The Effect Of Vitamin D On Olfactory And Olfactory Marker Protein In Experimental Autoimmune Encephalomyelitis Mice

Objective:By evaluating the changes of olfactory sense and inflammatory demyelinating lesions in the olfactory bulb（Olfactory bulb,OB）of mice with experimental autoimmune encephalomyelitis（EAE）,the serum vitamin D level in mice was detected,and the expression of vitamin D in the olfactory bulb was affected.The distribution of vitamin D receptor（VDR）,the changes of olfactory marker protein（OMP）and VDR in the OB.To explore the effect of vitamin D on olfactory function in EAE mice and its possible mechanism.Methods:C57BL/6 mice were randomly divided into Normal group（Normal）,EAE group（EAE）,and EAE supplemented vitamin D group（EAE+VD）.The EAE model was established by immunizing C57BL/6 mice with myelin oligodendrocyte glycoprotein（MOG）35-55 polypeptide/complete Freund’s adjuvant,the EAE+VD group was fed every other day from the first day of immunization,the mice in the normal group were fed with the same volume of normal saline.During the whole experimental period,the Weaver’s 15-point method was used to score the neurological function of the mice;buried food pellet test experiment was used to observe the changes of the olfactory function of the mice;the small animal magnetic resonance imaging technology was used to scan the olfactory bulb lesions of the living mice;Hematoxylin-eosin staining（HE）was used to observe the infiltration of inflammatory cells in the spinal cord and olfactory bulb;Luxol Fast Blue（LFB）was used to observe the demyelination of the spinal cord;immunofluorescence double staining was used to detect the expression of VDR and OMP proteins in the olfactory bulb;Western blotting（WB）and Real-time quantitative polymerase chain reaction（RT-q PCR）The levels of VDR and OMP protin the OB.Results:The EAE model was successfully built,and the neurological function score was increased;compared with the EAE group,the neurological function score of the EAE+VD group was lower（ie,the clinical symptoms were lighter）;after vitamin D3supplementation,the serum vitamin D₃level in the EAE+VD group was significantly higher than that in the EAE group statistical significance（P=0.0108,P<0.05）;.in the food pellet burial test,there was no significant difference between the first and second day,and the incubation period of the EAE group was higher than that of the Normal group on the third day（ie:found The time required for food pellets）was prolonged（P=0.0238,P<0.05）;the latency in the EAE+VD group was shorter than that in the EAE group（P=0.0333,P<0.05）,and the differences were statistically significant;in the small animal MRI scan,T1WI images of EAE mice showed plaques in the olfactory bulb Patchy low signal lesions,high signal lesions appeared in the olfactory bulb of EAE mice on T2WI images,and no obvious lesions appeared in the Normal group and the EAE+VD group;in the pathological staining of the spinal cord:the EAE+VD group had less inflammatory cell infiltration and less demyelination than the EAE group.In the pathological staining of olfactory bulb:EAE group saw multiple neuron degeneration and necrosis,and the neuron degeneration and necrosis in EAE+VD group was lighter than that in EAE group;olfactory bulb immunofluorescence double staining showed no co-expression of VDR and OMP proteins in olfactory bulb,and VDR was distributed in the olfactory nerve layer,and the VDR-positive cells in the EAE group were decreased compared with the Normal group,and the number of VDR-positive cells in the EAE+VD group was higher than that in the EAE group.In OB,compared with the Normal group,the VDR protein expression in the EAE group was decreased,and the difference was statistically significant（P=0.0120,P<0.05）.The VDR protein expression in the EAE+VD group was decreased compared with the Normal group,but was significantly lower than that in the EAE group.Compared with the Normal group,the VDR m RNA expression was down-regulated in the EAE group and the difference was statistically significant（P=0.0189,P<0.05）,and the VDR m RNA expression in the EAE group was down-regulated compared with the EAE+VD group,and the difference was statistically significant（P=0.0260,P<0.05）.In OB,the expression of OMP protein in the EAE group and EAE+VD group showed a downward trend.Compared with the Normal group,the OMP m RNA expression in the EAE group was down-regulated（P=0.0017,P<0.01）,and the OMP m RNA expression in the EAE+VD group was down-regulated compared with the Normal group（P=0.0205,P<0.05）.Conclusions:EAE mice have behavioral impairment of olfactory function,and objective olfactory bulb lesions can be detected;vitamin D intervention in EAE improves the clinical symptom to MS of EAE mice,and reduces inflammatory infiltration and demyelination in objective lesions;VDR expression in olfactory bulbs Compared with EAE+VD,the EAE group was down-regulated,and the expression of OMP in the olfactory bulb was down-regulated in the EAE group compared with the EAE+VD group.The protective effect of vitamin D on the olfactory function of EAE mice may be related to the expression of VDR and OMP in the olfactory bulb.