Effect of fish scale ointment on diabetic foot ulcer by inducing ferroptosis via the nuclear factor E2-related factor 2 pathway

Abstract

BACKGROUND

Excessive oxidative stress plays a key role in the development of diabetic complications, including impaired ulcer healing. Previous studies have shown that fish scale ointment can promote wound healing.

AIM

To preliminarily investigate the effect of fish scale ointment on wound healing in a diabetic foot ulcer (DFU) rat model by examining its regulation of the nuclear factor E2-related factor 2 (Nrf2) pathway and induction of ferroptosis.

METHODS

Fish scale ointment (collagen product) was prepared from 500 g of silver carp scales. A diabetic rat model was induced by high-fat and high-sugar feeding combined with intraperitoneal streptozotocin injections. For the DFU rat model, ulcer wounds were created by removing dorsal foot hair and cutting the skin to the fascia. The diabetic rats were randomized into five groups: Model, fish scale collagen (FSC), control + liproxstatin-1 (Lip-1), model + Lip-1, and FSC + Lip-1. In each group, treatments were administered once daily by topical application and intraperitoneal injection for 14 days. Wound healing was evaluated on days 7 and 14 after treatment. Hematoxylin and eosin staining was used to assess wound injury and capillary formation. Basic fibroblast growth factor (bFGF) and CD31 levels in wound tissue were measured by immunohistochemistry. Additionally, malondialdehyde (MDA), glutathione (GSH), ferroptosis-associated genes, and iron ion concentrations were quantified using assay kits. Protein levels of Nrf2, heme oxygenase-1 (HO-1), and glutathione peroxidase 4 (GPX4) were determined using Western blotting.

RESULTS

Compared with the control group, the model group showed slower wound healing, reduced angiogenesis, decreased bFGF and CD31 levels, increased iron ion concentration and MDA levels, reduced GSH levels, and decreased Nrf2, HO-1, and GPX4 protein expression (all P < 0.05). The FSC, model + Lip-1, and FSC + Lip-1 groups showed increased wound healing and angiogenesis, elevated bFGF and CD31 expression, lowered iron ion concentration and MDA levels, increased GSH levels, and enhanced Nrf2, HO-1, and GPX4 protein levels compared with the model group (P < 0.05). Improvements were more pronounced in the FSC + Lip-1 group compared with the FSC group (P < 0.05).

CONCLUSION

Fish scale ointment promotes angiogenesis and wound healing in DFU rat models by inhibiting ferroptosis, possibly through the activation of the Nrf2 pathway.

Key Words: Fish scale ointment; Nuclear factor E2-related factor 2 pathway; Ferroptosis; Diabetic foot ulcer; Fish scale collagen

Core Tip: Fish scale ointment accelerates necrotic cell shedding during wound healing, promotes capillary formation, enhances fibrous tissue and epidermal regeneration, facilitates wound repair, and exhibits anti-infective properties. This study selected the nuclear factor E2-related factor 2 signaling pathway as the target for observation. From the perspective of ferroptosis regulation, it investigated the mechanism by which fish scale ointment promotes diabetic foot ulcer healing in rat models.

INTRODUCTION

Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia resulting from impaired insulin secretion or action[1]. In 2019, the International Diabetes Federation estimated that 425 million people worldwide were living with DM[2]. Diabetic ulcers are a common complication, typically manifesting as local infection, circulatory disorders, and gangrene. These ulcers are referred to as diabetic foot ulcers (DFU) because they mostly occur in the foot[3]. The global incidence of DFU has continued to rise, with an estimated annual incidence of 6.3% among individuals with DM, affecting millions worldwide[4,5]. Once DFU is formed, the risk of progression increases, potentially leading to amputation or death in severe cases[6]. In addition, impaired glucose metabolism in diabetic patients causes hyperglycemia in ulcer wounds, further delaying DFU healing[7].

Several molecular mechanisms underlying diabetic ulcer wound healing have been identified. During DM development, excessive oxidative stress contributes significantly to diabetic complications, including impaired ulcer healing[8]. Among these, nuclear factor E2-related factor 2 (Nrf2) reduces apoptosis and promotes cell migration, proliferation, and differentiation by regulating the adaptive response to oxidative stress[9]. Exosomes from adipose-derived stem cells overexpressing Nrf2 have been reported to significantly reduce the ulcer area in DFU rats, accompanied by increased granulation tissue formation, angiogenesis, and growth factor expression, as well as a decrease in inflammation and oxidative stress-related proteins[10]. Consequently, Nrf2 is also a key target in DFU progression and treatment. Ferroptosis, a recently identified form of regulatory cell death, is implicated in various pathological processes, including diabetes, cancer, and neurodegenerative diseases[11-13]. In diabetic rats, high iron doses can lead to advanced glycation end-product accumulation in the liver[14]. Advanced glycation end-product accumulation can lead to the formation of glycosylated collagen and heightened oxidative stress, thereby impairing wound healing[15]. Therefore, ferroptosis plays a critical role in DFU wound healing; however, its underlying mechanisms require further investigation.

Fish scale ointment is prepared from fish scale glue, sesame oil, and beeswax. Traditionally, it has been used for clearing heat, removing necrotic tissue, promoting granulation, and regenerating muscle and skin[16]. Formulated with sesame oil and beeswax, the ointment preserves the active ingredients of fish scale collagen (FSC) and enables slow release, representing a fusion of traditional medicine and modern technology. During wound healing, fish scale ointment facilitates necrotic cell shedding, promotes capillary formation, enhances the regeneration of fibroblasts and epidermis, promotes wound repair, and exhibits anti-infective effects[17]. However, its specific mechanism remains unclear. Therefore, we selected the Nrf2 signaling pathway as the observation target for our study and investigated the mechanism by which fish scale ointment promotes DFU wound healing in rat models from the perspective of ferroptosis regulation.

MATERIALS AND METHODS

Experimental animals

Eight- to ten-week-old SD rats of specified pathogen-free grade [Vital River Laboratory Animal Technology Co., Ltd., Beijing, China, Production License No. SYXK (Beijing) 2022-0052], weighing 250-320 g, were fed adaptively for 1 week under controlled conditions (temperature: 21-25 °C, humidity: 40%-60%, 12/12 hours light/dark cycle) with free access to food and water.

Fish scale ointment preparation

Silver carp scales (500 g) were used as the raw material. Decalcification was carried out through selection, impurity removal, cleaning, air drying (water content: 17%), soaking in an ethylenediaminetetraacetic acid solution, neutral washing, and air drying. At 4 °C, the decalcified scales were soaked in a 0.1 mol/L sodium hydroxide solution for 6 h (material-to-liquid ratio of 1:8) to remove non-collagenous proteins. The prepared scales were then immersed in 0.5 mol/L acetic acid solution at a specified ratio, with pepsin added to extract collagen. The extract was filtered through a 300-mesh filter cloth. Sodium chloride was added until its concentration reached 0.9 mol/L, and salting out was carried out for 24 hours, followed by centrifugation to obtain the collagen precipitate. The precipitate was dialyzed for 24 hours in a dialysis bag (molecular weight cut-off: 10 kDa) and then lyophilized to obtain the collagen product.

Diabetic foot model construction and animal grouping

Rats were divided into the following groups: Control (normal feeding), model (DFU induction without treatment), FSC (DFU induction with fish scale ointment), control + Lip-1 (normal feeding with intraperitoneal injection of the ferroptosis inhibitor Lip-1), model + Lip-1 (DFU induction with intraperitoneal injection of Lip-1), and FSC + Lip-1 (DFU induction with fish scale ointment and intraperitoneal injection of Lip-1). Each group contained five rats.

To establish the diabetic rat model, rats in the model group were fed a high-fat diet containing 21% fat and 0.15% cholesterol for 4 weeks after 1 week of acclimatization. They then received a single intraperitoneal injection of streptozotocin (90 mg/kg) following 12 h of fasting. The control group received the same volume of sodium citrate buffer (10 mL/kg). Three days after streptozotocin administration, blood was drawn from the tail tips to detect fasting blood glucose. Successful diabetes induction was defined as fasting blood glucose ≥ 16.7 mmol/L, an absence of obvious weight loss, and the presence of polyphagia, polydipsia, and polyuria. Thirty rats were used to establish the diabetic rat model. After modeling, only three rats exhibited no obvious characteristics of diabetes; thus, the success rate of diabetes induction was 90.0% (27/30). Successfully induced diabetic rats were selected, and the DFU model was further established 2 weeks later. All rats were anesthetized through intraperitoneal injection with 2% pentobarbital sodium. After shaving the dorsal right hind foot and disinfecting the area with iodophor, a 6 mm full-thickness skin wound was excised and covered with a waterproof dressing. DFU modeling was confirmed by wound healing assessment. The model rats were randomly divided into five groups, with five rats per group. Interventions began 12 hours after DFU modeling and continued for 14 days. Rats in the model group received no treatment. The FSC group was treated with fish scale ointment (0.5 g/day) applied to the wound and fixed with medical gauze and adhesive plaster. The control + Lip-1 group received intraperitoneal Lip-1 injections (10 mg/kg) while being normally fed. The model + Lip-1 group underwent DFU induction plus intraperitoneal Lip-1 injections (10 mg/kg). The FSC + Lip-1 group received fish scale ointment treatment (0.5 g/day) plus intraperitoneal Lip-1 injections (10 mg/kg).

Detection of wound healing rate

On days 7 and 14 of the intervention, wound healing was evaluated visually. A transparent grid film was placed over the wound, and the images were scanned. Wound areas were quantified using Adobe Photoshop CS6, and the wound healing rate was calculated as follows: (wound area at baseline - wound area after treatment)/wound area at baseline × 100.

Hematoxylin and eosin staining

After intervention, the rats were anesthetized by intraperitoneal injection of pentobarbital sodium. Wound tissue and surrounding granulation tissue were isolated and processed for hematoxylin and eosin (HE) staining through a series of steps, including fixation, dehydration, embedding, sectioning, dewaxing, and hydration. Stained sections were examined under a microscope to evaluate capillary formation.

Immunohistochemical assay

Rats were anesthetized as described above, and wound and granulation tissue were collected. After fixation, dehydration, embedding, sectioning, dewaxing, hydration, and hydrogen peroxide treatment, the sections were blocked with goat serum for 35 minutes. The cells were then incubated overnight at 4 °C with primary antibodies against basic fibroblast growth factor [basic fibroblast growth factor (bFGF); No. 46879S, 1:500] and CD31 (No. 77699S; 1:200), followed by incubation with a secondary antibody (No. 8114) at 37 °C for 60 minutes. All antibodies were supplied by Cell Signaling Technology. After 3,3’-diaminobenzidine and HE staining, six random fields per section were observed under a light microscope, and the positive cell rate was analyzed using Image-Pro Plus.

Determination of ferroptosis-related indicators

Wound tissue and surrounding granulation tissue were collected, weighed, and washed with precooled phosphate-buffered saline to remove blood. The tissue was cut into small pieces and transferred to a grinding tube. For malondialdehyde (MDA) and glutathione (GSH) detection, a radioimmunoprecipitation assay lysis buffer was added, and the tissue was homogenized manually. All procedures were performed on ice. MDA and GSH levels were detected using commercial kits (Wuhan Fine Biotech Co., Ltd, Wuhan, China), and iron ion concentration was measured at 520 nm using a colorimetric assay kit (Beijing LABLEAD Trading Co., Ltd, Beijing, China) according to the manufacturer’s instructions.

Western blotting

Plasmosin was isolated from wound tissue homogenates, and protein concentrations were determined using the bicinchoninic acid method. After preparing the separation gel, 25 μg of the protein was loaded for electrophoresis and membrane transfer operations, followed by sealing for 2 hours at room temperature with 5% skim milk. Membranes were then incubated overnight at 4 ℃ with rabbit anti-rat primary antibodies against Nrf2 (No. 12721, 1:1000), heme oxygenase-1 (HO-1) (No. 70081, 1:1000), glutathione peroxidase 4 (GPX4; No. 52455, 1:1000), and β-actin (No. 4967, 1:1000), all from Cell Signaling Technology, United States. After washing, the membranes were incubated for 2 hours at room temperature with horseradish peroxidase-conjugated secondary antibody (No. 58802, 1:1000, Cell Signaling Technology, Shanghai, China). Protein bands were visualized using electrochemiluminescence. Grayscale analysis was performed using ImageJ, and the relative expression levels were normalized to β-actin as the internal reference gene.

Statistical analysis

All data are presented as mean ± SD. Statistical analyses were performed using SPSS 25.0 and GraphPad Prism 8.0. Comparisons between two groups were conducted using the t-test, while one-way analysis of variance followed by the Least Significant Difference test was used for multiple group comparisons. A P-value < 0.05 was considered statistically significant.

RESULTS

Effect of fish scale ointment on wound healing in rats

As shown in Table 1, the model group exhibited a significantly lower wound healing rate than the control group (P < 0.05). Compared with the model group, wound healing rates were significantly higher in the FSC, model + Lip-1, and FSC + Lip-1 groups (P < 0.05). Moreover, the FSC + Lip-1 group showed a greater increase in wound healing compared with the FSC group (P < 0.05).

Table 1 Comparison of wound healing rate.

Group	Wound healing rate (%)
	Day 7	Day 14
Control (n = 5)	45.58 ± 5.83	82.76 ± 3.06
Model (n = 5)	18.32 ± 1.62a	24.54 ± 3.09a
FSC (n = 5)	39.08 ± 5.63a,b	69.08 ± 3.48a,b
Control + Lip-1 (n = 5)	44.94 ± 2.74	79.64 ± 4.59
Model + Lip-1 (n = 5)	35.46 ± 3.04a,b	64.92 ± 5.82a,b
FSC + Lip-1 (n = 5)	42.54 ± 5.21b,c	73.94 ± 3.27a,b,c
F	27.84	140.2
P value	< 0.0001	< 0.0001

^aP < 0.05 vs control.

^bP < 0.05 vs model.

^cP < 0.05 vs fish scale collagen.

FSC: Fish scale collagen; Lip-1: Liproxstatin-1.

HE staining of rat wound tissue

In the control group, wound surface epithelial cells were arranged in an orderly manner, with abundant fibroblasts and capillaries. The model group displayed incomplete wound tissue structure, with extensive inflammatory cell infiltration and reduced fibroblasts and capillaries. The FSC group showed a more complete tissue structure compared with the model group, characterized by reduced inflammatory cell infiltration and increased fibroblasts and capillaries. The FSC + Lip-1 group exhibited a more complete tissue structure, fewer inflammatory cells, and more fibroblasts and capillaries compared with the FSC group (P < 0.05). See Figure 1 and Table 2 for details.

Figure 1 Hematoxylin and eosin staining of the new blood vessels in rat wound tissue. FSC: Fish scale collagen; Lip-1: Liproxstatin-1.

Table 2 Comparison of the number of new blood vessels in rat wound tissue.

Group	Number of new blood vessels
Control (n = 5)	22.60 ± 1.14
Model (n = 5)	6.20 ± 0.84a
FSC (n = 5)	18.60 ± 2.07a,b
Control + Lip-1 (n = 5)	22.40 ± 2.30
Model + Lip-1 (n = 5)	16.60 ± 2.07a,b
FSC + Lip-1 (n = 5)	20.80 ± 1.30a,b,c
F	64.71
P value	< 0.0001

^aP < 0.05 vs control.

^bP < 0.05 vs model.

^cP < 0.05 vs fish scale collagen.

FSC: Fish scale collagen; Lip-1: Liproxstatin-1.

Immunohistochemistry of bFGF and CD31 in rat wound tissues

Compared with the control group, the positive rates of bFGF and CD31 were significantly reduced in the model group (P < 0.05). The FSC, model + Lip-1, and FSC + Lip-1 groups exhibited significantly higher positive rates of bFGF and CD31 than the model group (P < 0.05). Moreover, the FSC + Lip-1 group showed higher positive rates of bFGF and CD31 than the FSC group (P < 0.05; Figure 2 and Table 3).

Figure 2 Immunohistochemistry staining of basic fibroblast growth factor and CD31 protein. FSC: Fish scale collagen; Lip-1: Liproxstatin-1; bFGF: Basic fibroblast growth factor.

Table 3 Comparison of basic fibroblast growth factor and CD31 expression in rat wound tissue.

Group	Positive rate of bFGF (%)	Positive rate of CD31 (%)
Control (n = 5)	30.40 ± 3.50	21.78 ± 1.15
Model (n = 5)	12.88 ± 0.97a	8.92 ± 0.64a
FSC (n = 5)	23.88 ± 4.18a,b	15.38 ± 1.18a,b
Control + Lip-1 (n = 5)	29.56 ± 2.26	20.30 ± 0.76
Model + Lip-1 (n = 5)	20.66 ± 3.63a,b	13.50 ± 1.18a,b
FSC + Lip-1 (n = 5)	26.76 ± 1.70b,c	17.44 ± 0.96a,b,c
F	24.81	110.3
P value	< 0.0001	< 0.0001

^aP < 0.05 vs control.

^bP < 0.05 vs model.

^cP < 0.05 vs fish scale collage.

FSC: Fish scale collagen; bFGF: Basic fibroblast growth factor; Lip-1: Liproxstatin-1.

Levels of ferroptosis-related indexes

As shown in Table 4, iron ion concentration and MDA levels increased, while GSH levels decreased in the model group compared with the control group (P < 0.05). In the FSC, model + Lip-1, and FSC + Lip-1 groups, iron ion concentrations and MDA levels were significantly lower, and GSH levels were significantly higher than in the model group (P < 0.05). Additionally, compared with the FSC group, the FSC + Lip-1 group showed lower iron ion concentration and MDA levels and higher GSH levels (P < 0.05).

Table 4 Comparison of ferroptosis-related indexes in rat wound tissue.

Group	Iron ion (μg/g)	MDA (mmol/g)	GSH (μmol/g)
Control (n = 5)	0.04 ± 0.01	3.10 ± 0.28	4.15 ± 0.36
Model (n = 5)	0.27 ± 0.01a	6.95 ± 0.31a	2.00 ± 0.08a
FSC (n = 5)	0.16 ± 0.02a,b	4.80 ± 0.22a,b	2.84 ± 0.22a,b
Control + Lip-1 (n = 5)	0.04 ± 0.02	2.99 ± 0.13	3.94 ± 0.09
Model + Lip-1 (n = 5)	0.20 ± 0.03a,b	5.08 ± 0.09a,b	2.66 ± 0.12a,b
FSC + Lip-1 (n = 5)	0.07 ± 0.02a,b,c	4.09 ± 0.07a,b,c	3.10 ± 0.14a,b,c
F	118.0	253.4	88.36
P value	< 0.0001	< 0.0001	< 0.0001

^aP < 0.05 vs control.

^bP < 0.05 vs model.

^cP < 0.05 vs fish scale collagen.

MDA: Malondialdehyde; GSH: Glutathione; FSC: Fish scale collagen; Lip-1: Liproxstatin-1.

Ferroptosis-related protein levels

As shown in Figure 3, Nrf2, HO-1, and GPX4 protein expression levels were significantly lower in the Model group than in the control group (P < 0.05). The expression levels of these proteins were significantly higher in the FSC, model + Lip-1, and FSC + Lip-1 groups compared with the model group (P < 0.05). Furthermore, the FSC + Lip-1 group showed significantly higher Nrf2, HO-1, and GPX4 expression compared to the FSC group (P < 0.05).

Figure 3 Levels of ferroptosis-related proteins in rat wound tissues. A: Western blot bar plot of the proteins; B: Nuclear factor E2-related factor 2 protein expression level; C: Heme oxygenase-1 protein expression level; D: Glutathione peroxidase 4 protein expression level. ^aP < 0.05 vs control; ^bP < 0.05 vs model; ^cP < 0.05 vs fish scale collagen. GPX4: Glutathione peroxidase 4; HO-1: Heme oxygenase-1; Nrf2: Nuclear factor E2-related factor 2; FSC: Fish scale collagen; Lip-1: Liproxstatin-1.

DISCUSSION

DFU is a chronic wound often associated with delayed healing due to circulatory disorders, comorbidities such as obesity, DM, and adverse environmental conditions[18]. Hyperglycemia impairs wound healing by increasing pro-inflammatory cytokine levels and decreasing growth factor availability, blood circulation, and cellular proliferation and migration at the wound site[19]. Fish scale ointment is composed of fish scale collagen, sesame oil, and beeswax, with collagen as the major component. Modern studies report that fish scales are rich in glial proteins, which protect the wound, promote hemostasis, and exhibit anti-inflammatory and analgesic properties, thereby supporting tissue cell growth[20]. The research team led by Professor Chen XG at Ocean University of China has made significant progress in fish scale product development[20]. They extracted collagen from fish scales using freeze–thaw technology, which preserves the amino acid structure of protein polypeptides. When combined with sesame oil and beeswax, the resulting fish scale ointment maintains the active components of collagen and enables slow release, representing a fusion of traditional medicine and modern technology[21].

This study investigated the wound repair mechanism of fish scale ointment in DFU rats. Compared with the model group, the FSC group showed statistically significant increases in wound healing, new blood vessel formation, and positive rates of bFGF and CD31. Angiogenesis is essential for wound repair, as it supplies blood vessels that provide progenitor cells, oxygen, and nutrients to maintain proliferation and remodeling at the wound site[22]. bFGF has been shown to promote angiogenesis and cell proliferation in injured tissues, thereby accelerating wound healing[23]. CD31, an endothelial cell marker, reflects the degree of angiogenesis[24]. In this study, fish scale ointment enhanced bFGF and CD31 expression in wound tissue, promoted capillary formation, and accelerated wound healing. Nrf2 is a key antioxidant factor that translocates to the nucleus to induce the expression of antioxidant enzymes such as HO-1 and plasma glutathione peroxidase[25]. Bitar et al[26] reported that oxidative stress is a major contributor to chronic diabetic complications and is closely linked to Nrf2 deficiency. Hayashi et al[27] reported that Nrf2 participates in corneal epithelial wound healing by accelerating cell migration and may serve as a treatment for diseases such as dry eye and chronic corneal epithelial defects. Compared with the model group, the FSC group exhibited significantly reduced MDA levels, elevated GSH levels, decreased iron ion concentration, and altered expression of Nrf2, HO-1, and GPX4 proteins in rat wound tissues. Ferroptosis is a recently identified form of iron-dependent cell death characterized by lipid peroxide accumulation[28]. It is primarily induced by three mechanisms: Iron ion accumulation, oxidative stress imbalance resulting from GSH depletion, and lipid peroxidation[29,30]. Ferroptosis occurs when cellular redox homeostasis is disrupted[31]. Nrf2 plays a central role in antioxidant defense by binding to antioxidant response elements and activating the transcription of downstream genes[32]. Numerous studies indicate that Nrf2 plays a pivotal role in regulating ferroptosis due to its diverse functions in iron, lipid, and amino acid metabolism[33]. Ferroptosis is associated with various pathological conditions. In many cancers, abnormal suppression of ferroptosis promotes disease progression and metastasis[34]. During tissue injury, the repeated activation of ferroptosis can cause extensive cell death and subsequent organ dysfunction[35]. Consequently, targeting Nrf2-related signaling pathways to modulate ferroptosis has emerged as a promising therapeutic strategy for cancer, neurodegenerative disorders, and ischemic diseases.

GSH synthesis and reduction are central to amino acid metabolism pathways that regulate ferroptosis. Insufficient GSH impairs the detoxification of peroxidized lipids generated by oxidative stress. Disruption of the cystine/glutamate antiporter (system Xc–) inactivates GPX4, preventing the metabolism of lipid oxides through the GPX4-catalyzed GSH reductase reaction. This leads to Fenton-like reactions that oxidize lipids and generate excessive reactive oxygen species[36]. Lipid peroxidation alters the fluidity and permeability of cell membranes, ultimately disrupting cellular structure and function. Many studies have shown that activation of the Nrf2/HO-1 pathway can prevent renal injury in diabetic nephropathy mice and alleviate inflammation and oxidative stress[37]. Similarly, in DFU, tissue biopsies from patients with chronic wounds exhibited reduced Nrf2 and HO-1 mRNA expression[38]. Elevated Nrf2 levels can temporarily upregulate vascular gene expression, thereby accelerating angiogenesis and chronic wound healing[39]. In this study, administration of ferroptosis inhibitors, in addition to fish scale ointment, further enhanced wound healing, supporting the role of ferroptosis in impaired wound healing in DFU.

This study had several limitations. First, the small sample size may have introduced bias, and the use of multiple experimental groups highlights the need for larger samples in future animal research. Second, Nrf2 expression was not dynamically monitored, and long-term data on the efficacy and safety of fish scale ointment were lacking. Furthermore, the mechanistic exploration of ferroptosis pathways remained preliminary, with no further assessment of potential alternative pathways or changes in upstream and downstream gene expression. To address these issues, future studies should adopt well-designed, large-sample, long-term studies. Safety evaluations will also be essential for clinical translation.

CONCLUSION

In conclusion, fish scale ointment may inhibit ferroptosis in DFU rat models through the Nrf2 signal pathway, thereby enhancing their antioxidant capacity and promoting wound healing. However, whether fish scale ointment also acts through additional signaling pathways requires further investigation.