| Recent studies found that nicotinic acetylcholine receptors were expressed not only in neuron cells, but also in non-neuron cells, such as endothelial cells, macrophages, epithelial cells and tumor cells. The expression of nicotinic acetylcholine receptor is involved in regulating the function of these cells and related to pathological and physiological changes in some diseases.α7 nicotinic acetylcholine receptor agonists has been used to treat many diseases.Object:Firstly, the aim of our study is to investigate the indirect and direct effect of nicotine on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs) and analyze the pathway mediated by nicotinic acetylcholine receptor.Scecondly, gx-50, an ingredient from Zanthoxylum, has been suggested to effectively active alpha7 neuronal nicotinic acetylcholine receptor by structure-based drug design methods. In the present study, we investigated whether alpha7 neuronal nicotinic acetylcholine receptor was involved in the neuroprotective effect of gx-50 on beta-amyloid (Aβ)25-35-induced neuronal damage in primary cultured cortical neurons. Methods:According to the concentration of nicotine in the serum of smokers, Ana-1 (macrophages) was challenged by different concentration nicotine, the supernatant of Ana-1 cells were collected for 3 h, 6 h, 12 h, 24 h and 48 h. The expression of IFN-γ,TNF-α,IL-1β,IL-8 were detected by RIA or enzyme-linked immunosorbent assay (ELISA). Treatment of HUVECs with supernatant from Ana-1 stimulated by 6×10-5 M nicotine for 12 h, the adhesion molecules were determined by ELISA. The supernatant from Ana-1 challenged by nicotine was pre-neutralized with anti-TNF-α, anti-IL-1β, anti-IFN-γ, or anti-TNF-α+ anti-IL-1βantibody together, respectively. The value of adhesion molecules was detected with ELISA. Adherent cells were counted in 5 microscopic high power fields.HUVECSs were treated by different concentration of nicotine. At the end of the incubation time, cell supernatants were collected. Levels of PGE2 were determined using enzyme immunoassay kit. The level of COX-2 mRNA and protein were detected by Real time-PCR and Western blot. Then, the cells were pre-incubated with 1×10-6Mα-Bungarotoxin (BTX) or 5×10-5 M PDTC for 30 min prior to 1×10-6 M nicotine exposure, then the level of COX-2 mRNA and protein were measured by Real time-PCR and Western blot. The level of PGE2 was determined by ELISA. The effect of nicotine on NF-κB DNA binding activity is measured by EMSA. After NS-398 treatment, nicotine-induced ICAM-1 protein expression was measured by Western blot and Immunofluoresence staining analyses.Cortical neurons were treated with different concentration of gx-50 for 48 h, the viability were measured by MTT assay. The inhibitory effect of gx-50 on Aβ25-35-induced neurons cytotoxicity and apoptosis, MTT assay and PI/Hoechst 33258 dual staining method were used. Cells were incubated with 10-6 M gx-50 for 2 h then exposed to 2.5×10-5 M Aβ25-35 for 48 h. The expression of anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax was detected by Real-time PCR and Western blot. The competitive binding assay was performed to verify gx-50 as an agonist ofα7 nAChR.Results:The expression of TNF-αand IL-1βin Ana-1 was improved as the increasing of nicotine concentration till 6×10-5 M. Moreover, there was an expression peak of TNF-αat 24 h as well as IL-1βat 12 h. However, nicotine could not regularly affect the release of IFN-γand IL-8. The expression of sICAM-1 and sVCAM-1 were significantly decreased in the groups of anti-TNF-α, anti-IL-1βor anti-TNF-α+anti-IL-1βpre-neutralized, and blocked partly the attachment of mononuclear cells to HUVECs. TNF-αor IL-1βrelease could be affected by nicotine with time- and concentration-dependent manner.Nicotine increased COX-2 mRNA expression in a dose-dependent manner. The maximal level of COX-2 mRNA and protein were expressed after exposure to 1×10-6 M nicotine for 2 h or 6 h. It was found thatα-bungarotoxin significantly decreased the level of COX-2 mRNA and protein by about 61.3% and 70.2% respectively of the nicotine-treated group. In addition, PDTC prevented the nicotine-induced COX-2 mRNA and protein expression by 73.5% and 80.1%. Nicotine-induced ICAM-1 protein expression was remarkably decreased after NS-398 pretreatment.Pretreatment of the cells with gx-50 for 2 h prior to 2.5×10-5 M Aβ25-35 exposure caused significantly apoptotic rate decrease and viability elevation. Furthermore, a marked reduction of bcl-2/bax ratio was found after 2.5×10-5 M Aβ25-35 exposure, which could be partly reversed by pretreatment of gx-50.Conclusion:Nicotine could increase the expression of ICAM-1 and VCAM-1 mediating TNF-α,IL-1βrelease by macrophages. In addition,nicotine induced COX-2 expression through NF-κB activation which mediated by nicotinic acetylcholine receptor and the induction of COX-2 was related to ICAM-1 expression. Furthermore, nicotine could induce COX-2 expression through activating nicotinic acetylcholine receptor and NF-κB pathway which was related to ICAM-1 expression. In addition, our research revealed that gx-50, an potentialα7 nAChR agonist could protect neurons against amyloid-induced neurotoxicity.
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