Soya-cerebroside, an extract of Cordyceps militaris, suppresses monocyte migration and prevents cartilage degradation in inflammatory animal models

Abstract Pathophysiological events that modulate the progression of structural changes in osteoarthritis (OA) include the secretion of inflammatory molecules, such as proinflammatory cytokines. Interleukin-1beta (IL-1β) is the prototypical inflammatory cytokine that activates OA synovial cells to release cytokines and chemokines in support of the inflammatory response. The monocyte chemoattractant protein-1 (MCP-1/CCL2) is one of the key chemokines that regulate migration and infiltration of monocytes in response to inflammation. We show in this study that IL-1β-induced MCP-1 expression and monocyte migration in OA synovial fibroblasts (OASFs) is effectively inhibited by soya-cerebroside, an extract of Cordyceps militaris. We found that soya-cerebroside up-regulated of microRNA (miR)-432 expression via inhibiting AMPK and AKT signaling pathways in OASFs. Soya-cerebroside also effectively decreased monocyte infiltration and prevented cartilage degradation in a rat inflammatory model. Our findings are the first to demonstrate that soya-cerebroside inhibits monocyte/macrophage infiltration into synoviocytes, attenuating synovial inflammation and preventing cartilage damage by reducing MCP-1 expression in vitro and in vivo. Taken together, we suggest a novel therapeutic strategy based on the use of soya-cerebroside for the management of OA. Introduction Osteoarthritis (OA) is a degenerative joint disease characterized by slow progressive cartilage degradation and synovial inflammation1. Biochemical mediators found in OA synovial fibroblasts (OASFs) that affect the cellular functions of multiple joint tissues include cytokines, chemokines, growth factors, and matrix metalloproteinases (MMPs)2. Although the pathogenesis of OA is complicated and remains poorly understood, leukocyte trafficking from the vascular lumen to sites of inflammatory stimuli is essential for effective immune surveillance3,4. Activation and accumulation of mononuclear leukocytes to inflammatory sites is regulated by chemokines, such as monocyte chemoattractant protein-1 (MCP-1)5. MCP-1, also known as chemokine ligand 2 (CCL2), belongs to the CC chemokine family. MCP-1 is chemotactic for monocyte/macrophages and activated T cells6. Increased concentrations of MCP-1 are detected in the blood, synovial fluid and synovial tissue in patients with OA7. Injection of MCP-1 into rabbit joints causes marked macrophage migration into the affected joint8. In animals suffering experimentally also indicated that over-expressed MCP-1 could induce synovitis9. It has also been reported that treatment with an MCP-1 antagonist prior to disease onset prevents the development of arthritis in a mouse model10. These data are supported by further research suggesting that MCP-1 may play a crucial role as a proinflammatory agent during OA pathogenesis11,12,13. MicroRNAs (miRNAs) are evolutionarily conserved, small, non-coding RNAs (19–25 nucleotides in length) that post-transcriptionally modulate the expression of downstream target genes by repressing translation or accelerating mRNA degradation14,15. MiRNAs are increasingly implicated in cartilage homeostasis and OA pathogenesis, especially in chondrocytes expression of genes encoding catabolic factors, such as matrix metalloproteinases (MMPs) and aggrecanase-1 (ADAMTS4)16. Much evidence indicates that miR-432 expression is reduced in various tumors, such as hepatocellular carcinoma, cervical and ovarian cancer17,18. Recent findings show that miR-432 functions as a tumor-suppressive miRNA in hepatocellular carcinoma cells and may represent a prognostic parameter and therapeutic target for lung adenocarcinoma19. However, the exact etiological mechanism of miR-432 in monocyte infiltration and OA pathogenesis is largely unknown. Cordyceps militaris, an entomopathogenic fungus belonging to the Ascomycetes class, has been widely used as an herbal medicine for inflammatory diseases in humans20. Soya-cerebroside, an extract of C. militaris, showed significant anti-inflammatory activity by inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW264.7 cells21. However, the anti-inflammatory mechanism of soya-cerebroside in OA has not been completely elucidated. In the present study, we demonstrate how soya-cerebroside attenuates IL-1β-induced MCP-1 expression and promotes monocyte migration by up-regulating miR-432 expression via AMPK and AKT signaling pathways, as well as abolish macrophage infiltration and protect cartilage in inflammatory animal model. Our results show that soya-cerebroside is a potential therapeutic candidate for OA disease. Results Soya-cerebroside attenuates IL-1β-induced MCP-1 expression and monocyte migration Studies indicated that soya-cerebroside has anti-inflammatory effects21. We hypothesized that it might decrease monocyte migration and inhibit inflammatory responses in OASFs. Cells were cultured with different concentrations o (...truncated)