Dataset Information

Elevated Kallikrein-binding protein in diabetes impairs wound healing through inducing macrophage M1 polarization.

ABSTRACT:

Background

The accumulation of M1-polarized macrophages and excessive inflammation are important in the pathogenesis of diabetic foot ulcer (DFU). However, the underlying mechanism of DFU pathogenesis and the crucial regulators of DFU are less well known. Our previous study reported that kallikrein-binding protein (KBP), an angiogenesis inhibitor, was significantly upregulated in diabetic patients compared to its levels in controls. The effects of KBP on monocyte chemotaxis and macrophage M1 polarization were elucidated in this study.

Methods

Plasma KBP levels and monocyte counts were assessed by ELISA and flow cytometry. Wound closure rates in different groups were monitored daily. The phenotype and recruitment of macrophages were measured by real-time PCR, western blot and immunofluorescence assays. The expression of Notch and NF-κB signalling pathway members was determined by the methods mentioned above. ChIP and dual-luciferase reporter gene assays were employed to explore the binding and transcriptional regulation of Hes1 and iNOS.

Results

We found that plasma KBP levels and circulating monocytes were elevated in diabetic patients compared to those in nondiabetic controls, and both were higher in diabetic patients with DFU than in diabetic patients without DFU. KBP delayed wound healing in normal mice; correspondingly, KBP-neutralizing antibody ameliorated delayed wound healing in diabetic mice. Circulating monocytes and macrophage infiltration in the wound were upregulated in KBP-TG mice compared to those in control mice. KBP promoted the recruitment and M1 polarization of macrophages. Mechanistically, KBP upregulated iNOS by activating the Notch1/RBP-Jκ/Hes1 signalling pathway. Hes1 downregulated CYLD, a negative regulator of NF-κB signalling, and then activated the IKK/IκBα/NF-κB signalling pathway.

Conclusions

Our findings demonstrate that KBP is the key regulator of excessive inflammation in DFUs and provide a novel target for DFU therapy.

SUBMITTER: Feng J

PROVIDER: S-EPMC6558923 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAfter surgery, wound recovery in diabetic patients may be disrupted due to delayed inflammation, which can lead to undesired consequences, and there is currently a lack of effective measures to address this issue. Mesenchymal stem cell (MSC)-derived exosomes (Exo) have been proven to be appropriate candidates for diabetic wound healing through the anti-inflammatory effects. In this study, we investigated whether melatonin (MT)-pretreated MSCs-derived exosomes (MT-Exo) could exert superior effects on diabetic wound healing, and we attempted to elucidate the underlying mechanism.MethodsFor the evaluation of the anti-inflammatory effect of MT-Exo, in vitro and in vivo studies were performed. For in vitro research, we detected the secreted levels of inflammation-related factors, such as IL-1?, TNF-? and IL-10 via ELISA and the relative gene expression of the IL-1?, TNF-?, IL-10, Arg-1 and iNOS via qRT-PCR and investigated the expression of PTEN, AKT and p-AKT by Western blotting. For in vivo study, we established air pouch model and streptozotocin (STZ)-treated diabetic wound model, and evaluated the effect of MT-Exo by flow cytometry, optical imaging, H&E staining, Masson trichrome staining, immunohistochemical staining, immunofluorescence, and qRT-PCR (?-SMA, collagen I and III).ResultsMT-Exo significantly suppressed the pro-inflammatory factors IL-1? and TNF-? and reduced the relative gene expression of IL-1?, TNF-? and iNOS, while promoting the anti-inflammatory factor IL-10 along with increasing the relative expression of IL-10 and Arg-1, compared with that of the PBS, LPS and the Exo groups in vitro. This effect was mediated by the increased ratio of M2 polarization to M1 polarization through upregulating the expression of PTEN and inhibiting the phosphorylation of AKT. Similarly, MT-Exo significantly promoted the healing of diabetic wounds by inhibiting inflammation, thereby further facilitating angiogenesis and collagen synthesis in vivo.ConclusionsMT-Exo could promote diabetic wound healing by suppressing the inflammatory response, which was achieved by increasing the ratio of M2 polarization to M1 polarization through activating the PTEN/AKT signalling pathway, and the pretreatment of MT was proved to be a promising method for treating diabetic wound healing.Graphical abstract: MT-Exo promotes diabetic wound healing by regulating M1 and M2 macrophage polarization.