Proteomics and transcriptomics explore the effect of mixture of herbal extract on diabetic wound healing process

Abstract

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.

Introduction

The global prevalence of diabetes among 20-79-year-old individuals was estimated to be 10.5% (536.6 million people) in 2021, and this is projected to increase to 12.2% (783.2 million people) by 2045 (Sun et al., 2022). Diabetic foot ulcers (DFUs), one of the most common and intractable complications of diabetes, are the typical chronic refractory wounds. Patients are at risk of amputation if treatment is not timely or appropriate. Long-term refractory wounds of DFU not only cause suffering to patients, but also induce economic losses (Edmonds et al., 2021; Eming et al., 2014). Risk factors for DFUs include persistent hyperglycemia, peripheral neuropathy (Vinik, 2016), lower extremity vasculopathy (Reardon et al., 2020), wound infection (Dunyach-Remy et al., 2016), foot biological pressure changes (Fernando et al., 2016), genetics, and a decreased number of transition M2 macrophages (Louiselle et al., 2021). Normal wound healing stages include inflammation, new blood vessel formation, macrophage transition from M1 to M2 (Louiselle et al., 2021), migration and proliferation of keratinocytes, fibroblasts and endothelial cells, and extracellular matrix (ECM) remodeling repairing the dermis (Baum and Arpey, 2005). However, the wound healing process in DFUs is often long because of chronic inflammation and insufficient angiogenesis.

Angiogenesis plays an essential role in the normal wound healing process and is a dynamic balance between vascular proliferation and growth, as well as vascular maturation and quiescence (Okonkwo and DiPietro, 2017). In DUFs, the hyperglycemic state is able to disturb the balance and is the main reason of many micro and macrovascular complications which finally hinder new blood vessel formation (Altabas, 2015). Under circumstances of high blood sugar over a long period, endothelial cells (ECs) become dysfunctional, causing loss of integrity, as well as increased risk of apoptosis and detachment. This can influence ECs circulating in the bloodstream (Piconi et al., 2006; Yu et al., 2013). Insufficient angiogenesis prolongs the time of diabetic wound healing. Therefore, promoting angiogenesis may be key to improving treatment of DFUs.

In addition to the widespread concern of angiogenesis in the treatment of DFUs, the role of M2 macrophages in diabetic wound healing has also attracted increased research interest in recent years (Louiselle et al., 2021). Macrophages are derived from monocytes, and there are three distinct subtypes of macrophages, including M1-, M2-, and intermediate macrophages (Basu Mallik et al., 2018). As the microenvironment of monocytes changes, monocytes can polarize into different subtypes which perform different functions. For instance, M1 macrophages are often thought to be involved in the pre-inflammatory phase. M2 macrophages are thought to be associated with the pro-healing phase because their polarization and production process can release different growth factors including transforming growth factor (TGF) and insulin-like growth factor (IGF). Moreover, M2 macrophages are stimulated by anti-inflammatory cytokines, such as IL10 and IL4 (Basu Mallik et al., 2018; Louiselle et al., 2021). M1 macrophages occupy the main phenotype of macrophages in DFUs and play a crucial role in DFUs pathology. The reduction in the number of M1 macrophages transitioning into M2 macrophages will affect angiogenesis, collagen deposition and thus affect wound healing (Liechty et al., 2020; Mirza and Koh, 2011). Given that M2 macrophages can promote wound healing, novel drugs that can promote the polarization of macrophages to the M2 phenotype may be useful for DFU treatment.

The current methods of treating DUFs mainly include glycemic control, antibiotic therapy of the infected wound, enhancing vascularization, debridement, application of wound dressings, negative pressure wound therapy, maggot therapy, use of growth factors, and skin substitutes (Lim et al., 2017). Although these treatments are effective, some are expensive and have negative side effects. Inflammation is a key driver of the pathogenesis of DFUs, and its presence in the wound bed can impair the healing process. Medicinal plants have been considered as effective drugs for thousands of years. Especially the ethnopharmacologically relevant medicinal plants which play a crucial role in the discovery of molecules with a wide range of biological activities, including the ability to alleviate hyperactive inflammatory responses, anti-cancer, and neurodegenerative disorders (Quimque et al., 2021). Uvaria alba (U. alba), a Philippine endemic plant, by reducing cytoprotective NRF2 cascade and target the activation of proinflammatory NF-κB pathway. The flavonol-enriched butanol fraction (UaB) from U. alba was found to be highly selective and noncytotoxic. UaB inhibited proinflammatory mediators including COX-2, iNOS, TNF-α, IL-6 and IL-1β, thus decreasing the production of proinflammatory cytokines (Notarte et al., 2023).

In addition to the aforementioned anti-inflammatory effects, medical plants can also promote wound healing and protect wounds from infection without serious side effects (Budovsky et al., 2015). Recently, a variety of herbal extracts have been found to treat various diseases and promote wound healing (Maver and Maver, 2015). Bark of Bauhinia purpurea extract has been shown to have anti-inflammatory, analgesic, and antipyretic effects in animal models (Gupta et al., 2005). Cyanoglucoside, a chemical regent of bark of Bauhinia purpurea extract, exhibited the highest inhibitory activity of the cyclooxygenase-2 enzyme (COX-2) model (Muhammad and Sirat, 2013). P. rubrae extract was studied in a burn rad model and was found to effectively prevent disturbance of microcirculation from burns. Further, P. rubrae extract can inhibit vascular contraction and decrease microvascular aggregation of erythrocytes (Chu, 1990). The extract of A. dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., a common traditional Chinese medicine, was found to accelerated diabetic wound healing by promoting neovascularization and granulation tissue formation (Zhang et al., 2017). The efficacy of extract of A. calamus L was evaluated using the full thickness skin wound rat model. Data showed that the extract of A. calamus L promote collagen maturation, increase the tensile strength of wound, and reduce the levels of lipid peroxide (Ponrasu et al., 2014). A recent study indicated that sitosterols are important chemical components of R. A. biseratae. In vitro studies demonstrated that the extract of R. A. biseratae can inhibit inflammation in osteoarthritis and regulate the immune system (Chen et al., 2021). While there have been studies reporting the wound healing properties of herbal (Choi et al., 2018), there is lack of basic research on the effect of mixtures of five herbal extracts on wound healing in DFUs, including bark of Bauhinia purpurea, P. rubrae, A. dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., A. calamus L and R. A. biseratae.

The main purpose of this research was to investigate 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. The 'Omic' science explores the actions and functions of various molecules in living organisms through collective technologies and approaches. Omics technology can be categorized into several types, including genomics, epigenomics, transcriptomics, proteomics and metabolomics, each studying specific aspects of biomolecules (Calabrò et al., 2020; Cimbalo et al., 2022). Transcriptomics is a field of science that concentrates on studying gene expression at the RNA level. It offers a comprehensive understanding of the structure and function of genes on a genome-wide scale, enabling researchers to uncover the underlying molecular mechanisms involved in biological processes (Cimbalo et al., 2022). Proteomics focuses on identifying and quantifying all proteins present in a cell, tissue or other organism. It can complement other "omics" technologies like transcriptomics and genomics to help understand the functions and structures of proteins in an organism. Proteomics is essential in understanding complex regulatory systems that control protein expression levels (Aslam et al., 2017). Those computational techniques also in discovering bioactive molecules show us a promising potential. Computational methodologies have not only been used to screen natural products such as sterols, peptides, alkaloids, polyphenols, and terpenoids, but have also provided a better mechanistic understanding of drug interactions with specific viral targets (Quimque et al., 2022).

In this research, we aimed to elucidate the mechanism of MHE promoting rat diabetic wound healing by multi-omics methods, and UHPLC-QE-MS was performed to identify the chemical composition of mixture of herbal extract. Our data showed that this mixture promotes angiogenesis, increases transition of M2 macrophage and collagen synthesis. Therefore, this mixture of herbal extracts may be an effective treatment for diabetic wound healing.