| ObjectiveThis study aims to explore the effect and mechanism of fisetin on prevention and treatment of mouse brain microvascular endothelial cells injury induced by high glucose and palmitic acidMethods1.In vitro experiments,high glucose(HG,25 mmol·L-1)and palmitic acid(PA,200 μmol·L-1)were used to intervene in the mouse brain microvascular endothelial cells induced injury to establish the model of diabetic brain microangiopathy.Groups are divided into control group,model group(25 mmol·L-1 high glucose + 200μmol·L-1 palmitic acid,HG + PA)and fisetin high dose groups(fisetin 0.3 μg·mL-1,1.5 μg·mL-1,7.5 μg·mL-1,37.5 μg·mL-1),fisetin low dose groups groups(fisetin 1.5μg·mL-1,3 μg·mL-1,6 μg·mL-1,12 μg·mL-1)、VEGF group(20 ng·mL-1).The effect of fisetin on hyperglycemic and hyperlipid-induced cell damage was detected by Cell counting kit-(CCK-)8 assay,Cytotoxicity was detected by LDH2.Detection of apoptotic gene B-celllymphoma-2 gene(B-celllymphoma-2,Bcl-2)and Bax expression by Western Blotting3.After treatment,the changes of oxidative stress related factors(ROS,NO,iNOS,MMP-2,MMP-9,TIMP-1,MDA,SOD,CAT)were detected by ELISA4.Detection of epoxy chloropropane Kelch sample related protein-1(Keapl),nuclear factor erythroid-2 related factor 2(Nrf2),NADPH:quinoneoxidoreducase-1(NQO1),Hemeoxygenase-1(HO-1)by Western BlottingResults1.When BEND3 cells were stimulated by HG and PA,the cell viability decreased to(58.82±2.619)%(P<0.001).Compared with the HG+PA model group,the cell vitality was the highest with the fisetin 6 μg·mL-1 group,which is(73.73 ±5.89)%(P<0.001),and the cell vitality of VEGF group is(76.19±2.62)%(P<0.001);The results of LDH test showed that the LDH content gradient of BEND3 cells was decreased in the groups of 1.5μg·mL,3μg·mL and 6μg·mL-1,compared with the HG+PA model group,among which the LDH content of BEND3 cells was the lowest in the group of 6μg·mL-1(0.36±0.24)U/L(P<0.001)2.The expression of apoptotic factor showed:Compared with the blank group,the Bcl-2/Bax ratio of HG+PA model group was decreased 0.25±0.1(P<0.001);compared with the HG+PA model group,the Bcl-2/Bax ratio was significantly increased 0.59±0.23 in the fisetin dose group(P<0.05)3.The results of oxidative stress related indicators:Compared with the blank control group,the expression of ROS,NO,iNOS,MMP-2,MMP-9,TIMP-1 and MDA in the model group increased significantly(P<0.01).Compared with the model group,the expression of ROS,NO,iNOS,MMP-2,MMP-9,TIMP-1 and MDA were decreased in the fisetin dose group(P<0.01).Compared with the blank control group,the expression of SOD,GSH-PX and CAT in the model group decreased significantly(P<0.01).Compared with the HG+PA model group,the expression of SOD,GSH-PX and CAT were significantly increased in the fisetin dose group(P<0.01)4.Detection of protein expression in Keapl/Nrf2/ARE pathway:Compared with the blank control group,the expression of Nrf2 cytoplasmic protein was decreased(P<0.01),a small amount of Nrf2 was transferred to the nucleus.After the intervention of fisetin,the expression of Nrf2 protein in cells was nuclearly shifted(P<0.05);the expression of NQO1 and HO-1 protein was significantly upregulated(P<0.05,P<0.01)Conclusion1.6 μg·mL-1,12 μg·mL-1 Fisetin protects against high glucose and palmitic acid damage of microvascular endothelial cells in mice brain,among them 6μg·mL-1 is the best effect of fisetin;2.Fisetin regulates the Bcl-2/Bax ratio of apoptosis factor in rat brain microvascular endothelial cells induced by high glucose and palmitic acid;3.Fisetin can improve the cell model of diabetic cerebral microvascular lesions,which may be related to inhibite the production of endogenous ROS in cells;4.Fisetin may improve the damage of diabetic brain microvascular cells by regulating the activity of intracellular antioxidant enzymes and the level of oxidative stress;5.Fisetin promotes Nrf2 protein nuclear transfer and activates Nrf2 downstream associated antioxidant stress gene proteins to exert their antioxidant effects. |
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