两栖类皮肤抗菌肽chensinin-1b对巨噬细胞极化功能调控作用及其分子机制研究

Macrophages can polarize towards M1 or M2 activated phenotypes in the challenges of micro-environment. Sepsis is mainly caused by immune dysfunction, which is induced by the disturbance of M1/M2 polarization. Regulation of the polarization has been reported to be effective therapeutics for sepsis and other inflammatory diseases. Our previous study demonstrated that the antimicrobial peptide chensinin-1b suppressed the secretion of proinflammatory cytokines from M1 macrophages, and was also found to be able to penetrate mouse skin and effective in vivo in a mouse wound infection model, the effects of chensinin-1b on mouse wound healing in mice are more significant than the popular commercial product mupirocin ointment. Therefore, we supposed that chensinin-1b has the ability to induce macrophages switch from M1 to M2. In this proposal, the mechanisms of the macrophage polarization and immune regulation by chensinin-1b will be investigated at both cellular and molecular levels by using various techniques, including in vitro culture system of macrophages, immunology, molecular biology, etc. Meanwhile, a cecal ligation and puncture sepsis model in rats is used to investigate the effects of chensinin-1b on macrophages and the modulating mechanisms. From the above research, we are trying to present the theoretical basis for the development of new peptide drugs to treat sepsis or inflammatory diseases.

巨噬细胞在不同微环境下可极化分为M1和M2两种亚型，极化稳态失衡在脓毒症免疫功能紊乱中起重要作用，调节巨噬细胞极化过程是治疗脓毒症及其它炎症相关疾病的一种有效方法。本课题组前期研究发现抗菌肽chensinin-1b能够抑制M1型巨噬细胞分泌促炎因子，且在小鼠皮肤伤口模型中对皮肤伤口具有明显的M2型特征的促进愈合作用，推测chensinin-1b能够抑制巨噬细胞M1型极化而向M2型转化。本项目拟通过应用巨噬细胞体外诱导体系、分子生物学、细胞免疫学等技术手段，从细胞、分子水平，探讨 chensinin-1b调节巨噬细胞极化过程及其免疫功能的分子机制。同时利用CLP小鼠脓毒症模型研究chensinin-1b在脓毒症中对巨噬细胞极化状态的影响，从整体动物水平阐明其与脓毒症发生发展的相关性，为该肽作为治疗脓毒症及免疫调节药物研发提供理论依据。

败血症是一种由感染引起的免疫反应综合征，导致多器官功能障碍，其死亡率高，缺乏有效的治疗方法。巨噬细胞是先天免疫系统的重要细胞，可极化成M1/M2不同表型，并参与败血症的发病机理。巨噬细胞极化的调节已成为一种潜在的治疗策略。本项目探究了两栖类皮肤抗菌肽1b及其类似物W3对巨噬细胞M1/M2型极化的影响及其潜在机制，并在CLP诱导的脓毒症动物模型中研究了1b基于调控巨噬细胞极化对脓毒症的治疗作用。为两栖类皮肤抗菌肽作为脓毒症治疗药物的研发提供新的思路和依据。利用ELISA、流式细胞术、RT-qPCR及western blot技术评估了1b及W3分别对M1/M2型巨噬细胞的表面标志物、炎症因子、特征基因及相关通路蛋白表达的影响，发现在LPS诱导的M1型巨噬细胞模型中加入1b及W3干预后，M1型细胞表面标志物CD86及M1型促炎因子与介质TNF-α、IL-6、IL-1β和NO的表达均明显下调；同时上调了M2型抑炎因子IL-10和TGF-β1的释放，M2型特异基因Arg1和Ym1的表达明显升高。检测了极化相关通路蛋白的表达，表明1b和W3在巨噬细胞在向M1极化过程中，对NF-κB和MAPK通路中IκB、NF-κBp65、JNK、ERK和p38等蛋白蛋白的磷酸化水平有显著抑制作用；同时使M2极化相关通路STAT6通路中PPAR-γ和KLF4表达量明显升高，表明1b和W3在LPS诱导巨噬细胞M1活化过程中可抑制M1过度极化，促进巨噬细胞向M2型转化。在IL-4诱导的M2型巨噬细胞模型中，1b和W3同样能抑制巨噬细胞向M2型过度极化，促使巨噬细胞向M1型转化。具体表现在：下调IL-10和TGF-β1的释放，抑制Arg1、Ym1和CD206表达，但促进了TNF-α和IL-6的释放。KLF4、PPAR-γ、p-STAT6等蛋白表达水平被显著抑制，同时IκB和NF-κB磷酸化水平升高。检测了1b和W3对巨噬细胞迁移功能的影响。1b和W3处理LPS诱导的M1型巨噬细胞的上清液可促进肺上皮细胞MLE-12及人乳腺癌细胞MCF-7的迁移。与之相反，IL-4诱导的M2型巨噬细胞经两种抗菌肽作用后，其上清液能抑制MCF-7细胞的迁移。同样证实1b和W3可双向抑制巨噬细胞向M1/M2型过度极化。在CLP诱导小鼠脓毒症模型中，1b在体内能通过调控巨噬细胞极化改善小鼠脓毒症，提高脓毒症小鼠的整体存活率。

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