抗菌肽AHP12在人单核/巨噬细胞THP-1中效应蛋白或靶标蛋白鉴定及功能机制研究

Sepsis is one of the commonest critical illnesses caused by a dysregulated host response to an initial insult. Unfortunately, contemporary pharmacological and therapeutical approaches to severe sepsis are nonexistent. Therefore searching for a novel medication to alleviate the inflammatory injury and improve outcome of sepsis is urgent and desirable. AHP12, a novel synthetic antimicrobial peptide, not only exhibits direct pathogen-killing and LPS-neutralizing activities in vitro but modulates immunity and other biological responses of the host in vivo. Although AHP12 is a promising candidate to against sepsis, the mechanism underlining the immunomodulatory potential is intact. Our previous research showed that prophylactic injection of AHP12 into mice prior to bacterial infection remarkably enhanced survival and incubation of AHP12 and mononuclear macrophage lines THP-1 significantly reduced LPS-induced excessive TNF-α release, suggesting the proinflammatory and anti-inflammatory dual identity of AHP12 to immune system and the effector or target proteins mediating such functions. In this study, we are planning to firstly hunt and identify the effector or target proteins of AHP12 in THP-1 cells using peptide affinity chromatography, SDS-PAGE and mass spectrum, then validate and investigate the interaction of AHP12 and effector or target proteins by production recombination proteins and bioinformatic analysis. Thirdly the SiRNA and overexpression cell models of effector or target proteins will be constructed to study the immunomodulatory mechanism of AHP12. A better understanding of immune mechanism of AHP12 and effector or target proteins may present novel therapeutic theory and approach for sepsis.

脓毒症是宿主免疫系统炎性调节失控引起的临床危重病症，尚无特效药和治疗方法，改善其治疗和愈后成为研究热点。抗菌肽AHP12除优良的抗菌和抗LPS作用外，还可双向调节宿主免疫，有望成为抗脓毒症新药，但其靶点和机制不甚明确。课题组前期研究发现AHP12可适度激活先天免疫系统提高急性腹膜炎小鼠生存率；亦可与THP-1细胞直接结合并抵抗LPS诱发的TNF-α过度释放，提示AHP12可能存在效应或靶标蛋白来介导其对免疫系统的双向调节作用。本研究拟用生物素修饰AHP12，以多肽亲和色谱法、SDS-PAGE和蛋白质谱筛选鉴定AHP12在THP-1细胞中的结合蛋白；利用重组蛋白验证和确定AHP12的效应或靶标蛋白并研究两者结合模式和特性；构建SiRNA干扰和过表达细胞模型研究该蛋白在AHP12免疫调节中的作用。借此探明AHP12和效应或靶标蛋白的免疫调节机制，为抗脓毒症新药开发提供理论依据和新思路。

脓毒症是宿主免疫系统炎性调节失控引起的临床危重病症，尚无特效药和治疗方法。本课题组前期研究发现抗菌肽AHP12除优良的抗菌和抗LPS作用外，还可调节宿主免疫，有望成为抗脓毒症新药，但其靶点和机制不甚明确。为研究AHP12其免疫调节机制。本课题以抗菌肽AHP-12在重要免疫细胞THP-1中效应蛋白为切入点，展开深入研究。首先合成高纯度AHP-12并成功进行生物素修饰，通过抗菌活性、中和LPS和抑制TNF-α表达等对比研究验证了生物素修饰并未影响AHP-12的主要生物学效应。之后利用生物素修饰AHP-12，通过多肽亲和色谱法和蛋白质谱分析筛选和鉴定出TRIF蛋白为AHP-12在THP-1细胞中主要结合蛋白，并在胞外考察验证了TRIF蛋白可与AHP-12特异性结合，同时该结合还具有浓度依赖性、饱和性和受环境离子强度影响较小等特点。随后课题组成功构建了TRIF蛋白SiRNA干扰和过表达细胞模型，并进一步研究发现，AHP-12预处理细胞，可发挥与SiRNA干扰模型细胞类似的减少LPS诱导TNF-α表达水平的效应，且AHP-12可使SiRNA干扰细胞TNF-α水平进一步降低；此外，AHP-12还可显著抑制TIRF蛋白过表达细胞经LPS诱导产生大量TNF-α，表明AHP-12可通过TIRF蛋白发挥免疫调节作用。同时，研究还发现AHP-12对TNF-α的mRNA表达水平和稳定性、Tnfa基因转录和TNF-α蛋白分泌并无显著影响，提示AHP-12可能在转录后阶段影响TNF-α合成。NF-κB信号通路和MAPK信号通路为TRIF蛋白介导的影响TNF-α合成的两大重要途径，但后续研究表明AHP-12对NF-κB信号通路并无显著影响，而MAPK信号通路的p38、JNK和Erk三条途径均能被AHP-12所抑制并显著降低后续LPS诱导的磷酸化水平。该效应也在对MAPKs p38信号通路进一步研究中得到验证，MAPKs p38信号通路影响TNF-α生物合成重要激酶MK2的Thr222和Thr334两个磷酸化活性位点亦能被AHP-12抑制，从而下调MK2激酶介导的TNF-α生物合成。通过本课题的实施可以明确抗菌肽AHP12在人单核/巨噬细胞THP-1中效应蛋白或靶标蛋白，揭示AHP12通过该蛋白发挥免疫调节作用的机制，为研究开发预防和治疗脓毒症的新型药物和治疗方案提供理论依据和新思路。

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