A potent sphingomyelinase inhibitor from Cordyceps mycelia contributes its cytoprotective effect against oxidative stress in macrophages

A novel water-soluble polysaccharide fraction, CME-1, with a molecular mass of 27.6 kDa and containing mannose and galactose in a respective ratio of 4:6, was prepared from Cordyceps sinensis mycelia and identified by NMR and GC-MS. In the current study, we examined whether CME-1 has anti-inflammatory effects in RAW264.7 cells. The ability of CME-1 to inhibit H2O2-induced cell death in RAW264.7 cells was assessed by using an MTT assay and annexin V/propidium iodide double staining; we found that CME-1 protected cells against H2O2-induced injury. H2O2-induced intracellular oxidative stress and mitochondrial membrane depolarization were also diminished with CME-1 treatment. We evaluated the hydroxyl radical scavenging ability of CME-1 by using the DMPO-electron spin resonance technique, which indicated that CME-1 acts as an intracellular antioxidant in a concentration-dependent manner through a mechanism other than its scavenging activity. Activities of both neutral and acid sphingomyelinases (SMases) were assessed in vitro, and results showed that the CME-1 inhibited activities of both neutral and acid SMases in a concentration-dependent manner. CME-1 reduced H2O2 treatment-elevated C16- and C18-ceramide levels measured by LC/MS/MS in RAW264.7 cells.jlr Results suggest that CME-1 protects RAW264.7 cells against oxidative stress through inhibition of SMase activity and reduction of C16- and C18-ceramide levels.-Wang, S-H., W-B. Yang, Y-C. Liu, Y-H. Chiu, C-T. Chen, P-F. Kao, and C-M. Lin. A potent sphingomyelinase inhibitor from Cordyceps mycelia contributes its cytoprotective effect against oxidative stress in macrophages. J. Lipid Res. 2011. 52: 471-479.