Study On The Formation Of Neutrophil Extracellular Traps By Anthraquinones Extracts Of Rhubarb During Sepsis

ObjectiveTo investigate the effects of anthraquinone extracts of rhubarb,namely free anthraquinone(FA)and bound anthraquinone(CA),on the expression of proinflammatory factors and the formation of neutrophil extracellular trap(NETs)during sepsis,and to explore the potential mechanism of anthraquinone extracts of rhubarb to inhibit the formation of NETs.MethodsPart I: 40 male Balb/c mice were randomly divided into a control group(CG group),lipopolysaccharide(LPS)group,LPS+FA group,and LPS+CA group,10 mice in each group.FA and CA were prepared at a concentration of 10 mg/ml.Mice in the LPS+FA and LPS+CA groups were pre-gavaged at 100 mg/kg doses for 12 h.The CG and LPS groups were given 0.5 % sodium carboxymethyl cellulose(CMC)by gavage.After 12 h,the sepsis model was established by intraperitoneal injection of LPS(15mg/kg)in all groups except the CG group,which was given the same dose of sterile saline intraperitoneally.After the completion of the LPS injection and waiting for 2 h,the second gavage was performed according to the first gavage dose.The general status of the mice was observed,and blood collection and specimen collection were performed after 12 h.The levels of tumor necrosis factor-α(TNF-α)and interleukins-1β(IL-1β)in plasma were detected by ELISA.HE staining was used to observe the pathological damage of liver and ileum.Immunofluorescence staining and Western blot were used to detect the expression of citrullinated histone(Cit H3)and myeloperoxidase(MPO)in liver and ileum tissues.Part II: HL-60 cells were induced to mature granulocytes.The degree of maturation of HL-60 cells was detected by Wright-Giemsa stain solution and flow cytometry.The CCK8 assay was used to confirm the optimal concentrations of FA and CA for the intervention of cells.We set up CG group,phorbol ester group(PMA group),FA+PMA group,CA+PMA group and PMA+NADPH inhibitor(DPI+PMA group).Except for CG and PMA groups,the other groups were pretreated with drug FA、CA and DPI for 30 min.Then,except CG group,other groups were added PMA(final concentration of 100 n M)for 4 h.The intervention was terminated by changing the medium 4h later,and FA,CA and DPI were added for 24 h.Immunofluorescence and flow cytometry were used to detect ROS release in each group of cells;the expression of Cit H3 and MPO was visualized by cellular immunofluorescence staining.Results1.Compared with CG group,the expression levels of TNF-α and IL-1β in the blood of LPS group were significantly increased(P < 0.05).However,after FA and CA treatment,compared with LPS group,the expression levels of TNF-α and IL-1β were significantly decreased(P < 0.05).FA and CA decreased the expression levels of TNF-α and IL-1β respectively and did not differ.At the same time,FA and CA significantly improved the pathological damage of liver and ileum.2.Immunofluorescence staining visualization showed that only a small amount of Cit H3 and MPO were expressed in the liver and ileum of the CG group,while the expression of Cit H3 and MPO was significantly increased in the LPS group.The expressions of Cit H3 and MPO in LPS+FA group and LPS+CA group were lower than those in LPS group.The results of Western blot showed that the expression of Cit H3 and MPO in the liver and ileum of the CG group was only a small amount,and the expression of Cit H3 and MPO in the liver and ileum of the LPS group was significantly higher than that of the CG group(P < 0.05).The expressions of Cit H3 and MPO in LPS+FA group and LPS+CA group were lower than those in LPS group(P < 0.05).In addition,there was no difference in the inhibition of intrahepatic Cit H3 expression between FA and CA,and the same was true for MPO.3.Cell fluorescence staining and flow cytometry showed that only minimal ROS production was seen in the CG group,and ROS release was significantly increased in the PMA group compared with the CG group(P < 0.05).The amount of ROS release in the FA+PMA,CA+PMA,and DPI+PMA groups was significantly reduced compared with that in the PMA group(P < 0.05).In addition,the inhibition of ROS release in the FA+PMA and CA+PMA group was not statistically different compared with the DPI+PMA group(P > 0.05).4.Immunofluorescence staining showed that the expression of Cit H3 and MPO in the CG group was very low,and the expression of Cit H3 and MPO in the PMA group increased.The expressions of Cit H3 and MPO in FA+PMA,CA+PMA and DPI+PMA groups were lower than those in PMA group.Conclusion1.Free anthraquinone and combined anthraquinone of rhubarb can down-regulate the expression level of inflammatory factors,improve the pathological damage of liver and ileum,and inhibit the formation of NETs in the liver and ileum in septic mice.2.Free anthraquinone and combined anthraquinone of rhubarb can inhibit the release level of ROS and the formation of NETs in mature HL-60 cells.