In vitro and in vivo evaluation of Ulva lactuca for wound healing

Ulva lactuca (U. lactuca) is an important seaweed species. Some ingredients in these seaweeds are thought to accelerate wound healing. However, limited data on the use of seaweed for wound healing exists. This study examined whether ethanol or aqueous extracts of U. lactuca promote antioxidant and anti-inflammatory properties in vitro and wound healing in vitro and in vivo. Cell proliferation, antioxidation, and migration were observed in NIH3T3 cells treated with U. lactuca extract in vitro. Both U. lactuca extracts were examined for their ability to inhibit inflammatory cytokine synthesis in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In vivo experiments involved four groups of albino mice (BALB/c; 10 mice per group). One 1.0 cm2 wound was created via excision of full-thickness skin on the back of all mice. Mice in Group I mice were treated topically with the ethanol extract of U. lactuca (25 mg/mL) for 10 d. group II mice were treated topically with an aqueous extract of U. lactuca (12.5 mg/mL) for 10 d. Group III received topical application of phosphate-buffered saline solution. Group IV wounds were maintained without treatment. Both extracts significantly increased fibroblast proliferation. The antioxidant activity of the U. lactuca extract was determined using a total antioxidant capacity assay. Both extracts inhibited the release of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) from LPS-mediated inflammation in RAW 264.7 cells. These extracts also upregulated the expression of Th2 cytokines such as transforming growth factor beta 1 (TGF-β1) and interleukin 10 (IL-10) in RAW 264.7 cells under pro-inflammatory conditions. Both extracts enhanced the migratory ability of NIH3T3 cells. U. lactuca ethanol extract enhances wound healing properties in vivo. These results suggest that bioactive compounds derived from U. lactuca extract are beneficial for wound healing and anti-inflammatory therapies.


Introduction
The skin is the largest organ of the body and covers its entire surface.When injured, it should repair quickly and perfectly, regardless of the injury's size and severity of, to prevent further harm.Wound healing is divided into four stages: hemostasis, inflammation, proliferation, and maturation.After skin injury, blood extravasation causes platelet aggregation and coagulation, triggering an inflammatory response, which lays the foundation for repair [1,2].During the repair phase, epithelial cells, dermal fibroblasts, and vascular endothelial cells migrate, proliferate, and differentiate from adjacent uninjured tissues to the wound site, leading to the maturation phase [3].
Since ancient times, humans have empirically used many plant resources to treat wounds, including cuts, abrasions, and burns [4].These medicinal plants are safe, reliable, clinically effective, and low-cost [5], making them widely accepted by the public [5].Seaweeds have been used in traditional medicine and the food industry for many centuries [6].They grow widely along coasts and are readily available, prompting numerous studies to explore them as sources of novel therapeutic compounds [7].Seaweeds contain bioactive compounds such as tannins, triterpenoids, and alkaloids, which can affect various stages of wound healing [8].
Seaweeds can prevent tissue damage and stimulate wound healing.Despite extensive research on medicinal plants for wound healing, limited data on marine resources exist, including seaweeds [7].Developing convenient and low-cost wound dressings made from seaweed for wound healing is expected.
Ulva lactuca (U. lactuca), also known as green algae (sea lettuce), is a macroalga belonging to the phylum Chlorophyta.It contains secondary metabolites, such as alkaloids, triterpenoids, steroids, saponins, phenolic compounds, and flavonoids, which have antibacterial, anti-inflammatory, antioxidant, and anticoagulant activities that may accelerate wound healing.The extract from Sargassum ilicifolium has stronger wound-healing properties than that from U. lactuca [7].However, this was with low dose oral administration, Although there are no evident therapeutic results in vivo.In vitro, the aqueous extract of U. lactuca induced proliferation and migration of the mice fibroblasts compared to cells in the control group.This study aimed to investigate whether ethanol or aqueous extracts of U. lactuca can promote antioxidant and antiinflammatory properties and explore their wound healing via in vitro and in vivo studies.This study was conducted to explore the potential wound healing properties of the U. lactuca extracts.
The U. lactuca extracts may present a potential therapeutic opportunity in wound healing.

Preparation of Ulva lactuca ethanol extract
U. lactuca powder was provided by Hualien (Taiwan).Briefly, 100 g of dry U. lactuca powder was placed in 1000 mL ethanol, stirred (300 rpm) at 60 °C for 1 h, and mixed vigorously for 72 h at 25℃.The extract was filtered through Whatman No.1 filter paper and called the ethanol extract.The supernatant was concentrated and dried using the CES-8080 freeze dryer (Panchum, Taiwan) (1,1000 rpm, 37℃).The final concentrated U. lactuca extract was stored at -20 °C until further use [9].

Preparation of Ulva lactuca aqueous extract
U. lactuca was extracted using the hot water extraction method.Briefly, 100 g of dry U. lactuca powder was placed in 1000 mL sterile water, stirred (300 rpm) at 60 °C for 1 h, and mixed vigorously for 72 h at room temperature.The extract was filtered through Whatman No.1 filter paper and called the aqueous extract.The extract was further concentrated and dried using the CES-8080 freeze dryer (Panchum) (1,1000 rpm, 37 ℃).The final concentrated U. lactuca extract was stored at -20 °C until further use [10].

Cell culture
NIH3T3 and RAW 264.7 cells were purchased from the Taiwan Cell Line Bank (Bioresource Collection and Research Center, Food Industry Research).NIH3T3 is mouse embryonic fibroblast cell line, and RAW 264.7 is mouse macrophage cell line.Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Seromed, Berlin, Germany) supplemented with 10% fetal bovine serum (Biological Industries, Israel), 2 mM L-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin in humidified air with 5% CO 2 at 37℃ in an incubator (Panasonic Healthcare, Japan).

MTT assay
Cell viability was determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT ) assay, as previously described [11].Briefly, cells were seeded at a density of 1 × 10 3 cells/well in a 96-well plate and cultured with DMEM for 16 h.Thereafter, cells were treated with serial concentrations of the ethanol (6.25, 12.5, 25 and 50 mg/mL) or aqueous extracts of U. lactuca (6.25, 12.5, 25 and 50 mg/mL) for 24 h and 48 h.Treatments at each concentration were performed in triplicate.Thereafter, the medium was aspirated and cells were washed with phosphate-buffered saline (PBS).Cells were subsequently incubated with MTT solution (5 mg/mL) for 4 h at 37 ℃.The supernatant was removed, and formazan was solubilized in isopropanol and measured spectrophotometrically at 570 nm (Hach DR900, USA).The mean absorbance of triplicate readings was calculated.

Total antioxidant capacity test
The total antioxidant capacity of U. lactuca was detected using the CheKine™ Total Antioxidant Capacity (TAC) Assay Kit (KTB1500, Abbkine, CA, USA ) [12].Concentrations of 6.25, 12.5, and 25 mg/mL of the ethanol or aqueous extracts were evaluated for antioxidant activity.Briefly, 150 μL of substrate diluent and 15 μL of substrate were premixed.Thereafter, 15 μL of reaction buffer and 10 μL of samples were added.The mixture was subsequently incubated for 5 min at room temperature.Absorbance was measured at 593 nm using a microplate reader (Hach DR900, USA).All experiments were performed in triplicate (n = 3).

RNA extraction and real-time reverse transcriptase-polymerase chain reaction
Real-time reverse transcriptase-polymerase chain reaction (Real-time PCR) was performed as previously described [13][14][15].RAW 264.7 cells were incubated for 18 h with lipopolysaccharide (LPS) or vehicle in the presence or absence of U. lactuca.The total cellular RNA was isolated from cultured RAW 264.7 cells with the GENEzol™ reagent (Geneaid Biotech, New Taipei, Taiwan) and quantified using a spectrophotometer (Hach DR900, USA).Total RNA (5 μg) from each sample was reverse-transcribed (RT) using 5× RT buffer, 6 μL DTT, 2.5 μL dNTP The reverse transcription products (5 μL) were mixed with Eco™ Real-Time PCR System (Illumina, San Diego, CA, USA) and primers, resulting in a final volume of 10 μL.Real-time PCR was performed using SimplyGreen qPCR Master Mix, Low Rox (GeneDireX, Inc., USA) under standard thermal cycling conditions: polymerase activation at 95 ℃ for 2 mins, followed by 40 amplification cycles at 95 ℃ for 10 s and 60 ℃ for 30 s, and a melt cycle at 95 ℃ for 15 s, 55 ℃ for 15 s, and 95 ℃ for 15 s.GAPDH was used as the reference housekeeping gene, and each primer master mix consisted of one forward and one reverse primer (10 μM each) and sterile water.The primer sequences are listed in Table 1.
Table 1.Primer sequences used in transcript level analysis for inflammation markers of RAW 264.7 cells.

In vitro migration/wound healing assay
Cell migration was determined using the wound healing scratch assay [16].NIH3T3 cells were inoculated into 6-well plates (1 × 10 6 cells/well) and cultured for 24 h at 37 °C in 5% CO 2 to achieve 80-90% cell confluency.A scratch wound was created using a 200 μL pipette tip and wound debris was washed away using PBS.Ethanol.ethanol (25 mg/mL) or aqueous U. lactuca (12.5 mg/mL) extracts were added.The initial wounding and migration of cells in the scratched area after 12, 24, and 36 h were captured using a microscope (Nikon, Tokyo, Japan).

Animals and mouse wound healing model
The

Statistical analyses
Results were obtained from three independent experiments (n = 3), and data were expressed as means ± standard deviation.Student's t-test and one-way ANOVA, followed by the Bonferroni post hoc test, were used for comparisons between the two groups and multiple groups.
Statistical significance was set at p <0.05.

Ulva lactuca extract promotes cell proliferation
Cell viability was assessed using the MTT assay.As shown in Fig 1, the ethanol extract of U.
lactuca promoted cell proliferation at a concentration of 6.25 50 mg/mL.Aqueous U. lactuca promoted NIH3T3 cell proliferation at concentrations in the range of 6.25-12.5 mg/mL.
However, the aqueous extract of U. lactuca inhibited cell proliferation at concentrations in the range of 25-50 mg/mL.Thus, the aqueous extract of U. lactuca was weakly cytotoxic to NIH3T3 at high doses.Consequently, in the following research, the highest concentrations used were 12.5 mg/mL for the aqueous extract of U. lactuca and 25 mg/mL for the ethanol extract U. lactuca.

Antioxidant activity of Ulva lactuca extract
The total antioxidant capacity of U. lactuca was also examined and the results indicated that the ethanol extract at 6.25 mg/mL to 25 mg/mL showed total antioxidant capacity in a dose-

Anti-inflammatory properties of Ulva lactuca extract
To verify the anti-inflammatory activity of the U. lactuca extract, we explored whether the ethanol or aqueous extract protected against LPS-mediated inflammation in RAW 264.7 cells.
Incubation of RAW 264.

Ulva lactuca extract enhances cell migration
To assess the ability of U. lactuca extract to induce migration, a wound-healing scratch assay was performed in vitro using the 25 mg/mL ethanol extract or 12.5 mg/mL aqueous extract.
Cell migration in NIH3T3 cells was photographed at 12, 24, and 36 h following extract

Wound contraction effect in Ulva lactuca-treated mice
A 1.0 cm 2 cutaneous excisional wound was created on the backs of mice.Group I mice were treated with 25 mg/mL ethanol extract of U. lactuca three times daily.Group II mice were treated with 12.5 mg/mL aqueous extract of U. lactuca three times daily.Group III mice were treated with PBS on excisional lesions.Group IV (control) had untreated wounds.Quantitative measurements of wound size were routinely used to assess the initial wound size up to wound closure.Photographs and measurements of the wounds were taken on the day of wound creation (day 0) and on days 3, 5, 9, day 12 (Fig 5).The group treated with the ethanol extract of U. lactuca showed significantly reduced wound areas compared to the PBS treatment and control groups within three d (p <0.05).In the control group, the open wound area on the third day was 96.61 ± 8.21 mm 2 .For the PBS treated mice, it was 96.80 ± 9.32 mm 2 , whereas for the mice treated with ethanol extract of U. lactuca, it was 77.63 ± 7.43 mm 2 .The open wound area for mice treated with the aqueous extract of U. lactuca was 87.90 ± 8.62 mm 2 .By the 5th day, wound areas were reduced in the groups treated with aqueous extract and ethanol extract compared with those in the control.The ethanol extract of showed better wound healing activity than the aqueous extract on days 3 and 9. PBS treatment had no significant wound healing effect compared with the control group.Ethanol extract therapy demonstrated better wound repair results than aqueous extract therapy.
To confirm this, in vivo experiments were conducted using three groups of C57BL/6 mice (10 each).Mice in Group I were treated topically with the ethanol extract of U. lactuca (25 mg/mL) for 9 d.Group II received PBS, and group III (control) had untreated wounds.The progressive healing changes in the wounds of the mice in each group are shown in (Fig 6).Statistical analysis showed that by the end of the study period, treatment with the ethanol extract of U. lactuca caused a significant wound contraction compared to the PBS and control groups (p <0.01).The ethanol extract of U. lactuca possesses promising wound healing properties in vivo.

Discussion
In this study, both ethanol extract and aqueous extract of U. lactuca could reduce wound areas in vivo.Both extracts promoted antioxidation and anti-inflammatory in vitro.The antiinflammatory effects of U. lactuca demonstrated in this study are supported by studies showing that sulfated polysaccharides present in algae have anti-inflammatory properties [18].A study by de Araújo et al [19] showed that U. lactuca extract exerts anti-inflammatory effects by targeting bradykinin.Premarathna et al. demonstrated that the anti-inflammatory activity of U. lactuca extract can accelerate wound healing [20].They reported that after 7 d of treatment with this extract, the wound healing rate in rats with excisional wounds was 61.67%, compared to that in the untreated control (52.76%).In our study, wound areas were reduced in the group treated with aqueous and ethanol extracts compared to those of the normal wound control on the 5th day of treatment.The topical application of the ethanol extract of U. lactuca showed better wound healing activity than the aqueous extract on days 3 and 9.
The wound healing effects of U. lactuca are partly attributed to its rich polysaccharide content.
A preliminary study indicated that the aqueous extract is richer in polysaccharides than the ethanol extract [21].In our experiments, the ethanol extract of U. lactuca demonstrated promising wound healing properties in vivo.A high concentration of the aqueous extract of U. lactuca (12.5-50 mg/mL) was marginally toxic to NIH3T3 cells.Therefore, we selected a low concentration (12.5 mg/mL) for animal experiments.Both the concentrated aqueous (12.5 mg/mL) and ethanol extracts (25 mg/mL) significantly increased fibroblast viability.However, we used a high concentration (25 mg/mL) of the ethanol extract in animal experiments.
Although the ethanol extract showed better therapeutic effects, this may have been attributed to its higher concentration.Additional purification or removal of harmful substances from the aqueous extract of U. lactuca requires further investigation.Thus, the ethanol extract of U. lactuca may provide a potential therapeutic opportunity for wound healing.

Conclusions
Our results showed that both the concentrated aqueous (12.5 mg/mL) and ethanol extracts (25 mg/mL) of U. lactuca significantly increased fibroblast viability and cell migration.The antioxidant activity of the U. lactuca extract was assessed using a total antioxidant capacity assay.The in vivo wound healing results were statistically significant compared to those of the control group.We found that high concentrations (25 mg/mL) of the aqueous extract of U. lactuca are toxic to cells.Therefore, we used a lower concentration (12.5 mg/mL) for animal wound treatment.Compared to the aqueous extract, the ethanol extract of U. lactuca demonstrated promising wound healing properties in vivo.Better therapeutic effects may be attributed to higher extraction doses or different extraction methods.Future studies should analyze the components of the extracts from both alcohol and water extractions.In our study indicated that the ethanol extract of U. lactuca provides a new therapeutic approach for wound healing.

Fig 1 .
Fig 1. Ulva lactuca increases the proliferation rate of fibroblasts.One set of 96-well culture dependent manner.A similar trend was observed in the NIH3T3 cells treated with aqueous extract (Fig 2).

Fig 3 . 7 .
Fig 3. Ulva lactuca inhibits mRNA expression of pro-inflammatory cytokines in RAW treatment.As indicated in Fig 4, an obvious enhancement of cell migration in NIH3T3 cells treated with aqueous or ethanol extracts was observed.
excision model was established based on the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (approval number 2022005, dated September 23, 2022) from the Institutional Animal Care and Use Committee of Tzu Chi University of Science and Technology.All animal care and experiments followed the Animal Care Guidelines of the Animal Center at Tzu Chi University.Syngeneic BALB/c female mice (6-8 weeks old) were housed under a 12-hour dark/light cycle with free access to food and water.Forty syngeneic adult female mice were divided into four groups of ten mice each.Mice were anesthetized with [17]g/kg sodium pentobarbital (Merck KGaA, Darmstadt, Germany) via intraperitoneal injection.A cutaneous excisional wound (1.0 cm 2 ) was created on their backs.Ethanol or aqueous U. lactuca extracts in 0.1 mL PBS were applied to the excisional lesions three times daily.Wound sizes were measured using calipers on days 3, 5, 9, and 12.The in vivo wound healing results were statistically analyzed using SPSS software.Mean expression was compared between groups using one-way ANOVA (p <0.05 was considered statistically significant).The in vivo experiments were conducted using four groups (10 in each) of albino mice (BALB/c; 10 mice per group).Mice in group I were topically treated with the ethanol extract of U. lactuca (25 mg/mL) for 10 d.Mice in group II were topically treated with the aqueous extract of U. lactuca (12.5 mg/mL) for 10 d.Group III received topical application of PBS solution and group IV (control) wounds were untreated.Changes in wound size were measured and expressed as percentages of the original wound size[17].Wound healing was calculated as follows: percentage of wound healing = (total wound area − present wound area)/(total wound area) × 100.