The Effects Of Copper On Claudin-1,-3,-5 And Oxidative Damage In Rat Brain Microvascular Endothelial Cells

Copper is an essential trace element that is vital to all living things. In brain, copper involves in a large number of biological processes, plays animportant role for acting normal physiology function in body. However, a number of central nervous system diseases, in particular neurodegenerative diseases, are closely related with copper. In order to explore the close relationship between copper and brain, the test developed an in vitro modelof primary cultured brain microvascular endothelial cells(BMECs).Cu Cl2 incubated BMECs for 12 h with 6 different concentrations, which were 30 ?M, 60 ?M, 120 ?M, 180 ?M, 240 ?M and 300 ?M. Cytotoxicity, protein levels and gene levels of claudin-1,-3 and-5 and oxidative stress-relatedenzymatic activity were measured after incubation. The results showed that:1. Gradient centrifugation with bovine serum albumin and digestionwith type ? collagenase were proper method for separating primary BMECs. Immunofluorescencewhich determinedspecific antigen ? factor showed positive, and the cell purity of BMECs was more than 99% by flow cytometry,detectingcadherin CD144.2. The cell viability of BMECs increased at first and then decreased with the increase of concentrations of copper. Cell viability of the 120 ?M group was at peak(63%) among copper ions groups. The viability of extracellular lactate dehydrogenase was consistent with cell viability, they showed a similar trend. In addition, BMECs had high requirements for nutrition and livingenvironment, as the negative group made a significant decline of cell viability without adding in fetal bovine serum.3. With the concentrations of copper ions increased,it showed similar trends of protein expression of claudin-1,-3 and-5. They decreased the expression from low concentrations groups to middle concentrations groups. The 180 ?M group was at the low peak of protein expression, and then they increased expression in high concentrations groups.4. Compared to the negative group,althoughthe m RNA of claudin-1,-3 and-5had statistical difference in relative expression, there were no clear trends among various groups of copper ions concentrations.5. In the process of oxidative stress, Cu Zn-SOD wasthe main type of BMECs' superoxide dismutase(SOD).With the copper ions concentrations increased, Cu Zn-SOD activity increased, but there may be a copper loading critical point, Cu Zn-SOD activity decreased after 120 ?M groups. Overall, with the increase of copper ions concentrations, peroxidase(CAT) activity decreased, and the 300 ?M group bottomed out. Inducible nitric oxide synthase(i NOS) was the main type of BMECs' nitric oxide synthase(NOS). With the increased of copper ions concentrations, i NOS activity was at a peak in the 120 ?M group, and followed by a downward trend.Conclusion: Copper ions could cause cytotoxicity in BMECs, but there might be an optimal concentration of copper ions that they could assist BMECs' self repair. Copper ions could mediate the protein expression of claudin-1,-3 and-5, which were closely associated with the concentrations of copper.Copper ions would cause oxidative damage in BMECs.