| Objective: In order to provide a new insight for the demyelinating diseases, the effect of estrogen on remyelination and its related mechanisms were investigated.Method: In present study, sixty-six female C57 BL/6 mice were randomized into 6 groups with each 11 cases: normal group, demyelinated model, natural recovery group(demyelination followed 2 weeks natural recovery), estradiol treated group(demyelination followed 2 weeks estradiol treatment), ovariectomized group(demyelination plus ovariectomy and recovery for 2 weeks), sham operation group(demyelination plus sham operation and recovery for 2 weeks). Demyelination was induced by mixed chew containing 0.2% Cuprizone. The MBP(Myelin basic protein)expression in brain was examined by immunohistochemistry staining to evaluate the effect of estrogen on demyelination and remyelination. The influence of estradiol on movement was observed by behavioral experiments. In order to reveal the related mechanisms of estradiol promoting remyelination, the expression of MBP, NG2(nerve glial antigen 2), Olig2(oligodendrocyte transcription factor 2) and GFAP(glial fibrillary acidic protein) was assayed using western blot and immunohistochemistry.Results:1 Demyelination animal modelThe Immunohistochemical results indicated that the Corpus callosum area in normal group MBP staining deep, dense uniform structure(The average optical density value is 0.18±0.05) and MBP in mice of demyelinated model group has shallow staining, loose and sparse structure, maldistribution, and the expression of MBP decreased significantly compared with normal group(P<0.05). The results suggested that mice were demyelinaed. The ovariectomized demyelinating mice were manifested dioestrus performance: a large number of leukocytes and none keratinized epithelial cells in vaginal smear with HE staining measured for 5 consecutive days. The results suggested that ovariectomy operation succeed.2 The changes of body weightThe body weight of normal mice increased gradually, that of demyelinating mice during the inducing process with CPZ experienced a dramatic drop, and then undergone a slow increase. But, the body weight of demyelinated mice decreased compared with that of normal mice. Stopping CPZ treatment, the body weight of estradiol treated group mice increased significantly, compared with that of natural recovery and ovariectomized group mice(P<0.05).3 The test of movement abilityIn rotarod experiment, the retention time of demyelinated mice on the rod reduced significantly, compared with that of normal mice(P<0.05). The retention time of estradiol treated mice significantly raised compared with that of natural recovery group and ovariectomized group(P<0.05). The retention time of mice in ovariectomized group had no difference with that of natural recovery group(P>0.05). There was no difference between sham opration and natural recovery group(P>0.05).4 Results of immunohistochemistryThe corpus callosum area of normal group, demyelinated model group, natural recovery group and ovariectomized group were NG2 positive cells, the oligodendrocyte precursor cells. NG2 positive cells in demyelinated model was increased compared with those of normal group(P<0.05). NG2 positive cells in estradiol treated mice increased significantly compared with those of natural recovery mice(P<0.05), suggesting that estradiol promoted the proliferation of oligodendrocyte precursor cell. NG2 positive cells in ovariectomized group and sham opration group had no difference with those of natural recovery group(P>0.05).In the corpus callosum area of normal group, small amounts of Olig2 or GFAP positive cells were observed in the corpus callosum(P<0.05). In demyelinated group, Olig2 and GFAP positive cells were increased significantly, compared with that of normal group(P<0.05). Olig2 and GFAP positive cells in natural recovery and ovariectomized group decreased significantly, compared with that of demyelinated group, but still more than that of normal group. Olig2 and GFAP positive cells in estradiol treated group decreased to the normal level(P>0.05). There was no difference between sham opration and natural recovery group(P>0.05).5 Results of western blotNG2 protein was expressed in normal group, demyelinated group, natural recovery and ovariectomized group, The NG2 expression was higher in demyelinated group than that in normal group(P<0.05), while there was no diffence between demyelinated and nature recovery group(P>0.05). The NG2 expression in estradiol treated group increased significantly compared with natural recovery group(P<0.05). The NG2 expression in ovariectomized mice was lower than that in natural recovery mice(P<0.05).Olig2 and GFAP protein expression were observed in mormal mice. The expression of Olig2 and GFAP were much higher in the demyelinated group than that in normal group(P<0.05). After estradiol treatment, the expression of Olig2 and GFAP decreased significantly(P<0.05). Also,Olig2 and GFAP expressions in natural recovery and ovariectomized groups decreased compared with that in demyelinated group(P<0.05). There was no difference between sham opration and natural recovery group(P>0.05).Conclusion: Estradiol could accelerate remyelination through promting proliferation oligodendrocyte precursor cell and its differentiaton into oligodendrocyte. Meanwhile, estradiol facilitated the remyelination process by inhibiting astrocytes proliferation and glial scar formation. |
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