The Study Of Protection Mechanism Of Recombinant Bactericidal Permeability-increasing Protein 21 In Rat Endotoxemia

PrefaceUncontrolled inflammatory response and multiple organs function failure from endotoxemia are media cell diseases resulting from a lot of cytokines' chain reaction. Now people have clearly recognized that endotoxin (lipopolysaccharide in Grams negative bacteria cell wall) is the most important trigger factor, and the chain reaction of cytokines plays a vital role in the disease entire development process.Recombinant bactericidal permeability - increasing protein 21 is an artificial synthesis amino terminal segments of BPI. Large experiments in vivo and vitro have shown rBPI21 has no apparent toxicity and immunogenicity to organisms, and has the same reactive of bactericidal and neutralization LPS as BPI. Therefore rBPI21 has a favourable application prospect to uncontrolled inflammatory response and multiple organs function failure, which come from Gram s negative bacteria infection or LPS invasion.Cytokines are soluble protein or sugar protein of highly reactive secreted by nerve cell, endocrine cell and histiocyte. On the one hand, they take part in complex cell — cell regulatory network in their generation micro - environment directly through bypass secretion and/or in the intimate way. On the other hand, they enter into blood circulation and cause generalized effects. In endotoxemia, LPS may trigger target cells directly by itself or indirectly through LBP, resulting in a series of inflammatory mediator such as interleukin - 1 , in-terleukin - 6, tumour necrosis factor - α and so on, initiating uncontrolled inflammatory response. TNF - α which can directly promote neutrophilic granulo-cyte, monocyte entering into inflammatory occur and releasing cytokines by ad-here to vascular endothelial, takes part in and magnifies uncontrolled inflammatory response , therefore plays a vital role in initiating endotoxemia.Several scholars have reported the protection of rBPI21 in endotoxemia. But so far there isnt a report from varying injection volume of rBPI21. In our test, based on experience formerly, through injecting different volume rBPI21 in endotoxemia rats, we continuously observed the changes of LPS, LBP and TNF -a in blood of different group rats and investigated the protection mechanism of rBPI21 in rat endotoxemia.Material and methods66 male Wistar rats, weighing 250 to 300g, were randomized to a control group ( n = 6) , a endotoxin group ( n = 12) and four treatment groups ( n = 12 per group). The control group, according to the weight of rats, received pyrogen -free 0. 9% saline from 275 to 330 jxl throughout tail vain. The endotoxin group was injected 1% LPS intravenously at lOmg/kg body weight and pyrogen -free 0. 9% saline 25 ~30|jd, and the four treatment groups were administered rBPI21 intravenously at 0. 625, 1. 25, 2. 50, or 5. 0 mg/kg body weight after injected LPS intravenously at 10 mg/kg body weight.Blood was obtained at 1, 2, 4, 6 and 12 hours in the endotoxin and four treatment groups. And in the control group blood was drawn only at 1 hour. Centrifugate and store sample - 30Ti until determination of LPS, LBP and TNF - a.Data was expressed as x ± SD. ANOVA of completely random design was used analyse the data. Mortality rates were compared among the groups using Fishers exact test. And a p value (double -sided) <0.05 was considered significant.Results1. The ability of rBPI21 to neutralization LPS in endotoxemia ratsThe contents of LPS have no difference at 1, 2 and 4 hours between rBPI21treatment 1 group and the endotoxin group. But at 6 and 12 hours, the levels of LPS in rBPI21 treatment 1 group ( 593. 3 ± 357. 6EU/ml, 9. 8 ± 3. OEU/ml respectively) were significantly lower than those in endotoxin group ( 1466. 7 ± 386. 3EU/ml, 587. 8 ± 259.2 EU/ml respectively) at the same time. Compared with the endotoxin group, the levels of LPS in rBPI21 treatment 2, 3 and 4 groups markedly dropped at any time points. And with the increasing of rB-PI21, the contents of LPS in treatment groups were decreasing. The graphy of LPS trendance has shown that the contents of LPS were highest at 1 hour in any group and thereafter steadily decreased.2. The influence of rBPI21 to serum LBP in endotoxin ratsThe LBP peak was found at 6 hours after the injection of LPS or LPS/rB-PI21 in all rats, and began to decrease at 12 hours. Compared with endotoxin group, LBP levels in treatment groups significantly decreased at any time point. Among rBPI21 treatment 2, 3 and 4 groups, the contents of LBP have no difference at 1 hour, but at 2, 4, 6, 12 hours with the increasing of rBPI21 LBP contents steadily decreased.3. The influence of rBPI21 to serum TNF - a in endotoxemia ratsTNF - a peak was found at 4 hours after injection of LPS or LPS/rBPI21 in all rats, and began to decrease at 6 hours. Compared with endotoxin group, TNF - a levels in treatment groups markedly decreased at any time point. TNF - a peak values were statistically significant difference among treatment groups, and with the increasing of rBPI21 TNF - a levels decreased more. Strong negative correlation was found between rBPI21 treatment doses and serum TNF - a peak values, r = -0. 8133 ,y =4790 - 1025x.4. 24h survival rate in different group ratsCompared with endotoxin group, rBPI21 2, 3 and 4 groups that rBPI21 injection doses were 1. 25 , 2.5, 5. Omg/kg body weight individually could protect the rats from 10 mg/kg LPS challenge, and the survival rates were increased from 16. 7% ( endotoxin group) to 58. 3% , 91.7% , 100% ( rBPI21 treatment 2,3 ,4 groups) respectively. The dose of 5. Omg/kg rBPI21 can fully protect the rats from the challenge of 10 mg/kg LPS, and the 24h survival rate was 100% .DiscussionBPI is a member of cation antibacterial peptides, and primarily lies in azurophil granules of neutrophils, and is one of natural immune systems defen-sing LPS invasion to organisms. Many studies have shown, that BPI and its reactive segments have no apparent toxicity and immunogenicity, and play a strong protection in endotoxemia models of human beings or different animals.In our test, we continuously observed the changes of LPS, LBP and TNF -a in blood of endotoxemia group and treatment groups to investigate the protection mechanism of rBPI21 in rat endotoxemia.From the graphy of LPS trendance, we discovered that the levels of plasma LPS were highest at 1 hour in all rats, and in addition that LPS levels of endotoxemia group rats appeared second peak at 6 hours, the contents of LPS in the rest groups were all gradually reduced with time being on. In physiological circumstances , organisms have an effective removal of endotoxin and detoxication function. Liver, spleen and intestine are main organs for removing endotoxin, and the lipoprotein, cation antibacterial peptides and anti - endotoxin antibody are all important materials for detoxicating endotoxin. Based on the two premises that there isnt infection outside and invasion inside, LPS content in vivo should gradually reduce with time going on.The datas of serum LBP and TNF - a in all rats have shown, that the peaks of serum LBP, TNF - a appeared at 6 and 4 hours individually, and began to decrease at 12 and 6 hours respectively. In the rBPI21 treatment groups, with rBPI21 increasing the peaks of serum LBP and TNF - a were all gradually reduced.The contents of LPS have no difference at 1, 2 and 4 hours between rBPI21 treatment 1 group and the endotoxin group. But at 6 and 12 hours, the levels of LPS in rBPI21 treatment 1 group(593. 3 ±357. 6EU/ml, 9. 8 ±3. OEU/ml respectively) were significantly lower than those in endotoxin group ( 1466. 7 ± 386.3EU/ml, 587. 8 ±259. 2EU/ml respectively) at the same time. Serum LBP and TNF - a in rBPI21 treatment 1 group were both lower than those in en-dotoxin group at any time point. Compared with the endotoxin group, the levels of LPS, LBP, TNF - a in rBPI21 treatment 2, 3 and 4 groups markedly dropped at any time points. With the increasing of rBPI21 , the contents of LPS, LBP and TNF - a in treatment groups gradually decreased. The results of our test indicated that the protection of rBPI21 in endotoxemia rats was primarily achieved through neutralizing LPS and decreasing LPS reactive in vivo. With the increasing of rBPI21, the function of neutralization LPS gradually enhanced.The chain reaction of cytokines plays a vital role in uncontrolled inflammatory response and multiple organs function failure. The biological effects of cytokines in vivo are extremely complex. In a certain concentration, they can protect organisms from infection by enhancing cell immune. But when the concentration exceed a certain limit, they will damage organisms and promote inflammation response process. TNF - a plays a leading role in initiating endotoxemia.Strong negative correlation was found between rBPI21 treatment doses and serum TNF — a peak values, r = -0. 8133,y =4790 — 1025x. The result indicated that rBPI21 can raise the survival rate of endotoxemia rats by reducing serum TNF - a peak value and decreasing uncontrolled inflammatory response and MOF.The 24h survival rates of endotoxin group and treatment groups were 16. 7% , 25% , 58. 3% , 91. 7% , 100% individually. In treatment groups, with the rBPI21 increasing, the survival rate raised too. This result suggested that the protection of rBPI21 has dose dependence. With the increasing of rBPI21 dose, the protection in endotoxemia rats gradually enhanced.Above all, the protection of rBPI21 has dose dependence in endotoxemia rats. The protection of rBPI21 in endotoxemia rats was primarily achieved through neutralizing LPS , decreasing LPS reactive in vivo and inhibiting LBP , TNFa synthesis.Conclusions1. The protection of rBPI21 has dose dependence in endotoxemia rats.2. The protection of rBPI21 in endotoxemia rats was primarily achievedthrough neutralizing LPS, decreasing LPS reactive in vivo and inhibiting LBP, TNFa synthesis.