MEBT/MEBO上调HSP90α激活HIF-1α调控血管生成途径促进慢性难愈合创面修复的分子机制研究

体表慢性难愈合创面（慢创）修复是世界性医学难点。中医药外治法皮肤再生医疗技术（MEBT/MEBO）治疗皮肤溃疡临床疗效确切，前期研究证实其可提高创面组织VEGF等表达，调控血管生成促创面修复，但作用靶点与机制仍待阐明。研究表明慢创缺氧环境下激活HIF-1α途径促血管生成加速创面修复，其中HIF-1α分子伴侣HSP90α发挥重要作用；网络药理学分析显示该技术治疗慢创可能与调控HIF-1信号通路密切相关，我们亦发现其可上调创面组织HSP90α和HIF-1α表达。由此提出科学假说：MEBT/MEBO可能通过上调HSP90α水平，激活HIF-1α信号通路调控VEGF转录表达，促进血管生成进而加速创面修复。我们拟构建慢性难愈合创面大鼠模型和培养人脐静脉内皮细胞，予以MEBT/MEBO干预，运用分子与细胞生物学等技术，从HSP90α-HIF-1α-VEGF信号通路探讨该技术促进慢性创面修复的可能机制。

The treatment and care of patients with chronic refractory cutaneous wounds has been a major healthcare burden and medical challenge around the world. Despite a plethora of research into using growth factors as therapeutic agents, to date, the field has been disappointing. Chinese medicine has well-established histories of use for treatment of skin ulcers. One of the most important and representative Chinese herbal preparations in this field is moist exposed burn therapy/moist exposed burn ointment (MEBT/MEBO). Our previous studies have demonstrated that MEBT/MEBO exerts its therapeutic effects on cutaneous wound healing through increasing levels of VEGF mRNA and protein expression in the granular tissues, accelerating the proliferation and division of vascular endothelial cells, and promoting neo-angiogenesis. However, the exact mechanisms underlying MEBT/MEBO’s effects on the promotion of angiogenesis in the wound tissues remain unclear. Hypoxic environment is commonly seen in chronic wound, and HIF-1αhas been shown to play an essential role in the wound healing. Activation of HIF-1α signaling by HSP90α activates transcription levels of VEGF, thereby inducing angiogenesis in the wound tissues. We recently found that application of MEBT/MEBO markedly increased production of HSP90α as well as enhanced the expression of HIF-1α in the wound tissues as compared with the model group. Taken together, we would hypothesize that MEBT/MEBO may specifically target and increase the production of HSP90α which then stabilize the expression of cellular HIF-1α and consequently activate the HIF-1α/VEGF signaling to promote the induction of angiogenesis in the wound tissues..To prove our hypothesis, we will first establish chronic refractory cutaneous wound in SD rats, and different groups of application of HSP90α protein, specific inhibition of HSP90α, or HSP90α inhibition plus application of MEBT/MEBO are designed. After treatment, tissues are collected from each group and supernatant is separated from tissue specimens for measurement of HSP90α by the ELISA method. Also, protein expression of HIF-1α in wound tissues is detected by Western blotting. To investigate whether upregulation of HSP90α by MEBT/MEBO stabilizes the expression of cellular HIF-1α and consequently activate the HIF-1α/VEGF signaling to promote the induction of angiogenesis in the wound tissues, we will further design both in vitro and in vivo studies. Both normal and HIF-1α siRNA transfected HUVECs are seeded in 6-well plates and normoxically or hypoxically cultured in ECM, rb-bFGF+ECM or MEBO+ECM media. Cells are collected for in vitro tubule formation assay to test the angiogenesis of HUVECs, immunofluorescent assay of cellular expression and nuclear translocation of HIF-1α, and WB detection of levels of HIF-1α and VEGF proteins. Finally, in vivo experiments of specific HIF-1α activation, inhibition of HIF-1α or HIF-1α inhibition plus application of MEBT/MEBO will be designed and carried out. Wound healing time and rates are recorded and calculated. On days 3, 7, and 14 after initiation of treatment, tissues are collected from each group. Levels of angiogenesis in the tissue specimens are examined and quantitated by immunostaining of CD31. Expression of HIF-1α and VEGF mRNA in tissue specimens are examined by RT-qPCR. Furthermore, protein expression of HSP90α, HIF-1α, VEGF and VEGFR2s in tissue specimens is detected by WB. Our study aims to identify and confirm the pharmacological target of MEBT/MEBO and to shed light on the molecular mechanisms underlying its effects on angiogenesis in the wound tissues. The data obtained from our research will provide scientific evidence for clinical application and market promotion of MEBT/MEBO for therapy of chronic wound healing.