Global histone PTM quantitation revealed anti-inflammatory epigenetic mechanisms of liquiritigenin based on optimized super-SILAC strategy

Ping Liu ^1,2 Jun Zhang ^2,3 Jingdan Zhang ^4,5 Yucheng Yuan ^2,3 Zhiqing Liu ² Sixian Chen ^1,2 Kaifeng Chen ^2,3 Li Dong ⁵ Yinan Zhang ¹ Meiyu Geng ^1,2 Minjia Tan ^1,2,3,4,5* Wensi Zhao ^6,7* Dong Xie ^7*

• ¹ School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Liaoning Province, China
• ² Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai, Shanghai Municipality, China
• ³ University of Chinese Academy of Sciences, Beijing, Beijing, China
• ⁴ School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong Province, China
• ⁵ Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Zhongshan, China
• ⁶ Tongji University, Shanghai, China
• ⁷ Department of Thoracic Surgery, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Yangpu, Shanghai, China

Liquiritigenin (LIQ), as a dihydroflavonone monomer compound with planar ring structure, exhibits potent anti-inflammatory activity. Histone post-translational modifications (PTMs) are closely associated with inflammatory diseases as well. To explore the relationship between anti-inflammatory effects and epigenetic regulatory mechanisms of LIQ, we optimized the super-Stable Isotope Labeling by Amino acids in Cell culture (super-SILAC) method combined with a compound stimulation strategy. Moreover, we evaluated identification coverage and demonstrated high reliability and reproducibility of the optimized method at both peptide and cellular lysate levels, holding the promise in elucidating disease pathology and drug mechanisms. We further applied the method to system-wide characterization of histone PTMs in M1 macrophages treated with LIQ. Quantitative results showed that H4K5ac, H4K16ac, H3K9ac, H3K27ac, and H2BK12ac were significant upregulated.Furthermore, transcriptome analysis revealed that LIQ could exert its anti-inflammatory effects by modulating histone PTMs and regulating gene expression through the PPAR signaling pathway. Collectively, we provided a sensitive and universal strategy for epigenetic mechanism research of natural products, and facilitated the epigenetic understanding of LIQ in inflammatory therapy.