Preparation Of Two Recombinant Heparin-binding Domain Polypeptides Of Fibronectin And Study Of The Effect Of The Polypeptides On Sepsis In Mice

Sepsis,an acute severe syndromes has still high mortality,although there are so many wonderful antibiotics , consummate organ support and surveille techniques. Fibronectin(FN)is known to be a large multifunction glycoprotein with binding sites for many substances including heparin, collagen, fibrinogen, fibrin and integrin cell surface receptors.It is involved in a variety of cellular processes, including tissue repair, blood clotting and cell migration or adhesion. Most studies suggested FN purified from plasma have the effect on sepsis, but it might transmit infectious diseases, such as HIV.Since it was difficult to express the whole molecular of FN by genetic engineering. Thus,it is necessary to express the related function polypeptides and study the function of recombinant polypeptides on sepsis.In our previous studies,we had expressed and purified the recombinant cell-binding domain polypeptide of FN ( rhFN55 polypeptide ) and N-terminal heparin-binding domain polypeptide (FNNHBD),and confirmed they both had the effect on mice with sepsis Our previous studies had constructed the recombinant yeast expressed vector with the C-terminal heparin-binding domain polypeptide(FNCHBD).But, the effect of FNCHBD on sepsis is unclear.In this study, we express and purify FNCHBD and FNNHBD, investigate the effect of FNCHBD and FNNHBD on sepsis in mice.The FNCHBD and FNNHBD were expressed and purified and t were identified by western-blot and by passing the heparin affinity column. The molecular weights of FNCHBD is 30.5KD, and that of FNNHBD is 27.9KD by the mass spectrogram.According to A.-H. Kwon,s and our test condiction,the septic mouse model was established by the injection of 100ug/kg lipopolysaccharide(LPS ) and 400mg/kg D-Galactosamine ( GalN ) into the mice from abdomen.The result show that both FNCHBD and FNNHBD can arise the survival of the experimental mice by administrating intravenously 30 min before LPS/GalN injection. In the control group, the survival rate of the mice was 11.1%,and the survival rate was 33.3% , 55.5% ,66.7% respectively at 5mg/kg ,10mg/kg , 20mg/kg of FNCHBD in the FNCHBD-treated group and that was 27.8%,50% 61.1% in FNNHBD-treated group respectively. The levels of serum AST,ALT,LDH and TBIL increased in the control group, and decreased in the FNCHBD-treated group and FNCHBD-treated group with the increasing dosage of FNCHBD and FNNHBD. Histopathology of liver showed that mass denaturation, necrosis, congestion, haemorrhage and hyperemia in the control group, both FNCHBD and FNNHBD can inhibited the injury of liver in a dose-dependent manner. The level of TNF-α,IL-6,IL-1βin plasma determined by ELISA in the control group were significantly higher than in the normal control, while both FNCHBD and FNNHBD can significantly inhibited the expression of these apoptosis of hepatocyte in a dose-dependent manner.A lot of apoptosis of hepatocyte were showed in the control group by electron microscope and TUNEL. However,both FNCHBD and FNNHBD both can significantly inhibited hepatocyte apoptosis in a dose-dependent manner. The results also showed that increasing the times of administration of FNCHBD and FNNHBD both can not contribute to the survival rate of mice with sepsis after LPS/GalN injecting at the base of 20mg/kg of FNCHBD and FNNHBD and administration with FNCHBD and FNNHBD respctivly before LPS/GalN injecting had the better effect on sepsis in mice than after LPS/GalN injecting. Conclusion:In this study, FNCHBD and FNNHBD were expressed and purified and the model of sepsis in mice was constructed successfully. FNCHBD and FNNHBD were both demonstrated to have the effect on mice with sepsis in a dose-dependent manner The effect on mice with sepsis of two polypeptides have no significant deviationin statistics. Therapeutic mechanism is similar, both through inhibiting expression of TNF-αand other inflammatory cytokines and suppressing subsequent apoptotic processes in the liver. Increasing the times of administration of FNCHBD and FNNHBD after LPS/GalN injecting do not contribute to their effect on mice with sepsis at the base of 20mg/kg of FNCHBD and FNNHBD. Administration with FNCHBD and FNNHBD respctivly beforeLPS/GalN injecting had the better effect on sepsis in mice than after LPS/GalN injecting.