Decoding Oral Leukoplakia: Microbiome Dysbiosis and Inflammatory Dynamics Unveiled in a Rat Model

Zhiqin Sang^1,2Yufeng Zhang³Enoch Kao³Tingting Zhu¹Jiazhen Yang¹Zhenjiang (Zech) Xu⁴Shi Huang^3*Fei TENG^1*Wanchun Wang^1*

• ¹Qingdao Stomatological Hospital, Qingdao, Shandong Province, China
• ²Ocean University of China, Qingdao, Shandong Province, China
• ³Faculty of Dentistry, The University of Hong Kong, Sai Ying Pun, Hong Kong, SAR China
• ⁴State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi Province, China

Oral leukoplakia (OLK), a precancerous condition, is closely associated with oral microbiome dysbiosis and systemic inflammation. In this study, we used a 4-nitroquinoline-1-oxide (4-NQO)-induced rat model of OLK to characterize the longitudinal changes in oral microbiome and inflammatory during OLK development. Leveraging 2bRAD-M sequencing, a novel metagenomic approach with high sensitivity for low-biomass samples, we observed significant shifts in the oral microbial diversity, marked by elevated abundances of Streptococcus, Glaesserella, and Pseudomonas aeruginosa. Moreover, shifts in microbiota precede manifestation of clinical symptoms of OLK. Functional pathway analysis highlighted enrichment in metabolism, quorum sensing, and cancer-associated microRNA pathways. Serum levels of inflammatory markers TNF-α and IL-6 were significantly elevated in OLK and significantly correlated with specific bacterial taxa. This study demonstrates the utility of 2bRAD-M sequencing in overcoming traditional metagenomic limitations, offering a high-resolution view of microbiome dynamics in low-biomass environments such as the oral mucosa. These findings establish oral microbiota as candidate early biomarkers for OLK screening and prevention, opening avenues for precision diagnostics and targeted therapies to mitigate cancer risk associated with OLK.