The Effect Of Recombinant FN C-terminal Heparin-binding Domain Polypeptide On Macrophage Function Of CLP-induced Septic Mice

Aim:To investigate the protective effect of recombinant C-terminal heparin-binding domain polypeptide of FN(rh FNHC-36)in model of sepsis by cecal ligation and puncture(CLP),and elucidate the protective function from the angle of mononuclear phagocyte.Methods:1.The construction of the model of CLP.The technique involves midline laparotomy, exteriorization of the caecum, ligation the caecum and puncture of the ligated caecum,extrusion a drop of feces and sewing up the cut.2.We set up experimental group as follows:the normal group,the sham group,the CLP+PBS group and the CLP+rh FNHC-36 group.The CLP+rh FNHC-36 group mice were administrated with 40mg/kg of rh FNHC-36 via tail vein half an hour before CLP plus one and two hours after CLP.As a control,the CLP+PBS group mice should administrate the same volume of PBS. Observed the mouse mortality rate of sepsis 14 days and estimate the clearance rate of bacteria by macrophage in abdomen and blood through calculating bacterial colony. Calculate the index of thymus and spleen and observe tissue pathology and structure of liver、spleen and lung to access the protection of rh FNHC-36 on septic mice.3.The single cell suspension of murine peritoneal cells was prepared under sterile condition and after adherent culture for 4 hours they were identified as macrophages.The cells were stimulated with LPS at a final concentration of 1μg/m L, 18 hours later, the concentration of NO in serum and NO produced by macrophages was detected by Griess kit.The level of plasma IL-6 was detected by ELISA.Transwell was used to detect the chemotaxis of macrophages.The expression of PD-L1,which was the marker of inhibitory ligand on macrophage, was measured by flow cytometry(FCM).Results:1.The model of CLP was successfully constructed,verificated by identifying the kind of microorganism from the peritoneal cavity and blood.After CLP,the mouse is marked by low weight,furring,trembling,diarrhea,tachycardia,being alone, along with ischemia and necrosis of distal caecum and markedly abscess.While the situation of mouse treated with rh FNHC-36 is much more better,2.There were no deaths in the normal group and the sham group in 14 days.The mortality of mice in CLP+PBS group was 70%,while that of CLP+rh FNHC-36 group was only 50%,with a significant difference(P<0.05).There were no bacteria colonies in the normal group and the sham group whatever from the peritoneal cavity or blood.The colonies of bacteria from the peritoneal cavity and bloo d in CLP+PBS mice was 3.4×10^4±3.9×10^3/ml、3.9×10^4±1.0×10^4/ml respectively,while that of CLP+rh FNHC-36 group was 1.4×10^3±2.1×10^2/ml、4.0×10^3±3.3×10^3/ml respectively,with a significant difference(P<0.05).The spleen and thymas index of mice in the sham group were almost the same with those of the normal group.The index of mice spleen 6hours and 24 hours after CLP wa s 3.0±0.4、2.9±0.5 respectively,while that of CLP+rh FNHC-36 group was 3.6±0.5、4.2±0.6 respectively.The index of mice thymas 6hours and 24 hours after CLP was 1.6±0.3、0.8±0.2 respectively,while that of CLP+rh FNHC-36 group was 2.7±0.5、1.8±0.2 respectively,with a significant difference(P<0.05).rh FNH C-36 can lighten the damage of the liver,spleen and lung.3.The NO concentration stimulated by LPS of mice in the normal and the sham group was 4.2±1.6umol/L and 4.6±1.1umol/L.The NO concentration of mice in CLP+PBS group was 13.4±3.3umol/L,while that of CLP+rh FNHC-36 group was only 4.4±3.2umol/L,with a significant difference(P<0.05).The IL-6 concentration of mice in the normal and the sham group was(6.3±6.3)pg/ml and(181±42)pg/ml.The IL-6 concentration of mice in CLP+PBS group was(64000±29000)pg/ml,while that of CLP+rh FNHC-36 group was only(19000±19000) pg/ml,with a significant difference(P<0.05).The chemotaxis index of macropha- ges of mice in CLP+PBS group was 3.8±0.9,while that of CLP+rh FNHC-36 group was only 7.8±1.9,with a significant difference(P<0.05).The PD-L1 expression of mice by LPS in the normal group was 69.0±10.9%.The PD-L1 expression of mice in CLP+PBS group was 91.5%±3.2%,while that of CLP+rh FNHC-36 group was only 80.4%±3.9%,with a significant difference(P<0.05).Conclusion:rh FNHC-36 can protect the sepsis mice induced by CLP through reducing the level of IL-6 and NO stimulated by endotoxin, promoting the chemotaxis of macrophages and inhibiting the expression of PD-L1.