出席國際會議心得報告書
案號：
97-2314-B-040-021-MY3100年03月16-19日
報
告 人黃翠賢 單位
中山醫學大學中 文 ： 第 八 十 九 屆 世 界 牙 醫 學 會
(IADR)學術年度大會 國家：美國
會 議 名 稱
英文：89th International Association of Dental Research Annual Meeting
會議地點
城市：San Diego
中文： 低能二極體雷射降低細菌外毒素誘導骨細胞發炎
發表論文題目
英文： Low level diode laser therapy reduces lipopolysacharride (LPS) induced bone cell inflammation心 得 報 告
出席國際會議心得報告書
案號：
97-2314-B-040-021-MY3100年03月16-19日 口頭報告。
3
Abstract
569 Low-level diode laser therapy reduces lipopolysaccharide (LPS)-induced bone cell inflammation
Thursday, March 17, 2011: 2 p.m. - 3:15 p.m.
Location: Hall C (San Diego Convention Center) Presentation Type: Poster Session
T.-H. HUANG, C.-T. KAO, and Y.-C. LU, Chung Shan Medical University, Taichung, Taiwan
Objectives: The aim of this study was to investigate the cytologic effects of inflammatory bone cells after in vitro low-level laser therapy (LLLT). Methods: A human osteosarcoma cell line (MG63) was cultured, infected with lipopolysaccharide (LPS) and exposed to low-level laser treatment at 5 J/cm2 or 10 J/cm2 using a 920 nm diode laser. MG63 cell attachment was observed under a microscope, and cell viability was quantified by mitochondrial colorimetric assay (MTT). LPS-treated MG63 cells were irradiated with LLLT, and the inflammatory markers, iNOS, TNF-α and IL-1, were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and western blot. The data were collected and analyzed by one-way analysis of variance (ANOVA); p<0.05 indicated a statistically significant difference. Results:
Low-level laser treatment on MG63 cells increased their ability to attach and survive. After irradiation, the expression levels of iNOS, TNF-α and IL-1 in LPS-infected MG63 cells decreased over time (p<0.05). Conclusions: Low-level diode laser treatment increased the proliferative ability and decreased the expression of inflammatory mediators in MG63 cells.
Introduction
Low-level laser therapy (LLLT) has applied more in regenerative medicine and dentistry [1]. Photodynamic therapy is become a feasible medical technology in the 1980s. Laser diode is formed by doping a very thin layer on the surface of a crystal wafer. It is also known as an injection laser. Laser diodes differ from conventional lasers, such as small size and weight, low current, low intensity and wide-angle beam.
It is reported that LLLT appears to increase mitochondrial respiration and adenosine triphosphate (ATP) synthesis [2, 3] and possesses biostimulatory effects on wound healing, collagen synthesis [4], and fibroblast proliferation [5,6]. Literatures review showed that LLLT can accelerate bone formation by increasing osteoblastic activity, vascularization, organization of collagen fibers and ATP levels [7-9].
LLLT demonstrated with anti-inflammatory, analgetic and regenerative effects [10,11]. The photodynamic therapy can reduce the numbers of viable bacteria in periodontal diseases [1]. In orthodontic tooth movement, LLLT can reduce patient pain without interfering with the tooth movement [12]. The orthodontic pain is
derived from the secretions of inflammatory cell that stimulate the nerve. The release of pro-inflammatory cytokines and lysosomal enzymes can promote tissue resorption at orthodontic tooth movement compression site [13]. Prostaglandins, IL-1, IL-6, TNFα, and receptor activator of nuclear factor kappa B ligand (RANKL) are all elevated in the periodontal ligament during tooth movement [13]. Thus it can applied LLLT to reduce the orthodontic pain and increase the tooth movement.
The purpose of present study was to investigate the cytologic effects of inflammatory bone cell after LLLT treatment in vitro.
Materials and Methods
Laser conditions
The diode laser (Ga-Al-As ,
Arts-Laser. Arts International Biotechnology Inc.
German) 920 nm was used in this study. The beam was clipped to cover the entire area of the plate (3.3 cm2). On average, a power output of 0.5W was measured, and this was calculated to deliver 5 J/cm2 and 10 J/cm2 (experimental group).
Non-irradiated (0 J/cm2) cells were used as control group and were kept under the same conditions. Both irradiated and non-irradiated samples were re-incubated at 37°C in a humidified atmosphere of 5% CO2.
Immunofluorescent stain
MG63 cells were seeded to adhere to the 3 cm dish (Techno Plastic Products, 92006, Germany) at a density of 5 x104 cell/mL for 24 hours. The lipopolysacharride (LPS, Sigma Co.) were added to culture for four hours. Cultured MG63 were irradiated with the lids off in the dark, at room temperature, with 5 J/cm2and 10 J/cm2 at 920 nm.
Non-irradiated cells were used as controls and were kept under the same condition.
After tone hour and 12 hours cells were fixed with 10% methanol (Sigma, St Louis, MO) for 20 minutes. Samples were blocked in PBS supplemented with 5% bovine serum albumin (Sigma) and then incubated in blocking buffer containing rabbit anti-phospho-FAK primary antibody (FAK; Abcam, Interlab Co, Ltd, Taipei, Taiwan) (1:1000 dilution in PBS) and iNOS antibody (rabbit; Invitrogen). After washing the cells with PBS, the cytoskeleton and cell nucleus were stained by incubating the cells with the secondary antibody (Biotin-conjugated goat anti-rabbit antibody) and phalloidin- FITC (Invitrogen) (1:1000 dilution in PBS), for 1 hour and with DAPI (Invitrogen) (30 nmol/L in PBS) for 20 minutes, respectively [15]. The stained cells were visualized by using a Zeiss (Carl Zeiss, Oberkochen, Germany) fluorescent
5 previous method [16], cultured cells were lysed in lysis buffer [ RIPA lysis buffer (50 mM Tris HCl pH 7.4, 1% NP-40, 150 mM NaCl，1 mM EDTA，1 mM PMSF, 1 mM Na3VO
4, 1 mM NaF, protease inhibitor cocktail tablet)] on ice. Cell extracts were sonicated, and protein concentration was determined with a Coomassie® Brillrant blue G-250 (Bio-Rad, 500-0006, US). Ten micrograms of protein were loaded in each lane. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene diflouride membranes, (Immunoblot PVDF membrane, BioRad, 162-0177).
Membranes were blocked overnight in blocking buffer containing Tris-buffered saline [TTBS—50 mM Tris; 150 mM sodium chloride (NaCl), Separation Scientific, 7647-14-5] containing 0.1% Tween 20 and 5% non-fat milk. The membranes were then incubated in primary antibody [rabbit IgG anti-human iNOS (1:1000,upstate,07-520,US)、rabbit IgG anti-phospho-ERK (1:1000,cell signaling)、
mouse IgG anti- β-actin (0.5:1000, Chimecon,MAB1501,US)] diluted in blocking buffer (as above) at room temperature for 1 h. The membranes were washed in TTBS and then incubated in secondary antibody [HRP-conjugated goat anti-rabbit IgG (1:5000, Chemicon, AP132P)、HRP- conjugated goat anti-mouse IgG (0.5:5000, Chemicon, AP124P)] diluted in blocking buffer (as above) at room temperature for 1 h. The membranes were washed and stained with 1X TMB/H (3,3’,5,5’-tetramethylbenzidine, Chemicon, ES014). The blots were then exposed to Xray film (Kodak MXG, Rochester, USA, 326052). The films were developed and then used BioDoc system to take record and LabWorks 4.5 soft ware to detect the bend density.
Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay
MG63 cells were seeded to adhere to the 3 cm dish (Techno Plastic Products, 92006, Germany) at a density of 5 x104 cell/mL for 24 hours. The lipopolysacharride (LPS, Sigma Co.) were added to culture for four hours. Cultured MG63 were irradiated with the lids off in the dark, at room temperature, with 5 J/cm2and 10 J/cm2 at 920 nm.
Non-irradiated cells were used as controls and were kept under the same condition.
Procedures were followed our previous experiment method [16]. Total RNA was isolated according to the manufacturer’s instructions. MG63 cell were harvested by 0.25% tripsin-EDTA and extracted with TRIZOL reagent (Invitrogen, 15596-018, India) 10 minutes. Added 200 µl chloroform (Tedia, CR-0360, US) 10 minutes, at 12,000 rpm, 4 oC centrifugation for 15 minutes. Added 500 µl isopropanol (Fluka, USA) to supernatant 10 minutes, at 12,000 rpm, 4 oC centrifugation for 15 minutes.
Remove the supernatant and washed with -20 oC 75% alcohol twice. The RNA in aqueous solution was reserved in -20 oC and detected by spectrophotometry ate 260nm.
1 µg of total RNA in aqueous solution was reverse transcribed in a volume of 50 µl containing 400 µM of each NTP, 10 units of RNase inhibitor, 10 µl 5 x Tris buffer, 2 µl RT-PCR enzyme mix and 800 nM of TNF α and IL 1 primer.
Reverse transcription was carried out at 50 °C for 30 min. After an initial denaturation step at 95 °C for 15 min, 40 PCR cycles were run, each consisting of denaturation at 94 °C for 1 min, annealing for 1 min at the respective temperature, extension at 72 °C for 1 min, and finally elongation at 72 °C for 10 min. The PCR products were separated by electrophoresis of 20 µl of each reaction mixture in a 2% agarose gel at 100 V/cm in 1 % Tris acetate EDTA buffer. Following electrophoresis, the gels were stained with thidium bromide, destained in distilled water and photographed with a charge-coupled device camera. The Lab Works 4.5 soft ware was used to analysis band density.
All above measurements were made in duplicate and are expressed as mean ± SD of three independent experiments. In addition, one-way ANOVA and Tukey’s multiple comparison test were used to reveal significant differences between the different investigation groups. The level of significance was set at P < 0.05.
Results
MG63 cell attachment and viability of attached cells
At each time interval, cell density and distribution were examined by light microscopy. It is evident that the first or second hour observation of MG63 attachment was no obviously difference. The MG63 cell attachment were higher in 5 J/cm2 and 10 J/cm2 irradiated groups at 12 hours (Fig. 1).
In all samples the number of attached viable cells increased during the cultivation period. After one hour and twelve hour observation, the cell viability existed statistical difference (p<0.05) between the control and experimental group (Fig.2).
iNOS expression of MG63
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In fluorescent stain of MG63 treated with different LLLT showed iNOS expression decreased (Fig. 3). After one hour of LLLT showed red dots (iNOS marker) were decreased as laser power was increased (Fig. 3). After 12 hour of LLLT showed no iNOS expression in irradiated groups (Fig.3).
The irradiated groups’ iNOS expression were lower than control group after one hour and three hour time period (p<0.05) but no difference after 12 hours (p>0.05) in western bolt assay (Fig.4).
pErk expression of MG63
The MG 63 cell’s pErk expression showed irradiated groups were higher than non-irradiated group at different time period (p<0.05, Fig. 5).
TNF-α and IL 1 markers expression of MG63
The inflammatory markers TNF-α and IL-1expression were shown in figure 7 and 8.
The TNF-α expression level were lower in irradiated groups (p<0.05) after 1 or 12 hour treatment (Fig. 6).
The IL-1 expression level was lower in irradiated groups after 12 hour treatment (p<0.05), but found no difference between irradiated and non-irradiated groups in one hour treatment (p>0.05, Fig. 7).
Discussion
The present in vitro study evaluate the effects of LLLT on inflammatory MG63 cells and showed that LLLT on adherent and viability of MG63 cells were higher in cells treated with 5 J/cm2 and 10 J/cm2 . It is also found that LLLT can reduce the inflammatory markers iNOS, TNF- and IL-1 expression in infected MG63 cells.
The viability assay was irradiated after MG63 cell attached to dishes. The results showed that after irradiated 12 hours, the MG63 viability was significant difference with control (P<0.05 Fig. 2). It represented that LLLT do have biostimulatory effects. The present study showed similar results as previous studies findings that LLLT can enhancement of fibroblast and chondral proliferation (17,18). This result also demonstrated from present pErk marker expression (Fig.5).
The higher expression of pErk signal showed cell was in high proliferation state (19).
LLLT of diode laser can promote the cell to growth in present study.
To understand the effects of LLLT on inflammatory cell, present study applied the LPS into cell culture. This method is seldom seen in present LLLT studies.
Lipopolysaccharide (LPS) is one of the most powerful bacterial virulence factors in terms of proinflammatory properties (20). Endotoxin, a cell wall component of
Gram-negative bacteria, plays a central role in the pathogenesis of septic shock. In conjunction with these changes in organ function, a wide variety of inflammatory mediators are released which appear to contribute to these responses. These include the release of proinflammatory cytokines (e.g. tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-8) (21). In present study, to prove LLLT has anti-inflammatory activity (22), author compared TNF-α and IL-1expression of the LLLT effects on LPS induced inflammation MG63 cells. The present result agree with it and showed diode laser with 5 J/cm2and 10 J/cm2 irradiation to inflammation MG63 can reduce the TNF-α and IL-1expression as compared with control (Fig. 6 and 7).
In inflammation, proinflammatory cytokines and lipid mediators play an important role in triggering the expression of the inducible isoform of nitride oxide (NO) synthase (iNOS or NOS-2) in various types. It is proved that proinflammatory cytokines, such as the TNF-α and IL-1 can induce the expression of INOS in vitro or in vivo (23). Platelet activating factor (PAF) can induce iNOS and enhance lipoploysaccaharide (LPS) induced expression of iNOS in macrophages (24). With this mechanism, the present study was using LPS to induce the cell inflammation and detect iNOS expression by fluorescent analysis and western blot analysis. The results showed MG 63 cell after LLLT treated, the iNOS fluorescent strength were decreased (Fig.3) and iNOS protein expression were decreased too (Fig. 4). It is support that LLLT has anti-inflammation effects.
In dentistry, tissue engineering of bone is an important field in periodontal disease, implantology orthodontic tooth movement. This concept is an exciting technology in regenerative medicine. LLLT phototherapy can provide the biostimulation effects to tissue. To enhance using the LLLT in clinical application will be beneficial to patient.
Conclusion
By applying the diode laser with low level power can promote the cell adhesion, increase the MG63 cell proliferation, reduce LPS induce inflammation markers iNOS, TNF-α and IL-1 expression.
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