和厚朴酚调节BDNF-TrkB-CREB信号通路对脑出血小鼠神经损伤和认知功能的影响

doi:10.3969/j.issn.1673-5765.2024.09.010

摘要/Abstract

摘要：

目的探讨和厚朴酚对脑出血（intracerebral hemorrhage，ICH）小鼠神经损伤和认知功能的影响及其作用机制。

方法将C57BL/6J小鼠随机分为假手术组，模型组，和厚朴酚低、中、高剂量组，以及和厚朴酚+抑制剂组。除假手术组外，其余组小鼠均通过自体血注入右侧基底神经节构建ICH模型。于ICH前15 min和ICH后1 h，和厚朴酚低、中、高剂量组分别腹腔注射10 mg/kg、20 mg/kg、40 mg/kg和厚朴酚，和厚朴酚+抑制剂组腹腔注射40 mg/kg和厚朴酚与5 μg/kg脑源性神经营养因子（brain derived neurotrophic factor，BDNF）-酪氨酸激酶受体B（tyrosine kinase receptor B，TrkB）-环磷酸腺苷反应元件结合蛋白（cyclic adenosine monophosphate response element binding protein，CREB）信号通路抑制剂K252a，假手术组、模型组腹腔注射等量的二甲基亚砜和生理盐水。采用改良的神经功能缺损评分（modified neurological severity score，mNSS）评估小鼠神经功能；通过Morris水迷宫实验计算逃避潜伏期、跨越原平台次数、目标象限停留时间百分比3个指标用于评估小鼠空间学习和记忆能力；苏木精-伊红（hematoxylin-eosin，HE）染色观察海马神经元病理学改变；原位末端转移酶标记（terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling，TUNEL）染色检测海马神经元凋亡率；酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测血清BDNF、TrkB水平；免疫印迹法检测海马组织BDNF、TrkB、CREB、磷酸化CREB（phosphorylated CREB，p-CREB）蛋白表达情况。

结果与假手术组小鼠相比，模型组小鼠mNSS、海马神经元凋亡率升高，逃避潜伏期延长，跨越原平台次数减少，目标象限停留时间百分比降低，血清BDNF、TrkB及海马组织BDNF、TrkB、p-CREB/CREB水平降低（均P<0.001），海马神经元结构模糊、排列紊乱、数量减少、体积变小，且细胞核固缩；与模型组小鼠相比，和厚朴酚低、中、高剂量组小鼠mNSS（均P＜0.01）、海马神经元凋亡率（均P<0.001）依次降低，逃避潜伏期（均P<0.001）依次缩短，跨越原平台次数（P=0.007、P<0.001、P<0.001）依次增多，目标象限停留时间百分比（P=0.004、P<0.001、P<0.001）依次升高，血清BDNF（均P<0.001）、TrkB（P=0.001、P<0.001、P<0.001）及海马组织BDNF（P=0.008、P<0.001、P<0.001）、TrkB（P=0.001、P<0.001、P<0.001）、p-CREB/CREB（均P<0.001）水平依次升高，海马神经元损伤有所改善；与和厚朴酚高剂量组小鼠相比，和厚朴酚+抑制剂组小鼠mNSS、海马神经元凋亡率升高，逃避潜伏期延长，跨越原平台次数减少，目标象限停留时间百分比降低，血清BDNF、TrkB及海马组织BDNF、TrkB、p-CREB/CREB水平降低（均P<0.001），海马神经元损伤加重。

结论和厚朴酚可能通过激活BDNF-TrkB-CREB信号通路减轻ICH小鼠海马神经元凋亡和损伤，改善认知功能障碍。

文章导读： 和厚朴酚可能通过激活BDNF-TrkB-CREB信号通路减轻脑出血小鼠海马神经元凋亡和损伤并改善认知功能障碍，为脑出血后神经损伤和认知功能障碍治疗提供了一种有希望的治疗方法。

关键词: 脑出血; 和厚朴酚; 认知功能; BDNF-TrkB-CREB信号通路

Abstract:

Objective To investigate the effects of honokiol on neurological injury and cognitive function in mice with intracerebral hemorrhage (ICH) and its mechanism.

Methods C57BL/6J mice were randomly grouped into the sham operation group, the model group, the low, medium, and high dose honokiol groups, and the honokiol+inhibitor group. Except for the sham operation group, ICH models were established by injecting autologous blood into the right basal ganglia in other groups. At 15 min before ICH and 1 h after ICH, the low, medium, and high dose honokiol groups were intraperitoneally injected with 10 mg/kg, 20 mg/kg, and 40 mg/kg honokiol, respectively. The honokiol+inhibitor group was intraperitoneally injected with 40 mg/kg honokiol and 5 μg/kg brain derived neurotrophic factor (BDNF)-tyrosine kinase receptor B (TrkB)-cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway inhibitor K252a. The sham operation group and the model group were intraperitoneally injected with equal amounts of dimethyl sulfoxide and physiological saline. The modified neurological severity score (mNSS) was applied to assess the neurological function of mice. The Morris water maze experiment was applied to evaluate the spatial learning and memory abilities of mice by calculating the escape latency, the number of times of crossing the original platform, and the percentage of residence time in the target quadrant. Hematoxylin-eosin (HE) staining was applied to observe the pathological changes in hippocampal neurons. Terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was applied to detect the apoptotic rate of hippocampal neurons. Enzyme-linked immunosorbent assay (ELISA) was applied to detect serum levels of BDNF and TrkB. Western blot was applied to detect the expression of BDNF, TrkB, CREB, and phosphorylated CREB (p-CREB) proteins in the hippocampus.

Results Compared with the sham operation group, the mNSS and apoptotic rate of hippocampal neurons in the model group increased, the escape latency prolonged, the number of times of crossing the original platform decreased, the percentage of residence time in the target quadrant decreased, and the levels of BDNF, TrkB in serum and BDNF, TrkB, p-CREB/CREB in the hippocampus decreased (all P<0.001). The structure of hippocampal neurons was blurred, the arrangement was disordered, the number was reduced, the volume was smaller, and the nucleus was shrunk. Compared with the model group, the mNSS (all P<0.01) and apoptotic rate of hippocampal neurons (all P<0.001) in mice in the low, medium, and high dose honokiol groups decreased successively, the escape latency (all P<0.001) shortened successively, the number of times of crossing the original platform (P=0.007, P<0.001, P<0.001) increased successively, the percentage of residence time in the target quadrant (P=0.004, P<0.001, P<0.001) increased successively, and the levels of BDNF (all P<0.001), TrkB (P=0.001, P<0.001, P<0.001) in serum and BDNF (P=0.008, P<0.001, P<0.001), TrkB (P=0.001, P<0.001, P<0.001), p-CREB/CREB (all P<0.001) in the hippocampus increased successively, the damage of hippocampal neurons was improved. Compared with the high dose honokiol group, the mNSS and apoptotic rate of hippocampal neurons in the honokiol+inhibitor group were increased, the escape latency was prolonged, and the number of times of crossing the original platform was decreased, the percentage of residence time in the target quadrant was decreased, and the levels of BDNF, TrkB in serum and BDNF, TrkB, and p-CREB/CREB in the hippocampus were decreased (all P<0.001), and the damage of hippocampal neurons was aggravated.

Conclusions Honokiol may alleviate hippocampal neuronal apoptosis and damage, and improve cognitive dysfunction in ICH mice by activating the BDNF-TrkB-CREB signaling pathway.

Key words: Intracerebral hemorrhage; Honokiol; Cognitive function; BDNF-TrkB-CREB signaling pathway

中图分类号:

李阳阳, 方建, 王晓雪. 和厚朴酚调节BDNF-TrkB-CREB信号通路对脑出血小鼠神经损伤和认知功能的影响[J]. 中国卒中杂志, 2024, 19(9): 1048-1057.

LI Yangyang, FANG Jian, WANG Xiaoxue.

Effects of Honokiol on Neurological Injury and Cognitive Function in Mice with Intracerebral Hemorrhage by Regulating BDNF-TrkB-CREB Signaling Pathway [J]. Chinese Journal of Stroke, 2024, 19(9): 1048-1057.

参考文献

[1] MAGID-BERNSTEIN J，GIRARD R，POLSTER S，et al. Cerebral hemorrhage：pathophysiology，treatment，and future directions[J]. Circ Res，2022，130（8）：1204-1229.

[2] WAN Y F，HOLSTE K G，HUA Y，et al. Brain edema formation and therapy after intracerebral hemorrhage[J/OL]. Neurobiol Dis，2023，176：105948[2023-01-17]. https://doi.org/10.1016/j.nbd.2022.105948.

[3] KHATOON F，ALI S，KUMAR V，et al. Pharmacological features，health benefits and clinical implications of honokiol[J]. J Biomol Struct Dyn，2023，41（15）：7511-7533.

[4] ZHENG J W，SHI L G，LIANG F，et al. Sirt3 ameliorates oxidative stress and mitochondrial dysfunction after intracerebral hemorrhage in diabetic rats[J/OL]. Front Neurosci，2018，12：414[2023-01-17]. https://doi.org/10.3389/fnins.2018.00414.

[5] ZHENG X C，GAO J L，ZHAO M M，et al. Honokiol attenuates mitochondrial fission and cell apoptosis by activating Sirt3 in intracerebral hemorrhage[J]. Chin Med J（Engl），2023，136（6）：719-731.

[6] YE S，XIE D J，ZHOU P，et al. Huang-Pu-Tong-Qiao Formula ameliorates the hippocampus apoptosis in diabetic cognitive dysfunction mice by activating CREB/BDNF/TrkB signaling pathway[J/OL]. Evid Based Complement Alternat Med，2021，2021：5514175[2023-01-17]. https://doi.org/10.1155/2021/5514175.

[7] KO H R，AHN S Y，CHANG Y S，et al. Human UCB-MSCs treatment upon intraventricular hemorrhage contributes to attenuate hippocampal neuron loss and circuit damage through BDNF-CREB signaling[J/OL]. Stem Cell Res Ther，2018，9（1）：326[2023-01-17]. https://doi.org/10.1186/s13287-018-1052-5.

[8] 国家质量监督检验检疫总局，国家标准化管理委员会. 实验动物福利伦理审查指南：GB/T 35892-2018[S]. 北京：中国标准出版社，2018：1-19.

[9] National Research Council（US）Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the care and use of laboratory animals[S]. 8th ed. Washington，D.C.：National Academies Press（US），2011.

[10] 王雪，刘阳，徐剑峰，等. 脐带间充质干细胞来源外泌体对脑出血模型小鼠海马神经元的保护作用[J]. 中国组织工程研究，2022，26（31）：4928-4934.

WANG X，LIU Y，XU J F，et al. Neuroprotective effect of umbilical cord mesenchymal stem cell-derived exosomes on hippocampal neurons in mice with intracerebral hemorrhage[J]. Chinese Journal of Tissue Engineering Research，2022，26（31）：4928-4934.

[11] WANG Y，TIAN M，TAN J Y，et al. Irisin ameliorates neuroinflammation and neuronal apoptosis through integrin αVβ5/AMPK signaling pathway after intracerebral hemorrhage in mice[J/OL]. J Neuroinflammation，2022，19（1）：82[2023-01-17]. https://doi.org/10.1186/s12974-022-02438-6.

[12] WANG D M，DONG X H，WANG C Y. Honokiol ameliorates amyloidosis and neuroinflammation and improves cognitive impairment in Alzheimer’s disease transgenic mice[J]. J Pharmacol Exp Ther，2018，366（3）：470-478.

[13] ZHAO Y K，LUO D，NING Z P，et al. Electro-acupuncture ameliorated MPTP-induced parkinsonism in mice via TrkB neurotrophic signaling[J/OL]. Front Neurosci，2019，13：496[2023-01-17]. https://doi.org/10.3389/fnins.2019.00496.

[14] LI Z，KHAN S，LIU Y，et al. Therapeutic strategies for intracerebral hemorrhage[J/OL]. Front Neurol，2022，13：1032343[2023-01-17]. https://doi.org/10.3389/fneur.2022.1032343.

[15] SUN G W，DING T Y，WANG M，et al. Honokiol reduces mitochondrial dysfunction and inhibits apoptosis of nerve cells in rats with traumatic brain injury by activating the mitochondrial unfolded protein response[J]. J Mol Neurosci，2022，72（12）：2464-2472.

[16] HOU M Y，BAO W F，GAO Y Y，et al. Honokiol improves cognitive impairment in APP/PS1 mice through activating mitophagy and mitochondrial unfolded protein response[J/OL]. Chem Biol Interact，2022，351：109741[2023-01-17]. https://doi.org/10.1016/j.cbi.2021.109741.

[17] WANG D M，CAO L Y，ZHOU X，et al. Mitigation of honokiol on fluoride-induced mitochondrial oxidative stress，mitochondrial dysfunction，and cognitive deficits through activating AMPK/PGC-1α/Sirt3[J/OL]. J Hazard Mater，2022，437：129381[2023-01-17]. https://doi.org/10.1016/j.jhazmat.2022.129381.

[18] QU C，LI Q P，SU Z R，et al. Nano-honokiol ameliorates the cognitive deficits in TgCRND8 mice of Alzheimer’s disease via inhibiting neuropathology and modulating gut microbiota[J/OL]. J Adv Res，2021，35：231-243[2023-01-17]. https://doi.org/10.1016/j.jare.2021.03.012.

[19] 邓祥敏，阮仁余，朱星宇，等. 基于BDNF/TrkB/CREB信号通路探讨鼠尾草酸对大鼠抑郁样行为的改善作用[J]. 中成药，2022，44（12）：4009-4013.

DENG X M，RUAN R Y，ZHU X Y，et al. Study on the improvement of carnosic acid on depression-like behavior in rats based on BDNF/TrkB/CREB signaling pathway[J]. Chinese Traditional Patent Medicine，2022，44（12）：4009-4013.

[20] GUO C P，LIU Y，FANG M S，et al. ω-3PUFAs improve cognitive impairments through Ser133 phosphorylation of CREB upregulating BDNF/TrkB signal in schizophrenia[J]. Neurotherapeutics，2020，17（3）：1271-1286.

[21] 韩珂，张郧涛，王晓鹏，等. 芍甘木瓜汤通过BDNF/TrkB/CREB信号通路改善脑卒中偏瘫痉挛状态大鼠神经行为学研究[J]. 药物评价研究，2021，44（10）：2161-2169.

HAN K，ZHANG X T，WANG X P，et al. Effect of Shaogan Mugua Decoction on neuroethology and regulation mechanism of BDNF/TrkB/CREB pathway in rats with spastic hemiplegia after stroke[J]. Drug Evaluation Research，2021，44（10）：2161-2169.

[22] WANG H X，JIANG N，LV J W，et al. Ginsenoside Rd reverses cognitive deficits by modulating BDNF-dependent CREB pathway in chronic restraint stress mice[J/OL]. Life Sci，2020，258：118107[2023-01-17]. https://doi.org/10.1016/j.lfs.2020.118107.

[23] LI H T，SUN J M，WU Y L，et al. Honokiol relieves hippocampal neuronal damage in Alzheimer’s disease by activating the SIRT3-mediated mitochondrial autophagy[J/OL]. CNS Neurosci Ther，2024，30（8）：e14878[2024-08-05]. https://doi.org/10.1111/cns.14878.

[24] WANG D M，YUAN Q，LIU S W，et al. BDE-47 flame retardant exposure induces microglial pyroptosis and cognitive deficits by activating the mtROS-NLRP3 axis via Sirt3 downregulation and is salvaged by honokiol[J/OL]. Environ Pollut，2023，334：122158[2024-08-05]. https://doi.org/10.1016/j.envpol.2023.122158.

[25] WEI P H，JIA M，KONG X Y，et al. Human umbilical cord-derived mesenchymal stem cells ameliorate perioperative neurocognitive disorder by inhibiting inflammatory responses and activating BDNF/TrkB/CREB signaling pathway in aged mice[J/OL]. Stem Cell Res Ther，2023，14（1）：263[2024-08-05]. https://doi.org/10.1186/s13287-023-03499-x.

[26] WANG Q，YIN X H，LIU Y，et al. K252a suppresses neuronal cells apoptosis through inhibiting the translocation of Bax to mitochondria induced by the MLK3/JNK signaling after transient global brain ischemia in rat hippocampal CA1 subregion[J]. J Recept Signal Transduct Res，2011，31（4）：307-313.