Gastrodin Regulates Expression Of Angiotensin?-NOX2/SirT3 Signaling Pathway In Reactive Astrocytes Via Activated Microglia

Objective(s):This study investigates the expression changes of the angiotensin system(ACE,AT1),NADPH-oxidase2(NOX2),sirtuin 3(SirT3)and trophic factors(IGF-1,BDNF)in the TNC-1 astrocytes,treated respectively with conditioned medium from BV-2 microglia with or without pretreatment of gastrodin and lipopolysaccharide(LPS)in vitro.The effects of gastrodin treatment on AngII-NOX2/SirT3 signal pathways in the reactive astrocytes were analyzed in terms of its inhibitory effect and signal mechanisms.Methods:TNC-1 astrocytes cultured in vitro were randomly divided into control group,LPS direct activation group(LPS group)and TNC-1 cells treated directly with different dosage of Gastrodin(Gd50 mg/L,Gd100 mg/L,Gd200 mg/L)group,conditioned medium derived from BV-2 microglia(CM)group,conditioned medium derived from BV-2 microglia treated with LPS(CM+LPS)group,CM+LPS+Gastrodin indirect treatment at low dose(CM+LPS+GiL),CM+LPS+Gastrodin indirect treatment at high dose(CM+LPS+GiH),CM+LPS+Azisartan group and CM+LPS+GiL+Azisartan group.The effect of Gastrodin on the cell viability of TNC-1 astrocytes 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,NOX2,SirT3 and nutritional factors(IGF-1 and BDNF).At the same time,the effect of Gastrodin on the iNOS,TNF-? expression in activated TNC-1 astrocytes was followed.Results:(1)Results of Western Blot showed that expression of TNF-? was no statistic difference in LPS group compared with the control group,and that of TNF-?expression in CM+LPS group was significantly higher than that in CM group.(2)Western Blot analysis showed that TNC-1 astrocytes remained relatively unreactive to direct Gastrodin treatment in terms of expression of SirT3 when compared with the control cells.(3)The results of Western Blot showed that the expression of TNF-? and iNOS were significantly inhibited in CM+LPS+GiH and CM+LPS+GiL groups compared with the CM+LPS group(p<0.05).(4)Western Blot and double immunofluorescence labeling showed that the expression of ACE,AT1,NOX2 and SirT3 in activated TNC-1 astrocytes in CM+LPS group was significantly higher than that in CM group(p<0.05).Of note,the expression of ACE,AT1 and NOX2 was decreased significantly in both CM+LPS+GiL and CM+LPS+GiH groups(p<0.05);however,SirT3,IGF-1 and BDNF expression was further increased(p<0.05).(5)Western Blot results showed that compared with CM+LPS group,the expression of NOX2,iNOS and TNF-? in CM+LPS+A group were significantly inhibited and promoted the expression of SirT3.However,compared with CM+LPS+A group and CM+LPS+GiL group,NOX2,TNF-?,iNOS was significantly higher in CM+LPS+GiL+A group,but the expression of SirT3 protein increased significantly(p<0.05).Conclusions:?The expression of angiotensin system(ACE and AT1),NOX2,SirT3,inflammatory cytokines(iNOS and TNF-?)and nutrient factors(IGF-1 and BDNF)in reactive astrocytes were increased.?Expression of NOX2,iNOS and TNF-? in reactive astrocytes were effectively inhibited by the Azisartan;meanwhile,SirT3 expression was augmented.? Gastrodin inhibits the expression of ACE and AT1 and proinflammatory mediators,but promotes the production of large amounts of neurotrophic factors that conceivably would be neuroprotective for tissue repair and remodeling.? More importantly,we show here that the effects of Gastrodin on astrocytes is microglia-mediated suggesting a cross-talk between activated microglia and reactive astrocytes.