Bmal1可能介导了丁酸钠对帕金森病小鼠的神经保护作用

doi:10.12122/j.issn.1673-4254.2024.05.09

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (5): 876-884.doi: 10.12122/j.issn.1673-4254.2024.05.09

Bmal1可能介导了丁酸钠对帕金森病小鼠的神经保护作用

王飞霞¹^,²^,³(), 张政²^,³, 孙艳²^,³, 杨柳菁¹^,²^,³, 郭桐彤¹^,²^,³, 潘夜厅¹^,²^,³, 丁嵩涛¹, 蒋林¹, 刘含登¹^,²^,³()

^1.重庆医科大学，实验教学管理中心组织与细胞生物学实验室，重庆 400016
^2.重庆医科大学，神经科学研究中心，重庆 400016
^3.重庆医科大学，基础医学院，重庆 400016

收稿日期:2023-12-15 出版日期:2024-05-20 发布日期:2024-06-06
通讯作者: 刘含登 E-mail:fiexia@stu.cqmu.edu.cn;hdliu@cqmu.edu.cn
作者简介:王飞霞，在读硕士研究生，E-mail: fiexia@stu.cqmu.edu.cn
基金资助:
重庆市自然科学基金(cstc2017jcyjAX0113);重庆市青年创新人才培养计划项目(CY220404);重庆医科大学大学生导师制创新人才培养项目(LTMCMTS202202)

Bmal1 mediates the neuroprotective effect of sodium butyrate in a mouse model of Parkinson's disease

Feixia WANG¹^,²^,³(), Zheng ZHANG²^,³, Yan SUN²^,³, Liujing YANG¹^,²^,³, Tongtong GUO¹^,²^,³, Yeting PAN¹^,²^,³, Songtao DING¹, Lin JIANG¹, Handeng LIU¹^,²^,³()

^1.Laboratory of Tissue and Cell Biology, Experimental Teaching and Management Center, Chongqing Medical University, Chongqing 400016, China
^2.Department of Center for Neuroscience Research, Chongqing Medical University, Chongqing 400016, China
^3.School of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China

Received:2023-12-15 Online:2024-05-20 Published:2024-06-06
Contact: Handeng LIU E-mail:fiexia@stu.cqmu.edu.cn;hdliu@cqmu.edu.cn

摘要/Abstract

摘要：

目的探究肠道微生物代谢产物丁酸钠（NaB）通过肠-脑轴对帕金森病（PD）小鼠发挥神经保护作用及其潜在机制。方法 39只7周龄雄性C57BL/6J小鼠随机均分为对照组（NC）、模型组（PD）和NaB治疗组（NaB），13只/组。除NC组小鼠注射等体积生理盐水外，其余各组连续5 d腹腔注射1-甲基-4-苯基-1，2，3，6-四氢吡啶［MPTP，30 mg/（kg·d）］建立亚急性PD模型。造模完成后NaB组连续灌胃NaB［300 mg/（kg·d）］治疗14 d，其余两组小鼠灌胃等体积的生理盐水。治疗结束后进行行为学实验检测小鼠的运动功能。Western blot分别检测小鼠纹状体中酪氨酸羟化酶（TH）、α-突触核蛋白（α-syn）的表达和结肠中促炎因子核因子κB（NF-κB）、肿瘤坏死因子（TNF-α）、白介素6（IL-6）以及紧密连接蛋白ZO-1、occludin、claudin的表达。通过HE染色观察小鼠结肠的炎性浸润。RNA测序分析筛选出小鼠结肠组织的差异基因。结果行为学实验结果显示，NaB治疗可提高PD小鼠的运动速度（P<0.01）以及四肢拉力（P<0.05）。Western blot结果显示，NaB上调PD小鼠纹状体中TH（P<0.01）和下调α-syn（P<0.05）的表达，NaB上调PD小鼠结肠组织中的Occludin和Claudin的表达（P<0.05），下调了NF-κB、TNF-α和IL-6的表达（P<0.05）。HE染色结果显示，NaB改善PD小鼠结肠的炎性浸润。结肠RNA测序结果显示，节律基因Bmal1可能介导了NaB对PD小鼠的神经保护作用（P<0.05）。总结 NaB改善PD小鼠的运动障碍，减少纹状体的多巴胺能神经元的丢失，并改善PD小鼠的结肠炎症，其机制可能与Bmal1有关。

关键词: 丁酸钠, 帕金森病, 肠-脑轴, Bmal1

Abstract:

Objective To investigate the mechanisms that mediate the neuroprotective effect of the intestinal microbial metabolite sodium butyrate (NaB) in a mouse model of Parkinson's disease (PD) via the gut-brain axis. Methods Thirty-nine 7-week-old male C57BL/6J mice were randomized equally into control group, PD model group, and NaB treatment group. In the latter two groups, PD models were established by intraperitoneal injection of 30 mg/kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) once daily for 5 consecutive days, and normal saline was injected in the control group. After modeling, the mice received daily gavage of NaB (300 mg/kg) or an equal volume of saline for 14 days. Behavioral tests were carried out to assess the changes in motor function of the mice, and Western blotting was performed to detect the expressions of tyrosine hydroxylase (TH) and α‑synuclein (α‑syn) in the striatum and nuclear factor‑κB (NF‑κB), tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and the tight junction proteins ZO-1, Occludin, and Claudinin the colon. HE staining was used to observe inflammatory cell infiltration in the colon of the mice. RNA sequencing analysis was performed to identify the differentially expressed genes in mouse colon tissues, and their expressions were verified using qRT-PCR and Western blotting. Results The mouse models of PD with NaB treatment showed significantly increased movement speed and pulling strength of the limbs with obviously upregulated expressions of TH, Occludin, and Claudin and downregulated expressions of α-syn,NF-κB, TNF‑α, and IL-6 (all P<0.05). HE staining showed that NaB treatment significantly ameliorated inflammatory cell infiltration in the colon of the PD mice. RNA sequencing suggested that Bmal1 gene probably mediated the neuroprotective effect of NaB in PD mice (P<0.05). Conclusion NaB can improve motor dysfunction, reduce dopaminergic neuron loss in the striatum, and ameliorate colonic inflammation in PD mice possibly through a mechanism involving Bmal1.

Key words: sodium butyrate, Parkinson's disease, gut-brain axis, Bmal1

王飞霞, 张政, 孙艳, 杨柳菁, 郭桐彤, 潘夜厅, 丁嵩涛, 蒋林, 刘含登. Bmal1可能介导了丁酸钠对帕金森病小鼠的神经保护作用[J]. 南方医科大学学报, 2024, 44(5): 876-884.

Feixia WANG, Zheng ZHANG, Yan SUN, Liujing YANG, Tongtong GUO, Yeting PAN, Songtao DING, Lin JIANG, Handeng LIU. Bmal1 mediates the neuroprotective effect of sodium butyrate in a mouse model of Parkinson's disease[J]. Journal of Southern Medical University, 2024, 44(5): 876-884.

图/表 6

图1 NaB改善了PD小鼠的运动功能障碍和焦虑症状

Fig.1 NaB improves motor dysfunction and relieves anxiety symptoms in PD mice. A: Pole test. B: Beam test. C: Average tension of pull test. D: The maximum tension in pull test. E: Mean speed in open field test. F: Total moving distance in open field test. G: Time in the central area in open field test. H: Representative movement tracks in open field test. n=11 in NC group, n=10 in PD group, n=13 in NaB group. *P<0.05, **P<0.01, ***P<0.001.

图2 NaB逆转了PD小鼠纹状体中α-syn蛋白的表达增加和TH蛋白的表达下降

Fig.2 NaB reverses upregulation of α‑syn and downregulation of TH protein expression in the striatum of PD mice. A: Western blots of TH and α-syn proteins. B, C: Quantitative analysis of protein expression levels pf TH and α-syn （n=3）. *P<0.05, **P<0.01.

图3 NaB治疗减轻PD小鼠结肠炎症因子水平并提高了结肠紧密连接蛋白的表达

Fig.3 NaB treatment reduces colonic inflammatory factor levels and increases colonic tight junction protein expressions in PD mice. A: Western blots of NF‑κB, IL-6, and TNF‑α in mouse colon. B: Western blots of ZO-1, Occludin, and Claudin proteins. C: HE staining of mouse colon tissues (Original magnification: ×100). D-I: Quantitative analysis of the protein expression levels of NF‑κB, IL-6, TNF‑α, ZO-1, Occludin and Claudin (n=3). *P<0.05, **P<0.01.

图4 NaB相关的差异表达基因

Fig.4 Differentially expressed genes (DEGs) in PD mice with NaB treatment. A: Schematic diagram of RNA sequencing of mouse colon tissues. B: Heat map of DEGs. C: Scatter plot of DEGs between PD and NC groups. D: Scatter plot of DEGs between NaB and PD groups. FC>1.2, P<0.05.

图5 NaB相关的DEGs的GO和KEGG分析

Fig.5 GO and KEGG analysis of the DEGs in PD mice with NaB treatment. A: GO analysis of DEGs between PD and NC group. B: GO analysis of DEGs between NaB and PD groups. C: KEGG analysis of DEGs between PD and NC groups. D: KEGG analysis of DEGs between NaB and PD group. FC>1.2, P<0.05.

图6 Bmal1的RNA测序结果验证

Fig.6 Validation of RNA sequencing results of Bmal1. A： Venn diagram. B, C: qRT-PCR detection of relative expression of Npas2 and Bmal1 mRNA in mouse colonic tissues. D, F: Western blots of Npas2 and Bmal1 proteins. E, G: Quantitative analysis of the protein expression levels of Npas2 and Bmal1 (n=3). *P<0.05, **P<0.01, ***P<0.001.

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Bmal1可能介导了丁酸钠对帕金森病小鼠的神经保护作用

Bmal1 mediates the neuroprotective effect of sodium butyrate in a mouse model of Parkinson's disease

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