Effect of combination of photobiomodulation 904 nm superpulsed laser therapy and Hippophae rhamnoides L. on third‐degree burn wound healing

Burn is a traumatic injury and aesthetic scarless repair poses a great challenge in area of cosmetic dermatology. Focus on multimode therapeutic strategies to promote healing of burns by regulating various stages of healing is warranted. Photobiomodulation therapy (PBMT), a non‐invasive modality grabs the attention to repair impaired wounds. Seabuckthorn extract (SBTL‐ALE) is known to possess antioxidant, anti‐inflammation, and tissue‐repair abilities. Current study aims to assess the effect of combination treatment of PBM 904 nm superpulsed laser and SBTL‐ALE (2.5%) on repair of third‐degree burn in rats.


| INTRODUC TI ON
Burn injury is the most devastating complication that causes financial burden, morbidity, occasionally mortality, and poses considerable challenges in dermocosmetic research area. Burns obstruct the healing process due to lengthened, dysregulated inflammatory responses, epidermal disruption, reduced angiogenesis, growth factors, hypoxia, oxidative damage, metabolic alterations, enhanced proteases, and septicemia. [1][2][3] In order to effectively manage burn treatment, there is an urgent need to search for alternative therapeutic approaches that may influence the various phases of repair and accelerate burn wound healing. Phytomedicine obtained from herbal sources is in great demand as its potential phytoconstituents can cure various disorders. Traditional phytomedicines are attracting renewed attention as a safe, affordable, and cost-effective approach with limited side effects. 4 Seabuckthorn (SBT, Hippophae rhamnoides L.) is an evergreen plant growing under adverse climatic conditions in Europe and Asia. It has been widely used and studied for its pharmacological effects on inflammatory, respiratory, digestive, and skin disorders. 5,6 Earlier studies from our lab have demonstrated that Seabuckthorn leaf aqueous lyophilized extract (SBTL-ALE) possesses potent antioxidative, anti-inflammatory, and reparative properties, and helps to accelerate normal and delayed wound healing. 7,8 Photobiomodulation therapy (PBMT) is defined as a non-thermal, biophysical healing modality, which employs the non-invasive delivery of photons to elicit photophysical and photochemical events, which lead to the alteration of biological activities. The light sources can be coherent or non-coherent, typically in the region of the visible red and near-infrared (NIR, 600-1100 nm) spectrum. PBMT exploits the absorption of light (light-tissue interaction) and intracellular chromophore activation to affect the downstream signaling cascade. Distinctive properties of PBM have allowed its use in the treatment of various medical disorders, including enhanced healing of chronic wounds. 9 Operation mode of PBMT can either be continuous, pulsed, or superpulsed wave. Superpulsed laser consecutively emits relatively strong high-powered light in nano/picosecond pulses, and deeper tissue penetration in a non-thermal way. [10][11][12] Recent studies have manifested that 904 nm superpulsed laser dynamically improved impaired wound healing. 11,13 Multimode therapies with the binary or other combinations of pharmacological therapeutic interventions and biophysical modalities have been shown to effectively improve the cure rates of several diseases, including the healing of intractable impaired wounds.
Hence, currently, these combined therapies are a focus of investigation globally. Several studies use PBMT as one of the treatment strategies in combination with different bioactive compounds/natural products like arginine, vitamin A, lactate, pyruvate, coenzyme Q10 (Co-Q10), curcumin, and honey to improve the curing rate of the non-healing wounds. 10,[14][15][16][17][18][19][20][21] Studies have shown the positive impact of the integrated treatment of herbal bioactive agents and PBM on wound repair and regeneration. The combined effect of curcumin and 890 nm pulsed laser PBM significantly enhanced wound repair by modulating inflammatory response, fibroblast proliferation, and angiogenesis on the excision-type wound in STZ-induced diabetic rats. 16 Moradi et al. 17 demonstrated that the combined influence of pulsed 890 nm PBMT (80 Hz, 0.2 J/cm 2 ) with curcumin nanoparticles accelerated healing through enhanced wound contraction, tensile strength, and decreased S. aureus counts in full-thickness excision wound in mice. Similarly, the combined impact of superpulsed 904 nm PBMT and medicinal honey synergistically attenuated inflammation, pain, and increased cellular proliferation during burn wound repair in rats. 20 In view of the complications and multi-etiological factors associated with chronic burn injuries, the current study assesses the impact of combined treatment of PBM 904 nm superpulsed laser and SBTL-ALE on third-degree burn repair in rats. This scientific approach hypothesizes that combined application of superpulsed 904 nm PBMT with SBTL-ALE regimen may accelerate burn wound healing by reducing inflammation, maintaining redox homeostasis, enhancing proliferation, neovascularization, dermal hydration, collagen deposition, and bioenergetics activation.

| Burn wound creation
The rat was anesthetized using an intraperitoneal injection of ketamine and xylazine cocktail in the proportion of 90:10 mg/kg. As reported previously, a third-degree transdermal burn wound was created on the dorsal side of the rats under aseptic conditions, except for the uninjured skin group of rats. 10 Briefly, the hairs were removed from the dorsal surface of the rats, and the underlying skin was cleaned with ethanol (70%). A circular metallic probe (1.5 cm diameter) was heated to 85°C (in hot water) and kept for 20 s on the depilated skin of the rat, followed by resuscitation with intraperitoneal administration of sterile normal saline. The animals were administered buprenorphine as an analgesic following burn injury.
After 24 h of burn induction, burn tissues were removed using surgical. The transdermal burn wound was kept uncovered (without any dressing) for the entire period of the experiment.

| Study design and treatment schedule
Twenty-five rats were randomly distributed into five groups, including uninjured skin group, control group that received no therapy, SBTL-ALE group treated with SBTL-ALE ointment, PBMT group treated with 904 nm superpulsed laser, and combination group that received dual treatment. SBTL-ALE group received topical application of SBTL-ALE ointment (2.5%, w/w) twice daily in the morning and evening for 7 postwounding days. 7 PBMT group received superpulsed 904 nm laser for 10 min once a day for seven postwounding days, as previously mentioned. 10 In the combination group, the PBM treatment was given once daily in the morning and SBTL-ALE ointment topical application twice daily in the morning and evening. There was an interval of 1 h between PBM treatment and SBTL-ALE topical application in the morning ( Figure 1). All the rats were euthanized after 7 days and the wound tissues were collected for biochemical, molecular, histological, and immunohistochemical analyses.

| Wound area contraction
Wound surface area was measured by capturing the images of wounds along with a ruler using a digital camera (Nikon Coolpix S3400), as described previously. 11 The digital photographs were captured on 0 (day of injury), 4th, and 8th-day postwounding. A ruler kept adjacent to each wound was used for the precise measurement of the wound area. The known distance of the ruler was measured in the number of pixels per centimeter in each digital image using image analysis software (ImageJ, NIH, USA). The outline of the wound area was created using the 'polygon selection tool' and measured in mm 2 .
The percent change in wound contraction was estimated by the formula given below:

| Histological investigation
A 10% buffer formalin was used to preserve histological samples of the wound tissues. The Skin tissue section of 5μm was stained using hematoxylin and eosin. Histopathological alterations such as cellular proliferation, angiogenesis, re-epithelialization, collagen bundles, and inflammation were examined under the light microscope (Olympus BX61TRF, Japan).
%Change in wound area = (Initial wound area − Area on measurement day)∕Initial wound area × 100 F I G U R E 1 Image depicting the experimental setup for photobiomodulation therapy (PBMT) using superpulsed 904 nm laser in rats and the detail of the PBM parameters for the treatment of transdermal third-degree burn wound.

| Immunohistochemical analysis
The wound tissues were collected and processed for Immunohistochemical (IHC) analysis as described previously. 7 Sections were probed with the following primary antibodies:

| ELISA assays for inflammatory and oxidative stress markers
A 20% (w/v) skin homogenate was made using RIPA lysis buffer. After sonicating the homogenate, it was centrifuged for 20 min at 5000 g and then the supernatant was used for the ELISA quantification for various inflammatory markers: TNFα (ELR-TNFα), IL-1β (ELR-1 L-1b, Ray Biotech), IL-6 (ELR-IL6, Ray Biotech), and NOS-2 (E-EL-R0520, Elabscience) using ELISA assay kits according to manufacturer's instructions. The protein concentration was measured following the method of Lowry et al. 23 A 10% (w/v) skin homogenate made in KCL-EDTA buffer was used for assays of oxidative stress (ROS levels) and antioxidant catalase activity. 10 DCFH-DA (100 μM), a fluorescent dye was used to measure ROS levels in crude tissue homogenate. 24 The residual homogenate was used to estimate catalase activity as previously mentioned. 25

| Assays of energy metabolizing enzymes activities and ATP level
Samples for the estimation of enzymatic activity were prepared, as described previously. 26 Samples were used to measure the lactate dehydrogenase (LDH) and citrate synthase (CS) activities, as per the methods described earlier. 27,28 Further, a 20% (w/v) skin tissue homogenate was made using RIPA lysis buffer to determine cytochrome c oxidase (CCO) enzymatic activity and ATP contents. Samples were then centrifuged for 30 min at 9000 × g. CCO activity and ATP level were analyzed using assay kits, Cytocox1 and FL-AA (bioluminescent) (Sigma-Aldrich), respectively, according to the manufacturer's guidelines.

| Statistical analysis
The data of all parameters were expressed as mean ± SE. One-way ANOVA with Bonferroni's post-hoc analysis was performed through software (GraphPad Prism) to evaluate the statistical significance between the different groups. A p < 0.05 was regarded significant.

| Combination therapy additively enhanced wound contraction
The present findings revealed that SBTL-ALE, 904 nm laser, and combined therapy enhanced the healing process prominently as compared to the burn control. Visual observations showed a progressive repair without edema, pus, and less inflammation on the peripheral skin near the injury site in the combination group compared to the control and standalone treatments (Figure 2A). Indeed, the best wound repair outcome was detected in combination treatment as evidenced by significantly (p < 0.05) faster wound area contraction on the 4th and 8th day postwounding in comparison to the burn control ( Figure 2B).

| Combination therapy enhanced reepithelialization, proliferation, and collagen deposition
Findings of histopathological examinations revealed that maximal augmented healing was exhibited in the group receiving combined treatment in comparison to control as well as standalone treatments. The combined treatment exhibited improved fibroblast proliferation, neovascularization, accumulation of well-arranged collagen bundle, re-epithelialization, and a decreased infiltration of inflammatory cells. However, the control group exhibited a denuded epidermis, reduced collagen accumulation, and an increased number of inflammatory cell infiltrations ( Figure 3A).
Accumulation of collagen was also confirmed through IHC analysis, which showed significantly enhanced (fivefold change) expression of collagen type 3 in combined treatment as compared to the control group. However, uninjured skin showed a significantly lower level of collagen type 3 expressions as compared to the control ( Figure 3B).

| Combined treatment accelerated angiogenesis, dermal hydration, ECM deposition, and redox homeostasis
Immunohistochemical analyses of molecular markers pertaining to angiogenesis (CD31), dermal hydration (AQP3), and redox homeostasis (Nrf2 and HO1) revealed enhanced expression (p < 0.05) in However, the uninjured skin group exhibited substantially (p < 0.05) decreased levels of CD31, AQP3, and HO1 as compared to burn control, which indicated that burn induction elicited significant alterations in these markers.

| Combined treatment enhanced bioenergetics activation
SBTL-ALE, 904 nm laser, and combination treatment caused a significant alteration in the activities of key enzymes involved in energy metabolism. CS activity was found to be significantly increased (twofold change, p < 0.05) in combined treatment in comparison to the control. However, LDH activity was significantly reduced (52%, p < 0.05) in combined treatment in comparison to the control. Furthermore, the combined treatment group showed a maximum rise in CCO activity (85%) and ATP level (9.7-fold change) among all treated groups compared with the control. The LDH activity was substantially (p < 0.05) reduced in the uninjured group compared to the burn control (Figure 7).
(890 nm laser, 80 Hz) and topical application of arginine (2%) considerably improved wound strength and stereological markers compared to the control rats. Similar to this, Otterco et al. 15 investigated that vitamin A treatment combined with PBM (670 nm laser) improved acute wound healing in rats by regulating cellular pathways involved in the inflammatory phase and neo-vascularization.
In another study, pulsed PBMT (890 nm laser) in combination with metformin has been shown to synergistically promote wound healing by reducing inflammation, tensiometeric, and stereological parameters in type-2 diabetic rats. 18  The results of the present study revealed that combined treatment significantly decreased inflammation evidenced by decreased proinflammatory markers viz. TNFα, IL-6, IL-1β, and NOS-2 levels. SBTL-ALE was reported to possess potent antioxidant and anti-inflammatory properties owing to the presence of quercetin as a major flavonoid. 22 The present findings are in accordance with Ahmed et al. 33 who reported that a combination of quercetin and 632.8 nm PBMT enhanced dermal wound healing by increasing collagen synthesis, reducing inflammation and oxidative stress in non-diabetic and diabetic rats compared to either treatment alone. Additionally, our recent study showed that SBTL-ALE alone topical application reduced oxidative stress and inflammation by decreasing levels of TNFα, IL-6, IL-1β, NOS-2, and ROS during burn wound repair. 7 In agreement with our results, Yadav et al. 20 revealed that combined therapy of 904 nm superpulsed PBM with medicinal honey attenuated inflammatory responses evident by significantly decreased NF-κB, TNFα, and IL-1β levels during burn wound repair in rats. Likewise, Beserra et al. 34 also reported that lupeol (natural triterpene) topical treatment down-regulated NF-κB, TNFα, and IL-1β, IL-6 levels, and improved excision-type dermal wound repair in rats.
There is a need for a greater amount of energy for the proliferation, differentiation, and migration of cells during the tissue repair process. The glycolytic pathway is a major source of energy for fibroblast, which synthesizes myofibroblast and collagen. The findings of the current investigation showed that the combined treatment resulted in bioenergetics activation during burn wound healing. CCO functions as a complex-IV of the electron transport chain where it reduces oxygen molecules to produce ATP and water. The results of F I G U R E 6 Effects of Seabuckthorn leaf aqueous lyophilized extract (SBTL-ALE), 904 nm superpulsed laser photobiomodulation (PBM), and combination (904 nm PBM + SBTL-ALE) treatments on oxidative stress (ROS, catalase) and inflammatory markers (NOS-2, TNFα, IL-6, IL-1β) during burn wound healing in rats. Values are mean ± SE, n = 5 rats per group. *p < 0.05 compared with control.
this study revealed that the combined therapy enhanced the activity of CS and CCO along with increased ATP levels, and decreased LDH activity, which led to bioenergetics activation during burn repair.
Similar to this, Yadav et al. 13 claimed that superpulsed PBM 904 nm laser alone treatment accelerated burn wound healing by modulating aerobic energy metabolism for maximal energy output. Similarly, SBTL-ALE alone treatment modulated the activities of key energy regulatory enzymes like HK, LDH, CS, G6PD, CCO, and ATP levels to improve burn wound repair. 7

| CON CLUS IONS
Taken together, the findings of the present study indicated that the combined application of superpulsed 904 nm laser PBMT and SBTL-ALE extract markedly improves third-degree burn repair in comparison to control as well as standalone treatments.
Overall, combined treatment synergistically enhanced burn repair by accelerating cellular proliferation, neovascularization, collagen deposition, attenuating inflammatory responses, and reducing oxidative stress along with bioenergetics activation. Further, this dual treatment might have the immense potential to be topically used on full-thickness third-degree wounds as an effective therapeutic modality in burn care management.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
All experimental protocols in this study were approved by the Institutional Animal Ethical Committee (DIPAS/IAEC/2017/14/ F I G U R E 7 Effects of Seabuckthorn leaf aqueous lyophilized extract (SBTL-ALE), 904 nm superpulsed laser, and combination (904 nm PBM + SBTL-ALE) treatments on enzymatic activities associated with energy metabolism and bioenergetics status: lactate dehydrogenase, CS, cytochrome c oxidase; LDH, citrate synthase; CCO and ATP levels during burn wound healing in rats. Values are mean ± SE, n = 5 rats per group. *p < 0.05 compared with control.
Ext./2019), and the animal experiments performed complied with the guidelines of the CPCSEA, Government of India.