Effects Of Oxygenation/Antioxygenation Imbalance In Pathogenesis And Therapy Of Pulmonary Interstitial Fibrosis

Interstitial lung disease consists of more than 200 kinds of diseases with apotentially fatal prognosis and unclear pathogenic mechanism.There are some commoncharacterstics of the clinical manifestation,laboratory and pathological changes in themand respective characters in each one. The pathologic features of the pulmonary fibrosisare characteristic of alveolar epithelium injury and excessive accumulation of inflame-matory cells,fibroblast overproliferation,extracellular matrix deposition,fibroprolifer-ation foci and honeycomb lung.The bleomycin-induced may lead to acute lung injury and pulmonary fibrosis inrodents and this is a mature model of pulmonary fibrosis research. Recent studysindicate that the oxygenation/antioxygenation imbalance(oxidative stress) can activateNuclear factor-κB(NF-κB).However,true mechanism is unclear now.NF-κB is animportanttranscription factor and may regulate the transcription of many inflammatorycytokines.P65 is an important function subunit in NF-κB.Our previous research showedthat the expression of NF-κB significantly augmented in pathological process bybleomycin-induced pulmonary fibrosis.This experiment investigates the relationbetween NF-κB activation and oxygenation/ antioxygenation imbalance and the effectin interstitial pulmonary fibrosis,and study the possible mechanisms which N-acetylcysteine(NAC) treats pulmonary interstitial fibrosis.Material and Methods Experimental material1 .Experimental animal8 weeks old C57BL/6,female(weight:17-20g) were maintained in specificpathogen-free conditions(Beijing Weitong Lihua experimental animal technologycompany).2．Experimental drugs10%Chloralhydrate,Bleomycin(Tianjin Taihe pharmacy co,ltd),N-acetyl cysteine(Hainan Zanbang pharmacy co,Itd).3．Experimental reagentsP65 Immunohistochemical(IHC) assay kit(Beijing Zhongshan goldenbridgebiotechnology co,ltd) and P65 m-RNA in site hybridization assay kit(Wuhan Bosterbiotechnology co,ltd);IL-4 and TGF-β1 enzyme-linked immuno-sorbent assay(ELISA)kit(Shanghai Senxiong science and technology industry co,ltd);Hydroxyproline (HYP),Glutathione(GSH) and Malonyldialdehyde(MDA) assay kit(Nanjing Jiancheng bio-technology research institute).4．Experimental equipmentultraviolet light,visible light spectrophotometer (France,S500-P);full-automatedmicroplate reader(Austria,TECAN);ultrasound homo-genate machine(Germany,UPH-200) .Animal model,experimental groups and tissue preparation1 .Animal model establishing and experimental groupingEighty female C57BL/6 were randomly divided into three groups,Mice wereanesthetized with 10%Chloralhydrate by intraperitoneal injection and operated toexpose the trachea in sterile condition.①Model group:Thirty-five mice were treatedwith Bleomycin(BLM,5mg/kg weight in 20ul sterile isotonic saline) by intratrachealinjection on day 0．②Therapeutic group:N-acetylcysteine(NAC,250mg/kg) wasadministrated via esophagus 5 days before bleomycin treatment in thirty-five miceuntil sampling,once a day.③Control group:Ten mice were treated with sterile isotonic saline(20ul) by intratracheal injection on day 0,Mice were killed seven days later.2．Sampling and preparation of lung tissue specimensAt 1,3,7,14 and 28 days postinstillation,animals were euthanized by exsangu-ination from abdominal aorta.(1) Bronchonalveolar lavage Mice anesthetized were fixed supinely.More than70 percent of Brochoalveolar lavage fluid(BALF) was obtained by washing the rightlung three times with 1ml aliquots of saline through a tracheal cannula.Cellsuspensions were centrifugated at 3000r/min for 10 minutes.Supernatant and left lungwere frozen at-70℃until thawed for assay.Precipitated cells were centrifugatedwith low speed and made smear. Immunohistochemical(IHC) staining and in sitehybridization staining were completed to detect the expression of P65 and its m-RNAin the brochoalveolar lavage cells.(2) Pulmonary tissue fixation and preparation Mice were killed with exsanguin-ation.Chest was opened and left bronchus was ligated.Right lung was first perfused byits main bronchus with a fixative solution(4% paraformaldehyde),immersed in thefixative for 3 h and embedded in paraffin, and made tissue wax block.Histopathological assessment1 .Routine pathological manifestationLung tissue were dehydrated in a graded series of ethanols,cut into 5μm-thickserial sections,stained with haematoxylin-eosin and Masson's trichrome.2．Histopathological grading of extent of alveolar inflammation and fibrosis inlung parenchymaAccording to Szapie?'s method,grade 0 for normal tissue, grades 1～4 forpresence of pulmonary inflatmmation and fibrosis,the severity of lesions was graded as1(mild), 2(moderate), 3 (severe) and 4(severe inflammation accompanied by totaldistortion of structure).Index determined and methods1．The content of HYP in pulmonary tissue was measured by sample base- hydrolyzation according to manufacturer instructions.2．The contents of GSH,MDA and protein in lung tissue or BALF were determinedwith colorimetry according to manufacturer instructions.3．ELISA was used to measure the concentrations of IL-4 and TGF-β1 in BALFaccording to manufacturer instructions.All plates were read on a microplate reader.Typically,standard curves generated with this ELISA were linear in the 0 to 1000 pgIL-4/ml or 2000 pg TGF-β1/ml range.4．IHC staining and in site hybridization staining were respectively completed todetect the expression of P65 and its m-RNA in the brochoalveolar lavage cell accor-ding to manufacturer instructions.5．The weight of mice were observed too.Statistical AnalysisStatistical analysis was done with the Statistical Package for the Social Sciences(SPSS) 13．0．Values were expressed as mean±SD,T test were used to analyze thequantitative data. A value of less than 0．05 was considered statistically significant.ResultsResults of histopathological analysis in miceOn day 7 postbleomycin administration,lung from model group showed markedalveolar infiltration with inflammatory cells and effusion,and thickening of inter-alveolar septa. Remarkable thickening of interalveolar septa,inflammatory cells infiltra-tion and focal solidation,and excessive collagen sediment were observed on day 14．Onday 28 extensive solidation of lung,loss of normal alveolar structure and vast inflamm-atory cells infiltration,obvious fibrosis presence and abundant collagen sediment withMasson trichrome staining appeared.Compared to mice in model group, the extent ofalveolar inflammation and inflammatory cells infiltration in mice from treatment groupwas significantly reduced on day 7,interalveolar septa mild thickening and a littlecollagen presence compared with model group(p<0．05) .The extent of lung solidationwas significantly reduced on day 28．The destructed alveolar structure,obvious interalveolar septa thickening and collagen sediment,and inflammatory cells infiltrationwere also found.Histopathological grading of extent of alveolar inflammation and fibrosis in lungparenchyma1．Comparison of alveolar inflammation grading in miceOn day 7 and 14 postbleomycin administration,alveolar inflammation grading inmice from model group being respectively 3．5±0．6 and 3．0±0．5,treatment group beingrespectively 2．5±0．3 and 2．1±0．2,the result of comparison in these two groups showedthat NAC significantly reduced the extent of alveolar inflammation by BLM(P<0．01) .2．Coparison of lung parenchyma fibrosis in miceAfter bleomycin administration, the extent of lung parenchyma fibrosis in BLM group and treatment group gradually aggravated. On day 28 postbleomycin,the grading of lung parenchyma fibrosis in mice from BLM group being 3．8±0．5,treatment group being 2．2±0．6,the result of comparison in these two groups showed that NAC significantly reduced the extent of lung parenchyma fibrosis by BLM(P<0．01) . Results of hydroxyprolineThe lung hydroxyproline levels,on day 28,were increased approximately two-fold in bleomycin-treated mice(696．5±41．2μg/g) compared to mice(348．0±22．6μg/g) that received saline intratracheally(P<0．01) .The values in treatment group,being respectively 450．6±27．1μg/g,520．3±22．8μg/g and 575．6±23．4μg/g,significantly incre-ased compared to control group(P=0．045,0．03 and 0．015) .However,comared with corresponding model group (respectively being 495．4±23．6μg/g,595．4±37．8μg/g and 696．5±41．2μg/g),HYP contents significantly decreased(P=0．046,0．03 and 0．02) . This showed that NAC remarkably reduced the extent of lung fibrosis by BLM. Contents of MDA and GSH1．Content of MDA and GSH in lung tissue(1) GSH content in lung tissue On day 7 postbleomycin administration,GSH level(862±10mg/gprot) of lung tissue in model group significantly decreased.There was significantly difference when compared with control group(P<0．01) .GSH content (135．2±15 mg/gprot) of lung tissue in treatment group was higher than that in model group and difference reached significance (P<0．05) .On day 28,GSH of model group was lower than that of contron group and there was no significant difference.(2) MDA content in lung tissue After bleomycin administration,MDA level of lung tissue in model group gradually increased significantly.On day 7,MDA level (2．73±0．41nmol/mgprot) was highest.When compared with control group( 1．1±02 nmol/ mgprot),there was a significant difference(P<0．01) .On day 7,MDA content(1．91±036 nmol/mgprot)remarkably decreased compared to model group and difference reached significance(P<0．05) .However,on day 14 and 28,MDA level in model group and treatment group gradually ran down and normally recovered.2．Content of MDA and GSH in BALF(1) GSH content in BALF On day 7 postbleomycin administration,GSH level (343．0±31．6 mg/L) of BALF in model group decreased and there was significant difference(P<0．05) when compared with control group(441．6±43mg/L).GSH content (518．8±48．7mg/L) of BALF in treatment group was higher than that in model group and control group and statistical significance was reached(P<0．01) .(2) MDA content in BALF On day 7 postbleomycin administration,MDA level (327±036 nmol/ml) of BALF in model group remarkably increased and there was a significant difference (P<0．01) when compared with control group (1．55±023 nmol/ml). MDA content(2．82±0．14 nmol/ml) of BALF in treatment group was higher than that in model group and statistical significance was reached(P<0．05) . Results of bronchonalveolar lavage cell P65 immunohistochemical (IHC) staining and in site hybridization staining1．Results of bronchonalveolar lavage cell P65 immunohistochemical (IHC) stainingOn day 7 and 14 postbleomycin administration,levels of the expression of P65 in the brochoalveolar lavage cells from model group and treatment group were respectively (43．4±2．6) %,(21．2±1．7) % and (21．4±2．3) %,(12．7±0．7) %,being significan-tly higher than that of control group(6．6±1．1) %,(7．0±1．0) % (P< 0．05) . When compared with model group,P65 expression of treatment group respectively ran down(t=10．23 and 6．24, P<0．05) .2．Results of bronchonalveolar lavage cell P65 m-RNA in site hybridization stainingOn day 7 and 14 postbleomycin administration,levels of the expression of P65 m-RNA in the brochoalveolar lavage cells from model group and treatment group were respectively (42．2±1．4) %,(26．4±2．4) % and (25．2±3．2) %,(17．1±4．4) %,being significantly higher than that of control group(7．0±1．3) %,(7．2±1．2) % (P<0．05) .When compared with model group,P65 m-RNA expression of treatment group respectively ran down (t=9．69 and 7．65, P<0．05) . Correlation analysis in MDA,GSH and P65On day 7 postinstillation,a positive correlation was found between P65 expression in bronchoalveolar lavage cell and MDA in BALF(r=0．738,P<0．05) and a negative correlation between P65 expression and GSH(r=-0．450,P<0．05) .In addition,P65 m-RNA expression in bronchoalveolar lavage cells was positively related with MDA too(r=0．648,P<0．05) .Contents of IL-4 and TGF-β1 in BALF1．Comparison of IL-4 content in BALFAfter bleomycin administration,content of IL-4 in BALF from model group and treatment group increased and on day 1 and 7 was significant.The value were respectively 56．8±11．0pg/ml,47．8±11．9pg/ml and 47．4±11．3pg/ml,41．9±10．7pg/ml, being significantly higher than that of control group31．1±8．5pg/ml(t=4．763,4．012 and 4．038,3．439;P=0．01,0．02 and 0．02,0．03) .The expression of IL-4 remarkably ran down in treatment group compared to model group(t=3．682 and 3．517;P=0．02 and 0．03) , NAC significantly inhibited the IL-4 expression by BLM.2．Comparison of TGF-β1 content in BALFAfter bleomycin administration, content of TGF-β1 in BALF from model group and treatment group increased and on day 7 and 28 was significant.The value were respectively 460．4±29．6pg/ml,381．4±97．6pg/ml and 321．0±63．5pg/ml,324．2±71．7pg/ ml,being significantly higher than that of control group 239．4±29．6pg/ml(t=17．216, 10．903 and 6．416, 5．114;P=0．001, 0．002 and 0．007, 0．007) .The expression of TGF-β1 remarkably ran down in treatment group compared to model group(t=8．539 and 4．434;P=0．001 and 0．021) ,NAC significantly inhibited the TGF-β1 expression by BLM.The weight change of mice in three groupsThree days after intratracheal instillation,weight of mice significantly begined to decrease between model group and treatment group.At day 7 weight failed most significantly and gradually increased.Mice from control group gained weight all the while in experimental period.On day 7,14 and 28 postbleomycin administration,the value of weight in model group being respectively15．7±0．29g,17．5±0．5g and 20．2±0．29g,treatment group being respectively 17．5±0．5g,19．7±0．29g and 22．5±0．5g,control group being respectively 20±0．5g,22±0．5g and 23．5±0．5g.The weight of mice from model group and treatment group was significantly lower than control group(P< 0．05) .There was a statistical significance in weight between model group and treatment group and weight remarkably decreased(t=-5．5,-6．5 and-7;P=0．01,0．007 and 0．006) .This indicated that NAC restricted the weight loss of BLM-induced mice.Conclusions1．The oxygenation/antioxygenation imbalance is concerned with bleomycin-induced acute lung injury and pulmonary fibrosis process and play an important role in it.2．The oxygenation/antioxygenation imbalance may activate NF-κB,further regulating cytokines IL-4 and TGF-β1 releasing and leading to interstitial pulmonary fibrosis.3．To a certain extent NAC may attenuate the extent of bleomycin-induced acute lung injury and pulmonary fibrosis.The mechanism may be associated with that NAC may restrain NF-κB activation to reduce the production of fibrosis associated cytokines.