Protective Effect and Mechanism of Ramelteon on Lipopolysaccharide-Induced Elevation of Permeability of Blood-Brain Barrier Model
OBJECTIVE:To explore the protective effect and mechanism of Ramelteon on lipopolysaccharide(LPS)-induced increased permeability of blood-brain barrier(BBB)model,as well as its relationship with inflammatory and oxidative stress responses.METHODS:Twenty-four healthy C57BL/6 male mice were divided into the control group(0.9％sodium chloride solution),Ramelteon group,LPS group and LPS+Ramelteon group.The blood brain barrier injury of mice was induced by LPS and the endothelial cell injury was stimulated.Brain permeability was assessed by using the Evans blue staining method,while cell viability was determined by utilizing the cell counting kit-8(CCK-8).Expression of human tight junction protein 1(ZO-1)and occludin in brain tissue and endothelial cells was evaluated through immunostaining and Western blotting.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and enzyme-linked immunosorbent assay were employed to measure the levels of inflammatory factor interleukin-1β(IL-1β)and monocyte chemotactic protein 1(MCP-1).Malondialdehyde(MDA)concentration in brain tissue and endothelial cells was detected by using the thiobarbituric acid method.Expression of human nuclear factor E2 related factor 2(Nrf2)and quinone oxidoreductase(NQO1)in the cell nucleus was determined via Western blotting.RESULTS:(1)Compared to the control group,mice in the LPS group showed increased brain permeability and significantly decreased neurological function scores.In comparison to the LPS group,mice in the LPS+Ramelteon group exhibited decreased brain permeability and significantly increased neurological function scores,with statistically significant difference(P＜0.05).(2)Compared with the control group,the LPS group displayed decreased expression of ZO-1 and Occludin,along with increased expression of IL-1β and MCP-1;compared with the LPS group,the LPS+Ramelteon group showed increased expression of ZO-1 and Occludin,and decreased expression of IL-1β and MCP-1,the differences were statistically significant(P＜0.05).(3)Compared with the control group,the LPS group showed decreased expression of Nrf2 and NQO1,and increased levels of MDA;compared with the LPS group,the expression of Nrf2 and NQO1 in LPS+Ramelteon group increased,while the level of MDA decreased,with statistically significant differences(P＜0.05).(4)Compared with the control group,bEnd.3 endothelial cell viability decreased,permeability increased significantly,and expression levels of ZO-1 and Occludin were significantly down regulated in the LPS group,with statistically significant differences(P＜0.05).Conversely,the LPS+Ramelteon group showed increased endothelial cell viability,decreased permeability,and significantly increased expression levels of ZO-1 and Occludin,which were positively correlated with the dose of Ramelteon,with statistically significant differences(P＜0.05).(5)Compared with the control group,LPS group significantly inhibited the expression levels of Nrf2 and NQO1;compared with the LPS group,the expression level of Nrf2 increased in the LPS+Ramelteon group,and was positively corr-elated with the dose of Ramelteon,with statistically significant differences(P＜0.05).(6)Both Compound C and si-Nrf2 could inhibit the activating AMP-activated protein kinase(AMPK)/Nrf2 signaling pathway,inducing oxidative stress and weakening the protective effect of Ramelteon on the reduction of ZO-1 and Occludin induced by LPS and the increase in endothelial monolayer permeability.CONCLUSIONS:Ramelteon can improve LPS-induced increased permeability of blood-brain barrier in vitro by activating AMPK-Nrf2 signaling pathway,and its mechanism may be related to inhibiting inflammatory and oxidative stress responses.
万方数据
维普
