Protective Effect Of Three Ingredients Of Safflower Against Lipopolysaccharide Induced Acute Lung Injury And Formation Of Neutrophil Extracellular Trap

Background:Acute respiratory disease syndrome(ARDS)is a severe respiratory disorder in clinic,which leads to an increase in mortality in critically ill patients.Acute respiratory disease syndrome can be caused by a variety of factors,including severe pneumonia,burns,and sepsis.It is characterized by the injury of alveolar capillary endothelial cells and epithelial cells,acute diffuse inflammatory damage,alveolar congestion and edema,and decreased lung compliance.In 2012 Berlin Definition,ALI was categorited as mild ARDS,while the term of ALI was still used in animal modle of ARDS.Despite extensive studies about ARDS have been reported to date,there has been no effective medicine for ARDS.Xuebijing is a traditional Chinese medicine for the treatment of sepsis and MODS,which contains the following five main Chinese herbs:safflower,red peony root,Szechuan Lovage Rhizome,Radix Salviae Miltiorrhizae and Chinese angelica.A mass spectrometric test analyzed the active pharmaceutical ingredients in the serum of mice treated with Xuebijing,and found that the major components of Xuebijing related to safflower included Safflor yellow A(SYA),Hydroxysafflor yellow A(HSYA)and Anhydrosafflor yellow B(AHSYB),etc.Safflower is commonly used for improving microcirculation,relieving pain and inhibiting inflammation,we speculate that the effects of safflower on microcirculation and inflammation might be useful in relieving pulmonary inflammation in acute lung injury.It has been reported that safflower yellow A and hydroxysafflower yellow A can alleviate acute lung injury,but there is no report on the effect of anhydrosafflor yellow B on sepsis-related lung injury.The purpose of this study was to investigate the effects of safflower yellow A,hydroxysafflower yellow A and anhydrosafflor yellow B on lipopolysaccharide-induced acute lung injury and their impacts on the release of neutrophil extracellular traps,and related pathways.Methods:In vivo,8-12 weeks old male C57BL/6 mice,weighing 20g-25 g,were randomly divided into 5 groups: Control group,ALI group,ALI+SYA(30 mg/kg)group,ALI+HSYA(30 mg/kg)group,ALI+AHSYB(30 mg/kg)group.LPS(10 mg/kg)was injected intratracheally to establish a model of acute lung injury in mice,and then normal saline,SYA,HSYA,and AHSYB were injected into abdominal cavity.24 hours later,eyeballs were extracted for blood,and the serum was taken after centrifugation to evaluate the level of inflammatory factors and MPO-DNA complex.The concentration of total protein in bronchoalveolar lavage fluid was determined.Total cell numbers and the ratio of neutrophils in bronchoalveolar lavage fluid was analyzed by flow cytometry.Lungs were taken for the determination of wet-to-dry weight ratio,histopathological staining and immunofluorescence staining.And the expression of related proteins was assessed by Western blot after lysis.In vitro,HL-60 cells were cultured in RMPI+10% fetal bovine serum(FBS),differentiated into neutrophils with DMSO(final concentration 1.25%),then PMA(100 ng/ml)was added to stimulate the production of NETs,and 1640 medium,SYA,HSYA,AHSYB(160 mg/l)were separately administered.The culture supernatant was taken to detect the level of MPO-DNA complex 3 hours later.After cell lysis,the expression of related proteins was determined by Western blot.Results:1.24 hours after intratracheal injection of LPS,H & E stains of lung tissue showed that compared with control group,the lung tissue of mice in ALI group was congested,edema,pulmonary septum was thickened or damaged and inflammatory cells infiltrated significantly in alveolus and interstitial tissue;the SYA group,HSYA group,and AHSYB group showed less lung tissue damage.The pathological scores of SYA group,HSYA group,and AHSYB group were significantly lower than those of lung injury group,which indicated that SYA,HSYA,AHSYB could reduce lung tissue damage caused by intratracheal injection of LPS.2.After intratracheal injection of LPS for 24 hours,the concentration of TNF-α and IL-6 in plasma and bronchoalveolar lavage fluid increased significantly,while the concentration of TNF-a and IL-6 in plasma and bronchoalveolar lavage fluid in SYA,HSYA,AHSYB group were lower than those in ALI group obviously.3.After LPS treatment,the lung wet/dry ratio increased significantly in contrast to control group.However,HSYA and AHSYB reduced the wet/dry ratio of ALI mice while no significant effect of SYA was observed.The protein concentration of bronchoalveolar lavage fluid in mice increased significantly after LPS treatment.Nevertheless,SYA,HSYA,and AHSYB can decrease the protein concentration of bronchoalveolar lavage fluid in mice.In the lung injury group,the total number of cells and the proportion of neutrophils in the alveolar lavage fluid increased greatly compared with the control group whereas SYA,HSYA,and AHSYB can reduce neutrophil infiltration to a certain extent.4.After 24 hours of LPS treatment,the immunofluorescence staining of lung tissue showed that there were more MPO and histone in the lung tissue of the ALI group than that of the control group.The secretion of MPO and histone in the lung tissue of SYA,HSYA and AHSYB groups was more than that of the control group,but significantly lower than that of the ALI group.5.In vivo experiment,LPS significantly increased the level of MPO-DNA complex in plasma,while SYA,HSYA and AHSYB groups was lower than that in ALI group.In vitro,PMA stimulated HL-60 cells to produce MPO-DNA complex.SYA,HSYA and AHSYB treatment could reduce the formation of MPO-DNA complex.6.The phosphorylation of Raf,MEK and ERK in the lung tissue in ALI group was higher than that in control group,however SYA,HSYA and AHSYB could reduce the phosphorylation of Raf,MEK and ERK.The phosphorylation of Raf,MEK and ERK were significantly increased after PMA stimulation,while the phosphorylation levels of Raf,MEK and ERK were significantly decreased after SYA,HSYA and AHSYB treatment.Conclusion:SYA,HSYA and AHSYB can alleviate the severity of LPS-induced acute lung injury in mice,which might be related to the inhibition of Raf,MEK and ERK phosphorylation and the reduction of neutrophil NETs formation.