Mycophenolate mofetil reduces epidural fibrosis by regulating TGF-β1/Smad2/3 axis to repress the proliferation, migration and differentiation of fibroblasts

Abstract

Background Excessive fibroblasts proliferation is believed as a major reason in the process of epidural fibrosis, which is known as a troublesome complication of lumbar laminectomy clinically. Mycophenolate mofetil (MMF) is a kind of immunosuppressant, previous studies had shown that it has the function of preventing fibrosis, but the role and mechanism are still unclear. In this study, we determined the repressed effect of MMF on fibroblasts proliferation, migration and differentiation in surgical-induced epidural fibrosis and the underlying mechanism. Methods In vitro, the impacts of MMF on cell viability were measured by cell counting kit-8 (CCK-8) assay and the fibroblasts proliferation rates were determined by using EdU incorporation, cell cycle assays and western blot. Additionally, the impacts of MMF on fibroblasts migration and differentiation were analyzed by cell wound scratch assay, transwell assay, immunofluorescence analysis and western blot. In vivo, we built epidural fibrosis models in rats and locally applied MMF. Histological and immunohistochemical staining were used to determine the effect of MMF on reducing epidural fibrosis and fibroblasts functions. Results CCK-8 assay demonstrated that MMF repressed fibroblasts proliferation in a time- and a dose-dependent manner. EdU incorporation assay and cell cycle analysis proved the inhibition effect of MMF on the proliferation of fibroblasts. Cell wound scratch assay, transwell assay and immunofluorescence proved the inhibition effect of MMF on the migration and differentiation of fibroblasts. Western blotting analysis proved that MMF inhibited the expression of TGF-β1, p-Smad2 and p-Smad3. The expression of proliferation proteins, migration proteins and differentiation proteins were downregulated. In addition, histological and immunohistochemical stain showed that MMF reduces epidural fibrosis by repressing the proliferation, migration and differentiation of fibroblasts. Conclusion MMF could inhibit the proliferation, migration and differentiation of fibroblasts, it reduced surgical-induced epidural fibrosis as well. TGF-β1/Smad2/3 axis may be the latent mechanism in MMF reduced epidural fibrosis. It might provide a new notion for mitigating surgery-induced epidural fibrosis.