| PART 1 THE EFFECT AND MECHANISMS OF NMDA-R ANTAGONISM ON PROLIFERATION DIFFERENTIATION AND MYELINATION OF OLIGODENDROCYTESObjective:To investigate the effect and mechanism of N-methyl-D-aspartate receptors on the proliferation,differentiation and myelination of oligodendrocytes.Methods:The primary oligodendrocytes of C57BL/6J mice were randomly divided into control group and MK-801 treatment group.Cell proliferation was detected by CCK-8 and EdU methods.Western blot analysis was used to detect Olig2 and MBP protein expression to reflect cell differentiation and myelination.The expression of NMDA-R subunits NR1,NR2 A,NR2B,NR2 C,NR2D,NR3 A and NR3 B was detected by qRT-PCR.Results:The results of CCK-8 showed that the NMDA-R antagonist MK-801 reduced the number of viable primary cultured oligodendrocytes in a concentration-dependent manner,that is,as the concentration of MK-801 increased,the number of oligodendrocyte decreased.EdU test showed that the number of proliferating oligodendrocytes in MK-801 group(32.9±8.47)% was significantly lower than that in the control group(42.4±7.35)%(p=0.0154)after treatment with 10 ? mol/L MK-801 for 48 hours.Western blot results showed that compared with the control group,the expression of Olig2(1.000±0.206vs0.794 ± 0.167,p = 0.013 < 0.05)and MBP protein expression(1.000 ± 0.276 vs0.650 ± 0.237,p = 0.026 <0.05)in oligodendrocytes were significantly decreased after MK-801 treatment.The qRT-PCR results showed that MBP mRNA expression was also significantly decreased after MK-801 treatment(p<0.01).Compared with the control group,NMDA-R subunit NR1 subunit mRNA expression of MK-801 treated group(1.04± 0.29 vs 0.53±0.27,p = 0.01<0.05)and protein expression(1.00 ± 0.10vs0.79 ± 0.17,p=0.016<0.05)were significantly decreased.The mRNA expression of NR2A(1.00±0.27vs0.45±0.19,p=0.005<0.05),NR2B(1.00±0.15vs0.55±0.20,p=0.004<0.05)and NR3A(1.08 ± 0.15 vs 0.79 ± 0.25,p = 0.038 < 0.05)were also significantly decreased,but the mRNA expression level of NR2C(p =0.519 > 0.05),NR2D(p = 0.230)and NR3B(p = 0.164)mRNA had no significant difference between the two groups.Conclusions:NMDA-R is blocked by MK-801 and has an inhibitory effect on proliferation,differentiation and myelination of oligodendrocytes,which may be mediated by down-regulating NR1,NR2 A,NR2B and NR3 A subunits.PART2 ANTI-PSYCHOTIC DRUGS REGULATE OLIGODENDROCYTE PROLIFERATION THROUGH NMDA-R AND ITS UNDERLYING MECHANISMObjective:To investigate the effects of antipsychotic drugs quetiapine,haloperidol and risperidone on oligodendrocytes proliferation and its underlying mechanism by altering NMDA-R subunits,and to explore whether antipsychotic drugs can reverse the decrease of cell proliferation induced by NMDA-R blockade.Methods:The primary cultured oligodendrocytes of C57BL/6J mice were randomly divided into control group,quetiapine(QUE)treatment group,haloperidol(HAL)group,risperidone(RIS)group,MK-801 group,MK-801 combined with QUE group,MK-801 combined with HAL group and MK-801 combined with RIS group.The cell proliferation was detected by CCK-8 and Ed U methods.The expression of NMDA-R subunits NR1,NR2 A,NR2B,NR2 C,NR2D,NR3 A and NR3 B were detected by q RT-PCR.Results:Compared with the control group,the antipsychotic drugs(QUE,HAL,RIS)could increase the number of primary viable oligodendrocytes.However,the results of Ed U showed that compared with the control group of Ed U-positive cells(39.95 ± 9.05)%,the QUE group had(46.90±8.47)% of the Ed U-positive cells;The Ed U-positive cells of the HAL group were(58.44±10.47)%,which was significantly higher than the control group(p = 0.0451);The Ed U-positive cells in the RIS group were(50.41±7.11)%,which was significantly higher than the control group(p =0.049).The q RT-PCR results showed that the expression of NR1 m RNA was increased in the QUE group compared with the control group(p=0.002),while the expression of NR3 A was decreased(p=0.016).The expression of NR1 m RNA(p=0.007),NR2 A m RNA(p=0.001),NR2 B m RNA(p=0.012)and NR2 C m RNA(p=0.007)were increased and NR2 D m RNA was decreased in the HAL-treated group(p=0.025).The level of NR1(p=0.007),NR2A(p=0.006)and NR2C(p=0.011)subunit m RNA of oligodendrocyte were increased in RIS-treated group.The CCK-8 results showed that QUE and HAL can reverse the inhibitory effect of MK-801 on oligodendrocyte proliferation.Conclusions:QUE has no effect on the proliferation of primary oligodendrocytes,but it can reverse the decrease in cell proliferation induced by NMDA-R blockade possibly by regulating the expression of NR1,NR3 A subunits.HAL can promote the proliferation of primary cultured oligodendrocytes and reverse the inhibitory effect of MK-801 on oligodendrocyte proliferation,mainly by up-regulating the expression of NR1,NR2A-C subunits and down-regulating NR2 D.Although RIS can not reverse the inhibitory effect of MK-801 on cells,its role in promoting cell proliferation may be achieved through up-regulating of NR1,NR2 A,and NR2 C subunits expression. |
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