| Objective:To study the expression of RAS system-related proteins(ATO,ACE,AT1),Notch signaling pathway-related proteins(Notch-1,NICD,RBP-JK,Hes-1)and inflammatory factors(TNF-α,IL-1β)in BV-2 microglia activated by lipopolysaccharide(LPS)or oxygen glucose deprivation(OGD)in vitro,and the effects of gastrodin intervention.To explore the interaction between RAS system and Notch signaling pathway in activated BV-2 microglia and the effect of gastrodin intervention on these two pathways.Methods:BV-2 microglia was cultured in vitro and tested by different experimental methods and groups.Part I,microglial cells cultured in vitro were randomly divided into four groups:control group,LPS group(1 mg/L)/OGD group,LPS/OGD+0.17mM gastrodin group and LPS/OGD+0.34mM gastrodin group.The expression of RAS system-related proteins,Notch signaling pathway-related proteins and inflammatory factor TNF-α,IL-1β in LPS or OGD activated microglia in vitro were detected by double immunofluorescence staining and Western Blot,which was to explore the effects of gastrodin on RAS system and Notch signaling pathway.Part Ⅱ,(1)The BV-2 microglia cells were divided into six groups:control groups,AT1Ⅰ(50 μM)groups,LPS(1 mg/L)groups,LPS+AT1Ⅰ groups,LPS+gastrodin(0.34 mM)groups and LPS+AT1Ⅰ+ gastrodin groups.Western Blot was used to detect the expression of Notch signaling pathway related proteins(Notch-1,NICD,RBP-JK,Hes-1)and inflammatory factors(TNF-α,IL-1β)in each experimental group,which was to investigate the expression of Notch signaling pathway in activated microglia after blocking the RAS system and the effect of gastrodin intervention.(2)The microglial cells were stimulated by exogenous Ang II,which were divided into 0,12,24,36 h different action time points to determine the best time of action.Then the cells were randomly divided into control group,Ang Ⅱ(10μM)group,Ang Ⅱ+DAPT(10 μM)group and control group,Ang II(10 μM)group and Ang II+gastrodin(0.34mM)group.The expression of Notch signaling pathway related proteins(Notch-1,NICD,RBP-JK,Hes-1)and inflammatory factors(TNF-α,IL-1β)were detected by double immunofluorescence staining and Western Blot,which was to investigate the effect of exogenous activation of RAS system on Notch signaling pathway.Part Ⅲ,The cells were randomly divided into six groups:control group,DAPT(10 μM)group,LPS(1 mg/L)group,LPS+DAPT(10 μM)group,LPS+gastrodin(0.34 mM)group and LPS+DAPT+gastrodin group.Western Blot was used to detect the expression of RAS system-related proteins(ACE,AT1)in each experimental group.Thus,it was further clear whether gastrodin can regulate Notch signaling pathway through RAS system.Results:Part I,The results of immunofluorescence and Western Blot showed that:the expression of RAS system-related protein(ATO,ACE,AT1),Notch signaling pathway-related protein(Notch-1,NICD,RBP-JK,Hes-1)and inflammatory factor(TNF-α,IL-1β in LPS or OGD activated BV-2 microglia was significantly higher than those in the control group(P<0.05);The expression of Notch signal pathway-related protein(Notch-1,NICD,RBP-JK,Hes-1),RAS system-related protein(ATO,ACE,AT1)and inflammatory factor(TNF-α,IL-1β)decreased compared with those of LPS or OGD group after intervention with low and high concentrations of gastrodin(P<0.05).There was no significant difference between different drug concentrations.Part Ⅱ,(1)The Western Blot results showed that:compared with the LPS group,the expression of Notch-1,NICD,RBP-JK,Hes-1 and inflammatory factor TNF-α,IL-1β was reduced in LPS+AT1Ⅰ group(P<0.05).It suggested that AT1 receptor blockers could reduce the expression of Notch signaling pathway and inflammatory factors in activated microglia.(2)The Western Blot results showed that:after 12 h,24 h and 36 h stimulation with Ang II,the protein expression levels of Notch-1,NICD and RBP-JK in BV-2 microglia cells increased most significantly in the 12 h group(P<0.05).The expression of Hes-1 protein in 24 h group increased to the highest level(P<0.05).The results of immunofluorescence and Western Blot showed that:the expression of Notch signaling pathway-related protein Notch-1,NICD,RBP-JK,Hes-1 and inflammatory factor TNF-α,IL-1β in Ang Ⅱstimulated microglia was higher than that in control group.Compared with the simple Ang II group,the expression of Notch-1,NICD,RBP-JK,Hes-1 and inflammatory factor TNF-α,IL-1βwas significantly decreased in Ang II+DAPT group(P<0.05).These results indicated that Ang II stimulation increased the expression of Notch pathway-related proteins and inflammatory factors in microglia.(3)The Western Blot results showed that:compared with LPS+AT1Ⅰ group,the combination of AT1I and gastrodin had no effect on the expressions of Notch-1,NICD,RBP-JK and Hes-1,which are related to Notch pathway(P>0.05).However,the expression of inflammatory factor TNF-α and IL-1β was further decreased(P<0.05)in LPS+AT1Ⅰ+gastrodin group.Compared with the control group,the expression of inflammatory factor TNF-α and IL-1β was significantly increased after the stimulation of exogenous Ang II in BV-2 microglia,while gastrodin could decreased the expression of TNF-αand IL-1β(P<0.05).These results indicated that gastrodin could affect Notch signaling pathway through RAS system.Meanwhile,the inhibitory effect of gastrodin on inflammation was not entirely through the RAS system.Part III,the Western Blot results showed that:there was no effect on the high expression of ACE and AT1 in LPS-activated BV-2 microglia after Notch signaling pathway blocked by DAPT(P>0.05).In addition,gastrodin intervention could reduce the expression of ACE and AT1 after Notch pathway blocked by DAPT(P<0.05).However,there was no difference about ACE and AT1 expression in LPS activated microglia between LPS+DAPT+gastrodin group and LPS+gastrodin group(P>0.05).It was further demonstrated that gastrodin could not directly regulate RAS system through Notch pathway.Conclusions:(1)The expression of RAS system,Notch signaling pathway and inflammatory factors was significantly increased in LPS or OGD-activated BV-2 microglia in vitro.Meanwhile,gastrodin intervention could effectively inhibit the RAS system,Notch pathway and inflammatory response.(2)RAS system could regulate the expression of Notch pathway and inflammatory factors in LPS-activated microglia.(3)Gastrodin played a neuroprotective role by inhibiting the activation of Notch signaling pathway and inflammatory response in microglia through the RAS system. |
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