X-Paste improves wound healing in diabetes via NF-E2-related factor/HO-1 signaling pathway

Abstract

BACKGROUND

Diabetic foot ulcers (DFU), as severe complications of diabetes mellitus (DM), significantly compromise patient health and carry risks of amputation and mortality.

AIM

To offer new insights into the occurrence and development of DFU, focusing on the therapeutic mechanisms of X-Paste (XP) of wound healing in diabetic mice.

METHODS

Employing traditional Chinese medicine ointment preparation methods, XP combines various medicinal ingredients. High-performance liquid chromatography (HPLC) identified XP’s main components. Using streptozotocin (STZ)-induced diabetic, we aimed to investigate whether XP participated in the process of diabetic wound healing. RNA-sequencing analyzed gene expression differences between XP-treated and control groups. Molecular docking clarified XP’s treatment mechanisms for diabetic wound healing. Human umbilical vein endothelial cells (HUVECs) were used to investigate the effects of Andrographolide (Andro) on cell viability, reactive oxygen species generation, apoptosis, proliferation, and metastasis in vitro following exposure to high glucose (HG), while NF-E2-related factor-2 (Nrf2) knockdown elucidated Andro’s molecular mechanisms.

RESULTS

XP notably enhanced wound healing in mice, expediting the healing process. RNA-sequencing revealed Nrf2 upregulation in DM tissues following XP treatment. HPLC identified 21 primary XP components, with Andro exhibiting strong Nrf2 binding. Andro mitigated HG-induced HUVECs proliferation, metastasis, angiogenic injury, and inflammation inhibition. Andro alleviates HG-induced HUVECs damage through Nrf2/HO-1 pathway activation, with Nrf2 knockdown reducing Andro’s proliferative and endothelial protective effects.

CONCLUSION

XP significantly promotes wound healing in STZ-induced diabetic models. As XP’s key component, Andro activates the Nrf2/HO-1 signaling pathway, enhancing cell proliferation, tubule formation, and inflammation reduction.

Key Words: Diabetes mellitus, Wound healing, NF-E2-related factor-2/HO-1 signaling pathway, Andrographolide

Core Tip: Diabetic foot ulcer (DFU) is a severe complication caused by diabetes mellitus. The healing process and time of skin wounds in diabetic mice are visibly improved by the application of X-Paste (XP). The analysis conducted on XP unveiled its 21 main components, with Andrographolide (Andro) displaying a notable binding ability with NF-E2-related factor-2 (Nrf2) by high-performance liquid chromatography. Moreover, the addition of Andro at the cellular level effectively alleviated high glucose (HG)-induced proliferation, migration, vascular injury, and inhibition of inflammatory products in human umbilical vein endothelial cells (HUVECs). Mechanistically, the Nrf2/HO-1 signaling pathway is activated by Andro, leading to the relief of HG-induced damage to HUVECs.

INTRODUCTION

Diabetes mellitus (DM), a metabolic disorder characterized by elevated high blood sugar[1], results from impaired insulin secretion or action[2]. Chronic hyperglycemia leads to dysfunction in various tissues, especially feet, eyes, kidneys, blood vessels and nerves[3]. Diabetic foot ulcer (DFU) represents one of the gravest complications of diabetes, impacting over 18 million people worldwide every year[4]. DFUs significantly diminish quality of life, leading to functional status, infection, lower-extremity amputation, and even mortality[5]. Research indicates that DFUs-associated lesions correlate with endothelial dysfunction and reduced vascular growth[6]. Angiogenesis, critical in wound healing, involves the degradation of the extracellular matrix primarily by matrix metalloproteinases. As a result of angiogenic stimulation, endothelial cells (ECs) migrate and multiply, leading to the formation of tubular structures and blood vessels[7]. If inflammatory cells are limited and there is a lack of oxygen and nutrient supply in the wound area, both angiogenesis and wound healing are negatively affected[8]. In recent years, therapeutic approaches aimed at targeting angiogenesis have become the prevailing strategy for treating diabetic wound[9-11]. Within these approaches, strategies that focus on alleviating oxidative stress of ECs, reducing EC apoptosis, and promoting angiogenesis have demonstrated a significant therapeutic effect. Therefore, it is crucial to explore ways to further improve vascular EC homeostasis, alleviate oxidative stress damage and reduce EC apoptosis in order to enhance the treatment of diabetic wounds[12].

The transcription factor NF-E2-related factor-2 (Nrf2) is pivotal in defending against oxidative stress and inflammation[13]. It plays a protective role in various diabetic complications, including diabetic nephropathy (DN) and diabetic cardiomyopathy[13,14]. Under basal conditions, Nrf2 is sequestered in the cytoplasm through its interaction with Kelch-like ECH-associated protein 1 (Keap1), positioning it for proteasomal degradation[15]. However, oxidative stress disrupts this interaction, freeing Nrf2 from Keap1. Subsequently, Nrf2 migrates to the nucleus, where it regulates antioxidant enzymes and participates in glutathione biosynthesis[16,17]. In addition, patients with Nrf2 gene variants are more prone to develop complications of diabetes, including peripheral neuropathy, kidney disease, foot ulcers, and microangiopathy[18,19].

The use of traditional Chinese medicine (TCM) in wound treatment dates back to ancient times. According to ancient Chinese medical textbooks, the use of various herbal medicines is recommended to treat wounds[20]. These practices, developed over centuries, reflect a rich history of empirical knowledge and cultural wisdom in addressing wound care through herbal treatments[21]. It has been reported that Deoxyshikonin has beneficial effect on delayed wound healing in diabetic mice[22]. Bletilla striata polysaccharide also promotes wound healing by inhibiting NLRP3 inflammasome[23]. Andrographolide improves renal DN by reducing hyperglycemia-mediated oxidative stress and inflammation through the Akt/NF-κB pathway[24]. Astragalus membranaceus is an herbal medicine typically used to relieve the complications of diabetes and it also promote wound healing[25,26].

While individual Chinese herbal medicines offer valuable therapeutic properties, their effectiveness in treating DFUs can be limited when used in isolation. To overcome this challenge, we have employed TCM paste preparation methods to amalgamate the active components of various medicinal materials. This integrated approach has been observed and analyzed both in vitro and in vivo for its impact on diabetic wound healing. Our research aims to offer new insights and potential strategies for more effective DFU treatment.

MATERIALS AND METHODS

Preparation of paste

Adopting the traditional method of preparing traditional Chinese medicinal ointments. Arnebia euchroma, Bletilla striata, Andrographis paniculata, and Astragalus membranaceus after purification and add 10 times the total weight of the water soaked for 1 h, using the soaking liquid direct boiling, the first time takes 30 min, the second time takes 20 min, after filtering the two liquids together to stand, remove sediment from bottom. The cocoon shells are also made in the same way. The two filtrates were mixed and evaporated to make XP.

Animal experiment

All BALB/C mice were purchased from Shulaibao Biotechnology (Wuhan, Hubei Province, China). The total 30 mice ranging from 10 wk ± 2 wk of age and weighing 20 g ± 2 g were placed in a specific pathogen-free environment with a temperature of 23 °C ± 2 °C and a light-dark cycle of 12 h. They were not restricted from access to food and water. Twenty randomly selected mice were induced with diabetes by the intraperitoneal administration of streptozotocin (STZ, 40 mg/kg body weight in 0.1 M citrate buffer, pH = 4.5), once a day for five consecutive days. Ten mice were designated as a negative control (NC) group and administered an equivalent dose of citrate buffer solution. After a week of observation, 10 mice were selected from the 20 induced diabetic mice, based on their fasting blood glucose levels exceeding 280 mg/dL, to establish wound experiments[27]. In short, these 10 mice were randomly divided into two groups, with 5 mice in each group. A circular wound with a diameter of 4 mm was made on the dorsal surface of each mouse by using ophthalmological scissors to establish the wound healing model[28]. At the same time, following this method, NC group mice were also evenly divided into two groups, forming circular wounds with a diameter of 4 mm on the back of each mouse. Phosphate-buffered saline (PBS) and XP were respectively applied intradermally to the wounds in each group. Pentobarbital sodium (1%, 50 mg/kg, IP) was used as an anesthetic when necessary to reduce the pain of mice during the experimental process. Images of the wounds were obtained on days 0, 3, 7, and 14. On the 14^th d after injury, wound samples were measured the area and collected for histological stain analysis. The animal models established according to the above-mentioned method are divided into the following groups: NC + PBS group, NC + XP group, DM + PBS group, and DM + XP group.

Hematoxylin and Eosin and masson stain

For histology, tissue was harvested from mice and fixed with 4% paraformaldehyde before paraffin embedding. Paraffin-embedded sections (5 mm) were stained with hematoxylin and eosin (Solarbio, China). Masson staining was performed for collagen quantification with a Masson assay kit (Sigma, United States), following the manufacturer’s protocols. Images were captured using the Ci-L microscope (Nikon, Japan) and analyzed using the Image J software (version 1.8.0).

Immunostaining

For immunofluorescence assays, the frozen section are dried and cells section were fixed in a fixative solution for 10 min, then sections were permeabilized with Triton X-100 for 15 min and blocked with 5% bovine serum albumin at 37 °C for 1 h. Then, the sections were incubated with primary antibodies at 4 °C overnight, washed three times with PBS, and incubated with Alexa Fluor 488-conjugated secondary antibodies for 1 h at 37 °C. After washing three times with PBS, nuclei underwent staining using DAPI at ambient temperature for 5 min. The frozen section and cells section were then observed under a confocal microscope (Leica, Germany) under each experimental condition.

Molecular docking

The X-ray crystal structures of Nrf2 were retrieved from the Protein Data Bank. The protonation state of all the compounds was set at pH = 7.4, and the compounds were expanded to 3D structures using Open Babel[29]. AutoDock Tools (ADT3) were applied to prepare and parametrize the receptor protein and ligands. The docking grid documents were generated by AutoGrid of sitemap, and AutoDock Vina (1.2.0) was used for docking simulation[30,31]. The optimal pose was selected to analysis interaction. Finally, the protein-ligand interaction figure was generated by PyMOL. The Nrf2 protein is represented as a slate cartoon model, ligands, including 21 main ingredients from XP, are shown as a cyan stick, and their binding sites are shown as magentas stick structures. Nonpolar hydrogen atoms are omitted. The hydrogen bond, ionic interactions, and hydrophobic interactions are depicted as yellow, magentas and green dashed lines, respectively.

Immunohistochemistry

Capillary density in the wounds was observed by using CD31 (1:50, Abcom, United States), alpha-smooth muscle actin (α-SMA, 1:100, Abcom, United States), and VEGFA (1:50, Abcom, United States) antibodies and then staining with DAB. Nuclei were labelled with the fluorescent dye DAPI after 5 min of incubation. The frozen section and cells section were then observed under a confocal microscope (Leica, Germany) under each experimental condition.

TUNEL assay

Tissue sections were fixed in 4% paraformaldehyde (PFA) and incubated with a 50 μL TUNEL reaction mixture (Promega, United States) and 0.3% Triton X-100, in dark conditions at 37 °C, for 1 h. This was followed by three washes with PBS. To label the nuclei, sections were then incubated with the fluorescent dye DAPI for 5 min. Both frozen tissue sections and cell samples were subsequently examined under a confocal microscope (Leica, Germany) for each experimental condition.

5-ethynyl-2’-deoxyuridine assay

Human umbilical vein ECs (HUVECs) were seeded at a density of 2.5 × 10⁵/well in a 6-well plate and treated as described. After 48 h, cells were incubated with 5-ethynyl-2’-deoxyuridine (EdU) reagent for 2 h and then stained with the EdU staining kit (Beyotime, Shanghai, China) for immunofluorescence staining. DAPI was used to stain the cell nucleus for 30 min. The percentage of EdU-positive cells was defined as the proliferation rate.

Western blot

The concentrations of protein were determined using a BCA kit (Beyotime, Shanghai, China). Equal amounts of proteins were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly-vinylidene fluoride membranes. Antibodies against Nrf2 (1:1000, Proteintech, China), HO-1 (1:2000, Proteintech, China), VEGFA (1:1000, Abcam, United States), α-SMA (1:1000, Abcom, United States), interleukin-6 (IL-6, 1:1000, Proteintech, China), tumour necrosis factor alpha (TNF-α, 1:1000, Proteintech, China), and β-actin (1:5000, Proteintech, China) were used as the primary antibodies. The Goat anti-rabbit-HRP and Goat anti-mouse-HRP (Proteintech, China) were used as the secondary antibody. The gray value of the strips was processed by Image J software.

Cell culture and transfection

HUVECs were obtained from Guangzhou Saliai Stemcell Co., Ltd and were cultured in EC medium supplemented with 5% fetal bovine serum (Gibco, United States) and 1% penicillin–streptomycin (Beyotime, China) in a humidified incubator containing 5% CO₂ at 37 °C. HUVECs were then exposed to EGM-2 supplemented with high glucose (HG, 33 mmol/L) for 72 h, while using D-mannitol as an osmotic control for the HG condition. The small interfering RNA (siRNA) plasmid of Nrf2 and its corresponding control plasmid were obtained from Guangdong Ruibo Biotechnology Co., Ltd. Transfection efficiency of siRNA-Nrf2 was confirmed by Western blot.

Cell Counting Kit-8 assay

In this experiment, we use high sensitivity cell counting kit (Beyotime, China) to detect the quantity of 10⁵ (100 μL) cells per well and 10 μL of drugs per well. The control group was 100 μL quantitative cells + 10 μL DMSO. The survival rate of the cells is expressed by T/C, where T is the OD value of the added cells, C is the OD value of the control cells.

Tube formation assay

HUVECs cells were seeded in Matrigel-pretreated 24-well plates, and incubated for 48 h. Then, the formation of tubules was observed under an optical microscope.

Wound healing assay

HUVECs were added to 12-well plates. The cell monolayer was scratched with a pipette tip, forming a wound. Then, HUVECs were photographed at 0 h and 12 h post-wounding to assess the wound healing of the HUVECs.

Statistical analysis

Experimental data were analyzed using GraphPad Prism 9.0. Data are presented as mean ± SD. For normally distributed and homogeneous variance data, t-tests were performed to compare two groups. For multiple group comparisons Dunnett’s-T3 test was used based on the distribution and variance. P value less than 0.05 was considered to indicate a significant difference.

RESULTS

XP played a role in the healing of diabetic skin wounds

To further investigate the involvement of XP in the wound healing process of diabetes, the mouse model was utilized to simulate diabetic wound healing, and the impact of XP was assessed (Supplementary Figure 1A). The wounds on the back of mice in both the NC and DM groups were treated with either PBS or XP. Initially, at 3 d post-treatment, there was no significant difference in wound area among the four groups (Supplementary Figure 1B). However, wound healing was faster in the NC + XP and DM + XP group, slowest in the DM + PBS group at 7 d, with a more pronounced difference at 14 d (Figure 1A and B). Hematoxylin and Eosin (H&E) staining results showed that DM + XP group exhibited the higher re-epithelialization rate than DM + PBS. In addition, the Masson staining demonstrated that the collagen deposition in XP treatment group was more than that of other groups (Figure 1C). XP also effectively reduced oxidative stress and apoptosis in the wound area of DM mice (Figure 1D and E). CD31 and α-SMA were main indicators of blood vessels angiogenesis in the diabetic wound bed. As shown in Figure 1E, few blood vessels were observed in the wound tissues of DM + PBS group, while the treatment of XP improved vascular network formation. Vascular endothelial growth factor A (VEGFA) has a substantial role in angiogenesis. The results showed that DM + XP group had a better recovery DM + PBS group, which suggesting that XP treatment accelerated diabetic wound healing by facilitating wound re-epithelialization and angiogenesis.

Figure 1 X-Paste promotes wound healing in diabetic foot ulcers mice. A: Images of mice grouped in either normal control (NC), NC plus X-Paste (XP), diabetic wound plus phosphate-buffered saline or diabetic wound plus XP were captured at days 0, 3, 7 and 14; B: Wound tissue sections were stained with Hematoxylin and Eosin and Masson in different groups; C: The representative images of reactive oxygen species production in wound tissue sections from different groups; D: The representative images of TUNEL in wound tissue sections from different groups; E: The representative images of CD31, alpha-smooth muscle actin, and VEGFA from different groups. NC: Normal control; PBS: Phosphate-buffered saline; DM: Diabetic wound; XP: X-Paste; α-SMA: Alpha-smooth muscle actin.

Screening and analysis of effective substances in XP

To determine the active components in XP responsible for diabetic ulcer healing, we conducted High-performance liquid chromatography (HPLC) analysis on ten batches of the paste. This analysis identified twenty-one main active substances, and their components were consistently stable across different XP batches (Figure 2A). We then sequenced the transcriptome of the diabetic tissues before and after treatment and found significant differences between the two groups, in which the level of Nrf2 were significantly increased in DM + XP group than DM + PBS (Figure 2B). GO enrichment analysis showed that in the three parts of molecular function, biological process, and cell component, the gene was up-regulated in myofibril and myofilament development and transmembrane transporter activity were significantly increased in the XP treatment group (Figure 2C). KEGG pathway showed that XP significantly increased NF-κB signaling pathway (Figure 2D). Subsequently, we used Nrf2 to simulate molecular docking with these 21 monomers of XP. We analyzed the interactions between protein and ligand, all functional residues were identified and classified according to their interactions. There are multiple groups of residues used to form interactions between receptor protein and ligand, such as the hydrogen bond formed by NRF2 and ligand (Table 1). Among these, the binding energy between Nrf2 and andrographolide (Andro) was particularly notable at -8.8 kcal/mol, suggesting a strong interaction (Figure 2E). Given these findings, our subsequent studies will primarily focus on investigating whether Andro can promote diabetic wound healing through enhancing wound re-epithelialization and angiogenesis.

Figure 2 Screening and analysis of effective substances in X-Paste. A: High-performance liquid chromatography analyzed 10 batches of the X-Paste (XP); B: The volcano plot shows deferentially expressed genes (DEGs) between the XP treatment group and the diabetic foot ulcer group; C: GO enrichment analysis of DEGs; D: The KEGG enrichment analysis of DEGs; E: The molecular docking between Nrf2 and Andro. BP: Biological process; MF: Molecular function; CC: Cell component.

Table 1 The binding energy of NF-E2-related factor-2 docking with the 21 monomers of the X-Paste.

Protein	Main ingredients	CAS NO.	Binging energy
Nrf2	Cinnamic acid	621-82-9	-5.8
Nrf2	3,4-Dihydroxybenzaldehyde	139-85-5	-6.3
Nrf2	β-Sitosterol	83-46-5	-8.2
Nrf2	4-Hydroxycoumarin	1076-38-6	-5.8
Nrf2	Palmitic acid	1076-38-6	-5.6
Nrf2	Folic acid	59-30-3	-5.7
Nrf2	Caffeic acid	331-39-5	-5.8
Nrf2	Carvacrol	499-75-2	-7.2
Nrf2	Wogonin	632-85-9	-7.5
Nrf2	Daucosterol	474-58-8	-5.7
Nrf2	Eugenol	97-53-0	-8.4
Nrf2	Andrographolide	5508-58-7	-8.8
Nrf2	Neoandrographolide	27215-14-1	-8.4
Nrf2	Oroxylin	480-11-5	-7.8
Nrf2	Skullcapflavone II	55084-08-7	-8.1
Nrf2	(+)-Borneol	464-43-7	-7.8
Nrf2	Dipterocarpol	471-69-2	-5.9
Nrf2	Oleanolic acid	508-02-1	-5.3
Nrf2	Alphitolic acid	19533-92-7	-7.1
Nrf2	(1R,2S,4R)-rel-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-ol	507-70-0	-6.1
Nrf2	DL-isoborneol	124-76-5	-6.1

Nrf2: NF-E2-related factor-2.

The signaling pathway of Nrf2/HO-1 was activated by XP in vivo

We examined the tissues of four groups and found that XP treatment dramatically increased the expression of Nrf2 and HO-1 in the DM group. However, there was no significant difference between the control and NC + PBS group after XP treatment (Figure 3A and B). Next, we examined the expression of CD31, VEGFA, and α-SMA at the protein level, and found that on the XP treatment to diabetic mice wound surface, the expression all increased, especially the expression of α-SMA (Figure 3C and D). XP also protected wounds in mice that did not suffer from the DM. We detected the expression of TNF-α and IL-6 in those four groups by Western blot and found that IL-6 and TNF-α was up-regulated in STZ-induced diabetic mice, while the protein expression was down-regulated after treatment with XP (Figure 3E and F). The results indicated that XP treatment accelerated diabetic wound healing by promoting wound re-pithelialization, angiogenesis and reducing inflammation of the wound.

Figure 3 X-Paste promotes diabetic foot ulcers wound healing through the NF-E2-related factor-2/HO-1 pathway. A: The expression of NF-E2-related factor-2 (Nrf2) and HO-1 were measured by Western blot from skin wound tissues; B: Quantification of Western blot results; C: The protein of CD31, VEGFA, and alpha-smooth muscle actin were measured by Western blot from skin wound tissues; D: Quantification of Western blot results; E: Western blot analysis of interleukin-6 and tumour necrosis factor alpha protein expression; F: Quantification of Western blot results. Statistical significance is expressed as ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, P > 0.05 was not denoted. NC: Normal control; PBS: Phosphate-buffered saline; DM: Diabetic wound; XP: X-Paste; α-SMA: Alpha-smooth muscle actin; IL: Interleukin; TNF-α: Tumour necrosis factor alpha; Nrf2: NF-E2-related factor-2.

Effect of Andro on proliferation and metastasis of HUVECs

We next explored the effect of Andro on HUEVCs by treating cells with in vitro experiments and found that at 24 and 48 h, 5 μM Andro had a significant effect on cell proliferation, particularly at 24 h (Supplementary Figure 2A). At 24 h, 5 μM, 10 μM, and 25 μM Andro was found to have significant effect on cell proliferation (Supplementary Figure 2B). So, the concentration of Andro is fixed at 5 μM. We then used HG to simulate HUEVCs in vitro. Compared with the NC group, HG significantly inhibited cell viability, tubule formation, and promoted apoptosis in HUVECs (Figure 4A). Added Andro counteracted the negative impact of HG on the migration of HUVECs (Figure 4B). It also promoted the level of CD31 and α-SMA (Figure 4C), suggesting that Andro promotes cell proliferation and wound healing in cell model.

Figure 4 Effect of Andro on proliferation and metastasis of human umbilical vein endothelial cells. A: The representative images of 5-ethynyl-2’-deoxyuridine, tubule formation and TUNEL in wound tissue sections from different groups; B: The wound healing assay in 0 h and 24 h from different groups; C: Immunofluorescence staining of CD31 and alpha-smooth muscle actin (α-SMA) in different groups; D and E: Western blot analysis of NF-E2-related factor-2 and HO-1 expression in each group and quantification of Western blot results; F and G: Western blot analysis of CD31, VEGFA, and α-SMA expression in each group and quantification of Western blot results; H and I: Western blot analysis of interleukin-6 and TNF-α expression in each group and quantification of Western blot results. Statistical significance is expressed as ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, P > 0.05 was not denoted. HG: High glucose; NC: Normal control; PBS: Phosphate-buffered saline; IL: Interleukin; TNF-α: Tumour necrosis factor alpha; Nrf2: NF-E2-related factor-2; Andro: Andrographolide.

In the HG environment, Andro significantly promoted Nrf2 and HO-1 and upregulated CD31, VEGFA, and α-SMA levels (Figure 4D-G), while decreasing the expression of IL-6 and TNF-α (Figure 4H and I). Based on these findings, we propose that Andro is a key component of XP in promoting diabetic wound healing. In future research, we plan to further explore whether additional elements of XP also contribute to enhance wound healing in diabetes.

Andro accelerates wound healing in diabetes by activating the Nrf2/HO-1 pathway

To further investigate the effect of Andro on HUVEC bioactivity, siRNA-Nrf2 was transfected into HUVECs to knockdown Nrf2. We found the fragmentation of HUEVCs was more pronounced after knockdown of Nrf2. Moreover, Nrf2 knockdown resulted in more apoptosis than that only in HG environment and after Andro processing, recovery of cell morphologyis apoptosis is not apparent (Figure 5A). We believed that Nrf2 knockdown affected cell proliferation, tube formation, and wound healing in HG-induced HUVECs (Figure 5B). Knockdown of Nrf2 counteracted the positive impact of Andro on the expression of α-SMA and CD31 in HUVECs (Figure 5C). At the same time, after Nrf2 knockdown, the expression of the Nrf2 and HO-1 were restored in the HG + siRNA-Nrf2 + Andro group relative to the HG + siRNA-Nrf2 group (Figure 5D and E). After Nrf2 knockdown, Andro treatment significantly increased the expression of CD31, SMA, and VEGFA (Figure 5F and G). Through the above results and the expression of IL-6 and TNF-α was suppressed (Figure 5H and I). Taken together, we believe that Andro promotes the formation of small tubes, reduces cellular apoptosis, and facilitates wound healing through the Nrf2/HO-1 pathway.

Figure 5 Andro accelerates diabetic foot ulcers wound healing by activating the NF-E2-related factor-2/HO-1 pathway. A: The representative images of 5-ethynyl-2’-deoxyuridine, tubule formation and TUNEL in wound tissue sections from different groups; B: The wound healing assay in 0 h and 24 h from different groups; C: Immunofluorescence staining of CD31 and alpha-smooth muscle actin (α-SMA); D and E: Western blot of NF-E2-related factor-2 and HO-1 expression in each group and quantification of Western blot results; F and G: CD31, VEGFA, and α-SMA expression in each group and quantification of Western blot results; H and I: Western blot analysis of interleukin-6 and tumour necrosis factor alpha expression and quantification of Western blot results. Statistical significance is expressed as ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001, P > 0.05 was not denoted.

DISCUSSION

DFU rank among the most prevalent complications of DM, significantly diminishing the quality of life for patients while imposing a substantial burden on healthcare systems and society[32]. The conventional wound healing process involves encompasses various stages, including cell migration and proliferation, coupled with the activation of the extracellular matrix. In the DFU patients, the process of wound healing involves inflammation, migration and proliferation, and subsequent remodeling[33,34]. Several articles have reported that the delayed healing of wounds in DFU patients is thought to be caused by factors such as inadequate blood circulation, nerve impairment, infection, and inflammation[35]. Furthermore, it is crucial to comprehend the molecular biological changes to develop precise treatment approaches.

Natural products, with their intricate and varied structures and significant biological effects, are considered essential sources for groundbreaking pharmaceuticals[36]. In our study, we used the preparation method of TCM paste to integrate the active components of many kinds of natural products to prepare XP. We explore the therapeutic effect of XP on delayed wound healing in diabetic mice. Diabetic mouse models were induced by injection STZ. When XP was applied to full-layer dermal wounds created on the shaved skin of these mice, the mice’s wound closing time was significantly accelerated, and the open wound area was significantly reduced. In the skin wound tissue, XP treatment effectively normalized the elevated TNF-α and IL-6 levels caused by HG, crucially suppressed the generation of oxidative stress (ROS), and enhanced the expression of epidermal growth factor CD31, α-SMA, and VEGF.

Subsequently, to further understand specific mechanism of XP promoting diabetic wound healing, we performed transcriptome sequencing on wound tissues from the XP treatment group and the diabetes group. This analysis revealed a marked increase in the Nrf2 Level in the XP treatment group. Based on these findings, we hypothesize that XP primarily enhances wound healing in diabetic mice through the upregulation of Nrf2 expression. The Keap1-Nrf2/HO-1 pathway is widely recognized as a vital pathway for antioxidant stress response within the body and is an important focus for inflammation-related illnesses, including DFU[37,38]. Normally, Nrf2 remains at low levels within cells due to negative regulation by Keap1, which binds to and facilitates its breakdown. However, when cell is harmed, Nrf2 surpasses this inhibition, moves into the nucleus, and proceeds to activate different genes, including HO-1[38]. The knockout of the pancreatic β cell specific Keap1 gene can stimulate the Nrf2 gene in the islets[39]. Conversely, when the Nrf2 gene is knocked out, the expression of antioxidant enzyme genes in the islets decreases. These findings suggest that the Keap1-Nrf2 system has an influence on pancreatic β cells, and Nrf2 manages the transcription of antioxidant enzyme genes in the pancreas[40]. In the STZ-induced diabetic model, Nrf2-knockout mice exhibited impaired blood-retinal barrier function and neuronal dysfunction compared to wild-type mice, which suggested that the Nrf2 system plays a role in protecting against diabetic retinopathy[41]. An increase in Nrf2 expression may improve mitochondrial function in diabetes, enhance the resistance of endothelial progenitor cells (EPCs) to diabetes-induced oxidative damage, and improve the therapeutic effect of EPCs on diabetes-related limb ischemia[42]. In our study, a significant increase in Nrf2 and HO-1 expression was observed in the XP treatment group. Considering the multifaceted role of Nrf2 and the findings from our research, we hypothesize that XP expedites wound healing by promoting Nrf2 expression and reducing inflammation.

Subsequently, we proceeded to identify the key components of XP that significantly contribute to wound healing in diabetic mice. A total of 21 main active substances were identified by HPLC, and the active ingredients in different batches of the paste remained stable. Molecular docking studies revealed that Nrf2 exhibits strong binding affinity with Andro, prompting us to focus primarily on investigating Andro’s role in wound healing. In future research, we aim to further examine whether other components of XP also contribute to the healing process in DFUs. Andro is an active component extracted and purified from andrographis paniculata. It has many pharmacological effects, such as anti-inflammatory and anticancer activities[43-45]. Andro exerts antioxidant and anti-inflammatory functions by enhancing the Nrf2 signaling pathway in multiple diseases[46-49]. The metabolic abnormalities of diabetic mice can be improved, and the progression of DN can be prevented by the synergistic effect of andrographis paniculata polysaccharide and Andro[50]. A study has shown that Andro plays a protective role in the kidneys in DN by alleviating oxidative stress and inflammation under diabetic conditions[24].

In our research, the proliferation of vascular ECs is the initial step in angiogenesis, followed by migration and tube formation, ultimately culminating in the creation of novel blood vessels[51,52]. To determine the effect of Andro on angiogenesis, we evaluated its ability to facilitate HUVEC migration and tube formation. Subsequently, we aimed to further investigate whether Andro improves wound healing through the promotion of Nrf2. However, upon the knockout of Nrf2, the positive effects of Andro on cell growth and migration were diminished. This suggests a pivotal role of Nrf2 in mediating Andro’s therapeutic actions. In conclusion, as a principal component of XP, Andro effectively promotes the wound healing in diabetic mice by activating the Nrf2/HO-1 signaling pathway.

CONCLUSION

We used several kinds of TCM to treat diabetic mice. We found that the paste accelerated wound healing, and Andro, the main component of the paste, activated the Nrf2/HO-1 signaling pathway to affect cell proliferation, tubules are formed, and inflammation is reduced (Figure 6). Our findings offer novel insights and potential targets for the therapy of DFU.

Figure 6 Effects on the wound healing in diabetic mice by the X-Paste through the NF-E2-related factor-2/HO-1 signaling pathway. X-Paste (XP) obviously promotes the healing of skin wounds in diabetic mice, resulting in an accelerated healing process and shortened healing time. High-performance liquid chromatography was used to analyze the 21 main components of XP, among which Andro exhibited a strong binding ability with NF-E2-related factor-2 (Nrf2). At the cellular level, the addition of Andro alleviated high glucose (HG)-induced proliferation, migration, vascular injury, and inflammatory product inhibition in human umbilical vein endothelial cells (HUVECs). Mechanically, Andro relieved HG-induced damage to HUVECs by activating the Nrf2/HO-1 signaling pathway. HPLC: High-performance liquid chromatography; Nrf2: NF-E2-related factor-2; Andro: Andrographolide; HUVECs: Human umbilical vein endothelial cells.