Gastrodin Regulates Expression Of AngiotensinⅡ-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-α expression in activated TNC-1 astrocytes was followed.Results:(1)Results of Western Blot showed that expression of TNF-α was no statistic difference in LPS group compared with the control group,and that of TNF-αexpression 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-α 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-α 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-α,iNOS was significantly higher in CM+LPS+GiL+A group,but the expression of SirT3 protein increased significantly(p<0.05).Conclusions:①The expression of angiotensin system(ACE and AT1),NOX2,SirT3,inflammatory cytokines(iNOS and TNF-α)and nutrient factors(IGF-1 and BDNF)in reactive astrocytes were increased.②Expression of NOX2,iNOS and TNF-α in reactive astrocytes were effectively inhibited by the Azisartan;meanwhile,SirT3 expression was augmented.③ 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.④ 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.