| Objective:Acute lung injury(ALI)is an acute onset of noncardiac and progressive respiratory insufficiency syndrome,directly or indirectly mediated by a variety of injurious stimulis.Pulmonary function of paitients with ALI will continue to deteriorate,if an effective treatment is not taken,and ALI will progress to a most severe form,that is,acute respiratory distress syndrome(ARDS).Despite massive reports of investigation focused on mechanisms of ALI,effective treatment of ALI/ARDS remains unsolved problems of intensive care medicine.Now is generally accepted that,recruitment of neutrophils play a central role in progression of ALI/ARDS.As one component of innate immunity system,neutrophils are the first cells to be recruited to the site of inflammation and have a various potent antimicrobial armour with the release of a plethora of cytokines/chemokines,includingTNF-α,IL-1β,IL-8,IFN,IP-10,MIP-1.Recent studys reported that,in response to microbes,cytokines,activated mesh platelet,even heme and other inflammatory stimulis,neutrophils casting out a mesh structure,neutrophil extracellular traps(NETs).It was demonstrated that,NETs prossess ability of binding and killing microbes including bacteria,virus and fungus.Unregulated NETs formation,however,will injury host tissue.Emerging evidence suggests that NETs involved in the process of thrombosis,autoimmune deasise and transfusion-related acute lung injury(TRALI).HO-1,encoded by HMOX genes,are key enzymes that catabolize heme,in other word,ferriporphyrin iron protoporphyrin(IX),into equimolar amounts of labile Fe,carbon monoxide(CO),and biliverdin.Dispite,heme oxygenase-1(HO-1)has been shown to display anti-inflammatory and anti-apoptosis properties in variety of diseases,the role of HO-1 in the regulation of NETs formation is still to be understood.Using lipopolysaccharide(LPS)-induced ALI models,this study was designed to investigate the therapeutic potential of NETs formation down-regulation by HO-1 up-regulation based on LPS-induced ALI model,and inflammation attenuation.Methods: A total 120 C57BL/6 mice(body weight 16-18 g)were used in this study.All mice received adaptive feeding for 1 week before experiment and followed by randomized allocation.According to the usages of agent including LPS,CoPP and NS,animals were divided into NS group,LPS group,LPS+NS group and LPS+CoPP group.The ALI model was created by intraperitoneal injection of LPS(5 mg/kg),and mice in NS group received the same volume of NS.Prior to LPS injection,mice in LPS+NS group and LPS+CoPP group were injected with CoPP(5 mg/kg)or the same volume of NS.At 1,3 or 7 days,animals were sacrificed by exsanguinations with sodium anhydrous ether.Serum and bronchial alveolar lavage fluid(BALF)were obtained.The non-lavaged lungs were performed with pathological detection.Hematoxylin eosin staining(HE)was used to observe alveolar and interstitial inflammation.Using confocal microscopy,deoxyribonucleic acid(DNA),myeloperoxidase(MPO)and histone H2 B were used to investigate whether NETs formed in injuryed pulmonary tissues.Quantification of protein in BALF supernatant was performed with BCA protein assay kit.Quant-iT? PicoGreen? dsDNA reagent and kits was used to detect the content of dsDNA in BALF supernatant and serum.Chemokines including IL-8 and MCP-1 in the BALF and serum were measured by commercially available ELISA kits.LDH activity was determined by LDH activity kit.Total RNA was isolated from non-lavaged right lung tissues.The relative HO-1 mRNA expression level was normalized for expression of the β-actin and calculated using the formula 2-△△Ct method.Results:1 CoPP alleviated LPS-induced lung inflammatory response1)By HE staining,mice in LPS group and LPS+NS group were found to have developed alveolar thickening,obviously leukocyte and hemocyte infiltrates within the interstitial and alveolar spaces.The inflammation score(IS)showed a significantly higher degree of affected area in LPS group and LPS+NS group compared to the NS group(P <0.01),which was increased to a peak on day 3.Interestingly,compared to the LPS+NS group,lung tissues of mice treated with CoPP suffered less lesion,and inflammatory infiltration was attetunated on day 1(P <0.01),followed by unconspicuous pneumorrhagia or pulmonary edema on day 3,and IS was reduced significantly(P <0.01).On the day 7,inflammatory reactions were reverse and there were no significantly difference between each group.2)The data of protein quantification showed that,protein content in BALF supernatant of NS group was unconverted.In comparison with NS group,the protein content in BALF supernatant of LPS group and LPS+NS group were significantly increased at day 1 and day 3 after LPS challenged(P <0.01).Compared to the LPS+NS group,the protein concentration in BALF supernatant of LPS+CoPP group was significantly lower(P <0.01).On day 7,there were no significantly difference between the protein concentration in each group.3)The results of chemokine IL-8 and MCP-1 examination showed that,on day 1 and day 3,both the expression level of IL-8 and MCP-1 in BALF supernatant and serum are higher than NS group(P <0.01).Compared with LPS+NS group,CoPP treatment was associated with downregulated BALF supernatant and serum IL-8 both on day 1(P <0.01)and day 3(P <0.05)in LPS+CoPP group,while we only observe an insignificantly trend of down-regulation of BALF supernatant and serum MCP-1 on day 1.On day 3,MCP-1 expression level of LPS+Co PP group was significantly lower than LPS+NS group both at BALF supernatant and serum(P <0.01).4)The level of LDH activity was determined,which indicated that,compared with NS group mice,the levels of LDH activity were increased in LPS group and LPS+NS group on day 1(P <0.01),which respectively reached a mean of 268.653 U/L and 266.590 U/L,followed by a peak in day 3(P <0.01),which respectively reached a mean of 369.513 U/L,403.438 U/L.At the meantime,we observed that the LDH activity was alleviated in LPS+CoPP group compaired to LPS+NS group(P <0.01)on day 1and day 3.There was no significantly change of LDH activity among each group on day 7.2 CoPP decreased LPS-induced neutrophil infiltration in lung tissuesThe results of western blot showed that,3 days after molding,the relative expression levels of lung tissue MPO protein in LPS group and LPS+NS group were higher than NS group(P <0.01).Moreover,a significant reduce of lung tissue MPO protein was observed in LPS+CoPP group on day 3(P <0.01).3 CoPP induced a up-regulation of HO-1 mRNA in lung tissuesThe results of RT-PCR showed that,CoPP treatment induced increased HO-1 mRNA expression on day 1 and day 3,which all had statistical significance(P <0.01),and reached a peak on day 3.There were no significantly difference of mRNA expression level of HO-1 among each group on day 7.4 CoPP reduced LPS-induced NETs formation in lung tissues1)Using the laser scanning confocal microscopy,we determined the co-localization of DNA,histone H2 B and MPO,indicating the existence of NETs,in the lung tissue especially peripheral alveolar space from LPS-induced ALI mice on day,while we failed to found a similarly phenomenon in NS group.2)The levels of BALF supernatant and serum dsDNA/NETs in LPS group and LPS+NS group were significantly higher than NS group on day 1(P <0.01).Three days after Co PP treatment,we observed a reduction in the levels of BALF supernatant and serum dsDNA/NETs,compared with LPS+NS group.There was no significantly difference of the levels of BALF supernatant and serum dsDNA/NETs among each group on day 7.Conclusions: Treatment of HO-1 inducer CoPP initiates a clearance of heme and a down-regulation of IL-8,subsequently reduces the level of NETs in serum and pulmonary alveoli,accompanied by attenuation of LPS-induced pulmonary injury and inflammation through a up-regulation of HO-1 expression in the model of LPS-induced ALI model,and demonstrates a therapeutic potential of CoPP based treatment in clinical prevention and therapy of ALI. |
