HMGB1 Induces The Pathogenesis Of Idiopathic Pulmonary Fibrosis By NF-κB Activation Of TGF-β1

Objection:Idiopathicpulmonaryfibrosis(IPF)is a potentially mortality lung disease,the etiology is unknown,and the early diagnosis is very difficult because of non-specific clinical manifestation.So far,IPF led to poor prognosis and mortality rates were high as a result of lack of effective clinical treatment.High-mobility group box 1(HMGB1)is a recognized nuclear protein and architectural chromatin-binding factor that binds DNA and promotes protein assembly on specific DNA targets,is mainly active released from macrophages and monocytes under inflammatory cytokines stimulation.Recent studies have indicated that HMGB1 can be passively released from necrotic cells.Extracellular HMGB1 is a multifunctional cytokine involved in the processes of underlying infection,cell apoptosis,immune responses and inflammation.The present study have indicated that HMGB1 can induce pulmonary fibroblast proliferation and EMT production,increase expression of α-SMA and E-cadherin,indicating that HMGB1 may induce fibroblast to myofibroblast differentiation.Notably,numerous studies have indicated that HMGB1 may be involved in lung fibrotic disorders.However,the role of HMGB1 in lung fibroblast to myofibroblast differentiation remains unclear.Transforming growth factor beta1(TGF-β1)is a key factor that mediates fibroblast to myofibroblast differentiation,can start the fibroblast proliferation,cause great production of collagen,and then lead to histopathological changes by pulmonary interstitial fibrosis of collagen deposition.the present study further investigated TGF-β1 plays an important role in the development of human lung fibroblasts.NF-κB is a discovery recently of new member of transcription factors family,and is the most important nuclear transcription factor.It is plays a central role in transcriptional regulation by stimulation of cell information in many cells mediated,participating in expression and regulation of variety genes,which is a sign of cell activiation.HMGB1 induce intracellular signal transduction by bonding with cell membrane receptors,which can activate NF-κB by combined with RAGE or TLR,and then lead NF-κB transfer to nuclear,induce transcription of downstream genes.Through TGF-β1 gene sequence analysis,we found that the promoter contains a NF-κB binding sites,GGGACCCCCC.Therefore,we have reasons to deduce:extracellular HMGB1 combines with RAGE/TOLL receptors,activates NF-κB,resulting in nuclear transfer,binding with TGF-β1 promoter induces large expression of TGF-β1 thus mediating mesenchymal transformation of lung fibroblasts cells,and then ultimately leading to development of IPF.We are expected to find more effective molecular target inhibition of mesenchymal transformation of lung fibroblasts cells,and control the development of IPF.Methods:①Collect in patients with IPF normal lung tissue and lung fibrosis of lung tissue specimens,comparing the degree of lung fibrosis through HE dyed;and comparing the expression of HMGB1 by using of immunohistochemical staining.②The MRC-5 human lung fibroblast cell incubated with HMGB1.α-SMA and collagenI mRNA and protein expression levels were detected by using reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and Western Blot,TGF-β1 protein expression levels were detected by ELISA.③The MRC-5 human lung fibroblast cell incubated with HMGB1,adding the TGF-β1 blocking antibody,α-SMA and collagen I mRNA and protein expression levels were detected by using RT-qPCR and Western Blot.④The MRC-5 human lung fibroblast cell incubated with HMGB1,the protein expression levels of NF-κB was detected by using Western Blot.⑤The MRC-5 human lung fibroblast cell incubated with HMGB1,adding the NF-κB inhibitor-PDTC,the protein expression levels of TGF-β1 α-SMA and collagen I were detected by using Western Blot.⑤The MRC-5 human lung fibroblast cell incubated with HMGB1,adding the TGF-β1 blocking antibody,the protein expression levels of NF-κB was detected by using Western Blot.Result:①In patients with IPF normal lung tissue and lung tissue of pulmonary fibrosis are HE staining,pulmonary fibrosis of lung tissue is more significant in patients with pulmonary fibrosis,the expression of HMGB1 obviously increases in patients with pulmonary fibrosis by using immunohistochemical staining.②The MRC-5 human lung fibroblast cell incubated with HMGB1,The mRNA and protein expression levels ofα-SMA and collagen I were increased,and the expression peaked at 72 h following 10ug/mL HMGB1 stimulation,at the same time,the protein expression of TGF-β1 peaked at 72 h.③The MRC-5 human lung fibroblast cell incubated with HMGB1,adding the TGF-β1 blocking antibody,the mRNA and protein expression levels of α-SMA and collagen I were decreased.④The MRC-5 human lung fibroblast cell incubated with HMGB1,the protein expression levels of NF-κB was decreased.⑤The MRC-5 human lung fibroblast cell incubated with HMGB1,adding the NF-κB inhibitor-PDTC,the protein expression levels of TGF-β1,α-SMA and collagen I were decreased.⑥The MRC-5 human lung fibroblast cell incubated with HMGB1,adding the TGF-β1 blocking antibody,the protein expression levels of NF-κB was uninfluenced.Conclusion:This study confirmed that HMGB1 can influence the expression of NF-κB,NF-κB activation may mediate TGF-β1 release through combined with the promoter of the TGF-β1 inducing lung fibroblasts cells to myofibroblast differentiation and the development of IPF,and we find which is an upstream regulator of TGF-β1 release in HMGB1-stimulated lung fibroblasts.Collectively,these results indicated that HMGB1 may induce lung fibroblasts cells to myofibroblast differentiation via NF-κB mediated TGF-β1 release.