The Molecular Mechanism Of That Cystatin F Gene Ablation Exacerbates The Status Of Demyelination In Cuprizone Model Mice

Objective:Multiple sclerosis(MS) is a kind of chronic demyelinating diseases in central nervous system(CNS), its main pathological features includes neroinflammation, demyelination, gliosis and axonal injury. The majority of MS patients gradually develop into secondary-progressive from relapsing-remitting, during which the symptom is aggravating without remission stage, and most patients are died of complications. Because of MS pathogenesis is not clear, so there is lack of effective treatment method. Our previous studies found that the expression of cystatin F(cys F) was increased in the demyelinating diseases. In order to elucidate the functional role of cys F, we used the cys F gene knockout mice to construct cuprizone demyelinating model, and we found that ablation of cys F gene was aggravated obviously the status of demyelination, but the mechanism of cys F involved in demyelinating process in cuprizone model is unclear. The purpose of this study is to explore the molecular mechanism of that cys F gene ablation exacerbates the status of demyelination in cuprizone model mice.Methods:C57BL/6J wild-type mice, cystatin F knockout(cys F KO) mice and cathepsin C overexpression (Cat C OE) mice were treated 0.2% cuprizone for creating demyelinating model of central nervous system(CNS). C57BL/6J mice were used as blank control. Real-time PCR and enzyme-linked immunosorbent assay(ELISA) were used to analyze the expression of CXCL2 in cys F KO mice and wild-type mice in brain after cuprzione treated 4 weeks, respectively. Myelin basic protein(MBP) and CC1 immunohistochemical(IHC) staining were preformed to observe remaining myelin and oligodendrocyte in the brains of Cat C OE mice and wild-type mice littermates after cuprizone treated 5 weeks, respectively. CD45 IHC staining was used to observe the infiltration of inflammatory cells in cuprizone models of these two kinds of gene manipulation mice. In vitro, primary cultured glial cells were co-incubated with recombinant cathepsin C(Cat C) protein for 24 hours to analyze the expression of CXCL2 mRNA in cells and CXCL2 protein in culture medium by real-time PCR and ELISA, respectively. The data were evaluated for statistical significance with one-way ANOVA and linear regression. P<0.05 was considered significant differences.Results:The levels of CXCL2 mRNA and protein expression in the brain were both significantly higher in 4 weeks’cuprizone treated mice than untreated mice. Further comparision showed that expression level in Cys F KO mice was significantly higher than that in wild type mice. Simultaneously, a large number of CD45 positive cells was found in cuprizone treated mice, and compared with that in wild type mice, significantly more CD45 positive cells were found in cys F KO mice. In vitro study showed that Cat C could induce the production and secretion of chemokine CXCL2 in mixed cultured glial cells, and the expression of CXCL2 mRNA was increased in a dose-dependent manner. Compared with the blank control group, after cuprizone treated for 5 weeks, the remaining myelin area ratio and residual oligodendrocytes were significantly reduced, and a lot of CD45 positive cells were found in the demyelinated position. The remaining myelin area ratio and residual oligodendrocytes in Cat C OE mice was significantly less than that in wild type mice. However, the number of CD45 positive cells in Cat C OE mice was significantly more than that in wild type mice.Conclusions:1. Cathepsin C can induce the production of chemokine CXCL2 in cultured glial cells of CNS.2. Ablation of cystatin F results in loss of its inhibition of cathepsin C activity, which could up-regulate the expression of CXCL2 and lead to more inflammatory cells infiltration, finally aggravates the position of demyelination.