Introduction

Globally, diabetes poses a significant health concern. An estimated 537 million adults live with diabetes, with 6.7 million deaths annually attributed to the disease or its complications, accounting for 12.2% of the total deaths1. China faces a particularly high burden, with the number of diabetics rising from 90 million to 140 million in the past decade (2011–2021). Diabetic foot, one of the most common chronic wounds, is the main cause of hospitalization, amputation, and even death of diabetes patients2. About 30% of diabetes patients will have diabetic foot ulcers, which seriously affect the prognosis of diabetes patients and also bring a huge economic burden to the society.

Wound chronicity refers to the failure of normal, orderly and timely healing process to achieve anatomical and functional integrity of the wound, resulting in transitioning from an acute state to a delayed healing or non-healing chronic wound, such as a diabetic foot3. The delayed healing in diabetes involves complex mechanisms, including impaired blood vessel formation (neo-angiogenesis) local ischemia, reperfusion damage, and aging and cellular dysfunction. However, the contribution of inflammatory response to healing of acute diabetic wounds is largely ignored. Inflammation in wound healing acts as a double-edged sword, with a beneficial initial inflammatory response that clears debris and bacteria and attracts repairing cells to the wound area, as well as a harmful persistent and excessive inflammation that acts as the primary culprit for chronic wounds4. Many studies have implicated inflammatory bodies (inflammasomes) as an important cause of various diseases, while it also participates in wound healing process5. These multi-protein complexes play a critical role in innate immunity which activates by cell injury, tissue damage, and infections6. NLRP3 is a protein 3 containing NACHT, LRR and PYD domains, and its role in inflammation has been extensively studied. It uniquely recognizes the damage-associated molecular patterns (DAMPs)7, and during the acute inflammatory phase, NLRP3 is expressed in pro-inflammatory macrophages4, leading to the production of inflammatory mediators, macrophage recruitment, and polarization into pro-inflammatory mediators. NLRP3 has been shown to play a protective role in normal skin wound healing8, while its over-activation may significantly impede diabetic wound healing9.

Fibroblast growth factor (FGF) is a family of signaling molecules with diverse biological effects. It promotes tissue repair by stimulating the growth of blood vessels (vascular endothelial cells), collagen production, and the differentiation of various cell types essential for wound healing10. Among FGF family members, both acidic FGF (aFGF or FGF1) and basic FGF (FGF2) have been used in clinical practice, and their structures, binding receptors and biological functions are similar. Unlike its counterpart bFGF, aFGF retains well its activity and stability in the acidic environment typical of chronic wounds, and thus exhibits advantages for chronic wound treatment11,12,13. Recombinant human aFGF (rhaFGF) is a human protein obtained through genetic recombination technology. It is a low molecular weight, acidic (pH 5.1) single chain polypeptide with the same efficacy as aFGF. Previous studies have shown that rhaFGF can promote the healing of diabetic wounds14,15. It has also been proven that rhaFGF can accelerate the wound healing process and shorten the healing time in acute wounds including deep partial-thickness burns or skin graft donor sites. However, there is little evidence regarding the effect of rhaFGF on the transition from an acute wound to chronic one in diabetes and the precise mechanism remains to be elucidated16.

Building on the current literature, we hypothesize that rhaFGF may promote DM wound healing by inhibiting excessive inflammation and preventing the occurrence of acute wound chronicity.

Results

rhaFGF promotes diabetic wound healing

To assess the therapeutic effect of rhaFGF on wound healing, the wound closure rates were compared among the Con, Con + rhaFGF, DM, and DM + rhaFGF groups within 14 days after wound creation. Figure 1 showed the healing process of the wound in the hindlimb paw of the representative mouse in each group. The wound size was quantitated for each mouse at each indicated time point, and the data showed that the initial wound size (day 0) did not differ significantly among the four groups (Fig. 2A). As shown in Fig. 2B, the DM + PBS group exhibited slowest wound healing rate compared to Con, Con + rhaFGF, and DM + rhaFGF groups at all time points (*p < 0.05). No significant differences in wound size were observed among the four groups at day 3 (Fig. 2C), day 5 (Fig. 2D), and day 7 (Fig. 2E) post-wound creation (*p > 0.05). However, starting from day 7, the wound healing rate in the DM + rhaFGF group became significantly higher than the DM + PBS group, and this difference kept increasing over time (day 9, day 11, and day 14; *p < 0.05). Compared to the Con group, the DM + PBS group’s wound sizes were significant larger on day 9 (Fig. 2F), day 11 (Fig. 2G), and day 14 (Fig. 2H) (*p < 0.05). Notably, the Con + rhaFGF group showed no significant difference in wound size compared to Con at any indicated time point (*p > 0.05). These results suggested that prolonged rhaFGF treatment (longer than 7 days) could significantly promote wound healing in diabetic mice, while no significant effect was observed when the treatment duration was less than 7 days.

Fig. 1
figure 1

Representative photographs showed wound healing process in hindlimb paw of control mice (Con), control mice with rhaFGF treatment (Con + rhaFGF), diabetic mice (DM) and diabetic mice with rhaFGF treatment (DM + rhaFGF).

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Fig. 2
figure 2

The wound healing process of mice (A), healing rate of wounds at day 0 (B) and statistical chart of the healing rate at different time points (day 3, 5, 7, 9, 11 and 14) (C-H). *p < 0.05, **p < 0.01, and ***p < 0.001.

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rhaFGF promotes the proliferation and migration of wound healing-associated cells

We next tested whether rhaFGF improves wound healing through promoting the proliferation in wound healing-associated cells, including vascular endothelial cells and skin cells. Human umbilical vein endothelial cells (HUVEC), human dermal fibroblasts (HFF), and human keratinocytes (HaCat) were cultured and treated with rhaFGF. CCK-8 assays were performed to measure the proliferation of HUVEC (Fig. 3A), HFF (Fig. 3B), and HaCat cells (Fig. 3C) following exposure to rhaFGF at indicated concentrations. rhaFGF treatment increased the proliferation in all three cell types, and HUVEC displayed the strongest response to rhaFGF (Fig. 3A). These findings indicated that rhaFGF promoted the proliferation of all wound healing-associated cells in a dose-dependent manner, and the effects varied among three cell types.

Fig. 3
figure 3

The effects of rhaFGF on the proliferation of HUVEC, HFF and HaCat cells under control and HG + LPS conditions. The cells were incubated with rhaFGF for 48 h and cell proliferation was assessed by CCK-8 assay. A. proliferation following 48 h of incubation with various rhaFGF concentrations in normal culture medium (n = 3/group). rhaFGF treatment increased the proliferation of HUVEC (A), HFF (B) HaCat (C) under control condition in a dose-dependent manner(n = 3/group), The proliferation of HFF (D), HUVEC (E), HaCat (F) were inhibited under diabetic inflammatory conditions mimicked by high glucose and LPS-induced inflammation and rhaFGF treatment (optimal rhaFGF concentration of 20ng/ml,5ng/ml and 50ng/ml respectively) partially reversed this inhibition (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

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We further investigated the effects of rhaFGF on cell proliferation under simulated diabetic wound conditions (high glucose and lipopolysaccharide-induced inflammation). Compared to the control group, lipopolysaccharide (LPS) + high glucose medium significantly inhibited the proliferation of HaCaT (Fig. 3D), HFF (Fig. 3E), and HUVEC cells (Fig. 3F) (*p < 0.05). Conversely, rhaFGF treatment significantly increased proliferation of all three cell types (*p < 0.05).

To verify the effect of rhaFGF on HFF, HUVEC, and HaCaT cell migration ability, we performed cell scratch tests. The results showed that compared with the LPS + HG group, Hacat cells in the LPS + HG + rhaFGF group with 20ng/ml rhaFGF migrated significantly at 6 h, 12 h, and 24 h after migration (p < 0.05). The migration of HUVEC cells in the LPS + HG + rhaFGF group with 5ng/ml rhaFGF group was significant at 12 h after proliferation, and not significantly at 6–24 h (p < 0.05) compared with the LPS + HG group. And compared with the LPS + HG group, the HFF cells in the 50ng/ml LPS + HG + rhaFGF group migrated significantly at 12 h and 24 h (P < 0.05), but not significantly at 6 h (P > 0.05) (Figs. 4 and 5).

Fig. 4
figure 4

Migration of HaCat, HUVEC and HFF cells after scratch and treatment with 20 ng/mL, 5 ng/mL and 50 ng/mL rhaFGF, respectively. Images are acquired by optical microscope and are examples of different independent experiments.

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Fig. 5
figure 5

The statistical analysis of migration distances for HaCat, HUVEC and HFF cells. Cell migration was assessed via scratch and treated with 20 ng/mL, 5 ng/mL and 50 ng/mL rhaFGF, respectively. *p < 0.05 and **p < 0.01.

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These results suggested that high glucose and inflammatory stimulus impaired HFF, HUVEC, and HaCat cell proliferation and migration, which could be partially reversed by rhaFGF treatment.

rhaFGF reduces Pro-inflammatory factors in diabetic mice

To elucidate the link between impaired healing and inflammation in diabetes, ELISA was used to measure serological and wound tissue levels of pro-inflammatory factors IL-6 and TNF-α in all groups at day 7 after wound creation. Mice in the diabetic group (DM) exhibited significantly elevated serological (Fig. 6A, C) and tissue (Fig. 6B, D) levels of IL-6 and TNF-α compared to controls (*p < 0.05). Conversely, rhaFGF treatment (DM + rhaFGF group) significantly reduced IL-6 and TNF-α levels in both serum and tissues (*p < 0.05).

Fig. 6
figure 6

Serological and wound tissue levels of IL-6 and TNF-α were assessed by ELISA in control and diabetic mice. Diabetic mice (DM) exhibited significantly higher serological (A) and wound tissue (B) IL-6 levels compared to controls. rhaFGF treatment (DM + rhaFGF) significantly reduced IL-6 levels in both serum and wound tissues. Similar to IL-6, serological (C) and wound tissue (D) levels of TNF-α levels were significantly elevated in DM compared to controls. rhaFGF treatment (DM + rhaFGF) significantly decreased TNF-α levels in both serum and wound tissues. *p < 0.05 and ****p < 0.0001.

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rhaFGF suppresses NLRP3 inflammasome in diabetic mice

To investigate the mechanism underlying chronic wounds in diabetes, western blot analysis was performed to measure the protein levels of NLRP3, ASC, and other inflammasome-related molecules in all groups at day 7 after wound creation (Fig. 7A). Compared to controls (Con), diabetic mice (DM) exhibited significantly increased NLRP3 expression (Fig. 7B; *p < 0.05). rhaFGF treatment (DM + rhaFGF group) significantly reduced NLRP3 protein levels (*p < 0.05). Similarly, the expression of downstream inflammasome components ASC (Fig. 7C) and Caspase-1 (Fig. 7D) was significantly lower in the DM + rhaFGF group compared to DM (*p < 0.05).

Fig. 7
figure 7

The effects of rhaFGF on the protein levels of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in wound tissue in control and diabetic mice. (A) Representative immunoblots showed the protein levels of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in all groups. Band intensities were quantitated for NLRP3 (B), ASC (C), Caspase-1 (D), IL-1β (E), and IL-18 (F) protein after normalization to β-actin. Diabetic mice (DM) exhibited significantly increased expression of all measured proteins compared to controls and rhaFGF treatment (DM + rhaFGF) significantly reduced this increase, while there was no significant difference between Con and Con + rhaFGF groups. *p < 0.05 and ***p < 0.001.

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Furthermore, the levels of IL-18 (Fig. 7E) and IL-1β (Fig. 7F), two important markers of NLRP3 inflammasome activity, were significantly elevated in the DM group compared to controls (*p < 0.05). Interestingly, rhaFGF treatment (DM + rhaFGF) significantly reduced IL-18 and IL-1β expression (*p < 0.05), whereas no significant difference in IL-1β was observed between the control and Con + rhaFGF groups (*p > 0.05).

Discussion

There are many factors that lead to the difficulty in healing of diabetic wound. High glucose microenvironment plays an important role in the occurrence and evolution of diabetic wounds. Usually, a high blood sugar environment can lead to pathological and physiological abnormalities throughout the body. When a diabetic patient is injured, a series of changes appear in the local microenvironment of the wound, including prolonged duration of inflammation17, dysangiogenesis18, hypoxia induced oxidative stress19, neuropathy20, accumulation of advanced glycation end products21, and impairment of neuropeptide signal transduction22. Previous studies have confirmed that sustained inflammatory response is an important cause of chronic DM wounds23. The main manifestations are prolonged inflammation and impaired function of wound healing cells during the proliferative phase24.

rhaFGF can inhibit the chronicity of acute wounds in DM

In DM wounds, due to prolonged exposure to high glucose environments, macrophage function changes25 prevents the transition from the inflammatory phase to the proliferative phase and results in persistent inflammation and an excessive response. This leads to an increase in the secretion of pro-inflammatory cytokines, reducing their ability to clear infections and delaying the repair process26. In addition, changes in the function of wound healing cells lead to reduced migration and proliferation of endothelial cells and keratinocytes, resulting in insufficient re-epithelialization of the wound and affecting wound healing27,28,29. Under physiological conditions, fibroblasts can secrete aFGF, promote cell proliferation30 and accelerate wound healing. For the DM wounds, the high glucose environment and sustained inflammatory response affect the function of fibroblast cells31, preventing them from secreting sufficient aFGF to aid wound healing. In addition, inflammatory factors and growth factors are extremely unbalanced in the environment of diabetes, unable to reach the threshold value of growth factors required for normal wound healing, which also makes the wound heal slowly32.

Previous studies have shown that the secretion of endogenous aFGF increases 12 h after human skin injury, reaches its peak at 48 h, and declines in 4 days after injury. Compared with acute wounds, the expression of aFGF is significantly reduced in chronic wounds33. This indicates that aFGF is involved in the wound healing process and its expression is insufficient in chronic wounds. It has been shown that aFGF is beneficial for tissue repair and epidermal regeneration. It can induce fibroblast proliferation and generates collagen, promote the proliferation and differentiation of endothelial cells, facilitate the formation of new capillaries, increase blood supply to wounds and accelerate granulation tissue growth34. Other study has shown that aFGF has a promoting effect on the healing of skin and mucosal injuries35. aFGF can promote mitosis of cells derived from the mesoderm and ectoderm, promote proliferation of epithelial cells in the epidermis, and contribute to epithelialization of wounds36. This is consistent with our study findings. During the wound healing process, there was no significant difference in wound healing rate between the DM group and the Control group in 7 days after wound creation. However, as time went on, the wound healing in the DM group was significantly slower than that in the control group on the 9th, 11th, and 14th day. After 14 days of wound creation, the wound healing speed in the DM group significantly decreased. With the treatment of rhaFGF, the wound healing rate of DM group significantly increased. The single cell culture results also confirmed that under high glucose and inflammatory stimulation, the proliferation and migration of fibroblasts, endothelial cells, and keratinocytes were significantly inhibited. But the proliferation and migration inhibition of these three cell types was significantly reduced after adding rhaFGF. These results indicate that there is a significant trend of chronicity in DM wounds, and the application of rhaFGF can promote DM wound healing and avoid the occurrence of chronicity.

rhaFGF reduces acute wound chronicity in DM mice by inhibiting sustained inflammatory response

The wound healing process includes four stages: hemostasis, inflammation, proliferation, and remodeling37. Acute inflammation plays a crucial role in wound healing process25. After skin injury, immune cells and cytokines are activated and initiate the inflammatory process. The release of pro-inflammatory cytokines constitutes an effective chemical attraction signal, which recruits immune cells to the wound area and is responsible for clearing pathogens and cell debris, thereby promoting wound healing38. During acute inflammation, pro-inflammatory factors such as IL-6 and TNF-α promote the homing of inflammatory cells, triggering pro-apoptotic genes and epithelial repair39. However, continuous stimulation of pro-inflammatory factors can exacerbate the inflammatory response of DM wounds, delaying wound healing and leading to the transition from acute wounds to chronic wounds5. Furthermore, the sustained inflammation induced by a high blood glucose environment leads to immune system dysfunction, resulting in an imbalance between inflammatory and growth factors, which is also an important reason for delayed wound healing23,32,40. Due to the long-term exposure of the body to a high glucose environment, hyperglycemia and oxidative stress can lead to abnormal secretion of pro-inflammatory cytokines, growth factors, and adhesion molecules. The microenvironment of inflammatory cells and wound healing cells around the wound changes, causing acute wounds in DM to be unable to enter the proliferative phase from the inflammatory phase, resulting in persistent inflammation. This is also the reason for the chronicity of acute wounds in DM26. Therefore, preventing the transition from acute inflammation to chronic inflammation is a key step in preventing chronic wounds.

Previous studies have shown that aFGF prevents the development of various inflammatory diseases41. In obese and insulin resistant mice, FGF-1 has a positive effect on glucose intolerance, liver lipid accumulation, and insulin resistance. At the same time, it significantly inhibits the secretion of cytokines (TNF-α and IL-6) in serum, thereby reducing diet induced liver inflammation in obese mice[50]. Other studies have shown that aFGF plays an excellent role in inhibiting inflammation of weak acidic wounds12. In the process of wound healing in diabetes, the nanoenzyme hydrogel system loaded with aFGF can promote the polarization of macrophage phenotype from M1 to M2 and promote angiogenesis, thus improving the potential of diabetes wound from chronic inflammation to wound repair and proliferation42. aFGF also plays a protective role in the central nervous system. It can reduce chronic inflammation and neuronal dysfunction caused by excessive activation of astrocytes or the persistent presence of reactive astrocytes by inhibiting their activation43. In our study, there was no significant difference in the healing rate between normal wounds and DM wounds at 7 days, but with the prolongation of time, the DM group showed a significant delay in wound healing. The expressions of inflammatory factors in the wound tissues and serum of each group showed that there were increased levels of pro-inflammatory factors (TNF-α and IL-6) in the wound tissues and serum of the DM group. After using rhaFGF, the levels of inflammatory factors in the wound tissue and serum of DM group significantly decreased. It can be seen that aFGF has a certain inhibitory effect on sustained inflammatory responses.

rhaFGF May promote wound healing by inhibiting the NLRP3 inflammasome signaling pathway

NLRP3 has recently become a key regulatory factor in chronic inflammatory response44. Previous studies have shown that there is a link between NLRP3 and the development of chronic wounds in diabetes45. The findings of Zhang et al.46 suggest that high expression of NLRP3 may be responsible for the excessive inflammation seen in human diabetic wounds and in high glucose-induced macrophages. The activation of NLRP3 inflammatory vesicles is induced by endogenous and exogenous danger signals such as lipopolysaccharide (LPS) and high glucose (HG)47. Diabetic wounds are accompanied by excessive production of a large number of reactive oxygen species (ROS), and the excessive accumulation of ROS will destroy the cell tissues and trigger inflammatory reactions. ROS promote the expression and activity of metalloproteinases, leading to a state of excessive protein hydrolysis in the wound and significant destruction of the extracellular matrix, which results in the cessation of the repair process ROS48. Overproduction of ROS in cells also leads to loss of cellular homeostasis, oxidative stress and eventual cellular destruction of organs49. In addition, ROS accumulation increases NLRP3 inflammatory vesicle activation, leading to caspase-1 activation and subsequent activation of cytokines IL-1β/IL-18. Activated caspase-1 can cleave inactive storage forms of IL-1β and IL-18 into active cytokines, thus exacerbating the inflammatory response in diabetes, which further leads to insulin resistance and organ dysfunction, making wound healing delayed50,51,5. In the acute inflammatory phase of healthy wounds, NLRP3 is expressed by pro-inflammatory macrophages and has a protective role in wound healing[8]. However, in DM wounds, the inflammatory cascade response caused by a large number of activated NLRP3 inflammatory vesicles becomes an important risk factor for non-healing wounds52.

The use of exogenous rhaFGF can supplement the insufficient growth factors in DM wounds, inhibit the activation of NLRP3 in DM wounds, reduce the secretion of pro-inflammatory factors, weaken inflammatory reactions, prevent acute inflammation from turning into chronic inflammation, and promote wound healing. Sun et al. reported that aFGF can alleviate oxidative stress and inflammation in mitochondria by activating Wnt/β-catenin signaling pathway, thus preventing endothelial damage caused by diabetes53. The findings of Wang D et al.54 indicates that aFGF and its variants can improve chronic kidney disease through PI3K/AKT mediated oxidative stress and inflammation inhibition. Another study has shown that intraventricular injection of aFGF can activate the hypothalamic MAPK/ERK signaling pathway, thereby inhibiting oxidative stress damage caused by hyperglycemia, effectively correcting abnormal increases in blood glucose levels, and reducing the impact of the high glucose microenvironment on cells55. Fan et al.41 reported that aFGF can weaken the c-Jun N-terminal kinase signaling pathway, thereby improving insulin resistance and inhibiting inflammatory responses. In this study, 7 days after wound creation, compared with the Con group, the DM group had higher levels of NLRP3 related molecules and IL-18 expression in the wound tissue of mice. The application of rhaFGF can effectively reduce the expression of NLRP3 related molecules and downstream molecules in the wound tissue of DM group, and significantly accelerate wound healing. This indicates that the excessive production of NLRP3 is an important cause of delayed wound healing. rhaFGF can promote wound healing by reducing the expression of NLRP3 and downstream molecules.

Based on the results of this study, it can be concluded that there is a sustained inflammatory response in DM wounds after the end of the acute inflammatory phase, which is an important reason for the chronic difficult-to-heal DM wounds. The use of rhaFGF can effectively inhibit the excessive inflammatory response of DM wounds, prevent acute inflammation from turning into chronic inflammation, and promote DM wound healing. Its mechanism may be related to the inhibition of excessive activation of NLRP3.

Our study has some limitations. Firstly, we only utilized an animal model, and further investigations in human clinical wounds are warranted in the future. Secondly, the single-cell culture model does not fully capture the complex interactions between human umbilical vein endothelial cells (HUVEC), human keratinocytes (HaCat), and human dermal fibroblasts (HFF) within the in vivo human granulation tissue environment. Thirdly, the 14-day study duration limits our ability to assess rhaFGF’s long-term effects. Fourthly, in exploring the mechanism of NLRP3 inflammasome in chronic rhaFGF induced diabetic acute wounds, we did not use NLRP3 gene knockout or blockade assays, which need to be fulfilled in future study. Finally, the study focused solely on diabetic wounds, excluding other chronic wound types. Therefore, in future study, we hope to broaden the scope of the study, extend the study time, further explore the relevant mechanisms.

rhaFGF promotes the healing of DM wounds by reducing the secretion of pro-inflammatory cytokines, which can prevent acute inflammation from turning into chronic inflammation. The mechanism may be related to its inhibition of NLRP3 activation in DM wounds.

In summary, our study focused on specific stage of the transition from acute to chronic diabetic wound which was seldom mentioned in previous literatures. It has been proven in this study that exogenous rhaFGF could effectively prevent the persistence of inflammation and thus halt the transition of acute diabetic wounds to chronic wounds, which may be related to the inhibition of NLRP3 inflammasome pathway activation. In addition, rhaFGF was proved to suppress the decline in cell proliferation and migration ability under HG + LPS (simulating diabetic wound in vitro) environment. This study provided preliminary theoretical support for rhaFGF application in the prevention of the development of non-healing diabetic wounds targeting suppression of inflammatory chronicity.

Methods

Experimental instruments

Cell culture incubator (Keinda: KT95-115-340), fluorescence microscope (BM/Piaim: A0007606), water bath (SIMPLAB: DN-22 S), portable blood glucose meter (yuwell: LC2023126), centrifuge (Sigma: Z654728CH-1EA), ultrasonic grinder (Sonice, USA: vcx-130pm), developer (Shanghai Qinxiang: 100230179-ICQ).

Experimental reagents

DMEM (Gibco: 12491015), fetal bovine serum (Sigma-Aldrich: F8318), rhaFGF (PEPROTECH: 100–17 A), high glucose medium (Thermo Fisher: C11995500BT), lipopolysaccharide (ACMEC: AC12037), CCK-8 assay kit (DOJINDO: CK04), NLRP3 (abcam: ab263899), ASC (SANTA CRUZ BIOTECHNOLOGY: sc-514414), Caspase-1 (abcam: ab138483), IL-1β (CST: 12242 S), IL-18 antibody (proteintech: 10663-1-AP), mouse IL-6 ELISA assay kit (Multisciences: 0-EK206/ 3–96), mouse TNF-α ELISA detection kit (Multisciences: 70-EK282HS/3–96), lipopolysaccharide (ACMEC: AC12037), streptozotocin (Macklin: S6089-1 g).

Experimental animals and model establishment

A total of 44 male eight-week-old C57BL/6 mice were acclimatized for one week. Body weight and fasting blood glucose were measured. Mice were then randomly divided into four groups (n = 11 / group): (1) control mice with acute wound (Con), (2) control mice with acute wound plus rhaFGF treatment (Con + rhaFGF), (3) diabetic mice with acute wound plus PBS (DM + PBS), (4) and diabetic mice with acute wound plus rhaFGF treatment (DM + rhaFGF). Within each group, the mice were further allocated for specific experimental purposes: 6 mice for wound healing observation and 5 mice for cytokine detection. The mice in the Con group received a regular diet, while the DM group received a high-fat diet for 4 weeks with their blood glucose monitored weekly. After four weeks, the diabetic group received a daily intraperitoneal injection of streptozotocin (STZ) in citrate buffer (40 mg/kg for 5 days) to induce diabetes. The Con group received an equal volume of citrate buffer injection. Three days later, fasting blood glucose was measured again. Mice with blood glucose levels ≥ 11.1 mmol/L were considered as diabetic mice56. A circular full-thickness wound of approximately 4 mm in diameter was created on the dorsal surface of the hindlimb paw to establish the acute wound model57. All mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Peking, China) and housed in an asepsis condition with a controlled temperature of 23 °C and a standardized light - dark cycle.

RhaFGF treatment and wound healing recording

On the day after successful wound creation (day 0), rhaFGF was diluted in fucoidan buffer to 10 µg/mL and topically applied daily to the wound with a dose of 0.08 µg/cm² for consecutive 14 days. The control mice received the same dose of fucoidan buffer. Wound closure was documented photographically on days 0, 3, 5, 7, 9, 11, and 14 (n = 6 per group). Images were analyzed using ImageJ software to calculate the wound healing rate and expressed as [(initial wound area - wound area at a specific timepoint) / initial wound area] x 100%.

Quantitative protein analysis (BCA Assay)

After indicated treatment, wound tissue protein was extracted using RIPA buffer with a tissue and RIPA volume ration of 2 : 30 and the protein contents was assessed using a commercial BCA assay kit. In brief, 15 µL of diluted protein lysate and BSA standards (prepared according to the kit instructions) were mixed with BCA reagents in a 96-well microplate, and after 30-min incubation, the absorbance of each well was measured at 562 nm wavelength using a microplate reader in triplicate.

Western blotting analysis

Total proteins were extracted and separated by SDS-PAGE using 10% polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes, blocked with 5% non-fat milk for 2 h, and incubated overnight at 4℃ with primary antibodies at 1:1000 dilution against NLRP3, ASC, Caspase-1, IL-1β IL-18, or β-actin. The membranes were washed with tris-buffered saline (TBS) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution) at room temperature. Protein bands were visualized using a gel imaging system, and band intensity was quantified using β-actin as a loading control.

ELISA analysis of inflammatory markers

Serum and tissue levels of inflammatory cytokines IL-6 and TNF-α were measured using commercially available ELISA kits according to the manufacturer’s instructions (n = 5/group).

Cell culture and treatments

The Human fibroblasts (HFF), Human umbilical vascular endothelial cells (HUVEC), and Human immortalized keratinocytes (HaCat) cells were both purchased from ScienCell Research Laboratories (San Diego, CA, USA). The cell lines were cultured using DMEM medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified incubators gassed with 5% CO2 and 95% room air. Both cells in four- to- eight passages were used for experiments.

To mimic the diabetic inflammatory environment58 of diabetic wounds, the cells were exposed to a combination of high glucose (HG) and lipopolysaccharide (LPS). Three groups were established: control, LPS + HG, and LPS + HG + rhaFGF. The cells were treated as follows: the LPS + HG group was exposed to LPS (10 ng/mL) in high-glucose DMEM for 48 h. rhaFGF was additionally administered to the LPS + HG treatment in the LPS + HG + rhaFGF group. And the cells in control group received an equivalent volume of DMEM medium. Following the 48-hour incubation period, cellular proliferation activity was assessed using the CCK-8 assay. The ranges of aFGF concentrations for the three cells were determined separately based on previous studies59,60,61, manufacturer’s guidelines of rhaFGF and our pilot study, and then the optimal concentration of rhaFGF ( 20ng/ml,5ng/ml and 50ng/ml for HUVEC, HFF and HaCat respectively ) was determined according to the results of our pilot study.

Cell viability assay (CCK-8)

Cell proliferation was assessed using a CCK-8 assay after 48 h of rhaFGF treatment in HFF, HUVEC, and HaCaT cells. Cell viability was determined in triplicate by measuring the absorbance at 450 nm using a microplate reader (n = 6). The following formula was used to calculate cell viability: cell viability (%) = (OD[treated] - OD[blank])/(OD[control] - OD[blank]) x 100%.

Migration assays

Cell Migration assay was used to verify the effect of rhaFGF on the migration ability of HaCat, HUVEC, HFF cells. Cells were taken in good growth condition, digested and resuspended into a single cell suspension, and the cell density (5 × 105cells/mL) was adjusted. Add the cell suspension evenly to a 6-well plate, 2 mL per well, and gently shake to evenly distribute the cells. Place in a 37 °C, 5% CO2 incubator overnight to allow the cells to adhere to the wall and reach 80% and 90% confluence. Use a 200 U sterile pipette tip perpendicular to the bottom of the plate and make scratches in a straight line at one time, and after scratching, gently rinse with PBS 23 times to remove the exfoliated cells. The LPS + HG group was treated with 2 ml of serum-free medium, the LPS + HG + rhaFGF group was diluted with serum-free medium. Hacat, HUVEC and HFF in the LPS + HG + rhaFGF group was treated with serum-free medium containing 20ng/ml, 5ng/ml and 50ng/ml rhaFGF respectively. All the cells was cultured in a 37 °C, 5% CO2 incubator, and the cell migration was recorded at 6 h, 12 h and 24 h, respectively. All images were processed with ImageJ. Migration distance (µm) was calculated by subtracting the distance at 6 h, 12–24 h from that at 0 h.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 9.3.0 for Windows. Continuous data are presented as mean ± standard deviation. Two-group comparisons were performed using unpaired t-tests, and one-way ANOVA was used for comparisons between multiple groups. Statistical significance was set at p < 0.05.

Conclusion

rhaFGF promotes the healing of DM wounds by reducing the expression of pro-inflammatory cytokines, which can prevent acute inflammation from turning into chronic inflammation. The mechanism may be related to its inhibition of NLRP3 activation in DM wounds.