Anti-inflammatory and cell proliferative effect of the 1270 nm laser irradiation on the BALB/c nude mouse model involves activation of the cell antioxidant system

Recently, many interdisciplinary community researchers have focused their efforts on study of the low-level light irradiation effects (photobiomodulation, PBM) as a promising therapeutic technology. Among the priorities, a search of new wavelength ranges of laser radiation to enhance the laser prospects in treatment of autoimmune and cancer diseases commonly accompanied by disorders in the antioxidant system of the body. The laser wavelengths within 1265-1270 nm corresponds to the maximum oxygen absorption band. Therefore, PBM effects on a model organism within this spectrum range are of particular interest for preclinical research. Here, we report comprehensive biomolecular studies of the changes in the BALB/c nude mice skin after an exposure to the continuous laser radiation at the 1270 nm wavelength and energy densities of 0.12 and 1.2 J/cm. Such regime induces both local and systemic PBM effects, presumably due to the short-term increase in ROS levels, which in turn activate the cell antioxidative system. © 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Therefore, we propose a comprehensive biomolecular analysis of the changes in the BALB/c Nude mice skin after an exposure to the continuous irradiation at the 1270 nm wavelength.

Materials and methods
Immunodeficient BALB/c Nude mice ("Pushchino" Ltd., Russia) were selected for control and experimental groups as an object of the study.The skin of these mice does not require hair removing, thus eliminating risk of additional skin irritation. Two experimental groups include animals (n = 8) irradiated by Raman laser operating at the wavelength of 1270 nm and power of 1.96 mW. Animals were exposed to a single irradiation session for 2 min at the intensity of 10 mW/cm 2 and energy density (ED) of 1.2 J/cm 2 , and for 12 seconds at the intensity of 10 mW/cm 2 and ED of 0.12 J/cm 2 . Control group animals (n = 7) were not exposed to laser irradiation.
The next day after irradiation, the animals from control and experimental groups were taken under ether anesthesia. The blood samples were taken and heparin-stabilized animal blood was used for further biochemical and immunological studies. Skin sample with a spot diameter of 0.5 cm exposed to laser irradiation on the lateral surface of the body was studied. The same skin area was explored in the control group animals. Unirradiated skin segments of control animals and irradiated skin regions of experimental animals were fixed in 10% neutral formalin for histological examination and in RNA-later solution for further studies on microRNA expression. All experiments were performed in accordance with World Medical Association Declaration of Helsinki (1964Helsinki ( ) amended in 1975Helsinki ( , 1983Helsinki ( and 1989 and Ethical Approval Committee of Ulyanovsk State University (No. 9, September 15, 2017).

Morphological methods
The irradiated skin patch of 0.5 cm in diameter was taken for histological assessment. The biopsy material was fixed in 10% neutral formalin and series of skin sections were stained with hematoxylin-eosin. Morphometry of skin structures was carried out using a video-test system supplied with Motic microscope, JVC digital video camera (Japan), and density photometry software "Mekos-С1" (Russia) at 10 to 40 times magnification. The quantitative analysis was performed for a conventional unit of the sections. Morphometric data were recognized as statistically significant when P calculated by the two-sided Student t-test was <0.05.

Molecular genetics methods
The irradiated skin section 0.5 x 0.5 cm was fixed in the RNA-later solution (LLC "Eurogen", Russia) and then was cut using sterile tweezers for an isolation of total RNA employing the "Extraction 100" kit (LLC "Vector Best", Russia). The reverse transcription reaction was launched immediately after the RNA isolation (set MMLV Kit, LLC Eurogen, Russia) with miRprimer2 to design primers for the microRNAs examined. MicroRNAs miR-31, −130а, −191, −200с, −205 were used to investigate the expression pattern and its involvements in the skin physiological processes [35][36][37][38][39][40][41][42][43]. MicroRNA profile for the skin samples were evaluated with the CX96 nucleic acid PCR amplifier (BioRad, USA). To normalize the data, miRNA-191 was chosen as the skin-referred gene for skin cells.

Biochemical methods
The level of lipid peroxidation products (diene conjugates (DC), ketodienes (KD), Schiff bases (SB), malondialdehyde (MDA)) and activity of the antioxidant system enzyme, catalase and reduced glutathione (GSH) level in the red blood cell hemolysate (1:10) were assessed. The DC, KD, conjugate trienes(CT) and SB levels were spectrophotometrically determined by Volchegorsky method [44] in heptane-isopropanol extracts. The concentrations of these products were calculated from the ratios between the optical densities of the heptane phase of E232/220 (DK), E278/220 (KD and CT), E400/E220 (SB) and were given in oxidation index units (O.I.U.). The MDA level (μmol/L) was determined in the test with thiobarbituric acid according to [45]. The catalase activity and GSH level were determined as referred [46].
The statistical significance of the results was estimated using the nonparametric Mann-Whitney U-test. Differences between groups were considered significant at p<0.05.

Immunological methods
To assay the cytokine levels in the skin homogenate and blood plasma from the control and experimental animals, immunoenzyme kits were used to quantify the murine tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) (eBioscience, USA).The statistical significance of the results was estimated using the nonparametric Mann-Whitney U-test. Differences between groups were considered significant at p<0.05.

Results and discussion
The laboratory BALB/c Nude mice used in the experiments are well known hairless and immunodeficient model which makes them suitable for research in dermatology, oncology, and immunobiology. The mice skin thickness measured by histology has been about 500 µm. Since the present research highlights the laser radiation effect on physiological processes in skin at the wavelength of 1270 nm, its results allow estimating laser potential for melanoma treating. So, the BALB/cNude mice used as a model are quite suitable for the experiment.
I. The histological analysis has shown that the back skin of the control group animals has a fine-folded relief shape and thin epidermis.
Epidermis usually comprises 2-3 rows of grown cell layers. In the first row (basal layer), the basal cells with dark purple round nuclei are clearly visible (Fig. 1). The second row (thorny layer) comprises 1-2 rows of keratinocytes. Next granular layer includes one row of strongly flattened cells with uneasily distinguished nuclei. However, the dark-violet keratohyalin granules are easily observed in the cytoplasm. The stratum corneum is thin and tightly adjacent to the granular layer peeling off in some places. A slight diffuse lymphoidleukocyte infiltration is observed in dermis. The dermis papillary layer is smoothed and papillae are sparse. Loose fibrous unformed connective tissue located sub-epidermally consists of thin collagen fibers and transfers into reticular dermis layer with no sharp boundaries.
The reticular dermis layer is composed of a dense fibrous unformed connective tissue consisting of multidirectional thick collagen fibrils. The connective tissue cells are mainly fibroblasts, macrophages, tissue basophils and some other differentiated cellular elements.
Noteworthy, histological analysis of skin samples in the second experimental group irradiated at the wavelength of 1270 nm and E = 0.12 J/cm 2 demonstrated no differences from the control group animals (Fig. 2). After laser exposure at the wavelength of 1270 nm and E = 1.2 J/cm 2 the mice skin has also a fine-folded relief and the skin structure is not changed (Fig. 3). The epidermis was not thickened over the normal size. The growth layer comprises 2-3 rows of keratinocytes with definablycolored rounded nuclei which have clearly visible nucleoli. In some places, the growth layer cells are overlapping each other. In these regions, the nuclei are elongated. The granular layer and stratum corneum are kept unmodified. Like in the control group, the stratum corneum is thin with no signs of damage, peeling off, and vacuolization. The dermis papillary layer is not modified clearly indicating that the stratum corneum does not show any pathological signs. In the interlobular connective tissue of subcutaneous adipose tissue and in endomisis, perimisia (intermuscular connective tissue), moderate diffuse lymphoid-leukocyte infiltration was visualized.
In the first experimental group, the morphometric analysis reveals a significant proliferation of keratinocytes in the basal layer compared to the control group (Table 1). An increase in the number of basal epitheliocytes is probably due to a higher level of cell mitotic activity in this layer after exposure to 1270 nm laser irradiation. In the material, there are few mitotic figures, since the experimental material is taken 24 hours after irradiation. Mitotic division lasts some 6-8 hours. Thus, 24 hours after laser exposure more nuclei are observed per unit area. In the second experimental group, no significant differences from the control group were registered.
As it was reported, the PBM experiments on dog epidermal keratinocytes, at the energy densities of 0.1, 0.2, 1.2 and 10 J/cm 2 (He-Ne laser, λ = 650 nm) stimulated the proliferative activity of skin cells. Therefore, these doses were chosen for experimental study on healing wounds in vivo models [19].
II. Experimental studies show the role of microRNAs in maintaining homeostasis of skin as an integral organ through their participation in regulation of proliferation, differentiation and apoptosis [16,35,36]. MicroRNAs are a relatively new class of endogenous, non-coding single-stranded small RNAs (18-22 nucleotides) participating in regulation of gene expression at the posttranscriptional level by inhibiting translation or destruction of specific transcripts of matrix RNA [37]. The pattern of microRNA expression has some tissue specificity. The expression pattern is studied for microRNAs, whose role in the physiological processes of skin has been demonstrated [39,47,48], namely, miR-31, −130а, −191, −200с, −205. For endogenous control, TaqMan Advanced miRNA Assays User Guide (ThermoFisher Scientific, USA, Cat. #A25576) recommends microRNA-191 to be used.
MicroRNA-31 is a key regulator of the proliferation and migration of keratinocytes (dividing cells of basal layer), differentiation and hair growth [38]. Increased expression of mir-31 appeared to be in some skin diseases characterized by excessive proliferation of keratinocytes, e.g., psoriasis, chronic inflammatory skin disease, long-term wound healing [39,40], and squamous cell carcinoma [41].
It has been demonstrated that miR-130a level increases in chronic wounds and delayed reepithelization [35]. Both MicroRNAs-200c and −205 participate in regulation of the cell cycle [42], as well as in the epithelial-mesenchymal transition and cell differentiation to the embryonic phenotype through direct (miRNA-200c) and intermediary (miRNA-205) regulation of ZEB1 and ZEB2 gene expression which suppresses E-cadherin gene transcription [43]. Thus, these miRNAs were chosen to study the genetic mechanisms involved in regulating the response to PBM 1270 nm.
Real-time PCR is used to assay miRNA expression in irradiated skin areas and intact area from the opposite side after laser exposure at E = 1.2 J/cm 2 . Figure 4 shows the expression of miR-31, −200c in skin after laser exposure.
In all skin samples irradiated with 1270 nm laser (E = 1.2 J/cm 2 ) a significant increase in miR-31 expression has been observed (Fig. 4), whereas microRNA-200c do not demonstrate such an overall change. Expression of these genes has only been observed in two animals (No.1, 4) in the experimental group. However, in most cases (75%), the mir-200c expression went down. The data are calculated for endogenous control of miR-191 and are given in relative units to compare expression dynamics in the irradiated and non-irradiated skin region of the same mouse.
No significant changes in the expression profile of miR-205 and miR-130a were observed in any sample.  In the second experimental group, there are no changes in miRNA expression. III. Biochemical studies on blood hemolysate of BALB/c Nude mice show that PBM of 1270 nm and E = 1.2 J/cm 2 leads to a significant increase of the reduced glutathione level (GSH) in erythrocytes of irradiated mice (Table 2), whereas the level of primary lipid oxidation products (diene conjugates) increases slightly and the levels of ketodienes and Schiff bases in contrast demonstrate significant decrease (Table 3). Among the products of protein oxidative modifications, the level of primary ketone groups (530 nm) decrease significantly in mouse erythrocytes ( Table 4). The obtained data assume the increase in the antioxidant status of mice red blood cells under PBM of 1.2 J/cm 2 .   This irradiation regime (1.2 J/cm 2 ) leads to protection of the membrane proteins and lipids, decrease in the pro-oxidant effect due to activation of antioxidant mechanisms, in particular, the endogenous glutathione system. The observed pronounced increase in GSH, as the main red blood cell antioxidant, could indicate an increase in its synthesis by glutathionedependent enzymes and through its participation in utilization of pro-oxidants. The most pronounced changes are observed in the level of oxidative modification of membrane proteins (Table 4). It can indicate a greater sensitivity of protein molecules to PBMat the wavelength of 1270 nm. Twenty-four hours after laser exposure, detoxification of reactive oxygen species intensifies that could indirectly indicate an increase of the singlet oxygen generation in response to laser exposure.
Thus, PBM causes an activation of the endogenous antioxidant defense systems and suppresses the action of reactive oxygen species generated in BALB/c Nude mice red blood cells immediately after laser exposure. This effect is consistent with the reported data highlighting the PBM-induced membrane-stabilizing effect and enhanced antioxidant blood properties reported earlier [49][50][51]. Thus, PBM stimulates the protective effect in red blood cells even at short exposure time and low irradiation dose (1.2 J/cm 2 ).
IV. The cytokines usually act through interaction with specific receptors at the plasma membrane rafts, which are expressed in small amounts on the cells [52]. They regulate the activation, differentiation and proliferation of immunocompetent and somatic cells. These regulatory proteins are involved in modulation both inflammatory and immune responses and are critical elements in the skin response to damage or viral-infection. IL-6 belongs to potent pro-inflammatory cytokines that stimulates B-cell proliferation and differentiation. It is involved in the production of multipotent colony-forming factors and megakaryocytes. IL-6 is found in normal-skin fibroblasts and sweat ducts [53]. It is shown that IL-6 is secreted by almost all cells, including keratinocytes, melanocytes and Langerhans cells after their stimulation with various factors, including cytokines, tumor necrosis factor (TNF), plateletderived growth factor (PDGF), bacterial and virus infection, and microbial lipopolysaccharides (LPS).
TNF-α is produced by monocytes, macrophages, T cells (Th1), keratinocytes, neutrophils, natural killers, endothelial, mast and myeloid cells, and lymphokine-activated killer (LAK) cells playing an important physiological role in immunoregulation [54]. TNF-α stimulates lymphocytes, neutrophils, eosinophils, endothelial cells, fibroblasts, inducing the production of IL-6 in many cell types [55]. However, it has been shown that TNF-α does not affect proliferation and differentiation of normal keratinocytes in a skin model [56]. TNF-α participates in inflammation and its level increases with the inflammation progression [57]. The experimental data report that PBM decreases production of IL-6 and TNF-α in the supernatant of activated mononuclear cells [58,59].
Twenty-four hours after exposure to a diode laser at 780 and 660 nm, in brain and blood tissues, a decrease in the level of IL-6 and TNF-α has been observed [60]. When analyzing the immunomodulatory PBM effect on the skin contact hypersensitivity reaction, no significant changes have been registered in TNF-α concentration [61]. Despite the progress achieved in studying the mechanisms of immunogenesis regulation, the immunomodulatory effect of laser radiation in vivo on the pro-inflammatory cytokine synthesis is still unclear.
The analysis of the results shows that the level of IL-6 in skin irradiated with λ = 1270 nm is significantly lower than its level in the non-irradiated area of the BALB/c Nude mouse skin, whereas TNF-α level increases slightly in response to laser exposure (Table 5). No significant differences are observed in the animal group irradiated at E = 0.12 J/cm 2 .  In the first experimental group the level of IL-6 decreases in blood plasma (51.26 ± 14.45 pg/ml versus 57.14 ± 12.82 pg/ml in the control group). TNF-α level in the blood plasma was the same in both experimental and control groups (Fig. 5). This is a positive dynamic, since laser radiation being 1.2 J/cm 2 does not induce the inflammatory reaction in the skin and the blood. Moreover, laser radiation reduces to some extent production of pro-inflammatory cytokines, in particularly, interleukin-6.

Conclusion
In the BALB/c Nude mouse model, PBM has been shown to induce a photo-stimulating effect of the 1270 nm radiation at the dose of 1.2 J/cm 2 in both an irradiated skin spot and peripheral blood cells. This effect is associated with biochemical reactions changing cell redox state, synthesis of signal molecules (cytokines and microRNAs) and also responsible for consequent physiological effects. In this case, the laser triggers cell protective effects through direct excitation of oxygen molecules. Nonetheless, low intensity and short exposure time of laser radiation cause pronounced effects on the membrane-protective action and enhanced antioxidant system. Also this irradiation decreases the synthesis of proinflammatory cytokines in blood plasma, mainly IL-6. Locally, in the region of laser exposure, the proliferative processes intensify with no signs of inflammation. Thus, in the BALB/c Nude mouse model the laser at the wavelength of 1270 nm and dose of 1.2 J/cm 2 produces local and systemic stimulation effect highly likely due to a short-term increase in the ROS levels which turns active the cell antioxidative system.
The further studies should be done with higher radiation doses for revealing whether 1270 nm radiation can treat the melanoma planted on BALB/c Nude mouse model.