菊淀粉型巴戟天寡糖降低肺炎链球菌脑膜炎小鼠的症状评分和死亡率

doi:10.12122/j.issn.1673-4254.2025.03.15

摘要/Abstract

摘要：

目的探究菊淀粉型巴戟天寡糖（IOMO）对小鼠肺炎链球菌脑膜炎（SPM）的治疗作用及可能的作用机制。方法将120只雄性C57BL/6J小鼠随机分为假手术组、SPM+生理盐水（Saline）组、SPM+IOMO 25 mg/kg剂量组、SPM+IOMO 50 mg/kg剂量组，n=30。从造模当天起（0 d），SPM+Saline组和SPM+IOMO组小鼠分别灌胃生理盐水或IOMO 25 mg/kg、50 mg/kg进行干预，持续7 d，记录症状评分和死亡情况；采用脑组织HE染色和尼氏染色评价病理状态和神经元损伤情况，qRT-PCR检测皮质中炎症相关分子mRNA水平，干湿重法测脑含水量和伊文思蓝染色评价脑水肿程度和血脑屏障通透性，Western blotting检测皮质中BBB相关蛋白水平，流式细胞分析检测浸润淋巴细胞IFN-γ水平；SPM造模后21 d采用旷场实验和新物体识别实验评价小鼠学习记忆功能。结果灌胃给予IOMO 50 mg/kg（1次/d，连续7 d）可降低SPM小鼠的症状评分和死亡率（P<0.05），缓解脑组织病理损伤，降低皮质炎症相关分子（IL-6、TNF-α、IL-1β、IL-18、IFN-γ、iNOS、NLRP3、ASC、Caspase-1、GSDMD）mRNA水平（P<0.05）； IOMO可以降低SPM小鼠脑含水量和伊文思蓝渗透量（P<0.05）；IOMO可以上调脑皮质血管内皮钙黏蛋白VE-Cadherin和闭合蛋白Occludin水平、下调水通道蛋白AQP4和BBB损伤的关键调控因子iNOS、IFN-γ水平（P<0.05）；建模21d后，与SPM + Saline组小鼠相比，IOMO可以改善SPM小鼠学习记忆能力（P<0.05）。结论首次发现IOMO可降低SPM小鼠的症状评分和死亡率，并改善其学习记忆能力，其作用机制可能与IOMO抑制过度炎症反应并保护血脑屏障有关。

关键词: 菊淀粉型巴戟天寡糖, 脑膜炎, 肺炎链球菌, 血脑屏障, 干扰素-γ

Abstract:

Objective To investigate the therapeutic effects of inulin-type oligosaccharides of Morinda officinalis (IOMO) in a murine model of Streptococcus pneumoniae meningitis (SPM) and explore its possible mechanisms. Methods A total of 120 male C57BL/6J mice were randomly assigned into Sham, SPM+Saline, SPM+IOMO (25 mg/kg), and SPM+IOMO (50 mg/kg) groups. After modeling, the mice received daily gavage of saline or IOMO at the indicated doses for 7 consecutive days, and the changes in symptom scores and mortality of the mice were monitored. Brain pathology and neuronal injury of the mice were assessed using HE and Nissl staining, and qRT-PCR was performed to detect mRNA levels of the inflammatory mediators. Brain edema and blood-brain barrier (BBB) permeability of the mice were evaluated by measuring brain water content and Evans blue (EB) staining; Western blotting was used to analyze the expressions of BBB-associated proteins, and flow cytometry was employed to detect IFN‑γ expression level in the infiltrating lymphocytes. Open-field test (OFT) and novel object recognition test (NORT) were conducted to assess learning and memory ability of the mice on day 21 after modeling. Results IOMO treatment at 50 mg/kg significantly reduced the symptom scores and mortality rate of SPM mice, alleviated brain damage, and downregulated mRNA levels of IL-6, TNF‑α, IL-1β, IL-18, IFN‑γ, iNOS, NLRP3, ASC, caspase-1 and GSDMD in the brain tissue. IOMO treatment also decreased brain water content and EB leakage, upregulated VE-cadherin and occludin expressions, and suppressed AQP4, iNOS, and IFN‑γ levels of the mice. IOMO-treated mice exhibited improved learning and memory compared with the saline-treated mice on day 21 after SPM modeling. Conclusion IOMO alleviates SPM symptoms, reduces mortality, and mitigates cognitive deficits in mice possibly by suppressing cerebral inflammation and protecting BBB functions.

Key words: inulin-type oligosaccharides of Morinda officinalis, meningitis, Streptococcus pneumoniae, blood-brain barrier, interferon-γ

李泽涵, 梁萌, 韩根成, 张学武. 菊淀粉型巴戟天寡糖降低肺炎链球菌脑膜炎小鼠的症状评分和死亡率[J]. 南方医科大学学报, 2025, 45(3): 577-586.

Zehan LI, Meng LIANG, Gencheng HAN, Xuewu ZHANG. Therapeutic effects of inulin-type oligosaccharides of Morinda officinalis on Streptococcus pneumoniae meningitis in mice[J]. Journal of Southern Medical University, 2025, 45(3): 577-586.

图/表 10

图1 实验流程

Fig.1 Schedule of treatment.

表1 qRT-PCR引物序列

Tab.1 Primer sequence used in qRT-PCR

Gene	Primer sequence (5'-3')
18S	F:TTGACGGAAGGGCACCACCAG
	R:GCACCACCACCACGGAATCG
TNF-α	F:CCCTCACACTCAGATCATCTTCT
	R:GCTACGACGTGGGCTACAG
IL-6	F:GATGGATGCTACCAAACTGGA
	R:TCTGAAGGACTCTGGCTTTG
IL-1β	F:TGAAGCAGCTATGGCAACTG
	R:AGGTCAAAGGTTTGGAAGCA
IL-18	F:CCAAATCACTTCCTCTTGGC
	R:GGCCAAAGTTGTCTGATTCC
IFN-γ	F:AAGCGTCATTGAATCACACCTG
	R:TGACCTCAAACTTGGCAATACTC
iNOS	F:GTTCTCAGCCCAACAATACAAGA
	R:GTGGACGGGTCGATGTCAC
NLRP3	F:ATTACCCGCCCGAGAAAGG
	R:TCGCAGCAAAGATCCACACAG
ASC	F:CTTGTCAGGGGATGAACTCAAAA
	R:GCCATACGACTCCAGATAGTAGC
Caspase-1	F:ACAAGGCACGGGACCTATG
	R:TCCCAGTCAGTCCTGGAAATG
GSDMD	F:CCATCGGCCTTTGAGAAAGTG
	R:ACACATGAATAACGGGGTTTCC

图2 IOMO降低SPM小鼠症状评分和死亡率

Fig.2 IOMO reduces symptom scores (A) and mortality rate (B) of SPM mice (Mean±SD, n=8). *P<0.05, #P<0.05.

图3 小鼠脑组织病理形态HE染色图

Fig.3 HE staining of brain histopathology of the mice (Original magnification: ×1.2, ×14).

图4 小鼠脑组织尼氏染色图

Fig.4 Nissl staining of the brain tissue of the mice (×1.2, ×32).

图5 IOMO对SPM小鼠脑皮质组织炎症相关分子mRNA水平的影响

Fig.5 Effects of IOMO on mRNA levels of inflammation-related molecules mRNA levels in the cerebral cortex of SPM mice (Mean±SD, n=4). *P<0.05 vs Sham group, #P<0.05 vs SPM+Saline group.

图6 IOMO对SPM小鼠脑组织含水量和BBB通透性的影响

Fig.6 Effects of IOMO on brain water content and BBB permeability of SPM mice (Mean±SD, n=6). A: Evans blue (EB) staining. B: Statistics of the results of EB staining. C: Statistics of brain water content. *P<0.05 vs Sham group,#P<0.05 vs SPM+Saline group.

图7 IOMO对SPM小鼠BBB通透性相关分子表达水平的影响

Fig.7 Effects of IOMO on BBB permeability-related molecular levels in SPM mice (Mean±SD, n=6). *P<0.05 vs Sham group,#P<0.05 vs SPM+Saline group.

图8 脑组织浸润淋巴细胞IFN-γ水平检测

Fig.8 Assessment of IFN-γ levels in the infiltrating lymphocytes (Mean±SD, n=4). *P<0.05 vs Sham group,#P<0.05 vs SPM+Saline group.

图9 IOMO缓解SPM引起的小鼠学习记忆功能障碍

Fig.9 IOMO alleviates SPM-induced learning and memory dysfunction in mice (Mean±SD, n=8). A, B: Traveling traces and statistics in open field test (*P<0.05 vs Crossing Training). C: Statistics of the results of new object recognition test (*P<0.05 vs familiar group).

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