Kallistatin调控巨噬细胞的数量和分化抑制糖尿病创伤愈合的作用及机制研究

M1 macrophages secrete inflammatory cytokines to induce inflammatory response in the early period of physiological wound healing. Along with the repair process, M1 macrophages change to be M2 macrophages which terminate inflammatory response through secreting anti-inflammatory cytokines. During diabetic statement, sustained activation of inflammatory response and M1 macrophages accumulation leads to delayed wound healing. Our previous study have shown that plasma Kallikrein binding protein (Kallistatin) was significantly upregulated and inhibited wound angiogenesis in diabetic patients. Pre-experiments revealed that Kallistatin transgenosis slowed down the wound healing process, activated inflammatory response sustainedly and increased the number of M1 macrophages, which suggested that upregulated Kallistatin may inhibit wound healing through regulating macrophages to induce inflammatory response abnormally. This study is aimed to illustrate the role and mechanism of Kallistatin in diabetic wound healing. Firstly, we will clarify the function of Kallistatin during the process of wound healing through overexpression or knock out Kallistatin gene. Further, we will definite the implications of whether Kallistatin regulates the numbers, homing and differentiation of macrophages to induce inflammatory response and inhibits wound healing in diabetic wound healing mice model. Overall, this study will elucidate the significant regulatory molecule involved in abnormal inflammatory response of diabetic wound healing, providing a new intervention target for the treatment of diabetic foot ulcer.

正常伤口愈合早期浸润M1型巨噬细胞分泌炎症因子诱发炎症反应，随着修复进程巨噬细胞由M1型转变为M2型，通过分泌抗炎因子终止炎症反应；糖尿病状态下炎症反应持续激活、M1型巨噬细胞积累，伤口愈合延迟。我们以往的研究表明糖尿病患者血浆激肽释放酶结合蛋白(Kallistatin)显著上调并抑制伤口血管新生；预实验显示Kallistatin转基因鼠伤口愈合减慢，炎症持续激活且M1型巨噬细胞数量增多，提示糖尿病情况下上调的Kallistatin还可能通过调控巨噬细胞诱导炎症反应异常从而抑制创伤愈合。本研究拟探讨：①过表达或敲除Kallistatin，进一步明确Kallistatin影响伤口愈合的功能；②明确糖尿病情况下Kallistatin是否具有调节巨噬细胞数量、归巢和分化的作用参与炎症反应和伤口愈合并阐明其分子机制。旨在明确糖尿病创伤修复炎症反应异常的关键调控分子，为治疗糖尿病足提供干预新靶点。

正常伤口愈合早期浸润M1型巨噬细胞分泌炎症因子诱发炎症反应，随着修复进程巨噬细胞由M1型转变为M2型，通过分泌抗炎因子终止炎症反应；糖尿病状态下炎症反应持续激活、M1型巨噬细胞积累，伤口愈合延迟。我们以往的研究表明糖尿病患者血浆激肽释放酶结合蛋白(Kallistatin)显著上调并抑制伤口血管新生；但Kallistatin是否可以通过调控巨噬细胞抑制创伤愈合尚无文献报道。本项目旨在探讨Kallistatin是介导糖尿病延迟愈合过程中的关键分子；明确Kallistatin是否具有上调巨噬细胞数量、促进伤口归巢及分化等功能发挥诱导炎症反应持续激活、抑制伤口愈合的作用；阐明Kallistatin调控巨噬细胞数量和分化的分子机制。为此本项目设计相关内容并取得以下重要结果：(1) 糖尿病病人血液中Kallistatin含量和单核细胞数目升高，其中糖尿病合并糖尿病足者较未合并糖尿病足者进一步升高。（2）Kallistatin抑制伤口愈合，而腹腔注射Kallistatin中和抗体可改善糖尿病小鼠伤口愈合。（3）Kallistatin使血液中单核细胞以及伤口组织中浸润的巨噬细胞数目增加。（4）Kallistatin促进巨噬细胞向M1型极化。（5）Kallistatin可通过激活Notch信号通路促进巨噬细胞M1极化。（6）Notch信号通路下游的靶基因Hes1可以结合到iNOS基因的promoter区上，但并不能直接激活其表达。（7）Kallistatin激活Notch信号通路后进一步激活NF-κB炎症信号通路促进巨噬细胞M1极化。（8）Kallistatin能上调M-CSF及MCP-1。该项目首次明确Kallistatin调控巨噬细胞型别与数量来延缓糖尿病伤口愈合的作用及机制。从体内外实验证实糖尿病状态下上升的Kallistatin可通过激活Notch信号通路来促进巨噬细胞向M1型极化，并且可上调M-CSF及MCP-1等与巨噬细胞分化来源和迁移趋化密切相关的关键分子来促进单核巨噬细胞的分化以及在伤口炎症部位的浸润聚集，从而使伤口组织处于一种长期的慢性炎症状态，为糖尿病足的治疗提供理论基础及可能靶点。