| Background and Objective: Schistosomiasis an endemic disease,is popular in tropical and subtropical regions.Currently,more than 240 million people worldwide infect with schistosomiasis and deaths approximate 500,000 to 800,000 are died each year.After schistosomiasis infection,patients appear severe pathological characteristic including liver fibrosis,portal hypertension,rupture of liver ascites,and varicose veins,which cause severe bleeding in the upper gastrointestinal tract.In the process,macrophage polarization plays an important role.In the early stage of this project,it had discovered the non-specifical bound peptide ZLW of Schistosoma japonicum with a certain anti-schistosomiasis effect.Therefore,this study analyzed the role of ZLW in host macrophage polarization shift,and aimed to explore the effect and mechanism of ZLW to improve liver fibrosis.Methods:(1)The structural features and functions of ZLW were analyzed with software such as PSIPRED,NetNGlyc 1.0,DISPHOS,andClustalW;(2)After RAW264.7 cells stimulated with 0.1,1,10,100,and1,000μg/ml ZLW,CD11 b,F4/80,CD86,and CD206 were used as macrophage markers,and the effect of ZLW on polarization shift of RAW264.7 cells was analyzed by flow cytometry.(3)Mice were randomly divided into a uninfected+NC group,a infected+NC group,and and infected+ZLW group,the infected+NC group and the infected+ZLW group were infected with Schistosoma japonicum through the epidermis of the abdominal cavity.After infection,the mice in the uninfected+NC group and the infected+NC group were injected with 100μl of 0.9%physiological saline every day,and the mice in the infected+ZLW group were injected with 100μl of 100μg Peptide ZLW every day;(4)After extracted mice macrophages from liver and spleen of each group at 3,24,33,42,and 49 d,macrophage was distinguished by the marker of CD11 b and F4/80,and identified M1 and M2 macrophage with CD86 and CD206 respectively,and the macrophage in all samples were analyzed by flow cytometry;(5)The liver tissue of mice in each group was taken at 3,24,33,42,and 49 d,and the pathological changes of liver tissue were detected by HE and Masson staining;(6)The liver tissue of mice in each group was taken at 3,24,33,42,and 49 d,CD68 was selected as a total macrophage marker,iNOS was selected as a M1 macrophage marker,CD206 and Arg1 were selected as M2 macrophage markers,and immunohistochemistry staining was used to determine the qualitative andlocalization of macrophages in liver tissues;(7)Blood was collected from the mouse’s eyeballs at 3,24,33,42,and 49 d,and the expression of IL-6and TNF-α in serum was detected by ELISA.Results:(1)Peptide ZLW is a peptide with 39 amino acid residues,rich in multiple phosphorylation sites,and highly homologous to the mouse Zfp36l2 protein.(2)Flow cytometry assay revealed that the percentage of M1 and M2 macrophages were at the highest(50.73%)and lowest(3.28%),respectively,while peptide ZLW concentration was 100μg/ml.(3)Flow cytometry assay showed that the ratio of M2/M1 in the liver was at the highest on 24 d and at the lowest on 49 d after peptide ZLW treated,and it was significantly lower than those in the uninfected+NC group and the infected+NC group;the ratio of M2/M1 in the spleen was at the highest on 33 d and close to the uninfected+NC group on 42 d,and there were significant differences with the infected+NC group(p <0.01).(4)HE and Masson staining analysis showed that the liver tissue lesions and inflammatory cell infiltration in the infected+ZLW group were significantly reduced and fibrous connective tissue proliferation was significant remission.(5)The results of IHC staining showed that CD68 cells in the liver tissue of the infected+NC group and the infected+ZLW group were significantly increased;CD206 cells in the liver tissue of the infected+ZLW group were significantly reduced compared with the infected+NC group;Theexpression of iNOS in the cytoplasm of hepatocytes increased significantly,while the expression of Arg1 decreased significantly.(6)The ELISA was used to detect the peripheral blood serum of mice in each group on 3,24,33,42 and 49 d.It was found that the IL-6 concentration in the infected+ZLW group was significant lower than that in the infected+NC group by 1.11,1.87,1.64,1.61,1.37 folds(p <0.05);the TNF-α concentration in the infected+ZLW group was significant higher than that in the infected+NC group by 1.05,1.57,1.44,1.52,2.79 folds at each detection time point except the 3d(p <0.05).Conclusion:(1)Peptide ZLW can induce M1 polarization in mouse liver and spleen macrophages,and has a certain inhibitory effect on M2polarization;(2)Peptide ZLW can affect the polarization of mouse liver macrophages by regulating arginine metabolism;(3)Peptide ZLW can improve or reverse the progress of liver fibrosis in schistosomiasis. |
