The Regulatory Effects Of RELMβ On Cigarette Smoke Induced Inflammation Of Airway Epithelial Cell 16HBE And Its Mechanism

Chapter 1 RELMβ was induced in human bronchial epithelial cell 16 HBE by cigarette smoke extraction and its regulatory role on pro-inflammatory factors Objectives: To detect RELMβ expression in human bronchial epithelial cell 16 HBE which was induced by cigarette smoke extraction(CSE)and its regulatory role on pro-inflammatory factors.Methods: 1.Human bronchial epithelium 16 HBE was cultured in vitro.CCK8 assay was used to determine the cytotoxicity of different concentrations of CSE on 16 HBE.2.16 HBE cells were stimulated by different dose of CSE for 24 h.The expression of RELMβ mRNA was detected by RT-PCR.Then 16 HBE cells were stimulated by different time and dose of CSE.RELMβ protein levels were detected by western blot.3.RELMβ-overexpressed cell line was built and identified.4.In RELMβ-overexpressed cell line and vector control,mRNA level of IL-1β,IL-8,IL-6 and TNF-α was detected by RT-PCR.The secretion of IL-1β,IL-8,IL-6 and TNF-α was detected was by MSD-ECL assay.5.After stimulating by recombinant human RELMβ(rhRELMβ),mRNA level of IL-1β,IL-8,IL-6 and TNF-α was detected by RT-PCR.The secretion of IL-1β,IL-8,IL-6 and TNF-α was detected was by MSD-ECL assay.Results: 1.CCK8 results showed that the cell survival rate in 15%CSE group was lower than that in the control group after 24 hours stimulation(P < 0.05).After 48 hours,the survival rate had no significant difference among the control group,2.5% CSE group and 5% CSE goup(P > 0.05).The cell survival rate of 7.5% CSE group,10% CSE group and 15% CSE group were significantly lower than that in the control group(P < 0.05).2.RT-PCR results showed that the expression of RELM in 5% and 7.5% CSE group were increased compared with that in the control group,and the difference was statistically significant(P < 0.05).WB results showed that RELMβ protein level was significantly upregulated in 7.5% CSE stumulated after 12 hours(P < 0.05).After 24 or 48 hours stimulation,RELMβ protein level in 2.5%,5% and 7.5% CSE group was significantly higher than that in the control group(P < 0.05).3.The mRNA and protein levels of RELMβ in the overexpression group were significantly higher than those in the vector group.It is demonstrated that the RELMβ overexpression cell line was successfully constructed.4.The results of MSD-ECL assay showed that the expression of IL-1β,IL-8,IL-6 and TNF-α was significantly higher in supernatant of RELMβ-overexpression group than those in the control group(P < 0.05).RT-PCR showed that the mRNA levels of IL-1β and IL-8 in the overexpression group of RELMβ were higher than those in the vector group(P < 0.05).The mRNA level of IL-6 or TNF-α had no significantly difference(P > 0.05).5.MSD-ECL assay showed that the secretion of IL-1β,IL-8,IL-6 and TNF-α was higher than those in the control group(P<0.05)after stimulating with different dose of rhRELMβ.In different time stimulation,the secretion of IL-1β,IL-8,IL-6 and TNF-α in supernatant increased in time-dependent manner(P<0.05).RT-PCR showed that the mRNA expression level of IL-1β in 50ng/mL group and 100ng/mL group was higher than that in the control group(P<0.05).IL-8 mRNA expression began to increase from 25ng/mL group and kept upreguating with the increasing dose of rhRELMβ(P<0.05).In different time groups,the mRNA levels of IL-1β and IL-8 were upreguated since 6 hour with rhRELMβ(P < 0.05).The mRNA levels of IL-6 or TNF-α had no significantly difference with control(P > 0.05).Conclusion: 1.CSE can induce the mRNA and protein expression of RELMβ in 16 HBE cells.2.RELMβ-overexpresstion 16 HBE cell line was successfully constructed.3.RELMβ can promote secretion of IL-1β,IL-8,IL-6 and TNF-α,and induce mRNA expression of IL-1β and IL-8 in 16 HBE.Chapter 2 Mechanism of RELMβ in regulating IL-1β and IL-8 Objective: To investigate the regulation mechanism of RELMβ in IL-1β and IL-8 by activating NF-κB signaling pathway.Method: 1.Western blot was used to detecte the expression of the related protein of NF-κB pathway in RELMβ-overexpression cell lines and rhRELMβ stimulation.2.To detect the expression of NF-κB/p65 in the cytoplasm and nucleus in RELMβ-overexpression cell lines and rhRELMβ stimulation by western blot analysis.3.Western blot was used to detecte the degradation of IκBα in RELMβ-overexpression cell lines and rhRELMβ stimulation.4.To detect the expression of IL-1β and IL-8 after NF-κB signal inhibitor PDTC or BAY 11-7082 by western blot analysis.Results: 1.The expression of IKKβ,IκBα and NF-κB/p65 had no significant difference between vector group and RELMβ overexpression groups.The expression of IKKα,p-IKKα/β,p-IκBα and p-NF-κB/p65 in RELMβ overexpression groups was higher than those in the pLV.0-NC group.After stimulating with different concentrations of rhRELMβ for 48 hours,the protein levels of p-IKKα/β,p-IκBα and p-NF-κB/p65 increased with the rising concentrations of rhRELMβ,while IKKβ,IκBα and NF-κB/p65 expression did not change significantly.With the concentration of 100 ng/mL rhRELMβ after different time,protein expression levels of p-IKKα/β,p-IκBα and p-NF-κB/p65 began to rise after 30 min stimulation.With the total NF-κB/p65 gradually reducing,IKKα,IKKβ and IκBα did not change significantly.2.The expression levels of NF-κB/p65 in cytoplasm had no significant difference between vector group and overexpression group.However,in the nuclear protein,the expression of NF-κB/p65 in the overexpression group was higher than that in the vector group.With the increasing of recombinant protein concentration,the expression of NF-κB/p65 in the cytoplasm gradually decreased,while the expression of NF-κB/p65 in the nucleus significantly increased in the 50ng/mL group.In 100ng/ mL group,NF-κB/p65 expression was reduced but still higher than that in then control group.After stimulating in different time with 100ng/mL of rh RELMβ,NF-κB/p65 in the cytoplasm gradually decreased,and its expression in the cell nucleus gradually increased in a time dependent manner.3.The expression level of IκBα in overexpression group with cycloheximide decreased at 8 hour.After rhRELMβ stimulation,IκBα degradation was not observed in the control group.In the experimental group,the expression level of IκBα at 6 hour was significantly lower than that at 0 hours,and the degration was observed at 8 hour.4.With different concentrations of PDTC and Bay-117082,p-NF-κB/p65 expressoin reduced with the decreased concentration of inhibitors in overexpression and vector control cells,and the protein expression of IL-1β and IL-8 also decreased significantly.Pretreated with PDTC and BAY 11-7082 for 2 hours,cells were stimulated with rhRELMβ.The expression of p-NF-κB p65、IL-1β and IL-8 was lower than those in the group which was only treated with rhRELMβ.Conclusion: 1.In 16 HBE bronchial epithelial cells,RELMβ is able to phosphorylate IKK,then makes IκBα phosphorylation and promotes its degration,and then phosphorylate NF-κ B/p65,so that NF-κ B/p65 translocates into cell nucleus,activating NF-κB signaling pathway.2.RELM regulates IL-1β and IL-8 expression via NF-κB signaling pathway.Summary 1.CSE can induce expression of RELMβ in 16 HBE cells.2.RELMβ-overexpresstion 16 HBE cell line was successfully constructed.3.RELMβ can promote the secretion of IL-1β,IL-8,IL-6 and TNF-α,and upregulate the mRNA expression level of IL-1β and IL-8.4.RELMβ is able to phosphorylate IKK,then makes IκBα phosphorylation and promote its degration,and then phosphorylate NF-κ B/p65,so that NF-κ B/p65 translocates into cell nucleus,activating NF-κB signaling pathway.5.RELM regulates IL-1β and IL-8 expression via NF-κB signaling pathway.