Evaluation of nanocellulose-based anthraquinone from marine fungi Penicillium flavidorsum as an alternative therapy for skin wound healing: histopathological and immunohistochemical evidences from a rat model

Background Wound healing represents a complex clinical challenge, necessitating the selection of appropriate wound dressings to facilitate an efficient healing process. This study aims to explore an effective approach to enhance wound healing by investigating the therapeutic potential of a nanocellulose‑based anthraquinone derived from marine fungi. Forty male Wistar rats were divided into five groups, including a control group and various four treatment groups. The wound healing process was assessed by measuring the wound area at different time points. Results The results showed promising outcomes in terms of wound healing progression. The group treated with anthraquinone and nanocellulose demonstrated the most favorable results, with normal epidermal architecture, marked hyperkeratosis, and minimal dermal edema. This study provides comprehensive evidence supporting the effi‑ cacy of this novel alternative therapy through histopathological and immunohistochemical analyses conducted on a rat model. Conclusion By addressing the limitations associated with conventional wound dressings, our research contributes to the development of innovative strategies for optimizing wound healing outcomes. The findings presented herein underscore the potential of nanocellulose‑based anthraquinone as a promising therapeutic option for promoting skin wound healing. Further investigations are warranted to elucidate the underlying mechanisms and establish the clini‑ cal viability of this alternative therapy.


Background
Wound healing involves a sophisticated physiological process that encompasses a series of intricate cellular and molecular mechanisms aimed at restoring damaged tissue integrity [1,2].The dynamic process of wound healing involves complex interactions among cells, components of the extracellular matrix, and biochemical signaling pathways [3,4].
Throughout the years, the progression of wound dressing technology has markedly advanced from the introduction of the first contemporary dressing in the mid-1980s.These dressings were designed to keep a moist environment, help absorb fluids, minimize infection, and promote wound healing and management [5].Modern wound dressings are now identified as interactive and bioactive remedies, merging the physical barrier offered by conventional dressings with the addition of specific bioactive molecules.These molecules promote cell renewal through the proliferation and migration of fibroblasts and keratinocytes, boost collagen production, fight against bacterial infections, and aid in drug delivery, all of which contribute to an effective healing process.A perfect wound dressing ought to preserve a high level of humidity at the wound location, absorb excess exudate, display non-toxic and hypoallergenic characteristics, permit oxygen transfer, guard against microbial intrusion, and be both comfortable and economical.Modern dressings take various forms, such as hydrogels, foams, sponges, films, and recently, nanofibrous mats [6][7][8].
There is a growing demand for sustainable, eco-friendly nanoscale materials.Biomass-derived polymers like cellulose are gaining attention due to their abundance, easy extraction, biocompatibility, non-toxicity, and biodegradability.Cellulose, in particular, is being considered for wound dressings, either as an additive or a primary material.Data indicate promising outcomes, especially in terms of cell proliferation and attachment [9,10].
Employing nanomaterials in wound therapy can address the shortcomings of conventional wound care and enhance the healing process [11].Consequently, there is a rising need for new treatment approaches to tackle these problems and speed up wound healing.In recent years, nanotechnology has made significant progress and holds great potential for numerous medical applications, including wound healing.[12,13].Nanomaterials offer specific advantages that could transform wound treatment because of their unique physicochemical properties and nanoscale dimensions [14].These materials can modulate biological processes at both the cellular and molecular levels due to their tailored surface properties, high surface-to-volume ratios, and other characteristics.As a result, nanomaterials have garnered significant attention as versatile tools for creating innovative wound healing therapies [15,16].
Fungi sourced from marine environments have a diverse biosynthetic machinery that allows them to produce a wide range of secondary metabolites from different chemical classes, such as alkaloids, polyketides, terpenoids, meroterpenoids, peptides, and steroids [17][18][19][20].
In light of wound healing demands for effective therapies to accelerate tissue regeneration, active biomolecules with healing abilities have been incorporated into nanostructured polymeric dressings.The synergistic effects of these combinations need to be investigated.This study aims to introduce a novel wound healing approach by exploring the therapeutic potential of nanocellulosebased anthraquinone derived from marine fungi, positioning it as a sustainable and effective alternative therapy for skin wound healing in a rat model through comprehensive histopathological and immunohistochemical analyses.

Experimental animals
Forty 10-week-aged male Wistar rats, weighing 110 ± 10 g, were purchased from a confined Animal House Colony.The rats were kept individually in plastic cages to prevent interferences such as biting and potential wound scratching from other animals.They were provided with a standard diet (Al Wadi Co., Giza, Egypt) [9] and had water ad libitum.The animals were kept in a laboratory maintained at a temperature of 25 ± 2 °C.A 1-week acclimatization period was observed before the commencement of the study to allow the animals to adapt to their new environment.

Ethical approval
Follow the normal operating procedures approved by the Institutional Animal Care and Animal Ethics Committee, Faculty Aquatic and Fisheries Sciences, Kafrelsheikh University, Egypt (IAACUC-KSU-005-2021).

Experimental design
Following the acclimatization period, excisional skin wounds of 1.5 × 1.5 cm in diameter were performed on the dorsal region of each rat (Fig. 1A, B).The experimental animals (n = 40) were then divided randomly into five experimental groups, with eight rats per group, as follows: I. Control group (G1): untreated skin wound group.II.Hydrogel treated group (G2): skin wound group treated with hydrogel only.
IV. Anthraquinone and nanocellulose treated group (G4): skin wound group treated with both anthraquinone and nanocellulose.
The amount used from hydrogel, anthraquinone, nanocellulose to cover the whole wound area.

Establishment of full thickness dermal excisional wound
To carry out the excisional wounds, the rats were anesthetized with a combination of ketamine and xylazine administered via intraperitoneal injection (ketamine 70 mg/kg and xylazine 7 mg/kg).The back hair of the rats was then shaved, and the area was disinfected using 70% ethanol.Full-thickness skin excisional wounds measuring 1.5 × 1.5 cm were created on the dorsal region of each animal, following the protocol described by Atiba et al. [36].The wound areas were measured then photographed using special size analysis software, NIH Image J software (available at http:// www.rsb.info.nih.gov/ ij), at various time points: 0, 3, 6, 9, 12, 15, 17, and 19 days post-wounding (Fig. 2).The alteration in wound dimensions was measured in terms of a percentage in comparison with the initial wound size (day 0), and the results are presented in Fig. 2. The medical dressings were changed for the excisional wound groups on the specified days, with careful inspection of the wound healing progress.

Isolation of bioactive compounds from marine fungi
All chemicals, solvents and reagents used were of analytical grade.The chemicals used from Sigma ® and Alpha for chemicals and were of the purest grade available.

Fungal material
A diverse array of marine fungi and other microorganisms collected from marine water from Alexandria beach at a depth of 3 m.The collected sample (from five different locations) was carefully placed into a clean plastic bag and immediately transported to the laboratory.Upon arrival, the sample was stored at −20 °C to preserve its integrity and ensure its suitability for further analysis.To facilitate fungal growth, a cul-ture medium was prepared using potato agar following a standardized protocol [37].

Extraction of fungal anthraquinone using ethanol
The extraction of anthraquinone from the fungal culture was performed using an ethanol-based extraction method [38].The resulting extract, as well as its fractions and sub-fractions, were subjected to monitoring through high-performance liquid chromatography (HPLC) to assess their composition and purity.Additionally, Fourier transform infrared (FTIR) analysis was performed in order to provide further insights into the chemical characteristics and structural properties of the extracted compounds.
The final extract contained 76.2% anthraquinone.conditions at room temperature in conical flask contains 300-ml Wickerham's medium for fermentation for one month.To stop the fermentation process, 300-ml EtOAc was added to the contents of the flask and centrifuge.Then, the content was filtered using Buchner funnel, and the filtrate was then transferred to separating funnel.Separate the aqueous phase and the EtOAc phase, the aqueous phase is then extracted several times with EtOAc till exhaustion.The combined EtOAc extracts was then evaporated under vacuum to obtain a solid or oily residue.The residue was then suspended in 90%MeOH fractionated with n-hexane, and water was added to render the MeOH 60% and then fractionated with ethyl acetate and butanol.Then, the extract was characterized by FTIR and nuclear magnetic resonance (NMR) spectroscopy [39].

Chemical test for anthraquinones detection 2.6.1 Detection of anthraquinone using Borntrager's test
To confirm the presence of anthraquinone in the extract, Borntrager's test, as described by Harborne et al. [40], was used.By preparing 1 g of anthraquinone, 10 ml of dilute HCl was boiled on water bath for 10 min and then filtered.The filtrate was extracted using benzene, and an equal volume of ammonia solution was added to the filtrate, followed by shaking.The emergence of a pink color in the ammoniacal layer indicates the presence of the anthraquinone moiety.

FTIR (Fourier transform infrared)
The FTIR analysis method uses infrared light to scan test samples and observe chemical properties (Riaz et al. [41]).The major parameters' settings for FTIR were the 4 cm −1 resolution and scan number of 64 times; the transmission spectrum within 450-4000 cm −1 was recorded.

Nanocellulose preparation
Nanocellulose was prepared using acid hydrolysis and mechanical process according to Peng et al. [42].Numerous methods have been established for extracting nanocellulose from cellulosic substances.These varied extraction techniques lead to differences in the types and characteristics of the nanocellulose produced.In this section, the primary methods of extraction are categorized into two types: acid hydrolysis and mechanical processing.
Cellulose underwent hydrolysis using 60% sulfuric acid at a ratio of 1:25 cellulose to sulfuric acid.This hydrolysis was performed under various concentrations of sulfuric acid, temperatures, and times.The process was halted by introducing a tenfold excess of distilled water (250 ml) into the mix.The resulting colloidal suspension was then centrifuged at 6500 rpm for 30 min.Following this, it underwent dialysis for 5 days to neutralize to a pH range of 6-7 and remove sulfate ions.The neutral colloidal suspension was subsequently sonicated for 10 min to ensure the uniformity of the produced nanocellulose [43].

Application of anthraquinone, hydrogel, and nanocellulose
Anthraquinone, hydrogel, and nanocellulose formulations (1:1) were topically applied to the injured wound area on the backs of the rats at specific time points: days 0, 3, 6, 9, 12, 15, 17, and 19 post-wounding.The application of these treatments aimed to assess their effects on wound healing progress.Additionally, the wound area was measured during each application to monitor any changes.

Collection of tissue specimens
At the conclusion of the study, specifically 19 days postwounding, all rats were euthanized in a humane manner through decapitation while under anesthesia, which was administered via an intraperitoneal injection of pentobarbital (500 mg/kg).The entire wound, along with a margin of approximately 5 mm of the surrounding healthy tissue, was excised.These tissue samples were then fixed in a 10% buffered formalin solution (pH 7.4) for a duration of 48 h.Following fixation, the samples were embedded in paraffin and prepared for subsequent histopathological and immunohistochemical (IHC) analyses.
After sample collection and termination of the study, all sacrificed rats, along with any remaining tissue samples as well as all bedding materials, were handled in accordance with strict hygienic protocols and properly disposed of in a controlled burial pit to ensure proper biosecurity measures.

Histopathological examination
The skin wound tissues were fixed in 10% formalin buffered solution (pH 7.4) for a period of 48 h.Subsequently, the fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).The histopathological assessment encompassed the evaluation of various parameters, including the extent of granulation tissue formation, necrosis, degree of epithelialization, connective tissue remodeling, and infiltration of inflammatory cells, as previously described by Tuan et al. [44].

Immunohistochemical analysis of TGF-β1 and VEGF
Immunohistochemical staining for TGF-β1 and VEGF was carried out on all tissue specimens, utilizing sections that were 4 μm thick and embedded in paraffin, following the protocol described by Saber et al. [45].The sections underwent deparaffinization in xylene and were rehydrated through a series of ethanol dilutions.
To retrieve antigens, the sections were submerged in a 0.05 M citrate buffer solution at a pH of 6.8.Endogenous peroxidase activity was blocked by incubation in a 0.3% H 2 O 2 in methanol solution for 20 min at room temperature (RT).To prevent non-specific binding, the sections were treated with Protein Block Serum Free for 30 min at RT. Immunolabeling of TGF-β1 was performed on all samples using an anti-TGF-β1 rabbit polyclonal antibody (BIOCYC GmbH & Co. KG, Im Biotechnologiepark TGZ I, 14,943 Luckenwalde, Germany) at a dilution of 1:50.The sections were incubated overnight in a humidified chamber at 4 °C.Similarly, immunolabeling of VEGF was performed using an anti-VEGF rabbit polyclonal antibody (49,026; BioGenix, Netherlands) at a dilution of 1:100.The sections were incubated overnight in a humidified chamber at 4 °C.Following this, all sections were washed with phosphate-buffered saline (PBS) and then incubated with a goat anti-rabbit secondary antibody (catalog number K4003, EnVision + ™ System Horseradish Peroxidase Labeled Polymer; Dako) for 30 min at room temperature.After washing with PBS, the sections were visualized using liquid DAB (3,3'-diaminobenzidine) and then washed in distilled water.Counterstaining was performed using Mayer's hematoxylin.Following counterstaining, the sections were dehydrated using an alcohol gradient, cleared with xylene, and mounted for examination under a light microscope.

Statistical analysis
Statistical differences between the various groups tested were evaluated using multiple t tests (unpaired two-tailed t test), employing the Holm-Sidak method to adjust for multiple comparisons.For statistical analysis, GraphPad Prism software version 8.00 (GraphPad Software, San Diego, California, USA) was employed.

Mortalities
Throughout the entire duration of the study, there were no recorded mortalities among the animals.

Characterization of anthraquinones
Figure 1 shows the FTIR analysis of both anthraquinone and nanocellulose where the bands or peaks were much closed and sharp spike which had the same characteristics bands.The shift vibration of C=O was detected at 1586 cm -1 and 1677 cm -1 .The small peaks shouldered just between 3449 and 3754 cm -1 .

Histopathological examination
The histological examination of the control group (G1) at day 19 post-wounding revealed distinct features indicative of impaired wound healing.Reactive mildly hypertrophied endothelial blood vessels were observed, extending into the markedly hyperplastic epidermis.The epidermis exhibited a clump of basal cells, hyperkeratotic features and frequently observed spongiosis.Within the epidermis, the keratinocytes appeared shrunken with pyknotic nuclei (necrotic) or swollen and rounded with vacuolated cytoplasm (Fig. 3A, B).
Group (G2), treated with hydrogel, exhibited subepidermal edema, and collagen fiber swelling associated with mild epidermal spongiosis.Additionally, inflammatory cell infiltration was evident, along with mild congestion of endothelial blood vessels (Fig. 3C, D).
In contrast, Group (G3), treated with anthraquinone, showed a different histopathological profile.The epidermis exhibited diffuse, severe irregularly hyperplastic features, accompanied by some epidermal spongiosis.Dermal edema was present, along with a few to minimal inflammatory cells infiltration.Notably, marked hyperkeratosis was observed, while the endothelial blood vessels appeared normal (Fig. 3E, F).
Group (G4), treated with anthraquinone and nanocellulose, exhibited a distinct histological pattern.The epidermal architecture appeared normal, with marked orthokeratotic hyperkeratosis.Minimal dermal edema and few perivascular inflammatory cell aggregations were observed (Fig. 3G, H).
Group (G5), treated with nanocellulose alone, exhibited specific histopathological features.The epidermis showed moderately psoriasiform hyperplastic characteristics, with prominent rete ridges and intercellular edema.Occasional epidermal necrotic cells were observed.Within the dermis, numerous inflammatory cells were present along with mild epidermal spongiosis (Fig. 3I, J).

Immunohistochemistry (IHC)
Immunohistochemical analysis was performed to evaluate the expression of both transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF) in the skin tissues of different treated groups.
On the other hand, Fig. 5 illustrates IHC analysis of vascular endothelial growth factor (VEGF) expression.In G1, intense staining of VEGF was observed in the dermal layer, extending toward the subcutaneous muscle (Fig. 5A).In G2, no epidermal staining was observed, but mild dermal staining for VEGF was detected (Fig. 5B).Notably, G2 exhibited intense immunostained dermal vessels, fibroblasts, and macrophages (Fig. 5C).G3 showed few immunostained areas around the hair shaft and dermis (Fig. 5D).In G4, both epidermal and dermal immunostaining for VEGF was faint (Fig. 5E).G5 exhibited faint immunostaining in the proliferated epidermis and hair follicles (Fig. 5F).

Discussion
Wound healing encompasses a multitude of cellular and molecular mechanisms, making it a complicated process aimed to restore the integrity of damaged tissues [46].This research explored the capabilities of a nanocellulosebased anthraquinone derived from marine fungi as an alternative therapy for skin wound healing in a rat model.The results demonstrated the histopathological and The choice of an appropriate wound dressing is crucial for successful wound healing.Traditional dressings have evolved over time to become interactive and bioactive solutions that promote cell regeneration, collagen synthesis, and fight against infections [47].However, the demand for sustainable and eco-friendly materials processable at the nanoscale is on the rise.Polymers derived from biomass, like cellulose and its derivatives, have attracted considerable interest because of their inherent characteristics [48].
In this study, a nanocellulose-based wound dressing loaded with anthraquinone derived from marine fungi was utilized.The experimental animals were divided into five groups, including a control group and various treatment groups.The wound healing process was assessed by measuring the wound area at different time points.The results showed promising outcomes in terms of wound healing progression.The group treated with anthraquinone and nanocellulose demonstrated the most favorable results, with normal epidermal architecture, marked hyperkeratosis, and minimal dermal edema.
Histopathological examination of the wound tissues supported the effectiveness of the anthraquinone and nanocellulose treatment.The control group exhibited mildly hypertrophied endothelial blood vessels, hyperplastic epidermis, and shrunken or swollen keratinocytes, which are indicative of prolonged wound healing [49].In contrast, the group treated with anthraquinone showed a significant improvement in wound healing, characterized by a severe irregularly hyperplastic epidermis and diffuse epidermal spongiosis.These findings suggest that anthraquinone treatment promotes enhanced tissue remodeling and regeneration [50].
Remarkably, the combination treatment of anthraquinone and nanocellulose resulted in the restoration of normal epidermal architecture with minimal dermal edema.This histological pattern suggests that the combination therapy enhances tissue regeneration and remodeling, which is consistent with previous studies demonstrating the beneficial effects of nanocellulose in wound healing [51].The presence of minimal dermal edema indicates improved vascularization and efficient drainage of the wound site.
To evaluate the expression of key growth factors involved in wound healing, IHC analysis was performed, focusing on transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF).TGF-β and VEGF play vital roles in wound healing process, especially during the early stages of healing process.TGF-β is a crucial regulator of cellular processes such as cell proliferation, extracellular matrix deposition, and tissue remodeling [52].It promotes the differentiation of fibroblasts into myofibroblasts, which are responsible for wound contraction and collagen synthesis [53].Additionally, TGF-β stimulates angiogenesis through induction of endothelial cells migration and proliferation [54].VEGF, on the other hand, plays a pivotal role in angiogenesis and neovascularization, essential for providing nutrients and oxygen to the healing tissue [55].The expression of TGF-β gradually declines during the later stages of wound healing as tissue remodeling and maturation occur, while VEGF level gradually decreases, reflecting the resolution of angiogenesis and the transition to the remodeling phase [56].
IHC staining revealed distinct patterns of TGF-β and VEGF expression among the treatment groups.In the control group, intense staining of TGF-β was observed in the proliferating dermal, suggesting its involvement in the prolonged wound healing process.In contrast, the group treated with anthraquinone showed no staining of TGF-β in epidermis, indicating a potential inhibitory effect on its expression, which is attributed to approximate epidermal growth.Importantly, the combination treatment group exhibited diffuse staining of TGF-β in dermal fibroblasts.Since TGF-β plays a crucial role in stimulating collagen synthesis and deposition by fibroblasts, promoting the formation of mature scar tissue, the combination therapy is suggested to contribute to improving wound healing outcomes [57].
Similarly, VEGF expression varied among the treatment groups.The control group showed intense staining of VEGF in the dermal layer, indicative of persistent ongoing angiogenesis during the healing process.In contrast, the group treated with anthraquinone exhibited no epidermal staining and only mild dermal staining of VEGF, suggesting a potential downregulation of VEGF expression.The combination treatment group showed faint epidermal and dermal staining of VEGF, indicating a potential modulation of VEGF expression and angiogenic processes in the wound bed.
Overall, histopathological and immunohistochemical findings provide valuable insights into the effects of anthraquinone and nanocellulose treatment on wound healing.The significant improvement in histopathological features, such as epidermal hyperplasia and spongiosis, as well as the modulation of TGF-β and VEGF expression, suggest the potential of this combination therapy in promoting tissue regeneration and angiogenesis.Further research is needed to clarify and explain the underlying mechanisms and optimize the dosage and application strategies for this promising therapeutic approach.
In conclusion, this research emphasizes the capabilities of a nanocellulose-based anthraquinone derived from marine fungi as a promising approach for enhancing skin wound healing.The observed histopathological improvements and modulation of TGF-β and VEGF expression support the efficacy of this novel therapy.Future studies should aim at improving the formulation and investigating its therapeutic efficacy in larger animal models, ultimately progressing to clinical trials.

Fig. 1 A
Fig. 1 A FTIR spectra of extracted anthraquinone.B H NMR data of anthraquinone compound in CDCL3 (400 MHz) obtained from marine fungi.C FTIR analysis for nanocellulose

Fig. 2 A
Fig. 2 A Wound area measurement.B Healing speed measurement