In Vitro and In Vivo Characterization of Oridonin Analogs as Anti-Inflammatory Agents that Regulate the NF-κB and NLRP3 Inflammasome Axis

Huiping Ou ¹ Zhanpan Wu ¹ Jinhua Ning ¹ Qiufeng Huang ² Wancun Wang ¹ Guochun Yang ¹ Yingxun Zhou ¹ Anguo Hou ¹ Lingyun Chen ¹ Peng Li ² Wenbin Jin ^1*

• ¹ School of Chinese Materia Medica, Yunnan University of Chinese Medicine, Kunming, China
• ² Shenzhen Polytechnic, Shenzhen, Guangdong, China

A collection of oridonin hybrids were designed and synthesized, and their in vitro anti-inflammatory activities were evaluated by screening their ability to inhibit NO production in RAW264.7 cells. The most promising compound, 4c, was selected for further evaluation via ELISA and WB analysis to determine its inhibitory effects on the expression of inflammation-related proteins, including phosphorylated NF-κB, phosphorylated IκB, NLRP3, IL-6, IL-1β, COX-2 and iNOS, after LPS stimulation.Notably, 4c also alleviated the symptoms of acute lung injury in mice. The RT-qPCR results suggested that 4c could reduce the LPS-induced expression of IL-6 and TNF-α in lung tissue at the mRNA level; moreover, the WB results indicated that 4c could significantly inhibit the expression of NLRP3, phosphorylated NF-κB and IL-6 in lung tissues. Docking simulations were performed to position compound 4c into the NLRP3 binding site and predict the covalent binding mode. Moreover, the RNA-seq results combined with the RT-qPCR data suggested that 4c could upregulate the genes Trdc, Stfa2 and Gsta2 and downregulate the genes Spib, Csf2 and Nr4a1. To conclude, this collection of oridonin hybrids may warrant further investigation as promising antiinflammatory lead candidates for the treatment of NLRP3-driven disorders.