Effects Of Breviscapine And Edaravone On MAPK Signal Pathway And Neuroinflammation Mediated By Activated Microglia

Objective:This study aimed to investigate the effects of Breviscapine and Edaravone on MAPK Signal Pathway and the expression of pro-inflammatory mediators in activated microglia.Along with this,we also explored the mechanism via which Breviscapine and Edaravone exerts their effects on microglia activation in vitro and in vivo.Methods:The focal cerebral ischemia model of MCAO in rats was established following a minimal invasive craniotomy.SD rats were randomly divided into sham operation group,MCAO group,MCAO+ Edaravone group(5 mg/kg),MCAO+Breviscapine group(100 mg/kg),and MCAO+ Edaravone(5 mg/kg)+Breviscapine group(100 mg/kg).Expression of MAPK signal pathway(p-P38?P38?p-JNK?JNK?p-ERK and ERK)and the proinflammatory mediators(iNOS?TNF-? and IL-1?)in the penumbral zones at 3d after MCAO was assessed by the immunofluorescence double staining and Western blot.The BV-2 microglia were randomly divided into control,LPS induced group,LPS+Scutellarin group,LPS+ Edaravone group,and LPS+Scutellarin+Edaravone group.The expression of above markers in activated microglia was also detected by the immunofluorescence double staining and Western blot.Results:(1)In vivo:? Western blotting assay showed that the protein expression of MAPK(p-P38,p-JNK and p-ERK)and the inflammatory mediators(iNOS?TNF-?and IL-1?)protein was significantly increased at 3d in the penumbral zones of MCAO rats,which was significantly different compared with the sham group(p<0.05).Compared with MCAO group,protein expression of p-P38,p-JNK,iNOS,TNF-? and IL-1? was markedly reduced by Breviscapine or Edaravone treatment or a combination of both drugs,whereas p-ERK protein was increased in these groups.This was conspicuous especially in MCAO+ Breviscapine + Edaravone rats in which iNOS expression in activated microglia was virtually abolished(p<0.05).?Immunofluorescence double staining with lectin(a specific marker for microglia)shows that resting microglia with small size and many branches express only a small amount of p-ERK,p-JNK and p-P38 in the penumbral zones in sham group.A large number of activated microglia with polygonal or oval cell bodies can be seen in the penumbral zones of MCAO rats,some of activated microglia co-expressed p-ERK,p-JNK and p-P38 immunoreactive cells.But in MCAO+Breviscapine group,MCAO+Edaravone group,and MCAO+ Breviscapine+Edaravone group,the number of activated microglia cells was obviously decreased,the number of p-P38 and p-JNK immunoreactive cells was significantly reduced,and p-P38 and p-JNK expression in microglia was decreased,whereas p-ERK expression in microglia was elevated.The expression of nonphosphorylated P38,JNK and ERK was mildly increased,but the results didn't show statistically significance.Some activated microglia co-expressed P38 can be seen in MCAO group.In MCAO+ Breviscapine group,MCAO+Edaravone group,and MCAO+ Breviscapine+Edaravone group,activated microglia cells were decreased,the number of P38,ERK and JNK immunoreactive cells was reduced,but there was no statistically significant under fluorescent quantitative analysis.(2)In vitro:? Western blotting assay showed that the protein expression of MAPK(p-P38,p-JNK and p-ERK)and the proinflammatory mediators(iNOS?TNF-? and 1L-1?)was significantly increased in LPS-induced BV-2 microglia(p<0.05).Compared with LPS group,the protein expression of p-P38,p-JNK,iNOS,TNF-? and IL-1? was markedly reduced by Scutellarin or Edaravone treatment or a combination of both drugs,whereas p-ERK protein expression was increased in these groups.This was conspicuous especially in LPS+ Scutellarin + Edaravone group in which iNOS and TNF-a expression in activated BV-2 microglia was virtually abolished(p<0.05).?Immunofluorescence double staining with lectin(a specific marker for microglia)shows that there was only a small amount of p-ERK,p-JNK and p-P38 in BV-2 microglia in the control group.The number of p-ERK,p-JNK and p-P38 immunoreactive cells was significantly increased in LPS group,significant difference in fluorescent quantitative analysis compared with the control group(p<0.05).But in LPS+Scutellarin group,LPS+Edaravone group,and LPS+Scutellarin+Edaravone group,p-P38 and p-JNK immunoreactive cells were significantly decreased,whereas p-ERK increased(p<0.05).And there was no distinct diference in expression of P38,JNK and ERK among these groups(p>0.05).?The expression of p-P3 8,iNOS and TNF-a can be blocked by using SB203580(a P38MAPK inhibitor),as well as Scutellarin,significant difference compared with the LPS group(p<0.05).But there was no significant difference among SB203580 or Scutellarin treatment or a combination of both treatment was found(p>0.05).Conclusions:(1)Expression of p-P38,p-JNK and p-ERK was significant increased in MCAO rats and LPS-simulated BV-2 microglia.(2)In vivo and in vitro,both Scutellarin and Edaravone markedly attenuated the expression of p38 and JNK in activated BV-2 microglia,but increased the expression of ERK.In addition,both drugs suppressed upregulated expression of inflammatory cytokines,iNOS,TNF-a and IL-1?.A combination of two drugs was more effective in regulating the MAPK signaling pathway and inhibiting the expression of pro inflammatory mediators(iNOS and TNF-a).(3)Scutellarin can inhibit the neuroinflammation in activated microglia by regulating the P38 MAPK signaling pathway.