Inflammation is a protective mechanism of the host in response to infection and cellular danger signals(Gilroy, 2021). However, excessive and uncontrolled inflammation can cause disease in the body, such as sepsis, Inflammatory Bowel Disease (IBD), atherosclerosis, type 2 diabetes and so forth(Bennett et al., 2018). During inflammation, macrophages produce a variety of pro-inflammatory factors, including tumor necrosis factor α (TNF-α), interleukin 18 (IL-18), interleukin 1β (IL-1β), nitric oxide (NO), etc, which are associated with chronic degenerative injury(Eltay and Van Dyke, 2023). Therefore, to find effective intervention methods of macrophage inflammatory and explore the regulatory mechanism has important clinical significance. LPS is a pathogen-associated molecular patterns (PAMP) and triggers a storm of inflammatory mediators and pro-inflammatory cytokines at cellular and systemic levels (Moriyama and Nishida, 2021). RAW 264.7 cells, BMDMs and PBMCs induced by LPS are widely used to explore new effective agents against inflammation(Wang et al., 2021). In this research, we screened and investigated the anti-inflammation effect of steroid natural compounds using both in vitro and in vivo models.

NLRP3 inflammasome is a kind of multi-protein complex composed of intracellular NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and protease caspase-1 as the core (Chen et al., 2023). Upon activation, NLRP3 inflammasome leads to the cleavage and activation of Caspase-1 to catalyzes the maturation of pro-inflammatory cytokines IL-1β and IL-18, thereby initiating an inflammatory response(Zhong et al., 2016). Activation of inflammasome to produce mature pro-inflammatory cytokine IL-β is mainly a two-step process. In the priming step stimulated by LPS, pro-IL-1β is synthesized through NF-κB activation. Pro-IL-1β cleavages into mature IL-1β by formed NLRP3 inflammasome in the activation step(Zhong et al., 2016). NF-κB is a ubiquitous transcription factor that activates inflammatory mediators and is involved in the occurrence and development of various inflammatory diseases(Liu et al., 2017). Therefore, modulating the activation of NF-κB and inhibiting the activation of the NLRP3 inflammasome is a possible therapeutic strategy for inflammatory diseases.

Mitochondrial damage has been recognized as a significant inner cause of NLRP3 inflammasome activation(Zhuo et al., 2024). The signals released by mitochondrial damage mainly include mitochondrial DNA (mtDNA) and mitochondria active oxygen species (mtROS). Reactive oxygen species (ROS) serve as a major signal to activate the NLRP3 inflammasome(Liu et al., 2021). Selective autophagy of mitochondria, termed mitophagy, is an important mitochondrial quality control mechanism that eliminates damaged mitochondria. Importantly, mitophagy restricts mtROS production, and remove damaged mitochondria, hindering the progression of NLRP3 inflammasome related inflammation response (Jiang et al., 2022; Zheng et al., 2022). Therefore, retention of healthy mitochondria by mitophagy can alleviate the occurrence of inflammatory response (Onishi et al., 2021).

AMPK functions as a pivotal signaling integration platform for regulating mitochondrial health. By sensing the intracellular energy status and mitochondrial damage signals, AMPK orchestrates the regulation of processes including mitophagy, thereby preserving mitochondrial integrity and function (Ahmed et al., 2024). The function of AMPK in mitophagy and NLRP3 inflammasome regulation was highlighted recently. For example, AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy(Lu et al., 2021). Meanwhile, researches indicated that AMPK/NF-κB signaling pathway has a vital role in the activation of NLRP3 inflammasome in lipopolysaccharide (LPS) -induced ALI rat model(Abd El-Fattah et al., 2022). Thus, the manipulation of AMPK activation could be promising approach for the prevention of inflammatory response.

Solidago canadensis L. (Asteraceae), commonly known as Canadian goldenrod, is a perennial herbaceous plant native to North America (Judžentienė et al., 2023). Currently, it is widely spread in Asia, Europe, and Australia and is considered as an invasive plant. It is used by Canadian Indians as a traditional medicine to treat inflammatory diseases, such as chronic nephritis, cystitis, and rheumatism(Wangensteen et al., 2012). Previous phytochemical investigations on this plant has reported the presence of terpenoids, flavonoids, phenolic acids and steroids, some of which exhibited anti-inflammatory activities. Steroids, being important natural products, play a crucial role acting as anti-inflammatory agents (Hanson, 2010). Although the anti-inflammatory effects of steroids were investigated before, it remains unclear whether steroids contribute to the anti-inflammatory activity of S. canadensis.

In our continuous anti-inflammatory research on S. canadensis, we contributed to search for novel bioactive steroids. In this study, we isolated three new and seven known steroid compounds isolated from S. canadensis, and found compound 10, named (22E)-Ergosta-6,9,22-triene-3β,5β,8α-triol, to be the most active scaffold in inhibiting the LPS-induced NO productions in RAW 264.7 cells. Compound 10, which was first identified from the linseed meal and structure-activity relationship analysis revealed that the double bond between C-9 and C-11 in compound 10 potentiated the NO inhibitory activity, thus exhibits potential anti-inflammatory properties(Shi et al., 2019; Song et al., 2014). However, the underlying mechanisms needs to be elucidated. Here in our study, we uncovered that compound 10 exhibited efficient anti-inflammation effect by suppressing NLRP3 inflammasome activation. Mechanistic study revealed that compound 10 alleviated LPS-induced NLRP3 inflammasome activation via AMPK/Mitophagy and the AMPK/NF-κB pathway. This is the first report on isolation and screen of steroids from S. canadensis with anti-inflammatory activity. Particularly, compounds 10 was found to be promising therapeutic agents in the treatment of inflammatory diseases.