Preliminary Study Of The Effect And Mechanism Of Substance P On Neisseria Meningitidis Antigen Induced COX-2Expression And PGE₂Secretion In Microglias

ObjectiveTo investigate the influence and mechanism of Substance P on Neisseria meningitidisantigen-induced COX-2expression and PGE2production in microglias.MethodsThe rat primary microglias was isolated and cultured in vitro, and divided into fourgroups according to the experimental purposes:(1) Control group (Equal volume ofDMEM/F12medium was added);(2) NMA group (Cells were treated with ofultrasonic treated crude antigen for8h);(3) SP group （microglias were incubatedwith1nmol/L SP for8h）(4) SP+NMA group（Cells were stimulated with10μg/mlantigen and1nmol/L SP for8h;(5) Antagonist group (microglias were pretreatedwith10nmol/L L-703,606，a SP receptor antagonist, for15min). Expression of COX-2mRNA was determined by real-time PCR, production of PGE2was detected byELISA, and nuclear translocation of NF-κB p65subunit and the expression of PGE2receptor EP2/EP4were analyzed by Western blot.Results1. Real-time PCR results show that the COX-2mRNA expression level in SP groupwas very low (0.24±0.061VS0.03±0.032, P<0.05). Compared with the controlgroup, NMA treatment only slightly increased COX-2expression（0.32±0.042VS0.03±0.032, P<0.05). However, the mRNA expression level was significantlyincreased when treated by combination of NMA and SP (0.80±0.052VS0.32±0.042, P<0.05). Furthermore, the cooperative effect of SP on COX-2mRNAexpression in microglias was reduced after treated by SP receptor antagonist L-703,606(0.43±0.086VS0.80±0.052, P<0.05).2. ELISA showed SP per se could induce production of PGE2to some degree(24.35±4.27pg/mL VS18.45±3.31pg/mL, P<0.05), and NMA could inducemicroglias production of PGE2(24.35±4.27) pg/mL VS (18.45±3.31) pg/mLP<0.05). While microglias were co-incubated with NMA and SP, PGE2secretionwas significantly increased (126.47±14.68pg/mL, P<0.01). However, PGE2secretion was decreased after pretreat with L-703,606, as compared with that inSP and NMA group （59.61±5.52pg/mL, P<0.05).3Western blot evidences that few NF-κB p65subunit translocate into nucleus afterantigen stimulation (P<0.05), but was significantly increased after antigen and SPstimulation (P<0.05). Similarly, p65translocation was reduced after pretreatmentof L-703,606.4There were no obvious changement of PGE2receptor EP2and EP4in NMA andSP group (P>0.05), but was significantly increased after NMA and SP (P<0.05).Likewise, L-703,606pretreatment could decrease EP2and EP4expression(P<0.05).Conclusion1. SP can synergize NMA to induce microglias transcription of COX-2mRNA andproduction of PGE2, and to induce expression of prostaglandin receptor EP2andEP4.2. The synergy effect of SP on the expression of COX-2may achieve by activationof NF-κB...