Oolong tea attenuates neuroinflammation by modulating the microbiota-gutbrain axis in a rat model of autism

Zhen Zheng¹Hongbo Zhao²Peng Zheng^2*Shaoqun Liu^2*

• ¹Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
• ²South China Agricultural University, Guangzhou, China

Abstract Background: Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder with limited effective treatments. Emerging evidence implicates dysregulation of the microbiota-gut-brain axis in ASD pathogenesis. Oolong tea (OT), a traditional Chinese tea with neuroprotective properties, may modulate this axis, but its effects and mechanisms in ASD remain unclear. We investigated whether OT attenuates neuroinflammation in a valproic acid (VPA)-induced rat model of autism via the microbiota-gut-brain axis and the TLR-4/IκB-α/NF-κB signaling pathway. Methods: An ASD model was established by prenatal VPA exposure (500 mg/kg, i.p., E12.5). Postnatal VPA-treated rats received OT (100, 200, or 400 mg/kg/d) for 4 weeks. Behavioral assessments included self-grooming, marble burying, and three-chamber social interaction tests. Nissl staining evaluated neuropathology. Gut microbiota composition was analyzed via 16S rRNA sequencing of fecal samples. Lipopolysaccharide (LPS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels were measured in the plasma, intestine and brain by ELISA. Intestinal and blood-brain barrier (BBB) integrity (claudin-1/5, occludin, ZO-1) and TLR-4/IκB-α/NF-κB pathway activation were assessed by Western blot/immunofluorescence. Microglial (Iba-1) and astrocytic (GFAP) activation and neuronal TLR-4 localization (co-staining with Neun) were examined. Antibiotic cocktail (ABX)-induced microbiota depletion validated gut microbiota dependency. Results: OT (400 mg/kg/d) significantly ameliorated repetitive behaviors (reduced self-grooming duration and marble burying), sociability deficits (improved sociability/social preference index), and attenuated cortical neuronal loss in VPA-treated rats. OT restored gut microbiota dysbiosis, specifically reducing pathogenic Ruminococcaceae and Bacteroides abundances. It decreased LPS, IL-6, and TNF-α levels in the plasma, intestine and cortex, while enhancing intestinal and BBB tight junction protein expression. OT suppressed TLR-4/IκB-α/NF-κB activation in both intestine and cortex, with TLR-4 predominantly localized to neurons, and reduced microglial/astrocytic activation. Critically, ABX treatment abolished OT’s neuroprotective effects and restored neuroinflammation. Conclusions: OT attenuates ASD-like phenotypes and neuroinflammation in VPA-treated rats by rebalancing gut microbiota, restoring intestinal/BBB barriers, and inhibiting neuronal TLR-4/IκB‐α/NF-κB signaling. This study highlights OT’s potential as a microbiota-targeted therapeutic strategy for ASD.