石菖蒲挥发油通过抑制TLR4/MyD88/NF-κB通路介导的小胶质细胞极化改善大鼠抽动障碍

doi:10.12122/j.issn.1673-4254.2026.03.18

摘要/Abstract

摘要：

目的探讨石菖蒲挥发油（VOA）通过TLR4/MyD88/NF-κB通路调控小胶质细胞（MG）抗抽动的机制。方法 48只3周龄SD大鼠，随机分为空白组（n=8）和模型制备组（n=40），采用腹腔注射亚氨基二丙腈诱导TD模型，将模型制备组大鼠随机分为模型组、硫必利组（47.91 mg/kg）、VOA组（51.12 mg/kg）、TAK-242组（3 mg/kg）和VOA+TAK242组（51.12 mg/kg+3 mg/kg），每组8只，1次/d，干预28 d。干预结束后，评估大鼠行为学变化情况，采用尼氏染色观察纹状体神经元形态改变，ELISA法检测大鼠血清、纹状体中TNF-α、C1q、TGF-β、VEGF含量，RT-PCR、Western blotting法检测观察大鼠纹状体中TLR4、MyD88、NF-κB p65的mRNA和蛋白的表达情况，免疫细胞荧光染色检测NF-κB p65入核情况、MG特异性标志物Iba1、M1型、M2型标记蛋白CD86、CD206的表达水平。结果与模型组相比较，VOA组大鼠抽动样行为评分降低（P<0.01），纹状体神经元损伤改善；血清及纹状体中TNF-α、C1q水平降低（P<0.01）、TGF-β、VEGF水平升高（P<0.01）；纹状体中TLR4、MyD88、NF-κB p65 mRNA和蛋白相对表达量水平降低（P<0.01），NF-κB p65 入核减少（P<0.05），Iba1、CD86相对荧光强度减弱（P<0.01），Iba1、CD206相对荧光强度增强（P<0.01）。结论 VOA通过降低MG的活化及极化状态，进而抑制脑内炎症水平改善抽动症状，其作用机制可能与抑制TLR4/MyD88/NF-κB通路有关。

关键词: 抽动障碍, 小胶质细胞, TLR4/MyD88/NF-κB通路, 石菖蒲挥发油

Abstract:

Objective To investigate the mechanism by which Acorus tatarinowii Schott volatile oil (VOA) alleviates tic disorder (TD) in rats. Methods Forty-eight 3-week-old SD rats were randomly divided into blank group (n=8) and TD model group with intraperitoneal injection of iminodipropionitrile (n=40). The rat models were further randomized into 5 groups (n=8) for treatment with daily gavage of saline (model group), tiapride (47.91 mg/kg) or VOA (51.12 mg/kg), intraperitoneal injection of TAK-242 (a TLR4 inhibitor; 3 mg/kg), or both VOA gavage and TAK242 injection for 28 days. Behavioral changes of the rats were assessed, and Nissl staining was used to observe neuronal morphology in the striatum. The levels of TNF-α, C1q, TGF-β, and VEGF in the serum and striatum were measured using ELISA. The mRNA and protein expressions of TLR4, MyD88, and NF‑κB p65 in the striatum were detected using RT-PCR and Western blotting, and NF‑κB p65 nuclear translocation and expressions of Iba1 (a MG-specific marker), CD86 and CD206 were analyzed using immunofluorescence staining. Results VOA treatment significantly reduced tic-like behavior scores and ameliorated striatal neuronal damage in the rat models, resulting also in lowered levels of TNF-α and C1q and increased levels of TGF-β and VEGF in both the serum and striatum, decreased mRNA and protein expression levels of TLR4, MyD88, and NF‑κB p65 in the striatum, and reduced nuclear translocation of NF‑κB p65. Immunofluorescence staining revealed significantly weakened expressions of Iba1 and CD86 and enhanced CD206 expression in the striatum of VOA-treated rats. All these ameliorative effects of VOA were significantly attenuated by treatment with TAK-242. Conclusion VOA alleviates tic symptoms in rats by inhibiting microglia activation and polarization to reduce neuroinflammation possibly through suppression of the TLR4/MyD88/NF-κB signaling pathway.

Key words: tic disorders, microglia, Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor kappa-B pathway, volatile oil of Acorus tatarinowii Schott

冯鹏, 王洁, 褚江伟, 王鹤霖, 田永衍, 张杰昌, 王明露, 王宇彤. 石菖蒲挥发油通过抑制TLR4/MyD88/NF-κB通路介导的小胶质细胞极化改善大鼠抽动障碍[J]. 南方医科大学学报, 2026, 46(3): 646-654.

Peng FENG, Jie WANG, Jiangwei CHU, helin WANG, Yongyan TIAN, Jiechang ZHANG, Minglu WANG, Yutong WANG. Volatile oil from Acorus tatarinowii Schott ameliorates tic disorder in rats by inhibiting TLR4/MyD88/NF-κB-mediated microglial polarization[J]. Journal of Southern Medical University, 2026, 46(3): 646-654.

图/表 8

表1 行为学评分表

Tab.1 Behavioral assessment form

Degree rating	Stereotypic behavior	Motor behavior
0	No stereotyped behavior	Quiet or normal activity
1	Rotation behavior (clockwise or counterclockwise rotation motion)	Be overexcited
2	Excessive head and neck movement	Exploring behavior increases
3	Excessive vertical movement of the head and neck combined with rotational behavior	Keep running
4	Head to side swing, combined with excessive vertical movement of the head and neck	Constantly jumping and startled jumping

表2 PCR引物序列

Tab.2 Primer sequences for RT-PCR

Primer	Forward	Reverse	Extension length (bp)
TLR4	TATCGGTGGTCAGTGTGCTT	CTCGTTTCTCACCCAGTCCT	167
MyD88	CCTGGGCACACATCTCAGTT	CTGGGGGCGGAATGTTTTTG	220
P65	TGGATGAGTAATGCGTCCAGG	GGACTGGCGGTGGAGAATAA	91
GAPDH	TGATGGGTGTGAACCACGAG	AGTGATGGCATGGACTGTGG	152

图1 第28天各组大鼠抽动行为评分

Fig1 Tic behavior scores of the rats in each group on day 28 (Mean±SD, n=8). a: motor behavior scores on day 28. b: Stereotyped behavior scores on day 28. A: Blank group; B: Model group; C: Tiapride group; D: VOA group; E: TAK-242 group; F: VOA+TAK-242 group. ##P<0.01 vs B group, ▲P<0.05, ▲▲P<0.01 vs C group, ■P<0.05 vs D group.

图2 各组大鼠纹状体尼氏染色

Fig.2 Nissl staining of the striatum of the rats in each group (Original magnification: ×200). A: Blank group; B: Model group; C: Tiapride group; D: VOA group; E: TAK-242 group; F: VOA+TAK-242 group.

图3 各组血清、纹状体中TGF-β、VEGF、TNF-α、C1q含量水平

Fig.3 Levels of TNF-α (a, e), C1q (b, f), TGF-β (c, g) and VEGF (d, h) in the serum and striatum of the rats in different groups (Mean±SD, n=3). A: Blank group; B: Model group; C: Tiapride group; D: VOA group; E: TAK-242 group; F: VOA+TAK-242 group. **P<0.01 vs A group; ##P<0.01 vs B group; ▲▲P<0.01 vs C group; ■■P<0.01 vs D group.

图4 各组大鼠纹状体中TLR4、MyD88、NF-κB mRNA相对表达

Fig.4 Relative expressions of TLR4 (a), NF-κB p65 (b) and MyD88 (c) mRNA in the striatum of the rats in each group. A: Blank group; B: Model group; C: Tiapride group; D: VOA group; E: TAK-242 group; F: VOA+TAK-242 group. n=3.**P<0.01 vs A group; ##P<0.01 vs B group; ▲▲P<0.01 vs C group; ■■P<0.01 vs D group.

图5 Western blotting 检测各组大鼠纹状体中TLR4、MyD88、NF-κB p65、p-NF-κB p65蛋白相对表达

Fig.5 Relative expression of TLR4, MyD88, NF-κB p65 and p-NF-κB p65 proteins in the striatum of the rats in each group detected by Western blotting. a: Results of Western blotting of the proteins. b-e: Relative expressions of TLR4, MyD88, NF-κB p65 and p-NF-κB p65 proteins. A: Blank group; B: Model group; C: Tiapride group; D: VOA group; E: TAK-242 group; F: VOA+TAK-242 group. n=3. **P<0.01 vs A group; ##P<0.01 vs B group; ▲▲P<0.01 vs C group; ■■P<0.01 vs D group.

图7 各组大鼠纹状中MG活化及极化表达情况

Fig.7 Expression of microglial activation and polarization markers in the striatum in each group. a: Immuneofluorescence staining for Iba-1 (red), CD86 (green) and DAPI (blue). b: Immuneofluorescence staining for Iba-1 (red), CD206 (green) and DAPI (blue). c, d: Fluorescence intensity of Iba-1 and CD86. d: Fluorescence intensity of Iba-1 and CD206. n=3. **P<0.01 vs A group,##P<0.01 vs B group, ▲▲P<0.01 vs C group, ■■P<0.01 vs D group.

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