Study On The Pharmacodynamics And Mechanisms Of TNFR1-selective Antagonistic Peptide Hydrostatin-SN10 In The Treatment Of Sepsis

BackgroundSepsis is a life-threatening organ dysfunction which is caused by host response disorder due to infection.It is the most common and dangerous disease in Intensive Care Unit（ICU）.Due to the elevated mortality rate,it has always been a serious clinical problemin Emergency and critical care Medicine.It is generally believed that the pathogenesis of sepsis is runaway inflammatory response,immune dysfunction,high metabolic status and multi-organ dysfunction induced by Lipopolysaccharide（LPS）after infection.The disorder of monocytes/macrophages plays a key role in the occurrence and development of sepsis.LPS can stimulate monocytes/macrophages to release a mass of inflammatory factors mainly tumor necrosis factor-α（TNF-α）.TNF-αexerts inflammatory effects via its downstream tumor necrosis factor receptor typeⅠ（TNFR1）.However,TNF-αinhibits inflammation and maintains immune balance when it binds to tumor necrosis factor receptor typeⅡ（TNFR2）.The balance shifting to TNF-α/TNFR1combined with a variety of inflammatory factors results in inflammation storm and rapid cascade expansion,causes systemic inflammatory response syndrome（SIRS）and multiple organ function impairment（MODS）.In the middle and late stage of sepsis,excessive immunosuppression is the main manifestation,and the body gradually enters the immune imbalance state.Inflammatory storm and immune imbalance caused by excessive release of inflammatory mediators are considered to be the main mechanisms that mediate septic organ injury and induce MODS,resulting in death and poor prognosis of patients.In addition,the process of immune imbalance is mainly related to TH17 and Treg cells.In the early stage of sepsis,TH17 cells play a dominant role in inflammation,while in the later stage,the overexpression of inflammatory factors exceeds the body’s ability to regulate,resulting in excessive consumption of TH17 cells and dominance of Treg cells,resulting in immunosuppression and immune paralysis,aggravating infection.There is lack of effective prevention,control drugs and methods.It is very important to suppress the outbreak of inflammation before the inflammatory storm occurred in sepsis patients.TNF-αis a cytokine with a variety of biological activities.TNF-αand its receptor TNFRs play key roles in the process of inflammatory response and immune regulation,which have been the important target for the pathogenesis of inflammatory diseases or autoimmune diseases,and also drug design.Currenttly,the monoclonal antibody drugs such as Infliximab（IFX）and Adalimumab,have achieved great success in the clinical treatment of inflammatory bowel disease（IBD）and rheumatoid arthritis（RA）,further proving that blocking TNF-αis an effective treatment.However,those anti-TNF-αmonoclonal antibodies completely block the biological function of TNF-α,leading to the influence of immune self-stability and immune surveillance functions of the body.Many patients are prone to tuberculosis infection,the generation of new autoimmune diseases and even induce tumor toxicity.TNF-αplays a proinflammatory role mainly through its receptor TNFR1,and its receptor TNFR2 plays an feedback regulation.Blocking TNFR1signaling pathway targetly will,neutralizing the pro-inflammatory activity of TNF-α,and maintaining TNFR2-mediated beneficial immune response（including immune regulation,tissue homeostasis and neuroprotection）has become a hotspot of drug research and development.It may be able to use TNFR1 as a drug target to specifically and specifically inhibit the function of TNFR1 can inhibit the inflammatory function of TNF-α,thus inhibit inflammatory storm.Meanwhile,opening TNF-TNFR2 pathway to maintain immune balance.The hypothesis may havegreat significance for the prevention and treatment of sepsis.In the early stage,TNFR1 was used as a specific target to panning the adenophage display library of Hydrophis cyanocinctus venom,and a target-specific active peptide Hydrostatin-SN10 was obtained.The anti-inflammatory peptide has achieved significant effect in the treatment of inflammatory bowel disease and rheumatoid arthritis mouse models.The indication of inflammatory bowel disease preclinical studies and pre-IND have been completed.Since SN10 can selectively and specifically bind to TNFR1 and block the TNF-TNFR1 pathway without interfering with the balanced immune function of TNF-TNFR2 pathway,it is suitable for the treatment of sepsis.This project intends to use in vitro cell inflammation models and sepsis animal models to perform pharmacodynamics and mechanism research of SN10,and perfect the pre-clinical research data of SN10.It lays a foundation for the research of SN10 as a First in Class innovative drug derived from Marine organisms with clear target.ObjectiveIn this study,the anti-inflammatory effect and mechanism of polypeptide Hydrostatin-SN10 in vitro were verified by LPS-induced inflammation models of Bone Marrow-Derived Macrophage（BMDM）and RAW 264.7 cell lines.Subsequently,Tnfr1^-/-and Tnfr2^-/-cell models were used to confirm that Hydrostatin-SN10 target to TNFR1.On the other hand,C57BL/6 mice sepsis models was established by Cecal Ligation and Puncture（CLP）and LPS to study the anti-inflammatory effect and mechanism of Hydrostatin SN10 in vivo.Subsequently,the specific selectivity of Hydrostatin-SN10 to target TNFR1 in vivo was verified by Tnfr1^-/-and Tnfr2^-/-animal models,and the mechanism of action of Hydrostatin-SN10 in sepsis was clarified.This will have important practical significance for developing selective TNFR1 antagonistic peptide Hydrostatin-SN10 into a first-in-class Marine innovative drug with independent intellectual property rights and clinically urgent needs with Chinese characteristics.Methods1.Study on the anti-inflammatory effect and mechanisms of Hydrostatin-SN10 in vitro1.1 On the basis of the inflammatory model of RAW264.7 and wild-type（WT）BMDM stimulated by 1μg/ml LPS.According to the preliminary experimental results,infliximab（IFX,TNF-αmab）was administered at 0.55μM（molecular weight:144.19KD,concentration:80μg/m L）,and SN10 was administered at 25μM,50μM and 100μM（molecular weight:1.25KD,concentration:31.25μg/m L,62.5μg/ml and 125μg/m L）was used to investigate the anti-inflammatory effect and mechanism of SN10 in vitro.1.1.1 The expression of inflammatory factor NO was detected by Griess reagent at 24h after LPS stimulation,and the expression of others inflammatory factors such as IL-6,TNF-αand IL-10 were detected by RT-PCR and flow cytometry at 6 h after LPS stimulation,to evaluate the anti-inflammatory effect of SN10.1.1.2 The phosphorylation levels of ERK,JNK,P65,P38 and IκB protein in the downstream TNFR1 pathway were investigated by Western blot,with the nuclear translocation of nuclear transcription factor（NF-κB）detected by immunofluorescence technique at 6 h after LPS stimulation to reveal the anti-inflammatory molecular mechanism of SN10.1.2 Based on the 1μg/ml LPS-induced BMDM inflammatory model of wild-type,Tnfr1 and Tnfr2 knockout C57BL/6 mice,confirming the anti-inflammatory effect of SN10 on different genotypes cells and its target selectivity against to TNFR1 at the cellular level.2.Study on the pharmacodynamics and mechanisms of Hydrostatin-SN10 in the treatment of sepsis2.1 Wild-type C57BL/6 mouse sepsis model induced by CLP and abdominal injection of LPS were performed,which for studing the pharmacodynamics and mechanisms of SN10 in the treatment of sepsis2.1.1 High-grade sepsis model of mice was established by ligation of 75%of the cecum or 15 mg/kg LPS.Continuous observation for 7 days for screening optimal drug concentration and evaluating treatment effect on sepsis of SN10 in accordance with the survival rate of sepsis mice intervened by different concentrations of SN10.2.1.2 Mid-grade sepsis model of mice was established by ligation of 50%of the cecum or 10 mg/kg LPS.24 h later,the absolute number of monocytes in peripheral blood,the expression levels of TNF-α,IFN-γ,GM-CSF,IL-1β,IL-6,IL-10 and other inflammatory factors in serum,as well as the organ function indexes of ALT,AST,BUN,Cr andα-AMS combined with the result of histopathological damage of lung,liver,kidney and spleen organs were analyzed by HE staining to evaluate the effect of 800μg/kg SN10on septic inflammatory storm and the protection of multiple organs in septic mice;The expression of Treg cells in spleen tissue and the activation of TNFR1 downstream inflammation-related signaling pathways were analyzed by Immunohistochemistry and Western blot,respectively,revealing the in vivo mechanism of SN10.2.2 Be based on the CLP-induced Tnfr1 and Tnfr2 knockout C57BL/6 mouse model of high-grade sepsis,IFX and different concentrations of SN10 were used for intraperitoneal injection treatment to verify the target selectivity of SN10 in vivo,which further explore the mechanism of action of SN10 in the treatment of sepsis through selective antagonism TNFR1.Results1.Study on the anti-inflammatory effect and mechanisms of Hydrostatin-SN10 in vitro1.1 SN10 significantly inhibited the expression of NO,TNF-α,IL-6 and other pro-inflammatory factors and the phosphorylation of TNFR1 pathway key proteins P65,IκB,JNK,ERK and P38 and NF-κB in the LPS-induced RAW 264.7 cell inflammation model.1.2 SN10 significantly inhibited the expression of NO,TNF-α,IL-6 and other pro-inflammatory factors and the phosphorylation of key proteins in NF-κB and MAPK signaling pathways downstream of TNFR1 in LPS-stimulated WT and Tnfr2^-/-BMDM inflammatory models.However,SN10 had no significant remission of the above indicators in Tnfr1^-/-BMDM.2.Study on the pharmacodynamics and mechanisms of Hydrostatin-SN10 in the treatment of sepsis2.1 The survival rate of mice treated by SN10 was significantly improved in CLP and LPS-induced severe sepsis model of WT C57BL/6 mice.800μg/kg group had the best effect.2.2 SN10 inhibited the absolute number of monocytes in peripheral blood of sepsis mice,biochemical indices of AST,ALT,CRE,BUN andα-AMS in serum,as well as the expression of inflammatory factors such as IL-6,TNF-α,IL-1βand IL-17,and increase the expression of anti-inflammatory factors such as IL-10.HE staining results showed that in the model group,inflammatory cells were infiltrated obviously,structures were damaged to varying degrees,and even tissue necrosis occurred.However,the inflammatory cell infiltration was significantly reduced and tissue destruction was not serious in the SN10and IFX groups,among which the SN10 was better.Compared with the model group,the expression level of Foxp3 and the phosphorylation of IκB,JNK,ERK and P38 was significantly inhibited in the SN10 and IFX groups,among which the SN10 was better,which showed by spleen immunohistochemistry and western blot,respectively.2.3 In CLP-induced severe sepsis model of Tnfr2^-/-C57BL/6 mice,the survival rate of mice treated with SN10 was significantly higher than that of model group,and the survival rate of mice in 800μg/kg group was 62.5%（P<0.001）,far better than IFX group（25%）;In the Tnfr1^-/-C57BL/6 mouse sepsis model,neither SN10 nor IFX significantly improved the survival rate of mice.ConclusionThis study confirmed that selective TNFR1 antagonistic peptide Hydrostatin-SN10can effectively inhibit the expression of NO,IL-6,TNF-αand other inflammatory factors at the gene level and protein level in vitro,playing an anti-inflammatory effect.The anti-inflammatory mechanism of Hydrostatin-SN10 was revealed by inhibiting the phosphorylation of key proteins in the NF-κB and MAPK pathways downstream of TNFR1.Furthermore,the specific selectivity of Hydrostatin-SN10 to TNFR1 was confirmed.Selective TNFR1 antagonistic peptide Hydrostatin-SN10 has a significant anti-inflammatory effect on septic animal models,which can prevent the occurrence of inflammatory storm and reduce the degree of organ damage.The mechanism of action is"selectively blocking the TNF-TNFR1 signaling pathway and blocking the inflammatory storm;Opening TNF-TNFR2 signaling pathway,regulating Th17/Treg,and maintaining immune balance".This has laid the foundation for the development of Hydrostatin-SN10as an innovative Marine drug with independent intellectual property rights and urgently needed clinical treatment for sepsis.