| Part 1.The effect of glucose and lysophosphatidylcholine on the interaction of mesangial cells and endothelial cellsObjective:To investigate the levels of Fn,ColⅣ, platelet-activating factor(PAF) and platelet activating factor receptor (PAF-R) of monolayer mesangial cell culture,endothelial cell and mesangial cell co-culture at different concentrations of D-glucose and lysophosphatidylcholine(LPC),explore the role of PAF produced by endothelial cells on the interaction of endothelial cells and mesangial cells.Methods:The model of monolayer mesangial cell culture, endothelial cell and mesangial cell co-culture were established respectively.The two models were subdivided into 6 groups:control, mannitol,high glucose,high LPC,high glucose and high LPC, BN52021.The levels of Fn,ColⅣand PAF were determined by ELISA in the culture media.The expression level of PAF-R mRNA in mesangial cells was determined by real-time fluorescence quantitative PCR(Real-time PCR).Results:1.The levels of Fn and ColⅣof high glucose,high LPC, high glucose and high LPC group were significantly higher than those of control or mannitol group both in monolayer cell culture model and co-culture model(P＜0.05),while the levels of Fn and ColⅣwere highest in the high glucose and high LPC group(P＜0.05).All these effects could be inhibited by BN52021(P＜0.05).2.In both cell culture models,the levels of PAF were increased in high glucose,high LPC, high glucose and high LPC group respectively,comparing with those in control or mannitol group(P＜0.05),and had the highest level in glucose and high LPC group.These effects couldn't be inhibited by BN52021. Meanwhile,the levels of PAF of different culture condition in co-culture were higher than those in monolayer cell culture(P＜0.05) 3.In both cell culture models,the expression level of PAF-R mRNA in mesangial cells of high glucose,high LPC,high glucose and group LPC,BN52021 group were higher than those of control or mannitol group(P＜0.05).The expression level of PAF-R mRNA in mesangial cells of the high glucose and high LPC group were higher than those of other groups(P＜0.05), and this effect couldn't be inhibited by BN52021.The expression level of PAF-R mRNA in mesangial cells of co-culture group were no difference with that of monolayer cell culture group.Conclusions:1.High glucose and high LPC can promote mesangial cells to secrete ECM.2.Exposed to high glucose and high LPC,endothelial cells and mesangial cells can interact with each other,which can promote the endothelial cells to secrete more PAF on the mesangial cells to produce more ECM,the increase of ECM stimulated by high glucose and high LPC is partly concerned with PAF.3.The expression of PAF-R in mesangial cells is promoted by high glucose and high LPC,which can enlarge the biological effect of PAF.Part 2.The mechanism of effects of PAF on the mesangial cells exposed to high glucose and high LPCObjective.To investigate the change of Fn,ColⅣ,TGF-β1,PKCβI of the mesangial cells under high glucose and high LPC,stimulator, inhibitor,explore the effects of PAF on the activation PKCβI/TGF-β1 signaling pathway to mediate ECM production in mesangial cells.Methods:The model of monolayer mesangial cell culture was established and divided into 8 groups:control,PAF,PAF+anti-TGF-β1 neutralizing antibody,high glucose and high LPC,PAF+ high glucose and high LPC,PAF+ high glucose and high LPC+anti-TGF-β1 neutralizing antibody,PAF+LY333531,PAF+ high glucose and high LPC+ LY333531.The levels of Fn and ColⅣwere determined by ELISA in the culture media.The expression levels of TGF-β1 and PKCβI mRNA in mesangial cells were determined by Real-time PCR. The expression levels of TGF-β1 and PKCβI protein of mesangial cells were determined by Western blot.The expression of PKCβI in mesangial cells was determined by immunofluorescence(IMF).Results:1.Comparing with the control,the levels of Fn and ColⅣof PAF group were increased(P＜0.05),these effects could be inhibited by anti-TGF-β1 neutralizing antibody and LY333531,respectively (P＜0.05).2.The levels of Fn and ColⅣof high glucose and high LPC group were higher than those of control group(P＜0.05) Comparing with high glucose and high LPC group,PAF could elevate the the levels of Fn and ColⅣ(P＜0.05),these effects could be inhibited by anti-TGF-β1 neutralizing antibody and LY333531,respectively(P＜0.05 ). 3.Comparing with the control,the expression levels of TGF-β1 mRNA and protein of mesangial cells of PAF group were increased(P＜0.05). These effects couldn't be inhibited by anti-TGF-β1 neutralizing antibody and could be inhibited by LY333531(P＜0.05).4.The expression levels of TGF-β1 mRNA and protein of mesangial cells of high glucose and high LPC group were higher than those of control group(P＜0.05) Comparing with high glucose and high LPC group,PAF could elevate the expression levels of TGF-β1 mRNA and protein of mesangial cells (P＜0.05) These effects couldn't be inhibited by anti-TGF-β1 neutralizing antibody and could be inhibited by LY333531(P＜0.05). 5.The expression levels of PKCβI mRNA and protein of mesangial cells of PAF group were higher than those of control group(P＜0.05).IMF revealed the expression of PKCβI in nucleus and cell membrane of mesangial cells and the evident nucleus transposition of PKCβI after mesangial cells exposed to PAF.These effects could be inhibited by LY333531,respectively(P＜0.05).6.Comparing with the control,the expression levels of PKCβI mRNA and protein of mesangial cells were higher in the high glucose and high LPC,PAF+ high glucose and high LPC group,respectively(P＜0.05).IMF revealed the expression of PKCβI in nucleus and cell membrane of mesangial cells and the evident nucleus transposition of PKCβI after mesangial cells exposed to high glucose and high LPC,PAF+ high glucose and high LPC.These effects could be inhibited by LY333531,respectively(P＜0.05).Conclusions:Exposed to normal glucose or high glucose and high LPC,PAF mediates ECM production through activating PKCβI/TGF-β1 signaling pathway in mesangial cells.Part 3.The effects of atorvastatin on the rnesangial cells exposed to high glucose and high LPCObjective.To investigate the changes of Fn,ColⅣ, PAF,PAF-R,TGF-β1,PKCβI of the mesangial cells under high glucose and LPC,atorvastatin,to explore the effects of atorvastatin on the mesangial cells under high glucose and high LPC.Methods:The model of monolayer mesangial cell culture was established and divided into 3 groups:control,high glucose and high LPC,atorvastatin.The levels of Fn,ColⅣ,PAF were determined by ELISA in the culture media.The expression levels of PAF-R,TGF-β1, PKCβI mRNA in mesangial cells were determined by Real-time PCR. The expression levels of TGF-β1 and PKCβI protein of mesangial cells were determined by Western blot.The expression of PKCβI in mesangial cells was determined by IMF.Results:1.The levels of Fn and ColⅣof atorvastatin group were lower than those of high glucose and high LPC group(P＜0.05) 2.The level of PAF of atorvastatin group was lower than that of high glucose and high LPC group(P＜0.05).The expression level of PAF-R mRNA in mesangial cells of atorvastatin group was lower than that of high glucose and high LPC group(P＜0.05).2.The expression levels of TGF-β1 mRNA and protein of atorvastatin group was lower than those of high glucose and LPC group(P＜0.05).3.Intervented by atorvastatin,the expression of PKCβI in nucleus and cell membrane of mesangial cells and the nuclear translocationof PKCβI of PAF group was significantly decreased.Conclusions:The renal protection of atorvastatin may be concerned with its down-regulated the expression of PAF and depression effect on the activation of the PKCβI /TGF-β1 signaling pathway of mesangila cells,which to decrease the secretion of ECM(Fn and ColⅣ) of mesangial cells under high glucose and high LPC.
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