Topical application of phycocyanin accelerates wound healing in STZ-induced diabetic rats via activation of the Nrf2 signaling pathway

Abstract

The present study aimed to evaluate the wound healing potential of phycocyanin (PC) in diabetic rats. STZ (55 mg/kg) was injected intraperitoneally in Wistar rats for the induction of diabetes. A sterile biopsy punch created a foot ulcer (5 mm). Diabetic rats were treated with topical PC (1.5% and 2.5%) for 2 weeks. Body weight and metabolic parameters were measured. Various biochemical estimations, including oxidative stress markers (LPO, PCO), antioxidant indices (GSH, SOD), collagen content, and anti-inflammatory markers (IL-1β, TNF-α, NF-κβ, IL-6), along with MMP-9, VEGF, Nrf2, and HO-1 levels were assessed in the wound tissue. MTT and in-vitro scratch assays were also performed. The results revealed that PC significantly increased wound closure and collagen content but failed to alter the STZ-induced increase in metabolic parameters. Furthermore, PC suppressed oxidative stress pro-inflammatory markers and MMP-9 levels and enhanced anti-oxidant defences, VEGF, Nrf2, and HO-1 levels in the wound tissue. In addition, in vitro studies demonstrated a significant increase in cell migratory activity in the wound-healing scratch assay. Collectively, this study suggests that PC accelerated wound healing by reducing oxidative stress and inflammation, possibly via upregulation of the Nrf2 signaling pathway, making it an effective approach for treating diabetic foot ulcers.

Introduction

Diabetes mellitus (DM) is a significant global health concern. According to the International Diabetes Federation (IDF) report in 2021, one in every 10 adults globally had diabetes, accounting for 537 million diabetics worldwide. Diabetic foot ulcer (DFU) is one of the most serious and prevalent complications of DM, contributing to a considerable global disability burden, and resulting in significant morbidity and mortality (Tan et al. 2019). According to epidemiological studies, the risk of developing foot ulcers is 2.5% per year and nearly 20% of the patients require hospitalization, resulting in lower limb amputation (Troisi et al. 2023). The etiology of DFUs is multifactorial, and the most common underlying cause includes poor glycemic control, which ultimately leads to neuropathy and peripheral vascular disease, resulting in lower limb infections and ulceration (Yang et al. 2023; Sa et al. 2024).

Wound healing represents a natural physiological reaction comprising hemostasis, inflammation, proliferation, and remodeling (Huang et al. 2023). It is described as a protracted inflammatory phase characterized by elevated levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. This causes higher tissue damage and delayed wound healing (Naseeb et al. 2023). Additionally, prolonged hyperglycemia is associated with an increase in reactive oxygen species (ROS), which leads to heightened oxidative stress and disrupts the balance between oxidative and antioxidative activities (Winarni et al. 2022). Moreover, an overabundance of ROS stimulates intracellular mediators, such as NF-κB, which leads to an increased release of inflammatory cytokines and a reduced activity of antioxidant enzymes (Cai et al. 2023). Nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2) is a transcription factor that regulates redox balance, anti-oxidant defense, inflammation, angiogenesis, and extracellular matrix (ECM) remodeling. It has been reported that Keap1 inactivates Nrf2 in the cytosol; however, when exposed to oxidative stress, Nrf2 is released and it translocates to the nucleus, forming a complex with Maf proteins, ultimately activating the antioxidant response elements (AREs), leading to an increase the production of antioxidative and cytoprotective proteins such as glutathione, heme oxygenase-1, and superoxide dismutase (Sun et al. 2020). Excessive hyperglycemia inhibits Nrf2-mediated antioxidant responses, thus leading to a prolonged inflammatory phase and hindering the normal wound-healing process in diabetic patients. Hyperglycemia reduces Nrf2 binding to Maf proteins and the production of antioxidant enzymes, increasing oxidative stress and inflammation, as well as decreasing cellular defensive mechanisms, resulting in an inability to heal wounds effectively (Yi et al. 2024). Traditional treatments for DFUs include glycemic control, infection management, surgical debridement, oxygen therapy, and various wound dressings. However, these treatments fail to address the cellular damage at the wound periphery or deliver active substances efficiently to the injured tissue site (Huang et al. 2023). Therefore, targeting oxidative stress and inflammation in the wound tissue may prove effective in accelerating wound closure in patients with DFUs.

Phycocyanin (PC; Fig. 1), a water-soluble, innate blue protein, is one of the phycobiliproteins extracted from cyanobacteria like Spirulina platensis (Fernandes et al. 2023). It has received considerable attention for its wide spectrum of pharmacological properties, such as antioxidant, anti-inflammatory, anti-diabetic, radical scavenging, hepatoprotective, anti-obesity, and immunity-boosting activities (Ziyaei et al. 2023). Oral PC has been demonstrated to exhibit anti-nociceptive effects in STZ-induced diabetic neuropathy in rats by inducing a significant reduction in pro-inflammatory cytokines (Abdel-Daim et al. 2020). Recent studies also suggest that PC enhances the proliferation and migration of human dermal fibroblast cell lines, improves wound re-epithelialization and vascularization, and promotes wound regeneration by improving fibroblast antioxidant mechanisms (Agustina et al. 2021). Moreover, studies have also revealed that PC activates the Nrf2 pathway thereby reducing oxidative stress and enhancing the activity of antioxidant enzymes in various pathological conditions (Liu et al. 2020; Althobaiti et al. 2024). Despite the evidence of PC’s therapeutic potential in different diseases, its role in diabetic-specific wound healing is largely unexplored. Therefore, considering the important pharmacological activities of PC, the current study evaluated the wound-healing potential of topical PC in STZ-induced diabetic foot ulcers in rats.

Fig. 1.

Chemical structure of Phycocyanin (PC)

Materials and methods

Experimental animals

Adult male Wistar rats (250–300 g) were obtained from the Central Animal House at Panjab University in Chandigarh. The rats were kept under conventional laboratory conditions: 25 ± 2 °C, 60–70% humidity, and a 12-h light/dark cycle. They had unlimited access to food and water provided ad libitum. The Institutional Animal Ethics Committee (IAEC) of Panjab University approved the experimental protocols (Approval No: PU/45/99/CPCSEA/IAEC/2023/830). All tests were carried out in accordance with the rules established by the Government of India's Committee for Control and Supervision of Experimentation on Animals (CCSEA).

Induction of diabetes

To induce diabetes, a single injection of streptozotocin (STZ; 55 mg/kg) dissolved in freshly made cold citrate buffer (0.1 M, pH 4.5) was administered intraperitoneally (Tombulturk et al. 2022; Dubey et al. 2023). Fasting blood glucose (FBG) levels were measured 72 h after STZ injection using a FreeStyle Optimum Neo digital glucometer (Abbott Diabetes Care Ltd., UK). Rats with FBG levels above 250 mg/dl were chosen for the experiment and monitored for an additional 7 days. FBG levels were tested on days 0 and 14.

Preparation of PC ointment

PC was gifted by EID Parry Nutraceuticals, India. The ointment was prepared at a concentration of 1.5% and 2.5% using a mixture of a 1:1 ratio of Lanolin and Vaseline (Sevimli-Gür et al. 2009). The foot ulcers were treated once daily with the topical application of PC ointment for 14 days.

Excision wound model and experimental design

Diabetic rats with a blood glucose level exceeding 250 mg/dl were used for wound induction. All rats were anesthetized through an intraperitoneal (IP) injection of ketamine hydrochloride (75 mg/kg) and xylazine (10 mg/kg). The skin surface of the footpad was shaved and then cleaned using a 70% ethanol wipe. Thereafter, a circular full-thickness wound was created on each rat's left foot, whose depth reached the fascia, using a 5 mm sterile biopsy punch (Sun et al. 2020). All animals received ketoprofen (5 mg/kg) for analgesia. Moreover, animals were closely observed for any infection, and those that showed signs of infection were replaced. To access the wound-healing property of PC ointment, the rats were grouped randomly into five groups (n = 8–10): Group I consisted of non-diabetic animals with a foot ulcer; Group II consisted of non-diabetic animals topically treated with 2.5% w/w PC ointment once daily for two weeks; Group III consisted of diabetic animals with a foot ulcer; Group IV & V consisted of diabetic animals topically treated with 1.5% and 2.5% w/w PC ointment once daily for two weeks (Sevimli-Gür et al. 2009). Throughout the study period of 14 days, the animals were carefully monitored for wound contraction. Upon completion of the treatment, rats were euthanized using cervical dislocation, and the wound tissues were collected for various biochemical and molecular estimations (Fig. 2).

Fig. 2.

Schematic representation of the experimental design. LPO lipid peroxidation, PCO protein carbonylation, MPO Myeloperoxidase, GSH reduced glutathione, SOD superoxide dismutase, CAT catalase, GPx glutathione peroxidase, TNF-α tumor necrosis factor alpha, IL-1β interleukin 1β, IL-6 interleukin 6, COX 2 cyclooxygenase-2, Nrf2 nuclear factor erythroid 2-related factor 2, HO-1 hemeoxygenase 1, VEGF vascular derived endothelial factor, MMP-9 matrix metalloproteinase-9, NF-κB Nuclear Factor kappa-light-chain-enhancer of activated B cells

Cell line-based assays

The L929 fibroblast cell line was obtained from the National Centre for Cell Science (NCCS) in Pune. The cell line was cultured and maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Himedia) with 10% Foetal bovine serum (FBS) (Himedia) and 1% antibiotic penicillin–streptomycin using a standard protocol and maintained in a CO₂ incubator. Once the cells attained confluency, it was trypsinized and utilized for the assays (Swathi et al. 2023).

Cell viability (MTT) assay

The cytotoxicity of PC on L929 cells was assessed using the MTT assay. MTT is a tetrazolium salt, and it undergoes enzymatic conversion into mysterious purple-tinted formazan crystals by the lactic acid dehydrogenase enzyme released by the mitochondria. L929 cells at a compactness of 2 × 10⁵ were seeded in a 96-well plate and cultured for 12 h at 5% CO2 and 37 °C (Li et al. 2022). After the cells adhere to the surface of the culture plate, the spent medium was replaced with Dulbecco’s medium, which contains various concentrations of PC (5, 25, 50, 100, and 200 μg/mL). The L929 cells were then incubated for 48 h in a CO2 incubator. Post incubation, cells were treated with 20 μL of 5 mg/mL MTT reagent and further incubated for 3 h at room temperature. The resulting formazan crystals were dissolved in 100 μL DMSO, and the absorbance was read at 580 nm in a microplate reader to reveal a purple color (Ren et al. 2020). The cell viability (%) was calculated using the formula:

$$\%\text{cell viability}=\text{Mean absorbance of test sample}/\text{Mean absorbance of negative control}\times 100.$$

In vitro wound healing (Scratch) assay

Each well of a 6-well tissue culture plate was seeded with L929 cells and these cells were incubated at 37 °C and 5% CO₂ for 24 h to allow cell adhesion and the formation of a confluent monolayer under DMEM high glucose media (4.5 g/L glucose) with different concentrations of PC (5, 25, 50, 100, 200 µg/ml) for 48 h (Ren et al. 2020). After incubation, the cell layer was scratched using a pipette tip, and the migration area was calculated at 0 and 24 h, and digitized images were captured with an inverted microscope (Nikon Eclipse TS2-Tokyo, Japan) (Zhou et al. 2022). The formula given below was used to calculate cell migration rates: Scratch wound area (%) = A_t/ A₀ × 100, where A_t The scratch area without migrating cells at different time points, and A₀: The scratch area at 0 h.

Wound closure

Percentage wound closure was calculated as the percentage of wounds that had been reduced from the original size of the wound on days 7 and 14. Wound surface areas were calculated and analyzed using Image J Software. Wound contraction was represented as a percentage of the healing wound area (Kant et al. 2014).

$$\%\text{Wound contraction}=\frac{\text{Wound area}\left(\text{day}0\right)-\text{Wound area}\left(\text{day 14}\right)}{\text{Wound area}\left(\text{day}0\right)}\times 100.$$

Biochemical estimations

After the treatment, rats were euthanized via cervical dislocation, and wound tissues were collected and homogenized in phosphate buffer (0.1 M, pH 7.4) followed by centrifugation at 12,000×g at 4 °C for 15 min. The supernatants obtained were subsequently utilized for assessing various parameters.

Protein estimation

The protein concentration was determined using the biuret method, with bovine serum albumin used as the standard (Gornall et al. 1949).

Hydroxyproline (HPR)

Collagen is the primary component of extracellular tissue, which provides essential structural support and strength. Hydroxyproline, a major part of collagen, is crucial for collagen stability (Egger et al. 2013). To analyze the content of hydroxyproline, tissues were dried in a hot air oven at 60–70 °C and then hydrolyzed in 6 N HCl at 120 °C for 4 h in sealed tubes. This was followed by oxidation with chloramine-T for 20 min. The reaction was stopped by the addition of 0.4 M perchloric acid and color development was achieved using Ehrlich reagent at 60 °C. The absorbance was then measured at 550 nm using a UV–visible spectrophotometer (Egger et al. 2013).

Hexosamine (HXA) and hexuronic acid (HUA)

To analyze the hexosamine fraction of wounded tissue, 0.05 mL of the sample was mixed with 0.5 ml of acetylacetone reagent and heated in a boiling water bath for 20 min, followed by cooling. Further, 1.5 mL of 95% alcohol was added, followed by 0.5 mL of Ehrlich’s reagent and kept for 30 min. To determine the hexuronic acid content, 0.125 mL of hydrolysate was diluted and carefully placed on the Borax-sulphuric acid mixture at 4 °C. The tubes were sealed and heated for 10 min in a furiously boiling water bath before cooling to room temperature. Then, 0.1 mL of 0.125% carbazole reagent in 100% alcohol was added to every tube. The color intensity was measured at 530 nm against a blank sample. The hexosamine and hexuronic acid content of the samples were determined using a standard curve prepared with D (+) glucosamine hydrochloride and D (+) Glucurono-6, 3-lactone (Murthy et al. 2013).

Lipid peroxidation (LPO)

Equal volumes (0.5 ml) of wound tissue homogenate and Tris–HCl were mixed and incubated at 37 °C for 2 h. After incubation, 1 ml of trichloroacetic acid (10%) was added, and the mixture was centrifuged at 1000×g for 10 min. Subsequently, 1 ml of 0.67% thiobarbituric acid was added to the mixture, which was heated in boiling water for 10 min. After cooling, 1 ml of distilled water was added. The absorbance was measured at 532 nm using a UV–visible spectrophotometer. Results were expressed as nmol of MDA/mg protein (Wills 1966).

Myeloperoxidase assay (MPO)

To measure MPO activity, 0.1 ml of wound tissue homogenate was mixed with 0.167 mg/ml o-dianisidine hydrochloride and 0.0005% hydrogen peroxide in 50 Mm of potassium phosphate buffer (pH 6). The change in absorbance was measured at 460 nm every 30 s for 4 min. MPO activity was expressed as units per gram of tissue (Bradley et al. 1982).

Protein carbonylation (PCO)

Wound tissue homogenate was mixed with 10 mM DNPH solution and vortexed. Subsequently, 20% TCA solution was added, and the mixture was incubated on ice for 15 min. After discarding the supernatants, the protein pellets were washed three times with a 1:1 mixture of ethanol and ethyl acetate by vortexing to remove any free DNPH. The protein pellets were then suspended in 1 ml of 6 M guanidine hydrochloride (dissolved in 50 mM phosphate buffer, pH 2.3) and incubated at 37 °C for 15–30 min, vortexing until fully dissolved. The carbonyl content was determined by measuring the absorbance at 366 nm (Colombo et al. 2016).

Superoxide dismutase (SOD) activity

A mixture of 0.1 mM EDTA, 96 mM nitroblue tetrazolium (NBT), and 50 mM sodium carbonate was prepared. An equal amount of hydroxylamine hydrochloride (pH 6.0) and tissue homogenate was then added to the mixture. The change in absorbance was observed at 560 nm at 30-s intervals for 2 min. The results were reported as SOD units per mg protein (Kono 1978).

Estimation of reduced glutathione levels (GSH)

Tissue homogenate was mixed with 5% sulphosalicylic acid and incubated, followed by centrifugation at 4500 × g for 10 min at 4 °C. To the resulting supernatant, 1.5 ml of 5,5. Dithiobis-(2-nitro benzoic acid) and 450 µl of phosphate buffer were added. The absorbance was recorded at 412 nm. Values were expressed as µM GSH per mg protein (Brehe and Burch 1976).

Enzyme-linked immunosorbent assay (ELISA) Estimation

TNF-α, IL-1β, and IL-6 (PeproTech, New Jersey, USA), Nrf2, HO-1, VEGF, MMP-9, and COX-2 (Elabscience Biotechnology, Inc, United States) levels were evaluated in the wound tissues of the animals using ELISA kits as per the manufacturer’s instructions and the absorbance value was recorded using a microplate reader at 450 nm.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Results were expressed as mean ± S.E.M. Biochemical and ELISA. One-way analysis of variance (ANOVA) followed by Tukey’s test was used to analyze biochemical and ELISA results. A p-value of < 0.05 was considered statistically significant.

Results

Effect of PC on % cell viability as assessed by the MTT assay

The effect of different concentrations of PC (5 − 200 μg/mL) on cell viability was assessed using an MTT assay (Fig. 3). As seen in the graph, the exposure of L929 cells to concentrations up to 200 µg/ml did not show noticeable toxicity as the cell viability remained above 80%.

Fig. 3.

Effect of PC on % cell viability. Cell viability is expressed as a percentage of untreated cells. Results are depicted as mean ± SEM of triplicate

Effect of PC on cell migration (scratch) assay

Cell migration is crucial for wound repair; thus, the scratch assay was carried out to observe the healing process. Following a 24-h exposure to PC, cells migrated towards the scratch area. The control group did not receive PC treatment and exhibited the least cell migration (Fig. 4). In contrast, the treatment group exposed to the highest concentration of PC (200 µg/ml) demonstrated the maximum increase in wound closure compared to the control group. This indicates a dose-dependent effect of PC on cell migration and wound repair. The significant enhancement in wound healing observed in the 200 µg/ml PC treatment group underscores the potential therapeutic benefit of PC in promoting cell migration and accelerating tissue repair.

Fig. 4.

Digital representation of the cell migration ability of L929 cells cultured with different concentrations of PC at 0 h and 24 h

Effect of PC on body weight, blood glucose, food intake, water intake, and urine excretion in diabetic rats

As shown in Table 1, diabetic animals exhibited a significant decrease [F (4, 25) = 4.556, p < 0.05] in body weight, while a significant increase in blood glucose [F (4, 25) = 259.2, p < 0.0001], food intake [F (4, 25) = 15.07, p < 0.0001], water intake [F (4, 25) = 78.87, p < 0.0001], and urine excretion [F (4, 25) = 106.9, p < 0.0001] as compared to the control group. However, treatment with PC (1.5% and 2.5%) failed to produce any statistically significant difference between the groups.

Table 1.

Data values are expressed as mean ± S.E.M and analyzed using one-way ANOVA following Tukey’s multiple comparison test (n = 6)

GROUPS	Blood glucose (mg/dl)	Body weight (g)	Food intake (g/rat/day)	Water intake (ml/rat/day)	Urine excretion (ml)
CONTROL	90.83 ± 6.93	265.83 ± 3.96	28.75 ± 2.21	54.16 ± 3.96	19.66 ± 2.45
PC (2.5%)	93 ± 6.65	269.16 ± 5.97	30.28 ± 2.71	56.33 ± 3.22	21.83 ± 2.94
STZ	416.5 ± 12.79*	241.67 ± 3.8*	48.98 ± 2.24*	123.66 ± 3.13*	95.83 ± 3.98*
STZ + PC (1.5%)	400.3 ± 8.49	247.5 ± 5.59	45.86 ± 2.49	113.83 ± 5.03	87.5 ± 4.28
STZ + PC (2.5%)	392.5 ± 12.83	254.16 ± 6.76	42.56 ± 3.05	109.5 ± 4.46	85 ± 3.22

Statistical significance is represented as *p < 0.05 as compared to the control group

Effect of PC on the wound closure percentage in diabetic rats

The representative images of wound closure at days 0 and 14 are shown in Fig. 5a. Moreover, the % wound closure [F (4, 15) = 56.72, p < 0.0001, Fig. 5b] was significantly delayed in STZ-induced diabetic rats when compared to the non-diabetic control rats. However, topical application of PC (1.5% and 2.5%) significantly improved % wound closure on day 14 when compared to the non-treated diabetic group (p < 0.0001).

Fig. 5.

a Digital representation of wound contraction on different days. b Effect of PC on the wound closure percentage in diabetic rats. Data values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA following Tukey’s multiple comparison test; (n = 4). Statistical significance is represented as *p < 0.05 as compared to the control group, ^#p < 0.05 as compared to the STZ group, ^αp < 0.05 as compared to the STZ + PC (1.5%) group. STZ Streptozotocin, PC Phycocyanin

Effect of PC on HP, HUA, and HXA levels in the wound tissue of diabetic rats

The collagen determinants were estimated in wounded tissue homogenate in rats. The hydroxyproline concentration directly measures collagen content, while HUA and HXA are matrix molecules to stabilize the collagen fibers. The hydroxyproline content [F (4, 25) = 34.63, p < 0.0001, Fig. 6a], HUA [F (4, 25) = 50.02, p < 0.0001, Fig. 6b), and HXA [F (4, 25) = 24.64, p < 0.0001, Fig. 6c] in wound healing tissue taken from DM rats was significantly decreased compared to that of control rats. Moreover, the results showed that PC (2.5%) treatment led to a significant increase in hydroxyproline, HUA, and HXA levels (p < 0.0001), but PC (1.5%) treatment failed to increase HXA levels when compared to the STZ group.

Fig. 6.

Effect of PC on a Hydroxyproline, b Hexuronic acid (HUA), and c Hexosamine (HXA) levels in the wound tissue of diabetic rats. Data values are expressed as mean ± S.E.M. and analysed using one-way ANOVA following Tukey’s multiple comparison test; (n = 6). Statistical significance is represented as *p < 0.05 as compared to the control group, ^#p < 0.05 as compared to the STZ group, ^αp < 0.05 as compared to the STZ + PC (1.5%) group. STZ Streptozotocin, PC Phycocyanin

Effect of PC on oxidative stress markers and antioxidant profile in the wound tissue of diabetic rats

As shown in Table 2, rats injected with STZ showed a significant increase in MDA [F (4, 25) = 14.62, p < 0.0001], PCO [F (4, 25) = 10.86, p < 0.0001], and MPO [F (4, 25) = 16.25, p < 0.0001] levels in wound tissue homogenate when compared to the control group. However, the results showed a significant reduction in all MDA (p < 0.01, p < 0.001) and MPO (p < 0.01, p < 0.0001) levels after topical application of PC (1.5% and 2.5%) compared to the STZ group. Also, PC (2.5%) enhanced PCO levels in the wound tissue (p < 0.05). Furthermore, diabetic rats also demonstrated a significant reduction in SOD [F (4, 25) = 19.22, p < 0.0001], and GSH [F (4, 25) = 25.64, p < 0.0001] in the wound tissues. Nevertheless, treatment with a higher dose of PC (2.5%) showed significant improvement in SOD (p < 0.01) and GSH (p < 0.001) levels.

Table 2.

Data values are expressed as mean ± S.E.M and analysed using one-way ANOVA following Tukey’s multiple comparison test n = 6

Groups	MDA (nmol/mg protein)	MPO(units/mg protein)	PCO (nmol/mg protein)	SOD (units/mg protein)	GSH (µmol/mg protein)
Control	0.94 ± 0.17	11.07 ± 2.06	11.73 ± 2.57	73.75 ± 4.54	49.18 ± 4.25
PC (2.5%)	0.65 ± 0.16	15.89 ± 5.21	18.19 ± 2.74	82.57 ± 4.87	54.35 ± 4.24
STZ	2.08 ± 0.13*	44.0 ± 4.62*	41.98 ± 3.91*	26.61 ± 5.66 *	12.52 ± 3.6*
STZ + PC (1.5%)	1.29 ± 0.14 #	23.38 ± 4 #	34.44 ± 5.37	38.80 ± 4.75	19.72 ± 3.17
STZ + PC (2.5%)	1.10 ± 0.13 #	20.96 ± 5.08#	25.50 ± 3.10#	57.62 ± 6.54 #	29.51 ± 3.28#

Statistical significance is represented as *p < 0.05 as compared to the Control group, ^#p < 0.001 as compared to the STZ group

Effect of PC on TNF-α, IL-1β, and IL-6 in the wound tissue of diabetic rats

The preventive effects of PC on inflammatory cytokines were investigated. Diabetic rats had significantly higher levels of TNF-α [F (4, 15) = 58.43, p < 0.0001, Fig. 7a], IL-1β [F (4, 15) = 9.945, p < 0.001, Fig. 7b], and IL-6 [F (4, 15) = 25.80, p < 0.0001, Fig. 7c] in wound tissue compared to the control group. Treatment with PC (2.5%) significantly lowered the levels of TNF-α, IL-1β, and IL-6, while PC (1.5%) significantly decreased the levels of IL-6 (p < 0.05) and TNF-α (p < 0.001) in diabetic rats.

Fig. 7.

Effect of PC on a TNF-α, b IL-1β, c IL-6 in the wound tissue of diabetic rats. Data values are expressed as mean ± S.E.M. and analysed using one-way ANOVA following Tukey’s multiple comparison test; (n = 4). Statistical significance is represented as *p < 0.05 as compared to the control group, ^#p < 0.05 as compared to the STZ group, ^αp < 0.05 as compared to the STZ + PC (1.5%) group. STZ Streptozotocin, PC Phycocyanin

Effect of PC on NF-κβ and COX-2 in the wound tissue of diabetic rats

As shown in Fig. 8a, b, STZ-injected rats showed significantly higher levels of NF-κβ [F (4, 15) = 42.21, p < 0.0001] and COX-2 [F (4, 15) = 22.20, p < 0.0001] activity as compared to the control group. However, topical application of PC dose-dependently reduced COX-2 activity, whereas PC (2.5%) was able to significantly lower elevated NF-κβ (p < 0.0001) levels.

Fig. 8.

Effect of PC on a NF-κβ levels and b COX-2 activity in the wound tissue of diabetic rats. Data values are expressed as mean ± S.E.M and analyzed using one-way ANOVA following Tukey’s multiple comparison test; (n = 4). Statistical significance is represented as *p < 0.05 as compared to the control group, ^#p < 0.05 as compared to the STZ group. ^αp < 0.05 as compared to the STZ + PC (1.5%) group. STZ streptozotocin, PC phycocyanin

Effect of PC on VEGF and MMP-9 in the wound tissue of diabetic rats

As depicted in Fig. 9a, b, the levels of VEGF [F (4, 15) = 25.51, p < 0.0001] were significantly decreased and MMP-9 [F (4, 15) = 110.1, p < 0.0001] were markedly elevated in STZ-induced diabetic rats when compared to the control group. However, the results of this investigation showed that topical application of PC (2.5%) significantly restored the levels of VEGF (p < 0.001). Moreover, PC application dose-dependently attenuated MMP-9 activity.

Fig. 9.

Effect of PC on a VEGF, and b MMP-9 in the wound tissue of diabetic rats. Data values are expressed as mean ± S.E.M. and analysed using one-way ANOVA following Tukey’s multiple comparison test; (n = 4). Statistical significance is represented as *p < 0.05 as compared to the control group, ^#p < 0.05 as compared to the STZ group, ^αp < 0.05 as compared to the STZ + PC (1.5%) group. STZ streptozotocin, PC phycocyanin

Effect of PC on Nrf2 and HO-1 in the wound tissue of diabetic rats

Since Nrf2 is known to play a critical role in maintaining redox homeostasis by recognizing antioxidant response elements and subsequently activating antioxidant genes, the Nrf2 level and its downstream protein HO-1 were determined accordingly. In the current study, a substantial decrease in the levels of Nrf2 [F (4, 15) = 54.92, p < 0.0001, Fig. 10a] and HO-1 [F (4, 15) = 21.83, p < 0.0001, Fig. 10b] levels was observed in the diabetes group when compared to the control group. In contrast, PC (2.5%) significantly increased the levels of both Nrf2 (p < 0.0001) and HO-1 (p < 0.001). Additionally, reduced Nrf2 levels in the STZ group were also significantly enhanced on the application of PC (1.5%).

Fig. 10.

Effect of PC on a Nrf2 and b HO-1 levels in the wound tissue of diabetic rats. Data values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA following Tukey’s multiple comparison test; (n = 4). Statistical significance is represented as *p < 0.05 as compared to the control group, ^#p < 0.05 as compared to the STZ group, ^αp < 0.05 as compared to the STZ + PC (1.5%) group. STZ streptozotocin, PC phycocyanin

Discussion

The management of wound healing in diabetic patients remains a major challenge that needs to be addressed. Recently, several natural compounds have been shown to possess immense potential in managing diabetes and its complications (Singh et al. 2022). PC has been shown to prevent diabetic complications by inhibiting free radical generation, glucose auto-oxidation, and protein glycation, thereby reducing AGEs formation (Nikolic et al. 2023). However, its role in DFU is still unexplored. Thus, the current investigation explored the protective effects of topical PC in diabetic wound healing in rats.

Streptozotocin (STZ) is commonly employed to induce diabetes in rodents by destroying pancreatic cell DNA, ultimately leading to a substantial reduction in insulin production (Prajapati et al. 2025; Rai et al. 2022). In the current study, in line with previous studies, a single dose of STZ (55 mg/kg) was used to induce diabetes, which significantly elevated the blood glucose levels in rats and also decreased body weight, increased food intake, urine excretion, and water intake (Ali et al. 2023). While PC has been reported to have a hypoglycemic effect in some studies, similar results were not obtained here due to the usage of a topical application, which did not produce systemic effects (El-Sayed et al. 2018). Also, PC led to an increase in body weight, but the results were not statistically significant. Moreover, there was no change in the metabolic parameters, including water intake, food intake, and urine excretion.

Fibroblast cells play a significant role in the formation of granulation tissue, extracellular matrix, and the synthesis of collagen, which collectively aid in wound closure (Narisepalli et al. 2023). To evaluate the wound healing property of PC, we employed the scratch assay, a robust and widely utilized in vitro technique used to evaluate cellular migration and proliferation during the wound healing process. Treatment with PC promoted cell migration, as the percentage of wound closure significantly increased after 24 h. Indeed, in an earlier study, PC has been shown to enhance the proliferation and migration of human dermal fibroblast cell lines, improving wound re-epithelialization and vascularization and promoting wound regeneration by improving fibroblast antioxidant mechanisms. Furthermore, STZ-induced diabetic rats also showed a significant decrease in the levels of hydroxyproline as compared to the control group. Collagen is a crucial component of the extracellular matrix (ECM), which plays a major role in the process of wound contraction (Nasiry et al. 2022; Nakhate et al. 2023). Hydroxyproline, an amino acid released during collagen decomposition, serves as a gold standard for routine measurement of collagen content (Andjić et al. 2022). However, topical application of PC led to a significant increase in the levels of hydroxyproline, suggesting accelerated wound healing due to enhanced collagen synthesis and tissue strength. Hexosamine and hexuronic acid are other important matrix molecules that play a vital role in wound healing (Preet et al. 2022). Decreased levels of these matrix molecules were observed in untreated diabetic wounds, indicating impaired wound healing, whereas post-treatment with PC led to a significant increase in their levels. The increase in collagen synthesis and matrix molecules may be attributed to a reduction in oxidative stress and inflammation, stimulation of fibroblast activity, modulation of growth factors, and improvement of glycemic control.

In the present study, the levels of IL-1β, IL-6, and TNF-α were increased significantly. Persistent and systemic inflammation is a major cause of non-healing diabetic ulcers (Zhang et al. 2022). Elevated blood glucose levels in diabetic foot ulcers trigger the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), which recruit immune cells to the wound site. Hyperglycemia impairs the function of immune cells, such as macrophages and neutrophils, compromising their ability to effectively clear pathogens and debris, thereby prolonging the inflammatory phase and delaying the wound-healing process (Chen et al. 2022). Also, prolonged hyperglycemia contributes to diabetic vascular complications by inducing oxidative stress and the formation of advanced glycation end products (AGEs), which activate NF-kB in vascular cells. Additionally, NF-κB activation upregulates the COX-2 enzyme, promoting inflammation and vascular dysfunction (Suryavanshi and Kulkarni 2017). Thus, the mechanism underlying the anti-inflammatory activity of PC may be attributed to its ability to suppress NF-κβ levels, a critical regulator of inflammation. Moreover, PC application also inhibited the cyclooxygenase-2 (COX-2) enzyme, which is responsible for the synthesis of pro-inflammatory prostaglandins. Interestingly, our results were in line with an earlier study that suggested the anti-inflammatory activity of Spirulina in STZ-induced diabetic nephropathy through the suppression of NF-κβ signaling cascade (Althobaiti et al. 2024). Moreover, the beneficial role of PC in diabetic wound healing could also be attributed to its multi-faceted anti-inflammatory actions, which include the downregulation of HMGB1, a key pro-inflammatory mediator, and inhibition of the NLRP3/NF-κB pathway, thereby reducing the levels of pro-inflammatory cytokines IL-1β and TNF-α (Alzokaky et al. 2020).

In the present study, an increase in LPO and PCO along with a decrease in SOD activity and GSH levels was observed in the diabetic wounds compared to normal wounds. In diabetic conditions, hyperglycemia leads to excessive ROS production through various molecular pathways such as polyol, glycolytic, hexosamine, protein kinase C, and advanced glycation end-product (AGE) pathways (González et al. 2023). This elevated ROS results in high levels of oxidative stress, which was evident in the current study. Interestingly, PC application attenuated these alterations by decreasing the levels of PCO & LPO and boosting SOD activity and GSH levels. These changes could be attributed to the activation of the Nrf2 signaling pathway. The Nrf2 pathway, a crucial regulator of cellular antioxidant defenses, plays an important role in diabetic foot ulcers. In response to oxidative stress, Nrf2 is activated and translocates to the nucleus, where it binds to antioxidant response elements (AREs), triggering the transcription of genes that encode various antioxidant enzymes and detoxifying proteins (Saha et al. 2020). In diabetic foot ulcers, deregulation of the Nrf2 pathway impairs antioxidant defenses, limiting the wound healing process and worsening the condition, whereas Nrf2 signaling activation is related to rapid wound healing via an increase in cell migration, especially in diabetic settings, making it a promising target for diabetic wound treatment. In the current study, the levels of myeloperoxidase (MPO) were significantly high in the non-treated diabetic rats, indicating increased neutrophil infiltration and inflammation. However, PC significantly reduced MPO levels, suggesting a reduction in neutrophil-driven oxidative damage and inflammation. The mechanism behind this effect may involve PC's ability to inhibit the activation of the NF-κB pathway, which is responsible for upregulating inflammatory mediators, including MPO. By reducing MPO levels, PC not only diminishes oxidative stress but also helps in modulating the inflammatory response, further contributing to accelerated wound healing in diabetic conditions.

The MMPs are a family of structurally related zinc endopeptidases that play an important role in initial wound debridement and epithelialization, angiogenesis, and extracellular matrix (ECM) remodeling (Patel et al. 2019). Enhanced activity of MMPs or imbalance between MMPs and their inhibitors results in ECM degradation and attracts more inflammatory cells into the site, hence worsening the chronic inflammatory conditions in the wound bed. In particular, MMP-9 degrades the specific biomarkers, i.e., VEGF, and makes them nonfunctional (Al-Romaima et al. 2022). Interestingly, studies have revealed that the MMP-9 levels in chronic wound fluid are almost 60 times higher than in acute wounds (Gary Sibbald and Woo 2008). MMP-2 and MMP-9 mRNA and protein levels are known to increase in diabetic wounds. This increase in protease activity destroys tissue and inhibits normal repair processes. In line with these findings, the results of this study showed significant upregulation in the MMP-9 levels in diabetic wounds. However, PC treatment significantly inhibited MMP-9 in diabetic rats. As inflammatory cytokines such as TNF-α and IL-1β are known to be key mediators in MMP-9 production, the anti-inflammatory properties of PC may be involved in the inhibition of MMP-9 expression.

VEGF is one of the key regulators of angiogenesis. In diabetic rats, a fundamental cause of the retardation of the diabetic wound-healing process is a reduction in VEGF expression at the wound site (Wang et al. 2018). Hypoxia-inducible factor 1-alpha (HIF-1α) is a transcription factor that regulates the expression of various genes, such as VEGF, which is involved in angiogenesis. In diabetic conditions, the activity of HIF-1α is often impaired due to chronic hyperglycemia, oxidative stress, and inflammation (Hwang et al. 2023). This impairment eventually leads to reduced VEGF expression, resulting in poor angiogenesis and delayed wound healing (Zhu et al. 2020). The current study clearly shows significantly decreased levels of VEGF in diabetic rats, which is regarded to be a primary cause of insufficient angiogenesis (Al-Romaima et al. 2022). Furthermore, the PC application boosted VEGF levels, efficiently increasing wound angiogenesis and collagen deposition. Our finding corroborates with an earlier study that demonstrated that PC modulated HIF-1α activity, which in turn regulated the expression of VEGF, thus enhancing angiogenesis and accelerating the wound-healing process (Braune et al. 2021).

The study had a few limitations that should be acknowledged. These include using only the STZ model to induce DM and not utilizing other models of wound healing for in-depth exploration. Also, lack of human tissue validation, smaller sample size, lack of suitable positive control, and inability to assess the wound tensile strength were other limitations of this study. PC was administered for a short period, and as such, it may have overlooked the long-term benefits or possible negative effects associated with continued usage. As a result, further studies are warranted, which will not only address these limitations but also demonstrate dose–response optimization, usage of mechanistic inhibitors of Nrf2, and provide head-to-head comparisons with clinically approved interventions, which will provide a better understanding of the therapeutic potential of PC in the treatment of DFUs.

Conclusion

In the current study, PC treatment drastically reduced the expression of inflammatory markers such as TNF-α, NF-κβ, MMP-9, and IL-1β while increasing VEGF expression and suppressing hyperglycemia-induced oxidative stress and inflammation through Nrf2 activation and NF-κβ suppression. This led to a marked improvement in wound healing in diabetic rats, suggesting the potential of PC as a promising therapeutic agent in the treatment of DFUs.

Funding

This experimental work did not receive any specific grant from any funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

Data will be made available on request.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval:

Compliance with ethical standards.