Experimental Study On The Effects Of Berberine On Mouse Microglia Cultured With Advanced Glycation End Products (AGEs)

[Objective]1.To explore the effects of AGEs on the activation of mouse microglia.2.To observe the effects of berberine on mouse microglia cultured with AGEs.[Methods]1.The BV-2 cells were first cultured with RMPI-1640 medium containing 10%FBS overnight with a cell inoculum density of 3 X 104/mL,then they were divided into five groups including control group,100?g/mL AGEs group,200?g/mL AGEs group,300?g/mL AGEs group and 400?g/mL AGEs group.After being cultured for 24h under different conditions,the morphological changes of microglia were observed by inverted phrase contrast microscope and cell viability was evaluated by MTT assays.Meanwhile,we adopted confocal laser scanning microscopy(CLSM)to monitor the expression of CD68,a surface marker of activated microglia,and Enzyme-linked immunosorbent assay(ELISA)to determine the concentration of TNF-a in the supernatant of cultured microglia.All of the tests were conducted to explore the optimal concentration of AGEs.2.After determining the optimal concentration of AGEs,the microglia were divided into three groups:control group,AGEs group(300?g/mL)and berberine group(10?mol/L).After exposure for 24h,the morphological changes were observed by inverted phrase contrast microscope,cell viability was evaluated by MTT assays and TNF-a concentration in the cell supernatant was examined by ELISA.Meanwhile,we used CLSM to detect the expression of CD68 and Western blot analysis to semiquantitatively detect the expression of CD68,RAGE and pNF-?B p65.[Results]1?The effects of AGEs and berberine had on the morphology of microgliaInverted phrase contrast microscope:ation:The adherence rates of microglia BV-2 cells in AGEs group was about 80-90%compared with 70-80%in control group.Most of the adherent cells in control group displayed fusiform or triangular cell bodies,the typical morphology of resting microglia in vitro,while after the exposure of AGEs for 24h,especially the higher concentration of AGEs,some changed into an enlaged cell body or amoeboid microglial cells,the typical morphology of activated microglia.The BSA group had the similar morphological changes to the control group.The cells in berberine group mostly displayed fusiform or triangular cell bodies and had less amoebocyte compared with control group,indicating a certain restoring effect on the AGEs-induced morphological changes.2.The effects of AGEs and berberine had on the cell viability of microgliaCompared with control group,the OD values of different AGEs groups and berberine group showed no significant difference(P>0.05).3.The effects of AGEs and berberine had on the expression of surface marker CD68Confocal laser scanning microscopy deected the locations and intensities of the green fluorescence:compared to the control group,the green fluorescence signal of CD68 in different AGEs groups expressed weaker,with the concentration of 300?g/mL the weakest.After the treatment of berberine,the green fluorescence intensities in berberine group increased.4.The effects of AGEs and berberine had on the concentration of TNF-?The concentration of TNF-a in the cell supernatant in different AGEs groups remarkably increased(P<0.001)compared with control group,while the difference between different AGEs groups had no statistical significance(P>0.05).Compared with control group,the The TNF-? concentration in the cell supernatant in BSA group showed no significant difference(P>0.05).Compared with AGEs group,the concentration of TNF-? in the cell supernatant in berberine group showed no statistical defference(P>0.05).5.The effects of AGEs and berberine had on the expression of cell activation related proteinsCompared with control group,the expression of CD68,RAGE and pNF-?B p65 in AGEs group(300?g/mL)were remarkably increased(P<0.01,P<0.05,P<0.01).Compared with AGEs group,the expression of CD68 and pNF-?B p65 in berberine group(100?mol/L)were remarkably decreased(P<0.01,P<0.05).The expression of RAGE in berberine group was decreased compared with control group though,the difference had no statistical significance(P>0.05).[Conclusions]1.AGEs(300?g/mL)could remarlably up-regulate the expression of CD68,RAGE and pNF-?B p65,thus increased the secretion of TNF-a in microglia,while had no effect on the viability of microglia.2.Berberine could down-regulate the expression of CD68 and pNF-?B p65 in mouse microglia cultured with AGEs(300?g/mL)for 24h,while had no effect on the expression of RAGE and the concentration of TNF-? in the cell supernatant.[Innovation]The goal of this study is to observe the effects of AGEs on the activation of mouse microglia and to explore the effects of berberine on microglia activation induced by AGEs,which have not been reported previously.