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

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.

Keywords: Bmal1; Parkinson's disease; gut-brain axis; sodium butyrate.

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.