Curative effects of sophorolipid on physical wounds: In vitro and in vivo studies

Abstract Early‐weaning syndrome is harmful to animals because an effect on growth in the early‐stage of life generally determines the overall growth rate. Sophorolipid (SPL), a surface‐active glycolipid compound, has been shown to exhibit antimicrobial activity and stimulate cell proliferation. Thus, in vitro and in vivo studies were conducted to evaluate the potential of SPL on the gut turnover after the wound. The in vitro experiment with HT‐29 cells showed the increased proliferation with increasing gene levels of collagenase‐1 and matrilysin‐1. Next, the 16‐day in vivo experiment was conducted with thirty rats (14‐day‐old), and the allocation was performed according to their body weight (BW) into three treatments: control diet (CON), 48 ppm of oxytetracycline‐supplemented diet (OTC) and 10 ppm of SPL‐supplemented diet (SPL). Dietary SPL accelerates the growth of rats in overall periods, and intestinal permeability was lower in SPL at day 16. Villus:crypt ratio and the goblet cell count were also higher in SPL than in CON at day 8. Caecal Streptococcus spp. were significantly reduced with dietary SPL and OTC at day 8 and 16, and total short‐chain fatty acid, acetate and butyrate levels were increased in the SPL at day 8. In conclusion, these data demonstrated that SPL could improve gut remodelling potential and modulate the gut environments, resulted in acceleration of post‐weaning growth. Therefore, SPL could have a potential as a feed additive aimed at promoting repair system after wound in animal's gut.

One such strategy is the use of antibiotic dietary additives as antibiotic growth promoters, which can have a significant effect on growth acceleration and feed efficiency improvement (Wegener, 2003). However, the use of antibiotics had been warned by World Health Organization (WHO) in 2014 because livestock can serve as a reservoir of antibiotic-resistant bacteria (Bates et al., 1994).
Thus, the livestock industry is seeking an alternative to antibiotics.
Previous studies have demonstrated that various feed additives have the potential to be a replacement of antibiotics (Markovi et al., 2009;Wang et al., 2010), but none of these have proven cost-effective in their growth-promoting effects or modulation of gut microbial populations (Niewold, 2007). A novel and eco-friendly alternative feed additive is therefore needed to substitute for antibiotics.
Recently, the antimicrobial properties of bio-surfactants have attracted attention owing to their low toxicity and high biodegradability. Of particular interest is sophorolipid (SPL), a glycolipid bio-surfactant produced by non-pathogenic yeast species such as Candida bombicola (Cho et al., 1999). Sophorolipid has lower toxicity and higher bio-degradability than other surfactants (Desai & Banat, 1997), and it displays many unique biological properties including antimicrobial activity, immune modulation, stimulation of skin dermal fibroblasts and collagen production (Concaix, 2003;Gross & Shah, 2003;Maingault, 1999). These properties suggest that SPL could have a potential to improve animal health and growth after weaning. Therefore, this study aimed to evaluate the beneficial effects of SPL on early-weaning syndrome.

| In vitro study (wound healing assay)
HT-29 cell line, human colorectal adenocarcinoma cell line, was achieved from the Korean Cell Line Bank. Cells were sustained in Roswell Park Memorial Institute 1640 medium with 10% heatinactivated fetal bovine serum (thawed at 4°C, then incubated at 65°C for 30 min) and 1% penicillin and streptomycin at 37°C and 5% CO 2 in a humidified chamber. Afterwards, a wound healing assay (cell migration test) was performed as previously described (Rodriquez et al., 2005), using three doses of SPL (1, 5 and 25 µg/ml). Briefly, HT-29 cells were seeded onto 6-well plates and grew until they reached 100% confluency. Randomly selected sites of the cell monolayer were then manually wounded by scratching with a pipette tip, and the cells were further incubated with/without SPL. Cells were photographed immediately after wounding (0 hr) and after 48 hr of incubation.

| mRNA analysis by quantitative real-time PCR
Trizol® (Invitrogen) was used to extract total RNA according to the manufacturer's procedure. And High-capacity cDNA Reverse Transcription kit (Applied Biosystems) was used to synthesize cDNA.
Amplications of target genes were determined using a RealHelix TM Premier qPCR Kit (NanoHelix) with a StepOnePlus Real-Time PCR System (Applied Biosystems). Primers are listed in Table 1. The 2 −ΔΔCT method was used to quantify the relative mRNA expression levels.

| Animal and experimental diets
All of the works related to animal was approved by the Korea University Institutional Animal Care and Use Committee (KU-IACUC). And all procedures for animal were conducted in accordance with the standard guidelines and protocols of Korea University (Approval number: KUIACUC-2020-0097). Thirty early-weaned male Sprague Dawley rats (14-days old) were used in a 16-day experiment (average body weight: 23.95 g). Rats were randomly allocated into three experimental treatment groups according to their initial body weight, with ten replications. The control diet was NIH-41 diet and its composition is presented in Table 2. Dietary treatments were as follows: (a) control diet, CON group; (b) control diet + 48 ppm oxytetracycline (OTC), OTC group; (c) control diet + 10 ppm SPL, SPL group. Feed was provided in powdered form and freely allowed to rats. All rats were housed in a clean and sanitary environment.

| Experimental procedures and sample collection
This experiment was conducted in two phases. Phase I (day 1-8) estimated the restoration effect and phase II (day 9-16) investigated

Gene name Sequence (forward, reverse)
In vitro experiment In vivo experiment

| Gut permeability test
Fluorescein isothiocyanate-dextran 4 (FD4; TdB Consultancy) was used to assess paracellular uptake. Food and water were removed from the cages 6 hr before euthanasia, and rats were given FD4 (0.44 g/kg BW) by oral gavage needle 4 hr before euthanasia. The optical density of FD4 was measured in each sample using a spectrophotofluorometer with excitation at 485 nm and an emission at 535 nm.  Table 1 showed the primers list. The concentration of total bacteria was used as a housekeeping control. The 2 −ΔΔCT method was used to quantify the relative bacteria levels.

| Gas chromatography-mass spectrometry for caecal short-chain fatty acid
The concentration of caecal short-chain fatty acid (SCFA; acetate, propionate and butyrate) was measured using gas chromatographymass spectrometry (GC-MS) (Furusawa et al., 2013). SCFA concentrations were quantified with peak areas and calculated with standards curves.

| Statistical analysis
Data were analysed using the analysis of variance procedure (ANOVA) with Statistical Analysis System 9.4 (SAS 9.4, 2011).
Significant differences between treatment means were determined using Duncan's multiple-range tests. p < .05 was defined as significant.

| In vitro experiment
Results and representative images of the wound healing assay 48 hr after SPL treatment are shown (Figure 1a,b). After 48 hr,

| In vivo experiment
Average BW at day 0 was 23.97 g and there was no difference between treatment groups. At day 8, the average BW of rats was similar among dietary treatment groups; however, the average BWs of animals in the OTC and SPL were numerically increased at day 16, respectively, compared to the CON group. In addition, the ADG was higher in the OTC and SPL than in the CON over the whole experimental period. The ADFI of the SPL was numerically increased when compared to the CON and OTC, respectively, from day 0 to day 16.
Villus height of rat fed 10 ppm of SPL-supplemented diet was significantly lengthened (p < .05) after 8 and 16 days when compared to other treatments (Figures 2c and 3c Figures 2g and 3g).  , 1992). Therefore, researchers in the livestock industry have begun to investigate ways to accelerate gut restoration during the post-weaning period in terms of nutritional and pharmacological aspects (Dirkzwager et al., 2005). One strategy being tested is dietary supplementation with antibiotics, which can alter the gut microbiota and help to maintain intestinal health and function (Adjiri-Awere & van Lunen, 2005;Taras et al., 2007). However, the livestock feed industry has been seeking alternatives to antibiotics because they can induce bacterial resistance by providing a reservoir for antibioticresistant genes (Woolhouse et al., 2015). In this study, the restoration effect of SPL was investigated in wound treatment using an in vitro assay. We also assessed the antimicrobial and therapeutic effects of SPL in early-weaned rats and evaluated whether SPL could substitute for the dietary efficacy of antibiotics.
The effects of glycolipid-type bio-surfactants on various cell types have been reported (Jezierska et al., 2018); however, the little information about relationship between SPL and intestinal epithelial cell was provided. In this study, we showed that SPL accelerated proliferation and migration in an in vitro wound model using HT-29 cells. Because rapid cell migration and proliferation play a key role in gut recovery following injury (Jung et al., 2009), our results suggest that SPL may be able to aid in the acceleration of intestinal wound healing. To elucidate the mode of action of SPL, we analysed the gene expression levels related to the wound healing process. Matrix metalloproteinases (MMP) are an enzyme family, which contains Zn ion and depends on Ca ion (Nagase & Woessner, 1999). And their functions are specifically related to angiogenesis, wound healing, tissue remodelling, cell migration and homing of inflammatory cells to the wound site (Parks, 1999). Particularly, MMP-1 is important in epithelial cell migration, while MMP-7 is involved in epithelial closure and membrane remodelling during gut wound repair (Pilcher F I G U R E 5 Relative short-chain fatty acid concentration (a), total short-chain fatty acid concentration (b), and the portion of acetate (c), propionate (d) and butyrate (e) of rats fed experimental diets for 8 days. CON, control; OTC, oxytetracycline; SCFA, short-chain fatty acid; SPL, sophorolipid. Control diet (CON), 48 ppm of OTC-supplemented diet (OTC) and 10 ppm of SPL-supplemented diet (SPL). Data expressed as mean ± SE. a,b Different superscripts in the same series indicate statistically significant differences (p < .05)

F I G U R E 6
Relative short-chain fatty acid concentration (a), total short-chain fatty acid concentration (b), and the portion of acetate (c), propionate (d) and butyrate (e) of rats fed experimental diets for 16 days. CON, control; OTC, oxytetracycline; SCFA, short-chain fatty acid; SPL, sophorolipid. Control diet (CON), 48 ppm of OTC-supplemented diet (OTC) and 10 ppm of SPL-supplemented diet (SPL). Data expressed as mean ± SE Saarialho-Kere et al., 1996). In the current study, the proper dosages of SPL treatment showed upregulating MMP-1 and MMP-7 expressions, suggesting that SPL treatment could improve intestinal healing by affecting epithelial cell migration and remodelling.
The single epithelial cell layer of the digestive tract is the widest area of contact between outside and inside of the body. Hence, intestinal barrier function is important because it is the organ that encounters the most external substances, including pathogens, antigens, toxins, as well as nutrients (Moeser et al., 2017).  (Maingault, 1999). At the same time, a thick layer of mucus, secreted by goblet cells, encloses the surface of epithelial cells and builds a barrier to extraneous toxic materials (Cornick et al., 2015), thus having a role as the first line of protection, particularly counter to pathogenic bac-  (Pulate et al., 2013). However, further studies will be needed to investigate more species within the intestinal microbiota.
The role of SCFA and its link to the gut microbiome has been extensively studied because SCFA is the end-product of gut microbiota fermentation with dietary non-starch polysaccharides which cannot be digested by animals (Le Poul et al., 2003). In this study, we found that the concentration of all SCFA was increased in rats given  (Maingault, 1999;Xu et al., 2019). Conversely, the SCFA modulation in gut might be able to regulate hepatic lipid metabolism and adipose tissue stabilization (Nishina & Freedland, 1990). In particular, acetate was found to promote lipogenesis and cholesterol genesis in the liver, while both were inhibited by propionate (Demigne et al., 1995).
It is thought that the ratio of acetate to propionate could be crucial to the relationship between liver and caecal SCFA concentrations (Morrison & Preston, 2016). Taken together, our results demonstrated that SPL as a dietary additive could modulate the gut microbiome and SCFA levels, and may also work to regulate general lipid metabolism in the host.

| CON CLUS ION
In conclusion, SPL appears to improve jejunal permeability and the gut defence system, as well as the caecal microbiota and SCFA levels, which in turn could aid in growth acceleration after weaning. The present study represents the first trial using SPL as a feed additive, and suggests that SPL could be a novel nutritional strategy aimed at overcoming early-weaning syndrome.

ACK N OWLED G EM ENTS
This work was supported by Pathway Intermediates (Shrewsbury, UK) and Korea University (Seoul, Korea).

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

A N I M A L WE LFA R E S TATE M E NT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes. All of the works related to animal were approved by the Korea University (KU) Institutional Animal Care and Use Committee (IACUC). All the procedures for animal were conducted in accordance with the standard guidelines and protocols of KU.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.481.