BML-275

Neuropathic pain is characterized by sustained pain hypersensitivity caused by nerve injury. The molecular mechanisms underlying this condition remain poorly understood. This study aims to elucidate the role of ACVR1 and its downstream pathways in mediating neuropathic pain through neuronal pyroptosis and neuroinflammation.

A spared nerve injury (SNI) model was established both in male and female mouse to induce neuropathic pain. Behavioral tests, Western blot, PCR, and immunofluorescence were used to assess the expression of ACVR1, p-Smad1/5/8, p-p38, and pyroptosis-related proteins (NLRP3, Caspase-1, and GSDMD-N). ACVR1, p38, and Smad1/5/8 were pharmacologically inhibited to evaluate their roles in neuropathic pain and pyroptosis.

Behavioral analysis confirmed successful SNI model establishment, marked by reduced paw withdrawal thresholds (PWT). Protein and mRNA expression analysis revealed significant upregulation of ACVR1, p-Smad1/5/8, and p-p38 in the spinal cord, particularly in neurons. Furthermore, SNI enhanced pyroptosis-related protein expression, including NLRP3, Caspase-1, GSDMD-N, IL-1β and IL-18. Inhibition of ACVR1 alleviated mechanical allodynia, reduced neuronal pyroptosis, and decreased serum IL-1β and IL-18 levels. Similarly, p38 inhibition mitigated NLRP3-induced pyroptosis without altering ACVR1 expression. In contrast, Smad1/5/8 inhibition by DMH-1 effectively reduced pyroptosis and inflammation via NLRP3 but had no effect on p38 phosphorylation. Combined p38 and Smad1/5/8 pathway inhibition synergistically decreased pyroptosis-related protein expression, highlighting their interactive roles in ACVR1-mediated neuropathic pain.

These findings suggest that ACVR1 exacerbates neuropathic pain by activating neuronal pyroptosis and neuroinflammation via the p38 and Smad1/5/8 pathways. Targeting ACVR1 and its downstream signaling pathways may offer novel therapeutic strategies for neuropathic pain.

Inflammation is a major risk factor for a variety of human diseases, such as sepsis, Inflammatory Bowel Disease (IBD) and also major cardiovascular disease including atherosclerosis. Solidago canadensis is used as a traditional medicine to treat inflammation-related diseases. However, the component with anti-inflammatory activity of Solidago canadensis is not clear. In this study, we aimed to search for new bioactive steroids from Solidago canadensis and investigate their anti-inflammatory activity both in vitro and in vivo. Lipopolysaccharides (LPS)-stimulated RAW264.7 cells, mouse bone marrow-derived macrophages (BMDMs) and peripheral blood mononuclear cells (PBMCs) were used to induce an inflammation response. Compound 10 outperformed other compounds for superior anti-inflammatory activity and significant inhibition of NLR family, pyrin domain containing 3 (NLRP3) inflammasome activation. Mechanistically, compound 10 induced mitophagy by activating AMP-activated protein kinas (AMPK) to suppress NLRP3 inflammasome activation. Inhibiting AMPK by inhibitor BML-275 significantly attenuated compound 10 induced mitophagy and subsequent the NLRP3 inflammasome. Besides, the NF-κB activation, key step in NLRP3 inflammasome priming, was also suppressed by compound 10 via activation of AMPK. In addition, the in vivo experiments showed that compound 10 could alleviate LPS-induced inflammatory and dextran sulfate sodium salt -induced colitis in C57BL/6 mice. Collectively, the present study, for the first time, shows that the steroids compound 10 exhibited anti-inflammatory effect via AMPK/mitophagy/NLRP3 as well as AMPK/NF-κB/NLRP3 signaling pathway, which strongly suggests the therapeutic potential of compound 10 in various inflammatory diseases.