Effects And Mechanisms Of Dendorbium Nobile Lindl.alkaloids On NF-κB/NLRP3 Pathway In Lipopolysaccharide-induced BV2

Objective: To investigate the anti-neuroinflammation of Dendorbium Nobile Lindl.alkaloids(DNLA)and its pharmacological mechanism via lipopolysaccharide(LPS)-induced BV2 cells.Methods: BV2 cells were co-operated with LPS performing as the model of neuronal inflammation.BV2 cells were divided into control,DNLA(350 ng/m L),LPS(100 ng/m L),LPS + DNLA(3.5 ng/m L),and LPS + DNLA(350 ng/m L).BV2 cells were pre-treated with DNLA(3.5 ng/m L or 350 ng/m L)for 1 hour,and then incubated with LPS(100 ng/m L).Proliferation of BV2 cells was detected by MTT assay,TNF-α in supplement was detected by ELISA kits.The m RNA expression of IL-1β was detected by RT-PCR.In addition,the protein expression of Iba1、NLRP3、ASC、caspase-1、NEK7、GSDMD in total protein solution and NF-κB p65,p-NF-κB p65,IκBα,p-IκBα respectively in cytosol and nucleus of BV2 cells were detected by Western Blot.Iba-1 and the NLRP3/NF-κB pathway were also proceeded to morphological analysis by immunofluorescence.Results:(1)MTT、ELISA and Western Blot together suggested that 100 ng/m L is the best concentration of LPS to make the inflammatory model,TNF-α production in cellular supernatant was at the maximum when LPS(100 ng/m L)induced BV2 cells for 24 h,and p-IκBα in cytosol and p-NF-κB in nuclear increased remarkably when LPS(100 ng/m L)induced BV2 cells for 1h.(2)ELISA assay suggested that after BV2 cells were treated with DNLA,TNF-α production in cellular supernatant was remarkably lower than the LPS group.DNLA suppressed LPS-induced microglial activation.DNLA decreased the LPS-induced TLR4,NLRP3,ASC and Caspase-1 expression.DNLA affected protein levels of IκBα,NF-κB and their phosphorylation products in cytoplasm and nucleus.Immunofluorescence staining assay indicated that DNLA inhabited NF-κB to transfer from cytoplasm into cell nucleus and inhabited NLRP3 inflammasome to activate.LPS increased Il-1β m RNA,while that increase was overturned by DNLA.(3)LPS and DNLA did not affect GSDMD obviously,but cleaved-GSDMD was increased by LPS(100 ng/m L)and it was decreased by DNLA(350 ng/m L).Conclusion: DNLA inhibited LPS-induced neuroinflammation in BV2 cells,and that effect might be connected with the NF-κB/NLRP3 pathway.