Background

The annual incidence of diabetic foot ulcers (DFUs) has been reported to vary from 0.2% to 11% in diabetes-specific clinical settings and less than 0.1% to 8% in community- and population-based cohorts. According to the International Diabetes Foundation, approximately 40 million to 60 million people worldwide are affected by DFUs, and a recent meta-analysis indicates a global prevalence of 6.3% among adults with diabetes, or about 33 million individuals. The cost of diabetes care is significant, amounting to $273 billion in direct and $90 billion in indirect expenses annually, in America. Foot complications in diabetes care excess annual expenditures ranging from 50% to 200% above the baseline cost of diabetes-related care. The cost of advanced-stage ulcers can be more than $50,000 per wound episode, and the direct expenses of major amputation are even higher. DFUs can be treated using various methods, including wound dressings, antibiotics, pressure-off loading, skin substitutes, stem cells, debridement, topical oxygen therapy, gene therapy and growth factors. For severe DFUs patients are at risk of amputation if treatment is not timely or appropriate. Amputating limbs not only causes physical pain to patients, but also brings economic burden due to lost productivity, and decreased employment linked to DFUs. Currently, long-term use of local antibiotics in clinical practice is prone to induce drug resistance, while growth factors do not effectively inhibit bacterial growth and control inflammation in wounds. Stem cell and gene therapies are still in the experimental stage. The method of local debridement combined with negative pressure therapy is expensive. Therefore, we urgently need an affordable, non-surgical method to treat diabetic ulcers. Extracts of bark of Bauhinia purpurea, Paeoniae rubrae, Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., Acorus calamus L, and Radix Angelicae biseratae have been used as traditional remedies to treat inflammation-related diseases and cutaneous wounds due to their anti-inflammatory properties and their ability to promote vascular renewal. However, there have been few studies on the mixture of these five herbal extracts on diabetic wound healing.

Purpose

This study was designed to assess the healing effect of a mixture of five aforementioned herbal extracts on diabetic ulcer wounds in rats, and to reveal the potential mechanisms behind any potential wound healing using transcriptomics and proteomics.

Study design

We designed the experiment to explore the effects of five herbal extracts on diabetic wound healing process through in vivo experiments and to investigate the underlying mechanisms through proteomics and transcriptomics.

Methods

We used a mixture of five aforementioned herbal extract to treat rat model of diabetic established by intraperitoneal injection of streptozotocin, and a 2 × 2 cm round full-thickness skin defect was created on the back of the rat. Staphylococcus aureus (1 ml of 1.5 × 10⁹ cfu/ml) was evenly applied to the wound. The wound was then observed for 72 h. The infected ulcer model of diabetic rats was considered to be successfully established if the wound was found to be infected with S. aureus. According to different medications, the rats were divided into three groups, namely mixture of herbal extract (MHE), Kangfuxin solution (KFS) and control (Ctrl). The effects of the medicine on wound healing were observed. HE staining and Masson staining were performed to evaluate the histopathological changes and collagen synthesis. IHC staining was used to assess the neovascularization, and M2 macrophage proliferation was determined by immunofluorescence staining. Proteomic and transcriptomic studies were performed to explore potential mechanism of five herbal extracts to promote wound healing. UHPLC-QE-MS was performed to identify the chemical composition of mixture of herbal extract.

Results

The study show that the mixed herbal extract promotes angiogenesis, proliferation of M2 macrophages, and collagen synthesis. Transcriptomics showed that rno-miR-1298, rno-miR-144-5p, and rno-miR-92a-1-5p are vital miRNAs which also play a significant role in role in regulating wound healing. Proteomics results showed that the following proteins were important in wounds treated with MHE: Rack1, LOC100362366, Cops2, Cops6, Eif4e, Eif3c, Rpl12, Srp54, Rpl13 and Lsm7. Autophagy, PI3-Akt and mTOR signaling pathways were enriched after treatment with MHE compared to other groups.

Conclusion

Herein, we have shown that MHE containing extracts of bark of Bauhinia purpurea, P. rubrae, A. dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., A. calamus L, and R. A. biseratae has significant wound healing effects in the diabetic ulcer wound rat model. These results suggest that local application of MHE in diabetic wounds can accelerate the wound healing process. Moreover, in vivo experiments revealed that the diabetic wound healing process was primarily mediated by angiogenesis and M2 macrophage transition. Therefore, this study may provide a promising and non-surgical therapeutic strategy to accelerate diabetic wound healing, thereby decreasing the number of limb amputations in diabetic patients.

中文翻译：

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蛋白质组学和转录组学探索混合草药提取物对糖尿病创面愈合过程的影响

背景

据报道，糖尿病足溃疡 (DFU) 的年发病率在特定糖尿病临床环境中从 0.2% 到 11% 不等，而在基于社区和人口的队列中则低于 0.1% 到 8%。根据国际糖尿病基金会的数据，全球约有 4000 万至 6000 万人受到 DFU 的影响，最近的一项荟萃​​分析表明，全球成人糖尿病患病率为 6.3%，即约 3300 万人。在美国，糖尿病护理的费用很高，每年直接费用达 2730 亿美元，间接费用达 900 亿美元。糖尿病护理中的足部并发症每年的额外支出比糖尿病相关护理的基线成本高出 50% 至 200%。晚期溃疡每次伤口发作的费用可能超过 50,000 美元，大截肢的直接费用更高。DFU 可以使用多种方法进行治疗，包括伤口敷料、抗生素、减压负荷、皮肤替代品、干细胞、清创术、局部氧疗、基因治疗和生长因子。对于严重的 DFU，如果治疗不及时或不恰当，患者有截肢的风险。截肢不仅会给患者带来身体疼痛，还会因生产力下降和 DFU 相关就业减少而带来经济负担。目前临床上长期使用局部抗生素容易产生耐药性，而生长因子不能有效抑制细菌生长和控制创面炎症。干细胞和基因疗法仍处于试验阶段。局部清创联合负压治疗的方法费用昂贵。因此，我们迫切需要一种负担得起的、非手术的方法来治疗糖尿病性溃疡。树皮提取物紫荆花、赤芍、白芷。& Hook.f. 前法国人。&节省。（霍夫姆。） 本斯。& Hook.f. 前法国人。&节省。, Acorus calamus L和 Radix Angelicae biseratae已被用作治疗炎症相关疾病和皮肤伤口的传统药物，因为它们具有抗炎特性和促进血管更新的能力。然而，关于这五种草药提取物的混合物对糖尿病伤口愈合的研究很少。

目的

本研究旨在评估上述五种草药提取物的混合物对大鼠糖尿病溃疡伤口的愈合作用，并使用转录组学和蛋白质组学揭示任何潜在伤口愈合背后的潜在机制。

学习规划

我们设计了实验，通过体内实验探索五种草药提取物对糖尿病伤口愈合过程的影响，并通过蛋白质组学和转录组学研究其潜在机制。

方法

我们采用上述五种草药提取物的混合物治疗糖尿病大鼠模型，通过腹腔注射链脲佐菌素建立，在大鼠背部造出一个2×2cm的圆形全层皮肤缺损。将金黄色葡萄球菌（1 毫升 1.5 × 10 ⁹ cfu/毫升）均匀涂抹在伤口上。然后观察伤口 72 小时。发现创面有金黄色葡萄球菌感染即认为糖尿病大鼠溃疡感染模型建立成功。根据用药的不同，将大鼠分为三组，即草药提取物混合液组（MHE）、康复新液组（KFS）和对照组（Ctrl）。观察药物对伤口愈合的影响。进行HE染色和Masson染色以评估组织病理学变化和胶原合成。IHC 染色用于评估新生血管形成，M2 巨噬细胞增殖通过免疫荧光染色确定。进行了蛋白质组学和转录组学研究，以探索五种草药提取物促进伤口愈合的潜在机制。采用 UHPLC-QE-MS 鉴定草药提取物混合物的化学成分。

结果

研究表明，混合草药提取物可促进血管生成、M2 巨噬细胞增殖和胶原蛋白合成。转录组学显示 rno-miR-1298、rno-miR-144-5p 和 rno-miR-92a-1-5p 是重要的 miRNA，它们在调节伤口愈合中也起着重要作用。蛋白质组学结果表明，以下蛋白质在 MHE 治疗的伤口中很重要：Rack1、LOC100362366、Cops2、Cops6、Eif4e、Eif3c、Rpl12、Srp54、Rpl13 和 Lsm7。与其他组相比，MHE 治疗后自噬、PI3-Akt 和 mTOR 信号通路得到丰富。

结论

在此，我们已经表明，MHE 含有紫荆树皮、红紫荆、白杨木(Hoffm.) Benth 的树皮提取物。& Hook.f. 前法国人。&节省。, A. calamus L , 和 RA biseratae在糖尿病溃疡伤口大鼠模型中具有显着的伤口愈合作用。这些结果表明，在糖尿病伤口局部应用 MHE 可以加速伤口愈合过程。此外，体内实验表明，糖尿病伤口愈合过程主要由血管生成和 M2 巨噬细胞转变介导。因此，这项研究可能提供一种有前途的非手术治疗策略来加速糖尿病伤口的愈合，从而减少糖尿病患者的截肢次数。