Estrogen Receptor α Suppressed Experimental Autoimmune Encephalomyelitis In Mice

BackgroundMultiple sclerosis (MS) is an autoimmune disease that features the infiltration of the central nervous system (CNS) by inflammatory cells around blood vessels, accompanied with multifocal demyelination. Currently, there are no effective therapies for MS patients. Clinical data shows that estrogen improves the relapsing-remitting of MS patients using biochemical and clinical indicators, which suggests that estrogen is a potential drug for the treatment of MS. The physiological role of estrogen is mediated by theαandβsubtypes of estrogen receptor (Er). Ers are indispensable for the biological effects of estrogen. Studies have found that the anti-inflammatory effects of estrogen are mediated by Erα. Therefore, Erαbecomes the focus of research in brain.ObjectiveWe constructed the recombinant lentivirus carrying C57BL/6 mouse Erα. Then, Erαrecombinant lentivirus was sterotaxically injected into the lateral ventricle of mouse. Overexpression Erαwas used to investigate anti-inflammatory effects of estrogen and Erαand its mechanism in experimental autoimmune encephalomyelitis (EAE) mouse model, and observe the efficacy of Erαrecombinant lentivirus.MethodsThe coding sequence (CDS) fragment of Erαgene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from C57BL/6 mouse ovary cells and put into pMD19-T vector, then verified by sequencing. Erαgene was inserted into the main virus vector LV-GFP-flag to construct recombinant lentiviral vector LV-Erα-flag that was confirmed by agarose gel electrophoresis (AGE) and DNA sequencing. Recombinant lentivirus V-Erα-flag was produced by 293T cells following the co-transfection of LV-Erα-flag with the packaging plasmids pHelper 1.0 and pHelper 2.0, and then the virus titer was examined. 293T cells were infected by V-Erα-flag and Erαprotein was detected by western blot. The neurons of mice were cultured using a serum-free culture medium, and then control lentivirus V-GFP-flag was used to infect the neurons. The infection efficiency and apoptosis rate were examined by flow cytometry (FCM) to obtain optimal multiplicity of infection (MOI). After that, V-Erα-flag with the optimal MOI was used to infect neurons, and the expression of ErαmRNA and protein in the neurons were detected by quantitative real-time PCR and western blot. We sterotaxically injected Erαrecombinant lentivirus into the lateral ventricle of mice brain, and then identified through H&E and flag immunofluorescence staining to obtain the optimal dose. 100 C57BL/6 mice were ovarectomized. After two weeks, EAE models were induced with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide, and then identificated of pathology and imaging methods. EAE mice were divided into five groups: estrogen group(treatment with estradiol), Erαagonist group(treatment with raloxifene), Erαrecombinant lentivirus group (Erαgroup)(treatment with Erαrecombinant lentivirus), empty virus group and normal saline (NS) group, then the clinical symptoms and body weight of each group were compared. The inflammatory response of the brain and spinal cord were studied by H&E staining. The expression of myelin basic protein (MBP) protein was detected by western blot and luxol fast blue (LFB)-H&E staining was used to analyze demyelination. Immunofluorescence staining identified matrix metallopeptidase 9 (MMP-9) expression in C57BL/6 mice CNS in each group. The expression of proinflammatory cytokines, such as MMP-9, tumor necrosis factorα(TNF-α), interferonγ(IFN-γ), interleukin (IL)-17, IL-23 and inhibition cytokine of inflammation, such as IL-4 mRNA and protein were examined by real-time quantitative PCR and enzyme-linked immunoabsorbent assay (ELISA).ResultsIt was confirmed by sequencing that the extracted CDS fragment of Erαgene had no mutation. LV-Erα-flag confirmed by AGE and DNA sequencing was successfully constructed, and packaged in 293T cells with a titer of 2×10~8 (TU/ml). The results of western blot showed Erαprotein expression in infected 293T cells and not express in uninfected 293T cells. C57BL/6 mice neurons were successfully primarily cultured. Neuronal purity was counted for >90% through neuron specific enolase(NSE) immunohistochemistry. MOI=5 control lentivirus V-GFP-flag could infect neurons, and the infection efficiency amounted to 89.8±4.03% but cell apoptosis rate was only 3.6±0.29% at the MOI of 5. Neurons could survive in culture for at least 8 weeks. During that period, the recombinant lentivirus was able to infect neurons with GFP sustained expression. The V-Erα-flag could also infect neurons at the MOI of 5, and the expression of ErαmRNA and protein were higher in infected neurons than uninfected neurons. Then, 15μl V-Erα-flag was sterotaxically injected into the lateral ventricle of mouse brain, which infected the CNS of C57BL/6 mice successfully. EAE models were constructed successfully through the identification of pathology and imaging methods. Comparison with empty virus group and NS group, treatment of estradiol, raloxifene and Erαrecombinant lentivirus could reduce the incidence, clinical symptoms and body weight loss of EAE mice, inhibit infiltration of brain and spinal cord by inflammatory cells and demyelination of nerve fibers, as well as decrease MMP-9, TNF-α, IFN-γ, IL-23, IL-17 expression (P <0.05) and increase MBP and IL-4 expression (P <0.05). Comparison of estrogen group, Erαagonist group and Erαrecombinant lentivirus group were no significant difference (P > 0.05).ConclusionsThe recombinant lentivirus carrying Erαgene has been constructed successfully, which infected 293T cells leading to express Erαprotein. V-Erα-flag is able to efficiently and stably infect neurons leading to significant upregulation of ErαmRNA and protein at the MOI of 5. 15μl V-Erα-flag was sterotaxically injected into the lateral ventricle of mouse brain, which infected the CNS of C57BL/6 mice successfully. The efficacy of Erαrecombinant lentivirus for the treatment of EAE mice is similar to estradiol and raloxifene. Erαrecombinant lentivirus can reduce incidence, clinical symptoms and body weight loss of EAE mouse, decrease inflammatory cytokines, such as MMP-9, TNF-α, IFN-γ, IL-17, IL-23 and increase inhibition cytokines of inflammation, such as IL-4 expression in CNS of mice with EAE. These results suggest that estradiol, raloxifene and Erαrecombinant lentivirus reduced inflammation and demyelination of EAE mice maybe through some mechanisms, such as reducing MMP-9, regulation the Thl/Th2 balance and inhibition of the IL-23/IL-17 axis. It may provide a new ideal for the treatment of MS.