The Study Of Synergistic Effect Of Insulin In Combination With Rosiglitazone On Clearance Of Aβ1-40from Brain Micro/Small Vessel By Regulation Of LRP-1in Type2Diabetic Rats

OBJECTIVE To investigate effects of insulin and rosiglitazone, alone or in combination on expression of Low-density lipoprotein receptor-related protein l(LRP-1) and amyloid beta(Aβ1-40) in diabetes rats brain micro/small vessel; To investigate the possible clearance mechanism of Aβ1-40on the brain micro/small vessel affected by drug intervention; To research effects of diabetes mellitus on expression of Aβ1-40in diabetes rats brain micro/small vessel.METHODS(1) Goto-Kakizaki (GK) rats were used as type2diabetic rats model. All GK rats were randomly divided into five groups:3-month-old GK rats group(DM3),6-month-old GK rats group(DM6), DM6treated with rosiglitazone group (DM6RSG), DM6treated with insulin group(DM6Ins), DM6treated with rosiglitazone in combination with insulin group(DM6Ins+RSG).(2) Expressions of LRP-1and Aβ1-40in the brain micro/small vessel were detected by immunohistochemistry(IHC); The content of LRP-1and Aβ1-40in the brain tissue was detected by enzyme-linked immunosorbent assays(ELISA); Expression of LRP-1mRNA was detected by RT-PCR.(3) All datas were analyzed by SPSS17.0and represented as mean±SEM. The ststistical signifance of differences was assessed by One-Way ANOVA test. Significance was considered as p <0.05.RESULTS(1) Immunohistochemical analysis of Aβ1-40expression in brain microvessels:Expression of Aβ1-40were visible in diabetes rats brain micro/small vessel of each group; The positive products of Aβ1-40were detected in the smooth muscle and endothelial cells. Vessels in DM6Ins、DM6RSG and DM3stained weakly positive compared with the faint staining seen in DM6; Compared to DM6, less number of capillaries that positively express Aβ1-40was detected in DM6Ins、DM6RSG and DM3(P<0.05).(2) Immunohistochemical analysis of LRP-1expression in brain microvessels:The positive products of LRP-1were detected in the endothelial cells. Vessels in DM6Ins+RSG and DM6RSG stained strongly positive compared with the faint staining seen in DM6. The number of microvessels positive expression of LRP-1was increased in the DM6RSG and DM6Ins+RSG compared with DM6(P<0.01). There were no difference about the number of microvessels positive expression of LRP-1between DM6and DM6Ins.(3) ELISA analysis Aβ1-40and LRP-1expression in brain tissue:The levels of Aβ1-40detected by ELISA in brain tissue were decreased in the DM6Ins、 DM6RSG and DM3compared with DM6(P<0.05);The expression of LRP-1in the brain tissue was increased in the DM6Ins+RSG and DM6RSG compared with DM6(P<0.01); The expression of LRP-1in the plasma membrane fraction of brain tissue was increased in the DM6Ins compared with DM6(P<0.01), but there was no difference about the expression of LRP-1in the brain tissue between them(p>0.05); The expression of LRP-1in the plasma membrane fraction of brain tissue was increased in DM6Ins+RSG compared with DM6Ins(P<0.01), but there was no difference about the expression of LRP-1in the brain tissue between them(p>0.05).(4) RT-PCR analysis LRP-1expression in brain tissue:Differences of ratio of LRP-1absorbance/GAPDH absorbance in GK brain. The expression of LRP-1mRNA in the brain was increased in the DM6Ins+RSG and DM6RSG compared with DM6(P<0.05). There were no difference about the expression of LRP-1mRNA between DM6and DM6Ins(p>0.05).CONCLUSIONS(1) With diabetes mellitus progression, the deposition of A(31-40in brain micro/small vessel increases obviously which suggests diabetic course can aggravate Aβ1-40deposition in brain micro/small vessel.(2) Insulin can prevent Aβ1-40deposition in the brain through a pathway involving intracellular translocation of LRP-1to the plasma membrane in diabetic brain tissue.(3) Rosiglitazone can upregulate expression of both LRP-1and LRP-1mRNA and then decrease accumulation of Aβ1-40in the diabetic brain tissue micro/small vessel.(4) In combination use, rosiglitazone appears to be able to sensitize/enhance insulin’s inducing the subcellular translocation of LRP-1to the plasma membrane in diabetic brain tissue.