Exploring microbial-derived chondroitin sulfate as a suppressor of microglial inflammation and pyroptosis

Neuroinflammation is a protective immune response in the central nervous system (CNS), primarily regulated by glial cells such as microglia, astrocytes, and oligodendrocytes. Microglia serve as the main innate immune cells, initiating responses to pathological stimuli through mechanisms including inflammasome activation. The NLRP3 inflammasome plays a central role in promoting inflammation and pyroptosis. Chondroitin sulfate (CS), a sulfated glycosaminoglycan found in the extracellular matrix, exhibits anti-inflammatory, anticoagulant, and antioxidant properties. This study investigates the anti-neuroinflammatory potential of microbial chondroitin sulfate (MCS), a novel compound developed by our team, in comparison to commercial CS (CCS). N9 mouse microglial cells were treated with MCS and CCS. We evaluated cytotoxicity and cell viability using LDH and CCK-8 assays. Levels of pro-inflammatory cytokines IL-1β and IL-18 were measured via ELISA. Western blotting and flow cytometry were used to assess the expression of NLRP3, caspase-1, GSDMD, and GSDMD-N, key proteins involved in inflammasome activation and pyroptosis. MCS significantly reduced LDH release and increased cell viability, indicating protection against cytotoxicity. It also suppressed IL-1β and IL-18 secretion and downregulated NLRP3, caspase-1, and GSDMD activation. Notably, MCS achieved effects comparable to CCS at doses approximately 200 times lower. This is the first study to demonstrate that MCS effectively inhibits NLRP3 inflammasome activation and pyroptosis in microglial cells. These findings highlight MCS as a potent anti-neuroinflammatory agent and a promising candidate for therapeutic development in CNS inflammatory disorders.