LPSHE accelerates re-epithelialization on burn-wounded skin

Skin wound healing is classically divided into three phases: inflammation, proliferation, and remodeling (Tiwari 2012; Burgess et al. 2022). In the second and third phases of wound healing, wounded skin undergoes re-epithelialization (Haase et al. 2003; Gurtner et al. 2008; Lim et al. 2023). To determine the re-epithelialization effect of the LPSHE, we established a burn mouse model and measured the burn wound area closure rates (Figure 2(A)). After 14 days, we observed that wounds treated with the LPSHE or Commercial ointment had significantly recovered in the wounded site compared with the non-treated group (Burn vs Burn+Commercial ointment: p<0.001, Burn vs Burn+LPSHE: p<0.001, Burn+LPSHE vs Burn+Commercial ointment: p=0.0150) (Figure 2(B)). To promote the re-epithelialization, a collagen layer must form and recover the skin wound site (Werner et al. 2007; Gurtner et al. 2008). The Commercial ointment or LPSHE had no effect on wound healing in burn-induced skin destruction until 3 days. However, after 14 days, we observed that the collagen formation in the dermis was further increased by treatment with the LPSHE or Commercial ointment (Figure 2(C)). To promote the wound re-epithelialization from the surrounding wound margins toward the center, keratinocytes increase the F-actin formation and increase keratinocyte migration (Krishnaswamy and Korrapati 2014; Rodrigues et al. 2019; Peña and Martin 2024). Phalloidin staining showed that the LPSHE or Commercial ointment treatment significantly enhanced F-actin distribution in the injured skin tissue after 3 days (Figure 2(D)). We further determined the expression of heme oxygenase 1 (HMOX1) and fatty acid-binding protein 4 (FABP4) in the wounded skin. HMOX1 has antioxidant and tissue-protective actions. FABP4 helps skin regeneration through fibroblast migration and extracellular matrix production (Lima et al. 2011; Furuhashi et al. 2014; Rivera-Gonzalez et al. 2014; Song et al. 2017). HMOX1 and FABP4 expression levels were increased by burn injury, and the LPSHE or Commercial ointment treatment further increased the FABP4 and HMOX1 levels (Figure 2(E)). Previous studies demonstrated that reducing ROS could be an attractive strategy for enhancing the wound-healing capacity (Parihar et al. 2008; Mittal et al. 2014). We confirmed that the increased ROS levels (nitrotyrosine and 4-hydroxynoneal expression) after burn injury were reduced by LPSHE or Commercial ointment treatment (Figure 2(F)). In addition, blood ROS levels also reduced by LPSHE or Commercial ointment treated groups (Figure 2(G)). Taken together, these burn wound-healing phenotypes demonstrated that LPSHE has burn wound-healing efficacy similar to Commercial ointment.

LPSHE attenuates hyperinflammation after burn injury

In the first phase of wound healing, acute and local immune responses activate the innate immune response (Engelhardt et al. 1998). However, hyperinflammation of multiple organs causes detrimental effects, disrupting the immune equilibrium and disturbing re-epithelialization (Evers et al. 2010; Burgess et al. 2022; Żwierełło et al. 2023). Therefore, hyperinflammation must be regulated to improve burn wound healing. The burn mouse model was treated with the LPSHE or Commercial ointment for three days after the burn injury (Figure 3(A)). To assess inflammatory responses, we examined the expression level of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the liver, lungs, spleen, kidneys, and skin. Increasing pro-inflammatory cytokines after burn injury was alleviated by LPSHE or Commercial ointment treatment (Figure 3(B–D)). The hematoxylin and eosin (H&E) staining results showed that inflammatory infiltrates in the multi-organs were reduced by the LPSHE or Commercial ointment treatment (Figure 3(E) and Figure S3A). In addition, IHC analysis demonstrated a reduction in CD3⁺ T cell infiltration following the LPSHE or Commercial ointment treatment (Figure 3(F) and Figure S4A). The inflammation score, which was assessed from 0 to 5 by analyzing the degree of infiltration in the H&E staining and IHC results, confirmed that the LPSHE or Commercial ointment regulated hyperinflammation (Figure 3(G) and Figure S4B). These results suggested that the LPSHE alleviates burn injury-induced hyperinflammation phenotypes.

Animal care protocol and establishment of a burn mouse model

Six-week-old male BALB/c nude mice (Orient Bio, Republic of Korea) were used for the in vivo experiments. The animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Pusan National University (Approval Number PNU-2022-0060). In vivo experiments were performed under the guidelines of the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. The mice were kept at the animal care facilities placed on temperature-maintained room (23±1°C) under a 12 h light/dark cycle and were fed commercial mouse chow and water. For burn injury, mice (n=5) were anesthetized and the dorsal hairs were clipped. The full-thickness burn wound was generated by 10 sec of contact with a 10-mm-diameter brass rod heated to 95°C. Mice were then administrated intraperitoneally with 1 mL of 37°C pre-warmed sterile isotonic saline to prevent shock symptom. After 1 day of burn wound generation, 500 mg of LPSHE and Commercial ointment was daily treated on mice burn-wounded area until the burn wound was fully recovered.

Treatment of LPSHE and commercial ointment

Purified LPSHE was prepared as a laboratory-grade ointment. 100 mg of LPSHE, glycerin, ethanol, and water per 1 g of ointment was appropriately mixed at room temperature. Burn-commercial ointment currently on the market, Madecassol Care Commercial ointment, was used as a positive control. Madecassol Care Commercial ointment mainly contains titrated extraction of Centella Asiatica 10 mg/g, neomycin sulfate 3.5 mg/g. And 500 mg of LPSHE and Commercial ointment was treated on mice burn-wounded area. Burn mouse group was treated with laboratory-grade ointment, which was excepted LPSHE as negative control.

Histological analysis

The tissue preparation protocol was validated as previously described (Son et al. 2019; Kim et al. 2023). At the end of the treatment period, animals were euthanized, and liver, lung, spleen, kidney, or skin samples were harvested. The samples were then fixed with formalin, dehydrated, and embedded in paraffin. Next, sections were cut into 4 mm and utilized for H&E, Masson’s trichrome staining, or immunohistochemistry (IHC) following standard procedures. Stained sections were observed under an Olympus IX71 microscope (Olympus Optical, Japan). Inflammation scores were obtained as previously described (Seno et al. 2015). The inflammation conditions were evaluated using at least three H&E stained tissue sections, and IHC sections of the liver, lung, spleen, and kidney per mouse followed by the instructions described below. Inflammation scores were determined as follows: 0, no inflammation; 1, cellular infiltration only in the perivascular areas; 2, mild cellular infiltration (less than one-third part of the sections is infiltrated with inflammatory cells); 3, moderate cellular infiltration (more than one-third part of the sections is infiltrated with inflammatory cells); and 4, infiltration of inflammatory cells is observed in the whole part of the sections.

Immunofluorescence and phalloidin staining

For HaCaT keratinocytes, the cells were fixed and permeabilized in cold acetone and then washed with cold PBS. For paraffinized tissue samples, deparaffinization was performed. After blocking with 1% BSA in PBS, the cells were incubated overnight with primary antibody at 4°C. Next, the cells were washed three times with cold PBS and incubated with DyLight 488-conjugated secondary antibodies (Thermo Fisher Scientific). After washing and counterstaining with DAPI (Sigma Aldrich), the glass slides were mounted with VECTASHIELD Hard-Set Mounting Medium (Vector Laboratories, Burlingame, CA) and visualized with an Olympus IX71 fluorescence microscope (Olympus Optical Co. Ltd., Tokyo, Japan). Phalloidin (Sigma Aldrich) staining was assessed according to the manufacturer’s protocol.

ROS quantification

The DCFDA Cellular ROS Assay Kit was used (#ab113851, abcam) for detecting ROS levels. Around 100 μl of mouse blood sample or 1×10⁴ of HaCaT cells were placed per well in 96-well plates. After appropriate treatments, incubated with the diluted DCFDA solution and Hoechst 33342 (#H3570, Invitrogen) for 1 h at 37°C protected from light. After incubation, the solutions were removed and 100 μl of buffer was added. Fluorescence was measured using a GloMax® Discover Microplate Reader at Ex/Em of 485/535 nm.

Cell culture

The culture methods of HaCaT cells were described in a previous study (Son et al. 2019). In detail, HaCaT cells were purchased from Korean Cell Line Bank (Seoul, Republic of Korea) and cultured in DMEM media consisting of 10% FBS and 1% penicillin (10,000 U/mL) – streptomycin (10,000 µg/mL) in a CO₂ incubator (37°C, 5% CO₂). Cells were free of mycoplasma contamination and were authenticated by short tandem repeat profiling within the past 12 months.

Wound-healing assay

Wound-healing assays were performed to measure changes in cell motility as previously described (Kim et al. 2016). In brief, HaCaT cells were cultured to 70% confluence and treated with LPSHE. The cell monolayers were then scratched with 200 mL pipette tips. Cells were then further incubated for 24 or 48 h. Photomicrographs were then taken at 100× magnification using the Olympus IX71 fluorescence microscope (Olympus Optical Co. Ltd., Tokyo, Japan).

Total RNA isolation and qRT-PCR

Total RNA was extracted from HaCaT cells or mouse tissue with AccuPrep® Universal RNA Extraction Kit (#K-3140, bioneer, Korea). Synthesize cDNA was analyzed by Real-time qRT-PCR using an Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems, USA). The qPCR primers sequences of the primers used are Mouse Il1β F: 5′-GCA ACT GTT CCT GAA CTC AAC T-3′, R: 5′-ATC TTT TGG GGT CCG TCA ACT-3′. Mouse Il6 F: 5′-GCT TAA TTA CAC ATG TTC TCT GGG AAA-3′, R: 5′-CAA GTG CAT CAT CGT TGT TCA TAC-3′. Mouse Tnf F: 5′-TCG TAG CAA ACC ACC AAG TG-3′, R: 5′-AGA TAG CAA ATC GGC TGA CG-3′. Mouse Hmox1 F: 5′-AGG AGC TGC ACC GAA GGG CTG-3′, R: 5′-CGT GGA GAC GCT TTA CAT AG-3′. Mouse Fabp4 F: 5′-TCA CCA TCC GGT CAG AGA GTA-3′. R: 5′-GCC ATC TAG GGT TAT GAT GCT C-3′. Mouse Gapdh F: 5′-CGA CTT CAA CAG CAA CTC CCA CTC TTC C-3′, R: 5′-TGG GTG GTC CAG GGT TTC TTA CTC CTT-3′. Human IL1β F: 5′-TTC TTC GAC ACA TGG GAT AAC G-3′, R: 5′-TCC CGG AGC GTG CAG TTC A-3′. Human IL6 F: 5′-TGA ACT CCT TCT CCA CAA GCG-3′, R: 5′-TCT TGG AGC TTA TTA AAG GCA TTC-3′. Human TNF F: 5′-CCG AGT GAC AAG CCT GTA GCC-3′, R: 5′-CCT TGA AGA GGA CCT GGG AGT AG-3′. Human HMOX1 F: 5′-CCA GGC AGA GAA TGC TGA GTT C-3′, R: 5′-AAG ACT GGG CTC TCC TTG TTG C-3′. Human FABP4 F: 5′-TTG ACG AAG TCA CTG CAG ATG A-3′, R: 5′-CAG GAC ACC CCC ATC TAA. Human GAPDH F: 5′-ATG ACA TCA AGA AGG TGG TG-3’, R: 5’-CAT ACC AGG AAA TGA GCT TG-3′. Each sample was assessed by triplication.

Western blot analysis

For acquiring the whole cell lysates, CETi Lysis Buffer with Inhibitors (TransLab, Korea) was used. The protein concentrations were determined by using a BioRad protein assay kit (BioRad Laboratories, USA). The protein samples were performed with SDS-PAGE, transferred to the NC (nitrocellulose) membrane, and blocked with 5% BSA in TBST for 2 h at room temperature. After incubation, membranes were treated with specific primary antibodies for overnight at 4°C. Washed with TBST and treated with peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, USA). The membranes were visualized using an ECL detection system (Roche Applied Science, USA) with iBright chemi-doc fl000 (Thermo Fisher Scientific, USA). Each analysis was assessed by triplication.

Cell viability assay

For cell viability assay, HaCaT cells were seeded at 10,000 cells per well in 96-well plates 1 d before treatment of LPSHE for 24 h. Cell viability was determined using CellTiter-Glo® Luminescent Viability Assay kit (#G7570, Promega).

We thank WoojungBio Analysis Center (Seoul, Republic of Korea) for analyzing LC/MS–MS and NMR data. We also thank Editage for English language editing.