Introduction

Skin is the first line of the body’s defense against environmental challenges. Its integrity plays a vital role in immunologic surveillance, direct protection against damage, and body homeostasis (Sorg et al. 2017; Kankam et al. 2022). Burn wounds are a worldwide issue. They cause 11 million injuries and around 180000 deaths annually (World Health Organization 2023). Skin burns have dreadful consequences, leading to several long-term pathological and emotional issues, such as higher risks of skin infection, cancers, and aesthetic impairments (Islam et al. 2022). Wound repair is a complex and delicate process that involves several stages. Burn wound cure requires reconstruction and substitution of damaged tissue; therefore, burn wounds are considered more complex and vulnerable than other wounds (Stubbe et al. 2019; Huang et al. 2022). Effective treatment of burn injuries is essential to minimize morbidity and mortality. Conventional therapies used for burn wound therapy are associated with unexpected side effects, allergies, low efficacy, and safety concerns (Pereira and Bártolo 2016). Therefore, there is an increasing interest in introducing new biocompatible natural alternatives for the treatment of burn injuries.

Microalgae are considered as promising sources of natural products, as they are easy to cultivate, have rapid growth, are rich in bioactive substances, and many can tolerate a broad range of salinity, pH, and temperature (Borowitzka 2018; Yousuf 2020). Microalgae is a term used to refer to a polyphyletic group of primarily unicellular oxygen-producing eukaryotes (microalgae) or prokaryotes (cyanobacteria). The bioactivity of cyanobacterial extracts for skin protection and regeneration has been reported in several studies (Tseng et al. 2021; Foo et al. 2023). The underlying mechanisms, however, have yet to be fully understood. Polysaccharides from cyanobacteria have recently attracted attention due to their anti-allergenic, anti-inflammatory, moisture-retention, UV-protection, and wound-healing properties (Demay et al. 2020; Costa et al. 2021; Tseng et al. 2021; Foo et al. 2023).

Among diverse extremophile cyanobacteria, some halophilic Cyanothece species were isolated from hypersaline marine and estuarine environments. Cyanothece strains are capable of fixing nitrogen, a process that is essential for the survival of many organisms (Fujita and Uesaka 2022; Scoglio et al. 2024). They thrive in extreme environments where they must constantly cope with a variety of stressors such as high salinity, UV radiation, a wide range of temperatures, and even extreme desiccation. Therefore, over the past 3 billion years, they have developed different physical and chemical mechanisms to survive various environmental conditions (Rosic 2021). Many of these adaptations are associated with polysaccharides and lipids of their supporting matrix.

The present study was designed to investigate the burn wound-healing properties of polysaccharides and fatty acids extracted from Cyanothece sp. isolated from a hypersaline lagoon (Lipar, Iran) in the rat model.

Material and methods

2.1. Cyanobacterial biomass

The halophilic unicellular cyanobacterium Cyanothece sp. was originally isolated from the back-barrier waters of hypersaline Lipar lagoon (25° 15′ 20″ N, 60° 49′ 43″ E ) located on the Chabahar, Gulf of Oman, Iran as previously described (Aminikhoei et al. 2022). The axenic colonies of Cyanothece sp. were grown in 2-L bioreactors using f/2 medium (Guillard and Ryther 1962) at 21°C, at a salinity of 90 ppt under constant aeration. The algae were grown under a 12h:12h light:dark cycle at approximately 80 µmol photons m-2 s-1 irradiance. The cells were harvested by centrifugation at 12000 ×g for 10 min and lyophilized prior to extraction.

Polysaccharide extraction

Dried algal biomass was ground to powder, then treated with 95% ethanol (1:10 g mL-1) with constant stirring (1000 rpm) for 2 h at 60°C. The suspension was centrifuged at 3000 ×g, the supernatant was removed, and the precipitate was washed with ethanol and acetone and then dried at room temperature. About 5 g of depigmented algal powder was used for ultrasound-assisted extraction. For this, the powder was suspended in a beaker containing distilled water (1:50 solid-to-water ratio) at pH=7 and ultrasonicated using a Bandelin HD 4200 (Germany) at ultrasonic power of 180 W, 66°C temperature and frequency of 50 Hz for 40 min. Following centrifugation at 9000 ×g for 10 min, the supernatant was collected, filtered, and concentrated on a rotary evaporator (IKARV10 Digital) to 60-70 mL of volume. Cold ethanol (-20°C) was added to the extraction (3:1) and precipitated polysaccharides were washed using ethanol and acetone, and finally dried at room temperature under a laminar flow hood.

2.2. Lipid extraction

The total lipid content of the algae was extracted and determined according to the protocol of Bligh and Dyer (1959) with some modifications. In brief, 50 g of dried algal powder was macerated in 1000 mL of chloroform and methanol (2:1 v:v) overnight. Solid residues were discarded by centrifuging at 12000 ×g for 10 min, and 500 mL of 4.5% NaCl solution was added to the supernatant and placed in a separating funnel. The saline phase was discarded and the solvent phase was rotary evaporated.

Experimental setup

The obtained polysaccharide and lipid complexes were weighed, and each was used to create 5% w/w Eucerin-based ointment. White Wistar male rats (n=40) weighing 300 and 350 g (average age: 3-4 months) were used to study the wound healing effects of polysaccharide and lipid complexes extracted from Cyanothece sp. The rats were housed in the animal laboratory of Bushehr University of Medical Sciences under an ambient temperature of 20-25°C, a humidity of 65-75%, and a 12 h dark/light cycle. The rats were allowed to free-feed on the standard commercial food pellets and provided with tap water during seven days of acclimatization and the experimental period. All experiments were conducted in compliance with the ethical principles for animal experiments established by the International Council for Animal Protection and were carried out under the supervision of the animal ethics committee at Bushehr University of Medical Sciences (Ethics committee code: IR.BPUMS.REC.1400.182).

Rats were randomly divided into four equal groups, each group consisting of ten rats: 1-Control group served as the negative control group, treated with Eucerin (Merck Co., Germany), 2-Standard group, served as the positive control group, treated with topical Alpha® Ointment (Fundermol, Iran), 3- Lipid group, treated with Eucerin-based microalgal lipid ointment (5%), and 4- The polysaccharide group treated with Eucerin-based microalgal polysaccharide (5%).

The dorsal fur of the rats was shaved with an electric hair clipper and disinfected with polyvinylpyrrolidone iodine. The wounds were induced after rats were intramuscularly injected with the mixture of 10% ketamine (50 mg kg-1 BW) and xylazine (10 mg kg-1 BW) for general anesthesia. Second-degree burn wounds measuring one square centimeter were induced on the left side of the rat’s back using an electric hot plaque (1 cm2) based on the procedure described by Farzadinia et al. (2016).

The surface of the induced wounds was treated topically with the relevant ointments twice a day in the first week at 12-hour intervals, once per day in the second week, once every two days in the third week, and without treatment in the fourth week of treatments. Wound healing percentage and other morphological evaluations were performed by photography on days 1, 7, 14, and 21. Tissue samples were obtained from rats (2 rats from each group) under general anesthesia on days 1, 7, 14, and 21. The biopsy samples (2-3 mm) were taken from the edges of wounded tissues using a biopsy puncher.

Histopathological evaluation

Histological examinations were performed at 7-day intervals throughout the experiments. The samples were fixed in 10% formalin, dehydrated with ethanol, and embedded in paraffin, then 5 μm microtome sections were prepared. The slides were stained with hematoxylin-eosin. Masson’s trichrome staining (Suvik and Effendy 2012) was used to better evaluate the collagen fibers during the healing period. The slides were photographed using a light microscope equipped with a Moticam Pro. digital camera. Quantitative calculations such as fibroblast cell counts, blood vessels, determining the area of the wound surface, necrotic tissues, and the thickness of the epidermis were performed in photomicrographs with the aid of online ImageTool (IT) program version 3.0 (developed by Willcox and coworkers at the University of Texas Health Science Center, San Antonio, Texas, USA). Morphological studies were also carried out to assess the appearance of the wound, evaluate the secretions, and compare the scar tissues on different days. Wound surface area was calculated by using the following equation:

$$\text{Percentage of wound area}=\left({\text{A}}_{0}-{\text{A}}_{\text{x}}/{\text{A}}_{0}\right)\times 100$$
(1)

in which A0 and Ax represent the wound area on the first day and the Xth day after burn wound induction.

To assess leukocyte counts both within experiments and across different treatments, a Sysmex cell counter (Sysmex K-1000 Hematology Analyzer, Japan) was utilized.

The histological evaluation of wound healing was carried out based on clinical criteria described in Table 1 at 5-day intervals (Keast et al. 2004; Farzadinia et al. 2016).

Table 1 Histological scoring of wound healing
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Biochemical assessment

Blood was collected from the animal's tails. Centrifuging at 1000 ×g for 29 min was used to collect the plasma from the blood samples. The levels of C-reactive protein, CRP (Biovender, USA), and inflammatory cytokines of TNFα (Biocompare, USA) and interleukins (IL)-2, 6, and 8 (BD Bioscience, USA), adrenocorticotropic hormone (ACTH), cortisol, testosterone, and tri-iodothyronine (T3) (Elabscience Biotechnology, China) were measured by quantitative ELISA methods according to the manufacturer’s guidelines.

Identification of cyanobacterial fatty acids constituents

The analysis of fatty acid components of the lipid extracts was carried out using a GC system (Varian CP-3800) equipped with a flame ionization detector (FID SGE Bpx, Australia) under the following conditions: capillary column (30 m×0.22 mm; film thickness 0.25 μm); carrier gas He at a pressure of 25 bar, detector temperature 250°C, injector temperature 270°C, temperature program: 125 °C hold for 30 s, 150 °C at 25 °C min-1 for 2 min, then 200 °C at 25 °C min-1 for 90 min. Before injection, methyl esterification of the fatty acids was performed based on the method of Ichihara et al. (1996).

Statistical analysis

Statistical analysis of quantitative data was carried out by using SPSS software (IBM Corp., 2013). Data normality was examined by running the Kolmogorov-Smirnov test and Levene's test to assess variances' homogeneity. The comparison of mean values was evaluated by parametric two-way ANOVA and Duncan test. In addition, qualitative data were analyzed using the Kruskal-Wallis method and the Mann-Whitney test.

Results

Figure 1 depicts the observed variations in the burn wound closure of the treated rats. Morphological results showed that on the seventh day after burn induction, the amount of secretions was higher in the control rats, with more purulent discharges and a greater extent of the wound and necrotic tissues compared to the treated groups. Furthermore, the transparent reddish appearance of wounds in the treated animals indicated partial wound healing. On the 14th day (Fig. 1), the color, extent, and appearance of the wound indicated a relatively healing phase, and the wound closure was significantly accelerated (p< 0.05) in the three treatment groups, especially in microalgal lipid and polysaccharide-treated groups compared to the control group. Recovered skin tissue was observed in all experimented animals on day 21; however, more improvement in microalgal extract treatments was evident, especially in lipid ointment-treated animals, where the wounds were fully closed. In addition, based on the wound healing criteria (Table 1) all examined rats showed significant healing scores in contrast to the control group (Fig. 4). Furthermore, the lipid ointment group on days 5 and 10 of burn, indicated significantly higher recovery scores compared to the standard Alpha-treated group.

Fig.1
figure 1

Skin photographs of burn-induced wound (arrows show the wound margins) in different experimental groups: (A) control group, (B): standard Alpha ointment, (C) Cyanothece sp. polysaccharide ointment, and (D) Cyanothece sp. lipid ointment in the rat model

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Microscopic (histopathological) evaluation of the wound showed a perfect epithelial layer with a completely normal tissue structure in all groups on the first day (Fig. 2). On the seventh day of wound induction, no clear epithelium was observed in the burnt skin tissue of all tested animals (Fig. 2). The epithelialization was not confirmed in the control group on the 14th day (Fig. 2), whereas, in other treated rats, the epithelium was relatively healed. Animals treated with lipids and polysaccharides showed normal histology of the dermis and thick collagen bundles (Fig. 3). On the contrary, in the control group, retarded healing, the lack of collagen maturation, and the absence of normal structure was evident (Fig 4). The results of the H&E-stained slides examination revealed regeneration of the epithelial layer and complete remodeling of the dermis on the 21st day (Fig. 2) with significantly increased epithelial thickness in microalgal ointment treatments (p<0.05).

Fig. 2
figure 2

Skin photomicrographs (hematoxylin and eosin staining; ×100) of burn-induced wound in different experimental groups(epithelium are shown by arrows) : (A) control group, (B): standard Alpha ointment, (C) Cyanothece sp. polysaccharide ointment, and (D) Cyanothece sp. lipid ointment in the rat model

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Fig. 3
figure 3

Skin photomicrographs (Masson’s trichrome staining, ×100) of burn-induced wound in different experimental groups: (A) control group, (B): standard Alpha ointment, (C) Cyanothece sp. polysaccharide ointment, and (D) Cyanothece sp. lipid ointment in the rat model

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Fig. 4
figure 4

Evaluation of wound healing activities of Cyanothece sp. extracted lipids and polysaccharides on second-degree burns in rats. The wound-healing scoring followed the method described by Keast et al. (2004) and Farzadinia et al. (2016). Data are expressed as mean ± SE (n = 3). *Significant difference compared with the control group (negative control). ** Significant difference compared with the standard group (standard control) (p<0.05)

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The results of measured inflammatory markers are presented in Table 3. The findings revealed a significant reduction of TNFα, IL-2, and IL-8 in all post-treatment groups compared to the control group (p<0.05) at day 7. Furthermore, a significantly greater decrease in pro-inflammatory cytokines resulted from microalgal ointment treatment compared to the standard Alpha ointment treatment, indicating a higher anti-inflammatory effect of microalgal lipid and polysaccharide treatments. No statistically significant variation was noticed between IL-6 levels of different treatments.

C-reactive protein (CRP) measurements and leukocyte counts are routinely evaluated as clinical markers of inflammation. Here, on the seventh day of measurement of these markers, we noticed that CRP levels and monocyte counts were significantly higher (p>0.05) in the control group than in the other treated groups (Table 4). This indicates that all tested ointments (standard Alpha, microalgal lipid, and microalgal polysaccharides ointments) significantly reduced burn-induced inflammation and associated markers. Additionally, the results of hormone measurements revealed that the levels of ACTH, T3, and cortisol were significantly reduced on day 7 in lipid and polysaccharide-treated animals compared to the control group (Fig. 5). No significant variations were detected in the testosterone levels (p<0.05).

Fig. 5
figure 5

Hormone analysis of burn-induced rats in different treatments. Data are expressed as mean ± SD (n = 3). a Significant difference compared to the control group. b Significant difference compared to the standard group

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GC-MS analysis results on extracted lipids are shown in Table 5 and Figure 6. Palmitic acid, 31.98%), octadecanoic acid (12.86%), and oleic acid (8.22%) contributed the highest percentage of extracted fatty acids from Cyanothece sp.

Discussion

The wound healing mechanism is a biological response for regenerating the damaged tissue by newly produced epithelial and connective tissue. The current study found promising wound-healing effects of cyanobacterial lipids in terms of morphological, morphometric, microscopic, and enzymatic evaluations. Furthermore, the examined ointment of polysaccharide-rich extracts from Cyanothece sp. was effective compared to the control group.

The appearance of the wound in terms of color, consistency, the amount and type of secretions, and the wound area was examined in consecutive weeks, all revealing the significant healing potential of Cyanothece ointment with an adequate regenerative efficacy of lipid ointment (Figs. 1, 2, 3 and 4, and Table 2).

Table 2 Morphometric analysis of burn wound healing effect of cyanobacterial lipids and polysaccharide ointments
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Re-epithelialization, fibroblasts, and angiogenesis indicate the proliferation phase. Quantitative assessment of the wound, such as the thickness of the skin epidermis and the number of blood vessels and fibroblasts, were investigated on different days. On day 14 post-treatment, the epithelium was not repaired in the negative control group. However, in other groups, the rat skin epithelium was repaired with a highly organized structure and a relatively appropriate thickness. Furthermore, on the 14th day, a significant increase in the number of blood vessels in the algal groups was noted, especially the lipid ointment group. This can be linked to the angiogenic effects of polysaccharides and fatty acids of Cyanothece sp. It has been widely accepted in the literature that angiogenesis plays a key role during wound repair (Senni et al. 2011; Komprda et al. 2020). Upregulation of angiogenesis marker gene Vascular Endothelial Growth Factor A (VEGFA) was observed in rats treated with sunflower, fish, and microalgae oils. Fatty acids from different sources have been confirmed to induce pro-angiogenic activity (Senni et al. 2011; Komprda et al. 2020). Marine polysaccharides have been shown to have a role in immunity modulation and macrophage stimulation (Senni et al. 2011; Kraiem et al. 2024). Macrophages generate vascular endothelial growth factor (VEGF), a potent pro-angiogenic factor that accelerates wound closure (Gacche and Meshram 2014). Furthermore, the higher wound healing activity and significantly greater vascularizing ability in cyanobacterial lipid ointment can be explained by the presence of fatty acids that are considered effective angiogenic stimulators (Majima et al. 2023).

In the present study, 14 days post-burn, it was found that in cyanobacterial treatments, greater wound-healing effects and increased collagen fibers were evident compared to those in control. In agreement with these findings, the cream prepared from an aqueous crude extract of Arthrospira (Spirulina) platensis showed a higher proliferation activity, collagen regeneration, and maturity than the control group (Gunes et al. 2017). In addition, the solvent extract (methanol: chloroform 50:50) of Microcystis aeruginosa revealed a significant role in diabetic rat wound healing (Hussein et al. 2019).

The results indicated significantly decreased levels (p<0.05) of inflammatory factors such as IL-2, IL-8, and TNFα on the seventh day post-burn (Table 3). Sacran, a sulfated polysaccharide extracted from the cyanobacterium Aphanothece sacrum exhibited significant anti-inflammatory activity through the inhibition of IgE, tumor necrosis factor α, interleukin 4, interleukin 5, and interferon γ production in mice (Ngatu et al. 2012). Similarly, burn-induced wound healing was enhanced by extracellular vesicles secreted from Synechococcus elongatus by promoting IL-6 expression and angiogenesis in mice (Yin et al. 2019).

Table 3 The levels of cytokines in the serum of studied rats in different experimental groups
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Proinflammatory cytokines such as interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α) play critical roles in animals’ immune response pathways (Culnan et al. 2018). Following the secretion of multiple inflammatory cytokines by cells of the innate immune system, various cell types, such as neutrophils, are recruited rapidly to the injured tissue, where they initiate the phagocytosis of microorganisms and cellular debris.

The number of neutrophils increased on the seventh day in the treated groups (Table 4), indicating the faster inflammation phase through the wound repair process (Peña and Martin 2024). The wound healing process is commonly followed by catabasis, or the resolution of inflammation where the leukocyte numbers diminish, and the wound goes through dynamic stages of remodeling and healing. Numerous studies demonstrated that following an injury, the inflammatory response events profoundly influence on the subsequent wound repair processes (Muire et al. 2020; Wilkinson and Hardman 2020).

Table 4 The number of leucocytes and CRP in different groups
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To further understand the wound healing process, the levels of adrenocorticotropic hormone (ACTH), cortisol, T3, and testosterone were explored (Fig. 5). Studies have confirmed that cortisol levels can be increased by stimulation with proinflammatory cytokine IL-1β or adrenocorticotropic hormone (ACTH) upon injury in both humans and rats (Fantidis 2010). The predominant effect of cortisol is to suppress excessive inflammation by limiting the initial proinflammatory response. Researchers have recently found that dietary A. platensis reduced rat stress by regulating some stress-related genes (Ardicli et al. 2022). In the current study, the serous levels of ACTH and cortisol decreased significantly on the seventh day of wound induction in all three groups compared to the control animals (Fig. 5). Tri-iodothyronine (T3) shows a significant difference with the control group 7 days post-burn induction. T3 directly stimulates the proliferation of epidermal keratinocytes and skin fibroblasts (Mancino et al. 2021), thus leading to the acceleration of wound healing.

From the study results, the Cyanothece sp. lipid ointment is significantly effective in healing burn wounds in rats. The study evidenced that the ointment resulted in immunomodulation, increased the speed of wound closure, and increased angiogenesis and blood vessel counts. Additionally, the ointment seemed to impact epithelial thickness and fibroblast counts, as well as other morphological, hormonal, and biochemical evaluations (Figs. 1, 2, 3, 4 and 5, and Tables 2, 3 and 4). In an in vitro evaluation, the aqueous crude extracts of S. platensis enhanced the proliferation of HS2 keratinocyte cell line as a wound-healing model (Gunes et al. 2017). In previous studies, Arthrospira (Spirulina) subsalsa lipid extracts showed strong anti-inflammatory effects (Shiels et al. 2022). Furthermore, microalgal lipids exert antimicrobial activity against a wide spectrum of microorganisms (Alves et al. 2020). The antibacterial activity of microalgal lipids plays an important role in accelerating the wound healing process (Farkha 2023).

These findings promisingly indicate that cyanobacterial lipids have the potential health benefits to treat inflammation-related disorders such as cardiovascular disease, cancers, and wounds (Shiels et al. 2022).

From the GC-MS data (Table 5), two alkanoic acids: palmitic acid (31.98%) and octadecanoic acid (12.86%) are the predominant compounds of Cyanothece sp. lipid extract, followed by oleic acid (8.22%), palmitoleic acid (4.89%), α-linolenic (4.5%), elaidic acid (4.14%), and decanoic acid (3.31%). Palmitic acid and octadecanoic acid were previously reported to exhibit wound-healing properties (Khalil et al. 2000; Grace Noviyanthi Sinambela et al. 2022). Palmitic acid (hexanoic acid) is a saturated fatty acid with potential antioxidant and anticancer properties (Sinambela et al. 2022). This medium-chain fatty acid plays a therapeutic role by accelerating oxidative metabolism (Castelli et al. 2023). Octadecanoic acid (stearic acid) is one of the most frequent fatty acids naturally occurring in various animal and plant body lipids with anti-bacterial and anti-inflammatory effects and is commonly used in the food and cosmetics industry (Chen et al. 2019). Furthermore, studies have proved the ability of linoleic acid and oleic acid to modulate the inflammatory response during the wound healing process, resulting in more rapid wound closure (Ribeiro Barros Cardoso et al. 2004; Cardoso et al. 2011; Bardaa et al. 2016).

Table 5 Fatty acid profile of extracted lipids from Cyanothece sp. revealed by GC analysis
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Natural fatty acids (FAs) have been proven to contribute to the regeneration of skin tissue, attributed mainly to their anti-inflammatory activity (Rojo et al. 2010; Mazutti Da Silva et al. 2018). In preliminary studies on fatty acids frequently found in natural oils such as oleic acid, linoleic acid, and γ-linolenic acid, potential anti-inflammatory and wound healing activities were reported (Ribeiro Barros Cardoso et al. 2004; Rojo et al. 2010); however, due to their low clinical efficacy, there has been growing interest in synergic combinations of fatty acid-rich extracts (Rojo et al. 2010; Silalahi and Surbakti 2015; Silva et al. 2018; Poljšak et al. 2020).

Conclusion

In conclusion, this study highlights the therapeutic efficacy of Cyanothece sp. extracts in promoting the healing of burn-induced wounds in rats. Utilizing cyanobacterial polysaccharides and lipids in wound treatment demonstrated notable enhancements in cell proliferation, epithelial thickness, fibroblast counts, and collagen density, leading to accelerated wound closure when compared to the control group. Moreover, the cyanobacterial ointment significantly reduced pro-inflammatory markers of TNFα, IL-2, and IL-8, as well as neutrophil counts, C-reactive protein (CRP), cortisol, and ACTH. In particular, the lipid extracts of Cyanothece sp. revealed higher wound healing potential that can be attributed to mediatory fatty acids such as palmitic acid, octadecanoic acid, oleic acid, palmitoleic acid, α-linolenic, and decanoic acid. These findings underscore the promising therapeutic application of Cyanothece sp. extracts, particularly its lipid components, in the field of wound healing.