The Effect And Mechanism Of SEH On Blood Brain Barrier And Cognitive Function Of DB/DB Mice

Objective1.To study the effect of type 2 diabetes on cognitive function and blood-brain barrier in db/db mice.2.To explore the role and possible mechanism of sEH in regulating blood-brain barrier and cognitive impairment in diabetic mice.3.To investigate the effect of 14,15-EET on the brain microvascular endothelial cells in high glucose culture and the mechanism of the regulation of AMPK / HO-1 signaling pathway.Method1.The mice were divided into normal control(NC)group,db /db group and db / db + sEHi group.At the beginning of 16 weeks,the mice in db / db+ sEHi group were given 2 mg / L of sEH selective inhibitor t-AUCB(about 0.6-0.8 μ g / kg body weight)in drinking water for 4 weeks,and the mice in NC group and db / db group were treated with vehicle.Mice brain microvascular endothelial cells(BMECs)were cultured underhigh glucose in vitro and treated with 14,15-EET,AMPK/HO-1 inhibitors respectively.2.The cognitive function was measured by Morris water maze.The permeability of BBB was detected by Evans blue staining and the structure of BBB was observed by transmission electron microscopy.3.Immunofluorescence and Western blot were used to detect the expression of junction proteins,epoxide hydrolase and AMPK / HO-1signaling pathway.4.The levels of 14,15-EET and 14,15-DHET in brain tissue were detected by ELISA and the levels of ROS in brain tissue sections and microvascular endothelial cells were detected by DCFH-DA analysis.Result1.The blood glucose level and body weight of db / db mice were significantly higher than those of NC mice(P < 0.01).t-AUCB could protect cognitive function of db/db mice;t-AUCB could significantly reduce the fasting blood glucose level of db / db mice(P < 0.05)and had no effect on body weight.2.The Evans blue dye exuded from the cerebral vessels of db / db mice was more detailed than that of NC mice(P < 0.01).Compared with untreated db / db mice,Evans blue leakage in t-AUCB group was significantly reduced(P < 0.05).In addition,the ultrastructure of BBB in cortex was observed under transmission electron microscope.In NC mice,the tight electron connection was shown as a tight zipper between adjacent endothelial cells,while in db / db mice,the tight electron density decreased with the formation of intercellular space.It was also observed that the tight junction extended into the microvascular cavity and formed a fish mouth structure in the diabetic brain.In the t-AUCB group,the endothelial cell gap closed with the reemergence of dark zipper like tight junction.3.The immunofluorescence and Western blot suggested that compared with NC mice,the expression of ZO-1 and VE-cadherin in db /db mice decreased significantly(P < 0.01).The expression of ZO-1 and VE-cadherin was significantly reversed by t-AUCB treatment(P < 0.01,P< 0.05).4.Compared with NC mice,the level of the brain sEH protein in db /db mice was higher(P < 0.05).However,t-AUCB treatment significantly inhibited the expression of sEH(P < 0.01).The change of ROS level is the same as the trend of sEH.The level of 14,15-EET in diabetic mice was lower than that in NC mice(P < 0.01),while t-AUCB treatment improved the level of 14,15-EET(P < 0.05).The protein expression of p-AMPK and HO-1 in db / db mice was lower than that in NC mice(P < 0.01),and the expression of AMPK / HO-1 in db / db mice was improved by the inhibition of sEH(P < 0.01).5.Immunofluorescence staining showed that compared with NG group,the immunofluorescence of protein VE-cadherin and ZO-1 in HGgroup MECs decreased significantly(P < 0.01).ROS accumulation increased in HG group(P < 0.01).The inhibition of AMPK and HO-1significantly reduced the protective effect of 14,15-EET on brain microvascular endothelial cells in HG group(P < 0.01).Conclusion1.The expression of sEH was up-regulated in db / db mice,and the inhibition of sEH could protect the cognitive function of diabetic mice and the permeability and structural integrity of blood-brain barrier.2.t-AUCB,an sEH inhibitor,can up regulate 14,15-EET,activate AMPK / HO-1 and reduce oxidative stress in the brain of db / db mice.3.14,15-EETmay inhibit oxidative stress and loss of connexin /adhesin of bmecs induced by high glucose through AMPK / HO-1pathway.