Expression Of Ang?-NOX2/SirT3 Signaling Pathway In Activated Microglia And Effection Of Gastrodin

Objective:This study investigates the expression changes of the angiotensin system(ACE?AT1?AT2),NADPH oxidase(NOX2)and Sirtuin 3(SirT3)in the activated microglia in the corpus callosum of newborn rats with experimentally induced hypoxic-ischemia brain damage(HIBD)as well as in LPS activated BV-2 microglia in vitro.The effects of Gastrodin treatment on Ang ?-NOX2/SirT3 signal pathways in activated microglia were analyzed in terms of its inhibitory effect and signal mechanism involved at the cellular,molecular and protein levels.Methods:To establish the HIBD model,one-day-old Sprague-Dawley(SD)rats were subjected to carotid artery ligation.After this,they were placed in a hypoxic chamber(8%O2,92%N2)for 2 hours.The newborn rats were randomly divided into the control group,HIBD group,HIBD+Gastrodin(100 mg/kg)group.The BV-2 microglia cells were randomly divided into control group,LPS group(1 mg/L),Gastrodia group with different concentrations of Gastrodin(50 mg/L and 100 mg/L)+LPS treatment group,LPS + Azisartan(10 ?M)group and Gastrodin(50 mg/L)+LPS+ Azisartan group and positive control group.The effects of Gastrodin on the cell viability of BV-2 microglial cells assessed by MTS.Western blot and double immunofluorescence labeling were carried out to investigate the effects of Gastrodin on the expression of angiotensin converting enzyme(ACE),AT1 receptor,AT2 receptors,NOX2 and SirT3 in vivo and in vitro.Along with this,the effects of Gastrodin on iNOS and TNF-a expression in activated microglial cells in vitro was followed.Results:In vivo experiment:Results of double immunofluorescence labeling showed that the number of activated microglia in the corpus callosum in the HIBD group was more numerous than the control group.Moreover,the immunofluorescence of AT1,NOX2 and SirT3 was evidently enhanced in the HIBD group(p<0.05).The immunofluorescence intensity of AT,and NOX2 in HIBD + Gastrodin group was significantly attenuated compared with that of the HIBD group(p<0.05).On the other hand,the immunofluorescence intensity of SirT3 in HIBD+ Gastrodin group was further enhanced in comparison to the HIBD group(p<0.05).Western blot analysis showed that ACE,AT1,NOX2 and SirT3 protein expression levels in HIBD group were significantly higher than the control group(p<0.05).ACE,AT1,NOX2 protein expression levels in the HIBD + Gastrodin group were decreased significantly.As opposed to this,SirT3 protein level was further augmented in HIBD + Gastrodin group(p<0.05).In vitro experiment:(1)Results showed that the iNOS and TNF-a expression was hardly detected in BV-2 microglia in the control group(p<0.05),but the expression of both proinflammatory mediators was markedly increased in LPS group.Remarkably,the increase of both biomarkers induced by LPS was suppressed by Gastrodin(p<0.05).(2)Western Blot and double immunofluorescence labeling showed that expression of ACE,AT1,AT2,NOX2 and SirT3 increased significantly in LPS group compared with the control group(p<0.05).Of note,ACE,AT1 and NOX2 expression was decreased significantly in both Gastrodin groups(p<0.05);however.AT2 and SirT3 expression was further increased(p<0.05).(3)Western blot results showed that NOX2,iNOS and TNF-a protein expression level was decreased in LPS+ Azisartan group compared with LPS group(p<0.05).A feature worthy of note was that SirT3 protein expression was increased in LPS + Azisartan group(p<0.05).In addition,in comparison with LPS +Gastrodin grouop and LPS +Azisartan group(p<0.05),NOX2,iNOS and TNF-a protein expression was reduced(p<0.05),but that of SirT3 was increased significantly in Gastrodin +LPS+Azisartan group(p<0.05)Conclusion:? Protein expression of angiotensin system(ACE,AT1 and AT2),NOX2,SirT3 and iNOS and TNF-a in the activated microglia in the corpus callosum in newborn rats with HIBD was elevated;likewise,the changes were observed in LPS activated BV2 microglia ? Expression of NOX2,iNOS,TNF-a and inflammatory cytokines in the activated microglia were effectively inhibited by the Azisartan;meanwhile,SirT3 expression was augmented.? It is suggested that the Ang?-AT1-NOX2/SirT3 signaling pathway is involved in the pathophysiological process of microglia activation.?Gastrodin can inhibit the activation of microglia and mediate the inflammatory response through AngII-AT1-NOX2/SirT3 signaling pathway.More importantly,Gastrodin plays a neuroprotective role through promoting the expression of AT2 receptor and SirT3.