The Role Of HMGB-1 In Silica-induced Pulmonary Inflammation And Fibrosis

Crystalline silica is one of the most abundant minerals on earth and a common particulate air pollutant in working and living environments.Occupational exposure to crystalline silica frequently occurs in a variety of industries,such as metal and coal mining,construction,whenever substances or materials containing silica.Living exposure to silica occurs in volcanic explosions and sandstorms.Prolonged exposure to crystalline silica has been known to be associated with increased risk for adverse health effects such as silicosis,lung cancer and other pulmonary diseases.Silicosis,identified by pulmonary parenchyma fibrosis,is a chronic occupational lung disease caused by long-term inhalation of silica dust in the workplace.Pathologic features of silicosis involve focal collections of dust and reticulin around the small airways,fibrotic lesions exhibiting irregularly arranged collagen,and lesions of massive fibrosis.Although prevention efforts have been required to implement for decades,silicosis is still one of high incidence occupational diseases worldwide,especially in China.Previous studies has showed that inhalation of respirable silica dusts could active Nalp3 inflammasome.Activation of the Nalp3 by silica also necessitates generation of inflammasome such as IL-1? etc.Furthermore,the uptake of silica particles could lead to cell rupture or death with concomitant release of intracellular silica particles that are re-ingested by other macrophages.It is well acknowledged that recurring cycles of macrophages phagocytosis may be one mechanism of the persistent and chronic inflammation elicited by silica particles.High-mobility group box protein-1(HMGB-1)is best known as an architectural transcription factor because of its ability to regulate gene activity through DNA bending.However,additional roles for HMGB-1 have been identified including the ability to bind to and modulate NF-?B,and as a secreted factor capable of regulating the expression of proinflammatory cytokines by binding to cell surface receptors,particularly the receptor for advanced glycation end products(RAGE)and the toll-like receptors 2 and 4.Those proinflammatory cytokines,such as tumour necrosis factor-?(TNF-?)and IL-?,induce HMGB-1 transform from cell nucleus to cytoplasm.Meanwhile,cell rupture or death,could release those proinflammatory cytokines to extracellular,lead to robust the inflammatory responses.This process was repeated and lead to the generation of chronic inflammation and fibrosis.So far,although numerous studies investigated the role of HMGB-1 induced by silica.However,those studies were just investigated the expression of HMGB-1 and its main receptor(RAGE)in silica-induced in vivo or vitro,and there is still unclear how HMGB-1 exert its function and the impact of HMGB-1/TLR4/NF-?B signaling pathway in silicosis.Therefore,we hoped to get better and comprehensively understanding the role of HMGB-1 in development of silicosis.The objective of this study was to explore the role of HMGB-1 in silicosis.In this study,we hypothesized that HMGB-1 has ability to regulate downstream signaling pathway,to robust the inflammation responses,eventually generated fibrosis.We designed mice experiments and case-control study to explore the function of HMGB-1 in silica-induced pulmonary inflammation and fibrosis.The main parts of this study are as follows:(1)To explore the expression of HMGB-1 induced by silica and the effect of HMGB-1 neutralizing antibody in mice.(2)To assess the role of HMGB-1 in silica-induced pulmonary inflammation and fibrosis.(3)A case-control study to investigate the association between HMGB-1/its main receptor and silicosis.Part I.The expression of HMGB-1 and the effect of HMGB-1 neutralizing antibody in silica-induced acute inflammatory responses in miceObjective:To assess the expression of HMGB-1 and the effect of HMGB-1 neutralizing antibody in silica-induced in mice.To determine the dose of antibody and the best observation time in the further studies.Methods:In this study,we generated an acute inflammatory response model by intratracheally instilled silica dust to mice and used HMGB-1 neutralizing antibody to directly neutralize the silica-induced HMGB-1 in the mice.A total of 120 C57BL/C mice were randomly divided into four groups as the normal saline group,silica group and silica+anti-HMGB-1 and silica+rmHMGB-1 groups.At 6h,1,2,3,7d after instilled treatment,6 mice of each group were sacrificed and the HMGB-1 concentrations in BALF were measure by enzyme linked immunosorbent assay(ELISA).Results:At day 1,2,3,7 after instilled treatment,compared to the NS groups,the exposure groups had a higher expression of HMGB-1 in BALF.Compared to the silica groups,the anti-HMGB-1 groups were significantly decreased.However,compared to the former days,on day 7 after treatment groups,the expression of HMGB-1 in BALF was decreased.There was no significantly difference between silica group and rmHMGB-1 group.Conclusions:HMGB-1 neutralizing antibody could neutralize the silica-induced HMGB-1 in mice;The expression of HMGB-1 decreased on day 7 after once instilled silica dust.Part ?.The effect of HMGB-1 in silica-induced inflammatory responses and fibrosis in miceObjective:To explore the role of HMGB-1 in silica-induced lung damage in mice;To explore the role of HMGB-1 in silica-induced pulmonary inflammatory;To investage the effect of HMGB-1 in silica-induced lung fibrosis.Methods:In this study,we generated an acute inflammatory response model by intratracheally instilled silica dust to mice and used HMGB-1 neutralizing antibody to directly neutralize the silica-induced HMGB-1 in the mice.A total of 192 C57 mice were randomly divided into six groups as the blank group,normal saline group,silica group,silica+IgY group and silica+anti-HMGB-1 and silica+rmHMGB-1 groups.At 2,7,28,84 day after instilled treatment,8 mice of each group were sacrificed;The 28 d and 84 d groups instilled treatment once a week;ELISA was used to detect the concentration of IL-6,HMGB-1,TNF-? in BALF in mice;RT-PCR was used to detect the relative expressions of IL-6,IL-1?,MCP-1,TNF-?,TGF-1?,COL1A1,COL3A1,Fibronectin,TLR4,NF-?B mRNA in lung tissues;H&E,Masson and Sirus red staining were observed to investigate the pathological changes and deposition of collagen fibers in mice.Results:?.Compared with blank and ns groups,the exposure groups had a higher expressions of TP,LDH,ACP,ACP in BALF in mice;There was no difference between anti-HMGB-1 groups and silica,silica+IgY groups in those indicators;?.The results of H&E staining showed that the significant inflammation response was observed in exposure groups.Compared to the silica and silica+IgY groups,anti-HMGB-1 groups' inflammation response were alleviated;Compared with control groups,the numbers of total cells,macrophages and neutrophils were increased in exposure groups,HMGB-1 neutralizing antibody could decrease those indicators;The concentrations of HMGB-1,IL-6,TNF-? were significant increasing in those exposure groups,and HMGB-1 neutralizing antibody could decrease those indicators;Compared between silica,silica+IgY groups and anti-HMGB-1 groups,the relative expressions of IL-6,IL-1? MCP-1,TNF-? mRNA decreased in anti-HMGB-1 groups.?.The Masson staining results showed that the significant fibrosis was observed in exposure groups.Compared to the silica and silica+IgY groups,anti-HMGB-1 groups fibrosis were alleviated;The RT-PCR results,compared between silica,silica+IgY groups and anti-HMGB-1 groups,the relative expressions of TGF-?,COL1A1,COL3A1,Fibronectin mRNA decreased in anti-HMGB-1 groups.Meanwhile,the HMGB-1 neutralizing antibody could decrease the relative expressions levels of TLR4,NF-?B mRNA.Conclusions:HMGB-1 neutralizing antibody can not alleviate the lung damage induced by silica dust;HMGB-1 neutralizing antibody decrease silica-induced pulmonary inflammation and fibrosis;HMGB-1/TLR4/NF-?B signaling pathway may be participant in progression of silicosis in mice.Part ?.The role of HMGB-1 and its main receptors in the development of silicosis:A case-control studyObjective:To invsetage the associations of HMGB-1 and its main receptors with silicosis.Methods:We recruited 133 silicosis patients and 125 age-matched healthy controls.All subjects were male;We recruited the personal information and occupational history and smoking status etc.ELISA was used to detect the concentrations of TGF-?1 COL1A1,COL3A1,MMP-2,MMP-9 in plasma,and RT-PCR was used to detect relative expressions of TLR2,TLR4,RAGE mRNA in peripheral blood.The correlations associations of HMGB-1 and its main receptors with silicosis were also analyzedResults:HMGB-1 and TLR2,TLR4,RAGE mRNA relative expression levels were up-regulated in silicosis patients;HMGB-1 was positively associated with TLR2 and TLR4(r=0.26,P=0.02;r=0.25,P=0.01);HMGB-1 was associated with silicosis,the OR(95%CI)for silicosis was 1.68(1.37-2.06);Compared with healthy controls,for detecting silicosis,the under the curve(AUC)was 0.84.Conclusions:Compared with healthy controls,HMGB-1 and its main receptors were up-regulated in silicosis patients;HMGB-1 was associated with silicosis;and may be a potential biomarkers for silicosis.