Laibin Zhang, Yuan Liu, Huanhuan Wang, Shuangyan Guo, Jieli Lv

School of Pharmacy, Xinxiang Medical University, Xinxiang, Henan, 453003, People’s Republic of China

Correspondence: Laibin Zhang; Jieli Lv, School of Pharmacy, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, Henan, 453003, People’s Republic of China, Email zhanglb@xxmu.edu.cn; jielilv2009@xxmu.edu.cn

Purpose: Angesinenolide B (ASB), a phthalide dimer with a peroxy bridge, is uniquely isolated from Chinese medicine Angelica sinensis radix and demonstrates significant anti-inflammatory properties. The objective of the current study was to evaluate the anti-inflammatory function of ASB and the potential mechanism in lipopolysaccharide (LPS)-stimulated macrophages and CuSO4-induced zebrafish models.
Methods: The level of nitric oxide (NO), a proinflammatory mediator, in LPS-stimulated RAW264.7 cells was quantified using Griess method. ELISA was employed to investigate the generation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), while qRT-PCR was utilized to analyze the mRNA expressions of TNF-α, IL-6, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Fluorescence microscopy and flow cytometry were employed for the determination of ROS generation. Western blot and immunofluorescence techniques were utilized to assess the impact of ASB on iNOS and COX-2, and on the NF-κB, MAPK and STATs signaling pathways. Moreover, the affinities between ASB and the target proteins were verified by molecular docking analysis. In vivo, ROS generation was explored using fluorescent probe DCFH-DA, and the TNF-α and IL-6 mRNA expressions were also evaluated in CuSO4-induced zebrafish inflammation model.
Results: ASB treatment was found to suppress the levels of NO, TNF-α, IL-6 and ROS, restrain the expressions of iNOS and COX-2 proteins and mRNA, as well as down-regulate MAPK and STATs signaling pathways in LPS-stimulated RAW264.7 cells. Furthermore, the administration of ASB effectively attenuated the overproduction ROS and the high mRNA expressions of TNF-α and IL-6 in a zebrafish model of inflammation induced by CuSO4.
Conclusion: ASB has the potentiality to reduce the levels of proinflammatory mediators and cytokines, decrease ROS production, and also down-regulate the MAPK and STATs signaling pathways, thereby exerting an anti-inflammatory effect. This implies that ASB could potentially serve as a viable approach for addressing inflammatory conditions.

Keywords: Angesinenolide B, anti-inflammation, signaling pathway, cytokine, ROS