The Mechanism Of LPS-induced Endoplasmic Reticulum Stress Inflammatory Response Under APS Intervention In BEAS-2B Cells

Increasing intensive cultivation density exacerbates the stress of poultry respiratory disease whose cellular mechanism involves multiple organelles,cytokines and signaling pathways in poultry lung injury.Endoplasmic reticulum stress(ERS)is an important pathway mediating the fate of airway structure cells that distinguish between mitochondrial and death receptor pathways.Epigenetic aims to explain clinical symptoms in molecular areas which participate in various life processes such as cell growth,differentiation etc.In this study,for providing a theoretical basis of the nutri-epigenetic regulation on pulmonary inflammatory response,we firstly established ERS model in vitro by stimulating of lipopolysaccharide(LPS),and then analyzed the expression of epigenetic modified enzyme and its biological function.Finally,we studied how astragalus polysaccharides(APS)regulate the production of LPS-stimulated pro-inflammatory cytokines.Experiment 1 Establishment of endoplasmic reticulum stress model stimulated by LPS in BEAS-2B cellsTo establish ERS model in vitro study,firstly,we detected the expression of the ERS marker immunoglobulin heavy chain binding protein(BIP)induced by LPS in BEAS-2B cells.Then,we analyzed the expression of unfolded protein response(UPR)signaling pathway proteins inositol requires enzyme 1α(IRE1α),protein kinase RNA-like endoplasmic reticulum kinase(PERK)and activation transcription factor 6(ATF6)to further explore the effect of LPS on ERS in BEAS-2B cells and its mechanism.The relationship of dose-effect or time-effect of ERS related protein expression was detected by Western Blot in LPS-induced BEAS-2B cells.The results of dose-effect indicated that the addition of 100 ng/m L,200 ng/m L and 500 ng/m L LPS increased the expression of BIP(P < 0.01)in BEAS-2B cells in a dose-dependent manner.200 ng/m L LPS and 500 ng/m L LPS could significantly improve the protein expression of PERK and IRE1α(P < 0.01)in BEAS-2B cells except ATF6(P > 0.05).The results of time-effect showed that 200 ng/m L LPS induced BEAS-2B cells for 0.5h,1h,6h,12 h and 24 h,up-regulated the protein expression of PERK(P < 0.05)after 6 h whereas BIP and eukaryotic translational initiation factor 2α(e IF2α)(P < 0.05)at later 12 h in BEAS-2B cells in a time-dependent manner,only increased Phosphorylated PERK(P < 0.05)at 6 h after LPS stimulation.Thus,LPS-stimulated BEAS-2B cells could successfully construct ERS model,and UPR signaling pathways PERK and IRE1α were selectively activated.Experiment 2 The effect of PRMT1 in BEAS-2B cell PERK-e IF2α signaling pathwayTo investigate the effect of PRMT1 on ERS signal pathway,the relationship of dose-effect or time-effect on protein arginine N-methyltransferase 1(PRMT1)expression in LPS-stimulated BEAS-2B cells was detected by RT-q PCR and Western Blot,and the subcellular localization of PRMT1 was observed by immunofluorescence assay.Also,methods of knockdown and overexpression of PRMT1 were utilized to study the potential role of PRMT1 in PERK-e IF2α signaling pathway.The results of dose-effect illustrated that the addition of 50 ng/m L LPS,100 ng/m L LPS,200 ng/m L LPS,and 500 ng/m L LPS could decrease PRMT1 m RNA expression(P < 0.001)in BEAS-2B cells compared with control while the expression of PRMT1 reduced only at 200 ng/m L and 500 ng/m L LPS induction in protein level(P < 0.05).The results of time-effect showed that the expression of PRMT1 protein was lower than that of the control after induction of BEAS-2B cells with 200ng/m L LPS for 1 h(P < 0.05),and it had a time dependent manner.Western Blot results demonstrated that 10 μM arginine N-methyltransferase inhibitor-1(AMI-1)inhibited the expression of PRMT1(P < 0.05)in BEAS-2B cells for 12 h,but it has no effect on phosphorylated PERK(P > 0.05);There exsited a significant increase in BEAS-2B cells PRMT1 transcription level(P < 0.001)and protein level(P < 0.01)after overexpression PRMT1,However,decreased expression of PERK-e IF2α signal pathway proteins phosphorylated PERK(P < 0.01)and its downstream e IF2α protein(P < 0.05).These results suggest that PRMT1 plays an important role in the upstream of PERK-e IF2α signaling pathway.Experiment 3 Anti-inflammatory activity of APS on BEAS-2B cells induced by LPS in Vitro studyIn order to clarify the functional significance of LPS-induced ERS and the anti-inflammatory activity of astragalus polysaccharides(APS)on LPS-induced BEAS-2B cells,we studied the effect of different LPS concentration on BEAS-2B cells proliferation and numbers,thereby to clear the amount of LPS additive for subsequent anti-inflammatory tests.Secondly,we utilized RT-q PCR technology to study the effects of LPS,APS,LPS + APS on BEAS-2B cells inflammatory cytokines expression.Experiment of MTT illustrated that addition 1 μg/m L LPS reduced BEAS-2B cells OD570 value(P < 0.001)after stimulating for 24 h,whereas 50 ng/m L,100 ng/m L,200 ng/m L and 500 ng/m L LPS additions have no significant effect(P > 0.05).Therefore,200 ng/m L LPS and 500 ng/m L LPS were selected for subsequent anti-inflammatory studies.The results of RT-q PCR showed that the addition of 200 ng/m L LPS and 500 ng/m L LPS could increase the expression of TNF-α(P < 0.05)in BEAS-2B cells,but there was no significant effect on TNF-α m RNA(P > 0.05)expression dealing with 200 ng/m L LPS + 200 μg/m L APS and 500 ng/m L LPS + 200 μg/m L APS.IL-1β m RNA(P < 0.01)expression was also increased by LPS induction,200 ng/m L LPS + 200 μg/m L APS had no significant change while 500 ng/m L LPS + 200 μg/m L APS significantly decreased the expression of IL-1β(P < 0.05)compared with 500 ng/m L LPS.All of which demonstrated APS had anti-inflammatory and protective effects on BEAS-2B cell stimulated by LPS,by decreasing the expression of inflammatory cytokines.Overall,PRMT1 regulates LPS-induced BEAS-2B cells ERS inflammatory reaction which could be alleviated by APS.