High-Frequency Near-Infrared Diode Laser Irradiation Attenuates IL-1β-Induced Expression of Inflammatory Cytokines and Matrix Metalloproteinases in Human Primary Chondrocytes

High-frequency near-infrared diode laser provides a high-peak output, low-heat accumulation, and efficient biostimulation. Although these characteristics are considered suitable for osteoarthritis (OA) treatment, the effect of high-frequency near-infrared diode laser irradiation in in vitro or in vivo OA models has not yet been reported. Therefore, we aimed to assess the biological effects of high-frequency near-infrared diode laser irradiation on IL-1β-induced chondrocyte inflammation in an in vitro OA model. Normal Human Articular Chondrocyte-Knee (NHAC-Kn) cells were stimulated with human recombinant IL-1β and irradiated with a high-frequency near-infrared diode laser (910 nm, 4 or 8 J/cm2). The mRNA and protein expression of relevant inflammation- and cartilage destruction-related proteins was analyzed. Interleukin (IL) -1β treatment significantly increased the mRNA levels of IL-1β, IL-6, tumor necrosis factor (TNF) -α, matrix metalloproteinases (MMP) -1, MMP-3, and MMP-13. High-frequency near-infrared diode laser irradiation significantly reduced the IL-1β-induced expression of IL-1β, IL-6, TNF-α, MMP-1, and MMP-3. Similarly, high-frequency near-infrared diode laser irradiation decreased the IL-1β-induced increase in protein expression and secreted levels of MMP-1 and MMP-3. These results highlight the therapeutic potential of high-frequency near-infrared diode laser irradiation in OA.


Introduction
Osteoarthritis (OA) is well-known articular joint disease often resulting in joint pain and disability in adults. Worldwide, 10% of people older than 60 years are estimated to manifest OA symptoms [1]. Furthermore, OA has been shown to be associated with aging, being overweight or obese, cartilage injuries, and genetic factors [2][3][4]. Mechanical stress-induced inflammation plays an important role in OA. Synovitis in the articular condylar surface followed by secretion of inflammatory cytokines such as interleukin 1β (IL-1β) from synovial cells is considered to be the initial stage of OA [5]. The secreted IL-1β infiltrates the cartilage and synovial fluid, causing chondrocyte inflammation [6]. The next stage of OA involves cartilage degradation, particularly extracellular matrix (ECM) destruction of the laser [18], and generates high peak output power. Furthermore, it does not cause excessive heat accumulation in the target tissue, thus overcoming an important concern for clinicians using laser therapies. Therefore, high-frequency near-infrared diode laser therapy may be considered a novel, effective, and safe therapy for OA. However, the effect of high-frequency near-infrared diode laser irradiation on OA cartilage has not yet been reported. Therefore, in this study, we investigated the effect of high-frequency near-infrared diode laser irradiation on the expression of inflammatory cytokines and MMPs in an in vitro OA model and attempted to highlight the potential of such laser devices in OA treatment.

Laser Irradiation
A high-frequency pulsed laser with 910 nm wavelength, 45 W maximum output power, 300 mW average output power, 200 ns pulse width, 30 kHz pulse repetition rate, and 9.6 cm 2 irradiation area (Lumix 2; USA Laser Biotech, Inc., Richmond, VA, USA) was used for the experiment. The depth of the medium in each culture dish was adjusted to 2 mm, and the laser was fixed at a height of 40 mm so that cells in one well of a 6-well plate at 1 degree. The following laser presets were used: program 1; pulse rate, 30 kHz; and overall duty cycle, 0.6%.

Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis
The mRNA levels of IL-1β, IL-6, TNF-α, MMP-1, MMP-3, MMP-9, and MMP-13 were determined by quantitative real-time PCR analysis using a LightCycler system (Roche Diagnostics, Basel, Switzerland) and QuantiTect SYBR Green PCR Master Mix (Qiagen, Tokyo, Japan). Briefly, total RNA was extracted from cultured NHAC-Kn cells using an RNeasy Mini Kit and reverse transcribed using the ReverTra Ace Kit (Toyobo, Osaka, Japan). Real-time PCR was performed using the Thunderbird STBR qPCR Mix (Toyobo) with specific primer sets ( Table 1). The relative mRNA levels were analyzed using the ∆∆Ct method and normalized to beta actin (ACTB) mRNA levels.

Two-Color Western Blot Analysis
Total protein was extracted from cultured NHAC-Kn cells using RIPA lysis buffer (Nacalai Tesque, Kyoto, Japan) supplemented with 1% (v/v) protease inhibitor cocktail. The cell lysates were centrifuged at 4 • C at 15,000 g for 20 min, and protein concentrations in the supernatant were measured using the bicinchoninic acid assay (BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein were electrophoresed on 10% polyacrylamide gels (e-PAGEL, ATTO, Tokyo, Japan) and transferred to PVDF membranes using the iBlot 2 Gel Transfer Device and iBlot 2 Transfer Stacks (Thermo Fisher Scientific), according to the manufacturer's instructions. Membranes were then blocked with 5% skim milk for 30 min at room temperature. The membranes were incubated overnight at 4 • C with primary antibodies against MMP-1 (Abcam, Cambridge, MA, USA; Cat# ab139332), MMP-3 (Abcam; Cat# ab52915), and β-actin (Wako, Osaka, Japan), and then incubated with Alexa Fluor 680-labeled antirabbit secondary antibody (LI-COR, Lincoln, NE, USA) or IRDye 800-labeled antimouse secondary antibody (LI-COR). Protein bands were detected using the Odyssey ® infrared imaging system (LI-COR); band intensities were quantitated using the ImageJ software and normalized to β-actin band intensity.

Enzyme-Linked Immunosorbent Assay (ELISA)
Here, 80% confluent NHAC-Kn cells were cultured in serum-and growth factor-free chondrocyte basal medium for 24 h and then treated with IL-1β for 12 h. Then, the cells were irradiated at 8 J/cm 2 and cultured for 12 h, and the culture supernatants were collected and stored at −80 • C until analysis. Levels of secreted MMP-1 and MMP-3 were measured using the MMP-1 (AB215083, Abcam) and MMP-3 (BMS2014/3, Thermo Fisher Scientific) ELISA kits, respectively, following the manufacturers' recommendations, followed by measurement of the absorbance at 570 nm using a microplate reader.

Statistical Analysis
Data are represented as mean ± SEM. Groups were compared using one-way ANOVA followed by Dunnett's post hoc test; P < 0.05 was considered to indicate statistical significance.

Effect of High-Frequency Near-Infrared Diode Laser Irradiation on Gene Expression of Inflammatory Cytokines in NHAC-Kn Cells
qPCR analysis showed that mRNA expression levels of IL-1β, IL-6, and TNF-α were significantly upregulated in NHAC-Kn cells treated with IL-1β for 4 h. However, the mRNA levels of IL-1β, IL-6, and TNF-α in the IL-1β-stimulated chondrocytes laser irradiated at 4 and 8 J/cm 2 were significantly lower than those in all the other cell groups. These results indicate that laser irradiation significantly attenuated the IL-1β-induced upregulation of these inflammatory cytokines ( Figure 1). qPCR analysis showed that mRNA expression levels of IL-1β, IL-6, and TNF-α were significantly upregulated in NHAC-Kn cells treated with IL-1β for 4 h. However, the mRNA levels of IL-1β, IL-6, and TNF-α in the IL-1β-stimulated chondrocytes laser irradiated at 4 and 8 J/cm 2 were significantly lower than those in all the other cell groups. These results indicate that laser irradiation significantly attenuated the IL-1β-induced upregulation of these inflammatory cytokines ( Figure 1).

Effect of High-Frequency Near-Infrared Diode Laser Irradiation on Gene Expression of MMPs in NHAC-Kn Cells
To evaluate the effect of high-frequency near-infrared diode laser irradiation on the expression of genes associated with cartilage destruction, we investigated MMP-1, MMP-3, MMP-9, and MMP-13 mRNA levels in irradiated NHAC-Kn cells. The MMP-1, MMP-3, and MMP-13 mRNA levels were significantly increased by IL-1β stimulation ( Figure 2). The MMP-1 and MMP-3 mRNA levels in IL-1β-stimulated cells irradiated at 4 or 8 J/cm 2 were lower than those in IL-1β-stimulated cells not exposed to radiation (Figure 2A and B); such an effect was not observed for MMP-13 ( Figure 2D). No statistically significant changes were observed in the MMP-9 mRNA levels of NHAC-Kn cells ( Figure  2C).

Effect of High-Frequency Near-Infrared Diode Laser Irradiation on Gene Expression of MMPs in NHAC-Kn Cells
To evaluate the effect of high-frequency near-infrared diode laser irradiation on the expression of genes associated with cartilage destruction, we investigated MMP-1, MMP-3, MMP-9, and MMP-13 mRNA levels in irradiated NHAC-Kn cells. The MMP-1, MMP-3, and MMP-13 mRNA levels were significantly increased by IL-1β stimulation ( Figure 2). The MMP-1 and MMP-3 mRNA levels in IL-1β-stimulated cells irradiated at 4 or 8 J/cm 2 were lower than those in IL-1β-stimulated cells not exposed to radiation (Figure 2A,B); such an effect was not observed for MMP-13 ( Figure 2D). No statistically significant changes were observed in the MMP-9 mRNA levels of NHAC-Kn cells ( Figure 2C). J. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 14 qPCR analysis showed that mRNA expression levels of IL-1β, IL-6, and TNF-α were significantly upregulated in NHAC-Kn cells treated with IL-1β for 4 h. However, the mRNA levels of IL-1β, IL-6, and TNF-α in the IL-1β-stimulated chondrocytes laser irradiated at 4 and 8 J/cm 2 were significantly lower than those in all the other cell groups. These results indicate that laser irradiation significantly attenuated the IL-1β-induced upregulation of these inflammatory cytokines ( Figure 1).

Effect of High-Frequency Near-Infrared Diode Laser Irradiation on Gene Expression of MMPs in NHAC-Kn Cells
To evaluate the effect of high-frequency near-infrared diode laser irradiation on the expression of genes associated with cartilage destruction, we investigated MMP-1, MMP-3, MMP-9, and MMP-13 mRNA levels in irradiated NHAC-Kn cells. The MMP-1, MMP-3, and MMP-13 mRNA levels were significantly increased by IL-1β stimulation ( Figure 2). The MMP-1 and MMP-3 mRNA levels in IL-1β-stimulated cells irradiated at 4 or 8 J/cm 2 were lower than those in IL-1β-stimulated cells not exposed to radiation (Figure 2A and B); such an effect was not observed for MMP-13 ( Figure 2D). No statistically significant changes were observed in the MMP-9 mRNA levels of NHAC-Kn cells ( Figure  2C).

Effect of High-Frequency Near-Infrared Diode Laser Irradiation on Protein Expression of MMPs in NHAC-Kn Cells
Next, we measured the protein expression of MMP-1 and MMP-3 using western blot analysis. The protein expression of MMP-1 and MMP-3 in the IL-1β-stimulated chondrocytes was significantly higher than that in the control cells. Similar to the qPCR results, protein expression of MMP-1 and MMP-3 in IL-1β-stimulated cells irradiated at 8 J/cm 2 was significantly lower than that in the nonirradiated IL-1β-stimulated cells (Figure 3). In addition, we performed ELISA to evaluate the levels of MMP-1 and MMP-3 secreted in the cell culture supernatant. Secreted MMP-1 and MMP-3 levels in the IL-1β group were considerably higher than those in the control group ( Figure 4). However, secreted MMP-1 and MMP-3 levels of the IL-1β-stimulated cells irradiated at 8 J/cm 2 were significantly lower than those of the nonirradiated IL-1β-stimulated cells (Figure 4).

Effect of High-Frequency Near-Infrared Diode Laser Irradiation on Protein Expression of MMPs in NHAC-Kn Cells
Next, we measured the protein expression of MMP-1 and MMP-3 using western blot analysis. The protein expression of MMP-1 and MMP-3 in the IL-1β-stimulated chondrocytes was significantly higher than that in the control cells. Similar to the qPCR results, protein expression of MMP-1 and MMP-3 in IL-1β-stimulated cells irradiated at 8 J/cm 2 was significantly lower than that in the nonirradiated IL-1β-stimulated cells (Figure 3). In addition, we performed ELISA to evaluate the levels of MMP-1 and MMP-3 secreted in the cell culture supernatant. Secreted MMP-1 and MMP-3 levels in the IL-1β group were considerably higher than those in the control group ( Figure 4). However, secreted MMP-1 and MMP-3 levels of the IL-1β-stimulated cells irradiated at 8 J/cm 2 were significantly lower than those of the nonirradiated IL-1β-stimulated cells (Figure 4).

Discussion
Recently, LLLT was shown to reduce inflammatory reactions both in vitro and in vivo by regulating proinflammatory gene expression [47]. LLLT (904 nm) diminished inflammatory cell migration in a dose-dependent manner in a mouse model of lipopolysaccharide (LPS)-induced periodontitis, with 3 J/cm 2 being the most effective dose [48]. Furthermore, LLLT at 780 nm (7.7 J/cm 2 ) suppressed IL-6 expression in a rat model of collagenase-induced tendinitis [49]; LLLT at 660 nm significantly reduced IL-6 and TNF-α expression in a rat model of carrageenan-induced pleurisy [50]; and LLLT at 660 nm (8 J/cm 2 ) reduced the expression of inflammatory markers such as IL-1β, IL-6, and TNF-α in LPS-stimulated human adipose-derived stem cells [51]. However, the precise mechanism underlying the effect of high-frequency near-infrared diode laser irradiation (904 nm) on cartilage inflammation remains unknown. In this study, we investigated the effect of high-frequency near-infrared diode laser irradiation on cultured human chondrocytes (NHAC-Kn cells). To mimic the degenerative effect of osteoarthritic cartilage, NHAC-Kn cells were stimulated using human recombinant IL-1β [52,53]. IL-1β is a proinflammatory cytokine that interferes with ECM turnover by accelerating cartilage matrix degradation and inducing chondrocyte apoptosis.
In this study, qPCR analysis revealed that IL-1β significantly upregulated IL-1β, IL-6, and TNFα mRNA levels in NHAC-Kn cells and that these levels were significantly attenuated after irradiation of the IL-1β-stimulated chondrocytes with a high-frequency near-infrared diode laser at 4 or 8 J/cm 2 . The gene expression of IL-1β, IL-6, and TNF-α in rat models of acute joint inflammation [54,55] and in the cartilage of rat models of OA [56] decreased significantly after LLLT (808 nm, 142 J/cm 2 ) and laser irradiation (808 nm, 71 J/cm 2 ), respectively. Moreover, photobiomodulation therapy (830 nm, 214 J/cm 2 ) effectively reduced the OA-induced IL-1β, IL-6, and TNF-α protein expression in experimental rat OA models [57]. Our results are consistent with these previous reports; however, the wavelengths, power numbers, frequency, and pulse numbers of laser irradiation differ.

Discussion
Recently, LLLT was shown to reduce inflammatory reactions both in vitro and in vivo by regulating proinflammatory gene expression [47]. LLLT (904 nm) diminished inflammatory cell migration in a dose-dependent manner in a mouse model of lipopolysaccharide (LPS)-induced periodontitis, with 3 J/cm 2 being the most effective dose [48]. Furthermore, LLLT at 780 nm (7.7 J/cm 2 ) suppressed IL-6 expression in a rat model of collagenase-induced tendinitis [49]; LLLT at 660 nm significantly reduced IL-6 and TNF-α expression in a rat model of carrageenan-induced pleurisy [50]; and LLLT at 660 nm (8 J/cm 2 ) reduced the expression of inflammatory markers such as IL-1β, IL-6, and TNF-α in LPS-stimulated human adipose-derived stem cells [51]. However, the precise mechanism underlying the effect of high-frequency near-infrared diode laser irradiation (904 nm) on cartilage inflammation remains unknown. In this study, we investigated the effect of high-frequency near-infrared diode laser irradiation on cultured human chondrocytes (NHAC-Kn cells). To mimic the degenerative effect of osteoarthritic cartilage, NHAC-Kn cells were stimulated using human recombinant IL-1β [52,53]. IL-1β is a proinflammatory cytokine that interferes with ECM turnover by accelerating cartilage matrix degradation and inducing chondrocyte apoptosis.
In this study, qPCR analysis revealed that IL-1β significantly upregulated IL-1β, IL-6, and TNF-α mRNA levels in NHAC-Kn cells and that these levels were significantly attenuated after irradiation of the IL-1β-stimulated chondrocytes with a high-frequency near-infrared diode laser at 4 or 8 J/cm 2 . The gene expression of IL-1β, IL-6, and TNF-α in rat models of acute joint inflammation [54,55] and in the cartilage of rat models of OA [56] decreased significantly after LLLT (808 nm, 142 J/cm 2 ) and laser irradiation (808 nm, 71 J/cm 2 ), respectively. Moreover, photobiomodulation therapy (830 nm, 214 J/cm 2 ) effectively reduced the OA-induced IL-1β, IL-6, and TNF-α protein expression in experimental rat OA models [57]. Our results are consistent with these previous reports; however, the wavelengths, power numbers, frequency, and pulse numbers of laser irradiation differ.
MMPs are Zn +2 -or Ca +2 -dependent endopeptidases that degrade ECM components. The MMP family comprises 24 members, 23 of which are found in mammals [58,59]. MMPs play a key role in pathological conditions such as inflammation, rheumatoid arthritis, OA, cardiovascular diseases, fibrosis, and cancer [59]. As an array of proteases, MMP-1, MMP-3, and MMP-13 degrade type II collagen and proteoglycans, causing cartilage proteolysis [60]. High concentrations of MMP-1 (collagenase-1), which is mainly produced by synoviocytes, were found in the synovial fluid of patients with joint lesions and OA. It degrades aggrecan and collagen types I, II, III, VII, X, and IX [61][62][63][64][65]. MMP-3 (stromelysin-1), which is produced by articular synovial cells, chondrocytes, fibroblasts, and macrophages, has the broadest substrate specificity among MMPs and degrades cartilage proteoglycans; collagen types III, IV, V, VII, and IX; laminin; and fibronectin. It has also been implicated in joint destruction in rheumatoid arthritis. MMP-9 (gelatinase B) is usually secreted by fibroblasts and inflammatory cells, which migrate to lesion sites, and can cleave native type IV and V collagens [66]. The presence of active MMP-9 in the synovial fluid has been reported. Because of limited substrate specificity, MMP-9 does not initiate direct joint destruction. As MMP-9 in synovial fluid is produced by inflammatory cells and platelets, MMP levels are influenced by inflammation and likely reflect joint inflammation [67]. MMP-13 (collagenase-3) is a member of the collagenase subfamily and is usually found in bone articular cartilage and chondrocytes [68]. MMP-13 was also found in synovial tissue, and has been associated with OA and degradation of cartilage proteoglycans and collagen types I, II, and III [69,70]. Moreover, MMP-13 has been suggested to be involved in pathways associated with MMP-9 activation [58,71]. Therefore, MMP-13 may be considered a critical component of the cellular machinery involved in regulating articular cartilage turnover and a potential therapeutic target for cartilage destruction [68]. Several studies have reported the effect of low-level laser irradiation on MMP synthesis and associated pathological conditions [66,[72][73][74][75].
In present study, high-frequency near-infrared diode laser irradiation at 4 and 8 J/cm 2 reduced MMP-1 and MMP-3 mRNA levels, compared with those in the IL-1β only group. However, the MMP-9 and MMP-13 mRNA levels were not altered by laser irradiation at 4 or 8 J/cm 2 . In addition, we assessed the protein expression and secreted levels of MMP-1 and MMP-3 by western blotting and ELISA, respectively. Laser irradiation at 8 J/cm 2 significantly reduced the IL-1β-stimulated increase in protein levels of MMP-1 and MMP-3. To our knowledge, this is the first report on the effects of high-frequency near-infrared diode laser irradiation on IL-1β-induced human articular chondrocyte inflammation in an in vitro osteoarthritis model. These results reveal the therapeutic potential of high-frequency near-infrared diode laser irradiation in inflammatory conditions of skeletal elements.
LLLT (830 nm, 1.5 J/cm 2 ) was reported to significantly decrease the gene expression of MMP-3 at weeks 6 and 8 and that of MMP-1 and MMP-13 at week 8 in a rabbit model of anterior cruciate ligament transection (ACLT)-induced OA. [76]. Furthermore, LLLT significantly decreased the protein levels of MMP-9 in a rat model of papain-induced cartilage injury [77]. Thus, the MMP-1 and MMP-3 levels observed in the current study are consistent with those reported previously. However, the MMP-9 and MMP-13 levels in this study are not consistent with those in the previous reports. This discrepancy may be attributed to the difference in the method for OA induction. In experimental animal models, OA is induced by ALCT or local papain administration. Our study was an in vitro study involving IL-1β stimulation in human articular chondrocytes. In addition, the power, wavelengths, and pulses of the high-frequency diode laser are different from those of LLLT; therefore, the outcomes of these two laser therapies cannot be compared directly. Histological evaluation of the effects of high-frequency diode lasers using OA animal models is required.
A previous study demonstrated that LLLT suppresses the inflammatory response of human adipose-derived stem cells by increasing intracellular cyclic AMP levels and subsequently decreasing NF-κB activity [51]. LLLT was also shown to suppress the activation of the NF-κB signaling pathway in LPS-treated mesenchymal stem cells by inhibiting phosphorylation of p65 and IκBα, thereby exerting an antiinflammatory effect [78]. It has been reported that the IL-1β-induced increase in MMP-13 expression requires p38 activity, JNK activity, and NF-κB translocation but does not involve MEK, whereas the increase in MMP-1 expression requires p38 and MEK activity but does not involve JNK and NF-κB [79]. MMP-1 and MMP-3 expression in IL-1β-stimulated chondrosarcoma cells was suppressed by p38 and ERK inhibitors but not by JNK inhibitor, and MMP-13 expression was suppressed by p38 and JNK inhibitors but not ERK inhibitor [80]. These findings indicate that the signaling pathway for IL-1β-mediated regulation of MMP-13 is different from that for IL-1β-mediated regulation of MMP-1 and MMP-3 and may not involve MEK/ERK activities. Previous studies indicated that high-frequency diode laser irradiation affected ERK activity but not p38 and JNK activity [41,42]. Our qPCR results show that high-frequency diode laser irradiation decreased the IL-1β-induced MMP-1 and MMP-3 expression but did not affect MMP-13 expression. Collectively, these findings suggest the involvement of the MEK/ERK signaling pathway in the IL-1β-and irradiation-mediated regulation of MMP-1 and MMP-3 expression in chondrocytes.
Because the biological effects of lasers are nonspecific, indirect, transient, and complex, it is difficult to ascertain the precise effect of laser irradiation in cellular signaling pathways. One study reported that helium-neon laser irradiation activated the Akt signaling pathway [81], whereas another study conducted using the same laser reported that laser irradiation inactivated this pathway [82]. Nevertheless, the detailed mechanism underlying the antiinflammatory effect of laser irradiation remains poorly understood. Further studies are necessary to elucidate the precise mechanism underlying the effect of high-frequency diode laser irradiation in chondrocytes.

Conclusions
The present study demonstrates that high-frequency diode laser irradiation ameliorated inflammation and production of MMP-1 and MMP-3 in IL-1β-stimulated human chondrocytes. Collectively, our findings highlight the potential applicability of high-frequency diode laser irradiation in reducing the inflammation and cartilage destruction observed in OA.