65±2.25^△	110.84±8.99^△	171.98±12.99^△	102.65±7.24^△

[1]	刘冠,陶贵周,王洪新. 黄芩苷对腹主动脉结扎诱导的大鼠心肌肥厚和细胞凋亡的作用及其机制[J]. 吉林大学学报(医学版), 2023, 49(4): 850-857.
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[6]	裴丹, 刘学. 抑制PDGFRα活化对小鼠脑损伤后胶质细胞增殖和瘢痕形成的影响[J]. 吉林大学学报(医学版), 2020, 46(05): 1023-1028.
[7]	李环, 曲振廷, 钱鸿昊, 徐晗, 曲莉, 张晶, 王洪艳, 徐小磊. 人参皂苷Rg1联合人参皂苷Rg3对雄性生殖功能损伤模型小鼠生殖功能的改善作用[J]. 吉林大学学报(医学版), 2020, 46(04): 707-713.
[8]	王叶, 王红, 朴丽贞, 杨山, 张书剑, 金正勇. 高氧诱导下新生大鼠脑损伤的发生机制和前列腺素E1的干预作用[J]. 吉林大学学报(医学版), 2019, 45(06): 1206-1211.
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[10]	王冲, 郝铮, 刘伟明, 赵丛海, 张金男. 颅脑损伤后硬膜外血肿骨化1例报告及文献复习[J]. 吉林大学学报(医学版), 2018, 44(03): 628-630.
[11]	王亚东, 李东朋, 郭德伟, 宋及时, 李红伟, 钱伟强, 杨波. 富血小板血浆对创伤性颅脑损伤大鼠神经功能的保护作用[J]. 吉林大学学报(医学版), 2016, 42(05): 910-914.
[12]	张恒, 秦治刚, 梁华新, 房晓萱. 重型颅脑损伤患者大骨瓣减压术后迟发性颅内血肿对预后的影响[J]. 吉林大学学报(医学版), 2016, 42(02): 362-365.
[13]	王茜，张辉，刘名，李琪佳，耿丽鑫，孙明宏，田清友，张宇新. 人参皂苷Rg1对帕金森病模型小鼠黑质区FADD和FLIP表达的影响及其意义[J]. 吉林大学学报(医学版), 2014, 40(05): 962-966.
[14]	罗军，伍胶，毛青花，曹发正，胡艳颖，徐俊晨，王齐. 山楂黄酮对微波辐射所致Wistar大鼠脑损伤的治疗作用[J]. 吉林大学学报(医学版), 2013, 39(2): 291-293.
[15]	房绍宽, 白晶, 吴江. 人参皂苷Rg1对小鼠全脑缺血致白质纤维损伤的保护作用及其机制[J]. J4, 2010, 36(5): 930-933.