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

Objective:This study aimed to investigate the effects of Scutellarin on MAPK signal pathway(extracellular regulated protein kinases ERK2,p38 mitogen-activated protein kinase p38,c-jun N-terminal Kinase JNK)and the expression of inflammatory mediators(iNOS,TNF-?)in activated microglia.Along with this,we also explored the mechanism via which Scutellarin exerts its effect on microglia activation in vitro and in vivo.Methods:The BV-2 microglia were randomly divided into control,LPS induced group and LPS+Scutellarin groups.Expression of MAPK signal pathway(ERK2,p38 and JNK)and the inflammatory mediators(iNOS,TNF-?)in activated microglia was detected by the immunohistochemistry,immunofluorescence staining and Western blot.The animal model of microglia activation in rats was established following intraperitoneally injection of LPS.Healthy 3-day-old neonatal SD rats were randomly divided into control,LPS induced,LPS+Breviscapine low-dose group(5mg/kg),LPS+Breviscapine medium-dose group(10mg/kg)and LPS+Breviscapine high-dose group(20mg/kg).Expression of MAPK signal pathway(ERK2,p38 and JNK)and the inflammatory mediators(iNOS,TNF-?)in activated microglia in activated microglia in the corpus callosum was also assessed by the immunohistochemistry and Western blotResults:?Immunohistochemistry results show that iNOS and TNF-a were expressed in cytoplasm,while P-p38 and P-JNK were in nucleus.The cells expressed a small amount of iNOS,TNF-? and P-p38 in the control.A large number of microglia were activated in LPS induced group,expression of iNOS,TNF-? and P-p38 was dramatically increased.However,in LPS+Scutellarin group,expression of iNOS,TNF-? and P-p38 was significantly decreased.In contrast,P-ERK1/2 immunoreactive cells were increased compared to LPS induced group.Expression of P-ERK1/2 can be observed in nucleus.? Immunofluorescence staining shows that expression of iNOS,TNF-?,P-p38 and P-JNK was negligible in the control.A large number of activated microglia with polygonal or oval cell body can be seen in LPS group.Expression of P-p38,P-JNK,iNOS and TNF-? was significantly increased in LPS induced activated microglia.But in LPS+Scutellarin treated group,the numbers of P-p38,P-JNK iNOS,TNF-? immunoreactive cells were significantly reduced.Expression of P-ERK1/2 was lower in control group while increased in LPS group,and P-ERK1/2 expression was enhanced in lectin labeled activated microglia when treated with Scutellarin.? Western blot assay showed that the protein expression of iNOS,TNF-?,P-p38 and P-JNK was significantly increased in LPS induced-activated BV-2 microglia There was significant difference compared with the control group(p<0.05).Expression of iNOS,TNF-?,P-p38 and P-JNK protein was significantly suppressed in cells of LPS+ Scutellarin group compared with the control(p<0.05).Expression of P-ERK1/2 was significantly increased in LPS+Scutellarin group compared with the LPS group(p<0.05).There was no significant difference in the protein expression of p38 and ERK2 among all groups in vitro(p>0.05).? Western blot assay showed that the protein expression of iNOS,TNF-a and P-p38 was significantly increased at 24h in corpus callosum in LPS induced rats compared with the control group(p<0.05).The protein expression ofiNOS,TNF-? and P-p38 was significantly reduced in LPS+Breviscapine high-dose rats,significant difference compared with the LPS group was expressed as p<0.05.However,there is no significant difference between low-dose,medium-dose and LPS groups(p>0.05).The protein expression of P-ERK1/2 was significantly increased in LPS+Breviscapine high-dose group rats compared with the control(p<0.05).There was no significant difference in the protein expression of p38,ERK2 among different groups in vivo(p>0.05).Conclusions:? Scutellarin can significantly reduce the protein expression of p38 and JNK of the active BV-2 microglia,but increase the expression of ERK2.Scutellarn may decrease the expression of inflammatory mediators iNOS and TNF-? in activated microglia through MAPK signal pathway,therefore exert its protective effect on microglia mediated inflammation.?Breviscapine can significantly reduce the protein expression of p38 and the inflammatory mediators iNOS and TNF-? in corpus callosum in LPS induced rats.