Volatile oil from Acorus tatarinowii Schott ameliorates tic disorder in rats by inhibiting TLR4/MyD88/NF-κB-mediated microglial polarization

doi:10.12122/j.issn.1673-4254.2026.03.18

Abstract

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

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.

Figures/Tables 8

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

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

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.

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.

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.

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.

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.

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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