Effect Of Simvastatin On The Permeability Of Rat Brain Microvascular Endothelial Cell Under Different Hypoxia Conditions

Backgroud and objective:Hypoxic-ischemic brain injury is the most common cerebral vascular disease, which can be further aggravated due to the reperfusion after recanalization. Thus, it is of great practical value to investigate the potential therapeutic strategies. Recently, Statins (3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors), which is known as a popular hypolipidemic drug in clinical application, has being used in ischemic cerebral vascular disease based on its non-antilipemic protective effects. It was demonstrated that Statins has a significant reversal function which can relieve many pathophysiologic changes during cerebral ischemia and reperfusion process. Furthermore, Statins can reduce the rat brain infarct size, relieve the brain edema and attenuate brain damage after acute stroke. Blood brain barrier (BBB) is an important structure to maintain the microenvirement of brain. Brain microvascular endothelial cells (BMECs) is the major cellular component of BBB, which make up the barrier between microenvironment and circulating blood.There are great differences in genes, proteome and physiological functions between peripheral vascular endothelial cells and BMECs.Therefore, injury of BBB and the BMECs could result in the disbalance of brain microenvirement, and then lead to dyregulation at functional, metabolism and structural levels for neurocytes.The alteration of permeability is the most basic and most direct signs in such BBB injuries.Thus, investigating the effects of Statins on the permeability of BMECs under different hypoxia conditions can elucidate potential mechanisms at cellular level directly. In this study, we built transient hypoxia (TH), continuous hypoxia (CH), reoxygenation (R) models of BMECs, and observed their permeability.We then detected the expression of matrix metalloproteinase (MMP-9), inducednitric oxide synthase (iNOS), hypoxia inducible factor-1α(HIF-1α) in BMECs treated with or without Simvastatin under different hypoxia conditions. Furthermore, we discussed the possible mechanism of the influence of Statins on the monolayer permeability of BMECs under different hypoxia conditions, and to supply some experimental evidence for explaining its effects of relieving cerebral ischemic injury at different levels.Methods:BMECs derived from SD rat were isolated by enzymes digestion followed by 20%BSA centrifugation.Normoxia, transient hypoxia, continuous hypoxia and reoxygenation models were built under different oxygen and glucose deficiency conditions.TH group was cultured with low glucose DMEM in a hypoxia incubator chamber (3%O2–5%CO2–92%N2) for 0.5h.CH group was cultured with glucose deprivation solution (Krebs solution) in hypoxia incubator chamber (3%O2– 5%CO2– 92%N2) for 12h. R group was cultured with Krebs solution in hypoxia incubator chamber (3%O2– 5%CO2– 92%N2) for 12h, followed by cultured with low glucose DMEM in a normal oxygen partial pressure incubator chamber (37℃,5%CO2 ) for 4h.BSA permeability experiment and transendothelial electrical resistance (TEER) technique were employed to evaluate the permeability across the BMECs monolayer and the changes after treated with Simvastatin (0.1μ, 1.0, 10μmol/L), iNOS inhibitor (SMT), MMP-9 inhibitor I (MI), and HIF-1αinhibitor (GEN). The cell proliferation activity was detected with CCK-8 kit.The iNOS activity was measured with iNOS kit and the expression of MMP-9 and HIF-1αin each group was detected by indirect immunofluorescence under confocal laser scanning microscopy.Results:Part one:1. BMECs grew from brain microvascular fragments appeared to be tight conjunctive cobblestone monolayer and were identified by vWF expression.Growth peak is ranged from the 3rd day to 6th day. Primary BMECs showed very strong barrier characteristic and the TEER declined significantly after the 4th generation (P<0.05).2. BSA permeability experiment indicated that the permeability rate reaching the peak after reoxygenation for 4h, and then declined gradually, which was in accordance with the result of TEER. Compared with group N, the amount of BMECs that rounded and sheded in group TH, CH, R was gradually increased and the intercellular space was gradually expanded.Simvastatin treatment could prevent the change of cell morphology to some extent. The TEER of group N, TH, CH, R were gradually decreased (P<0.05) and CCK-8 proliferation activity was gradually increased (P<0.05).Except 0.1μmol/L Simvastatin has no significant effects on R group, each concentration could improve the BMECs'TEER and proliferation activity obviously (P<0.05).Part two:1. 10μmol/L Simvastatin could improve the TEER and proliferation activity of group TH, CH, R (P<0.05).The TEER and proliferation activity of group TH treated with MI and GEN had no significant difference with control (P>0.05). The TEER and proliferation activity of group CH, R treated with MI /SMT/ GEN had significant differences with control (P>0.05).2. MMP-9-positive cells showed green fluorescence in endochylema.The expression of MMP-9 of control was gradually increased in group TH, CH, and R compared with group N (P<0.05). Simvastatin could reduce the expression of MMP-9 of group TH, but had no significant difference with control (P>0.05). Simvastatin significantly reduced the expression of MMP-9 of group CH, R (P<0.05).MI obviously reduced the expression of MMP-9 in each group (P<0.05). SMT/GEN significantly reduced the expression of MMP-9 of group TH, CH, R (P<0.05). The expression of MMP-9 in group TH, CH, R treated with SMT/GEN were lower than that with MI, and had significant difference in group CH, R (P<0.05). Pearson coefficient correlation between TEER and MMP-9 after treatment with Simvastatin was -0.875,p=0.000.3. The iNOS activity of control was gradually increased in group TH, CH, and R compared with group N (P<0.05).Simvastatin/MI/GEN could significantly reduce the iNOS activity of group CH, R (P<0.05).SMT obviously reduced the iNOS activity in group TH, CH, R (P<0.05).The iNOS activity of group TH, CH, R treated with GEN were lower than that with SMT, and had significant difference in group R (P<0.05). Pearson coefficient correlation between TEER and iNOS activity after treatment with Simvastatin was -0.652,p=0.001.4. HIF-1α-positive cells showed red fluorescence in nucelus.No HIF-1αexpression was detected in group N. HIF-1αwas gradually increased in group TH, CH, and R (P<0.05). Simvastatin/MI/SMT could increase the expression of HIF-1αin group TH (P<0.05), but had no significant difference in group CH, R (P>0.05). GEN obviously reduced the expression of HIF-1αin group TH, CH, R (P<0.05), and had. significant difference with that treated with Simvastatin/MI/SMT(P<0.05).Pearson coefficient correlation between TEER and HIF-lαafter treatment with Simvastatin was -0.836,p=0.000.Conclusion:1. On the basis of previous research, we isolated and cultured the BMECs derived from SD rat with enzyme digestion followed by 20%BSA centrifugation.TEER showed that cells before the 4th generation were suitable for permeability study. We built the transient hypoxia, continuous hypoxia and reoxygenation models in vitro with low glucose DMEM/Krebs solution and hypoxia conditions successfully. The permeability of three hypoxia modles was gradually increased and cell proliferation activity decreased.3. Except low concentration Simvastatin having no significant effects on BMECs with reperfusion, simvastatin could improve the permeability of BMECs under different hypoxia conditions in a dose-dependent manner.3. Inhibiting the iNOS activity can improve the permeability of BMECs under different hypoxia conditions.The expression of MMP-9 and HIF-1αwere related with the increasing permeability of BMECs under continuous hypoxia and reoxygenation conditions. On the contrary, increasing the expression of HIF-1αmay reduce the permeability of BMECs under mild hypoxia. The expression of MMP-9 and iNOS activity of BMECs under hypoxia conditions may have realationship with HIF-1α, and MMP-9 may be also regulated by iNOS.4. The improvement effects of Simvastatin on tne permeability of BMECs under transient hypoxia conditions may be related with the increasing expression of HIF-1α, and such effects under continuous hypoxia and reoxygenation conditions may be related with inhibition of MMP-9 and iNOS activity.