The Effects Of CRISPLD2 And The Bifunctional Fusion Protein CRISPLD2-anti-HMGB1 In The Treatment Of Septic Mice

Part I. The influence of CRISPLD2 on HMGB1 induced inflammation and its therapeutic effect for septic miceObjective:To determine the effect of CRISPLD2 on serum inflammatory factors levels in septic mice and the influence of CRISPLD2 on inflammatory factors transcription, and to investigate whether CRISPLD2 can improve the survival of septic mice.Method:THP-1 cells were treated by HMGB1 and harvested at 0h、1h、3h、6h、12h、 24h for CRISPLD2 expression analysis. To explore whether the transcription of inflammatory factors and chemokines were influenced by CRISPLD2, we stimulated THP-1 cells with HMGB1 (lug/ml) after knockdown the expression of endogenous CRISPLD2 using the most effective siRNA. We also added recombinant CRISPLD2 to in combination with HMGB1 to test the effect of CRISPLD2 on HMGB1 induced inflammation. To determine the effect of CRISPLD2 on serum inflammatory factors levels, septic mice were injected with recombinant CRISPLD2 and blood sample were drawn from vein at 24h for determination the concentration of cytokines. Further, the survival curves for septic mice injected with and without recombinant CRISPLD2 were recorded and analyzed.Results:HMGB1 can enhance the expression of CRISPLD2 in a time dependent and dose dependent manner. HMGB1 induced inflammation (TNF-a、IL-6、IL-8 and MCP-1) was magnified after endogenous CRISPLD2 was silenced. Correspondingly, HMGB1 induced inflammation was curtailed if recombinant CRISPLD2 was treated. The serum levels of TNF-a and IL-6 were reduced by CRISPLD2 administration in mice with sepsis. However, the survival duration was not prolonged in mice treated with CRISPLD2 compared to those treated with physiological saline. TConclusions:CRISPLD2 can alleviate HMGB1 induced inflammation but this effect cannot be translated into survival benefits.Part II. Synthesis and identification of the bi-functional fusion protein--CRISPLD2-anti-HMGB1Objective:First, to design and synthesis the bi-functional fusion protein (CRISPLD2-anti-HMGB1). Second, to determine its affinity with LPS and HMGB. Third, to calculate the value of thermal equilibrium dissociation constant [Kd(M)].Method:Using genetic engineering techniques, CR1SPLD2 was implanted into the Fc domain of anti-HMGB1 antibody, forming a fusion protein (CRISPLD2-anti-HMGB1). Coomassie brilliant blue staining was applied to identification the fusion protein after polyacrylamide gel electrophoresis. BiaCORE technique was used to determine the affinity of CRISPLD2-anti-HMGB1 with LPS and HMGB1, respectively. The Kd(M) was calculated by using Scatchard plot method. Competitive ELISA assay was performed to determine the affinity of CRISPLD2-anti-HMGB1 with LPS and HMGB1.Results:The molecular weight of CRISPLD2-anti-HMGB1 was about 250kD. The affinity of CRISPLD2-anti-HMGB1 with LPS was slightly less than that of CRISPLD2 with LPS. The Kd(M) for CRISPLD2-anti-HMGB1 and LPS was (3.11±0.21) *10-6. The affinity of CRISPLD2-anti-HMGB1 with HMGB1 was slightly less than that of anti-HMGB1 with HMGB1. The Kd(M) for CRISPLD2-anti-HMGB1 and HMGB1 was (2.02±0.68) *10-10. The competitive curves for CRISPLD2-anti-HMGB1 and CRISPLD2 binding to LPS, as well as that for CRISPLD2-anti-HMGB1 and anti-HMGB1 binding to HMGB1 were similar.Conclusions:CRISPLD2-anti-HMGB 1 can bind to LPS and HMGB1 due to its high affinity with them.Part Ⅲ. The influence of the bi-functional fusion protein (CRISPLD2-anti-HMGB1) on inflammation response and its therapeutic effect for septic miceObjective:First, to explore the pharmacokinetics of CRISPLD2-anti-HMGB1 in vivo. Second, to investigate its influence on inflammation response and inflammatory pathways. Third, to determine whether this fusion protein can protect septic mice from organ injury and death.Method:After a single dose of CRISPLD2-anti-HMGB1 (50mg/kg) was injected, mice blood samples were drawn from vein at lh, Id,3d,7d, 10d and 14d for determination the concentration of this fusion protein as time goes by. Co-incubation the mixture of LPS (0.1ng/ml) and HMGB1 (lug/ml) with CRISPLD2. anti-HMGB1 and CRISPLD2-anti-HMGB1 for 12h, respectively. Then, the prior co-incubated solutions were added into THP-1 culture mediums. THP-1 Cells were harvested 24h later and the expression of p-p65、p-p38、 p-pJNK1/2、p-pERK1/2 and the transcription of TNF-a, IL-6、IL-8、MCP-1 were tested. In vivo experiments, CRISPLD2-anti-HMGB1、CRISPLD2、anti-HMGB1 were injected for mice with sepsis. The serum inflammatory factor levels were tested 24 hours later after CRISPLD2-anti-HMGB1、CRISPLD2、anti-HMGB1 administration. The lung tissues from different intervention groups were also. The survival rates for different treatment groups were recorded every 12 hour until the end of the 10th day.Results:The half-life period of CRISPLD2-anti-HMGB1 is around 4 days. In vitro, this fusion protein can alleviate the mixed solution [LPS (0.1ng/ml) and HMGB1 (1ug/ml)] induced TNF-a、IL-6、IL-8 and MCP-1 transcription. In addition, this fusion protein can also mitigate the activation of NF-kb pathway and MAPK pathways by down regulation transcriptional factors such as p-p65、p-p38、p-pJNK1/2、p-pERK 1/2. In vivo, this fusion protein can reduce the concentration of inflammatory factors TNF-a and IL-6. Moreover, it can also reverse sepsis induced lung injury and protect mice against death events in a dose dependent manner.Conclusion:CRISPLD2-anti-HMGB1 can alleviate inflammation in vitro and in vivo, protect septic mice from organ injury and death.