Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells

doi:10.7518/hxkq.2023.2023133

Abstract

Abstract:

Objective This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients. Methods HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco’s modification of Eagle’s medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm²) and C2 and D2 (energy density: 5.625 J/cm²). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay. Results 1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05). Conclusion 1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm². Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.

Key words: high glucose environment, interleukin-6, tumor necrosis factor-α, osteoprotegerin, receptor activator of nuclear factor-κB ligand, human periodontal ligament cells, low-level laser

CLC Number:

R 783.5

Tang Meng, Cui Zhan-qin, Wang Yangyang, Chen Zengguo, Li Wenjing, Zhang Cuiping. Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells[J]. West China Journal of Stomatology, 2023, 41(5): 521-532.

Figures/Tables 12

Fig 1

Fig 2

Fig 3

Fig 4

Fig 5

Fig 6

Tab 1

Tab 2

Fig 7

Tab 3

Tab 4

Tab 5

References 35

1	中国医师协会内分泌代谢科医师分会, 国家代谢性疾病临床医学研究中心. 糖尿病分型诊断中国专家共识[J]. 中华糖尿病杂志, 2022, 14(2): 120-139.
	Endocrine Metabolism Branch of Chinese Medical Doctor Association, National Clinical Medical Research Center for Metabolic Diseases. Chinese experts’ consensus on the classification and diagnosis of diabetes ty-ping[J]. Chin J Diabetes Mellitus, 2022, 14(2): 120-139.
2	Zhu W, Qiu Q, Luo H, et al. High glucose exacerbates TNF-α-induced proliferative inhibition in human perio-dontal ligament stem cells through upregulation and activation of TNF receptor 1[J]. Stem Cells Int, 2020, 2020: 4910767.
3	Papadopoulou A, Todaro A, Eliades T, et al. Effect of hyperglycaemic conditions on the response of human periodontal ligament fibroblasts to mechanical stretching[J]. Eur J Orthod, 2019, 41(6): 583-590.
4	Yashima Y, Kaku M, Yamamoto T, et al. Effect of continuous compressive force on the expression of RANKL, OPG, and VEGF in osteocytes[J]. Biomed Res, 2020, 41(2): 91-99.
5	Wang L, Liu C, Song Y, et al. The effect of low-level laser irradiation on the proliferation, osteogenesis, inflammatory reaction, and oxidative stress of human periodontal ligament stem cells under inflammatory conditions[J]. Lasers Med Sci, 2022, 37(9): 3591-3599.
6	Carroll JD, Milward MR, Cooper PR, et al. Developments in low level light therapy (LLLT) for dentistry[J]. Dent Mater, 2014, 30(5): 465-475.
7	黄生高, 张建兴, 熊培颖, 等. 持续静压力对人牙周膜细胞OPG及ODF mRNA表达的影响[J]. 临床口腔医学杂志, 2006, 22(6): 347-350.
	Huang SG, Zhang JX, Xiong PY, et al. Effect of conti-nuously compressive pressure on the expression of OPG and ODF mRNA in human periodontal ligament cells in vitro [J]. J Clin Stomatol, 2006, 22(6): 347-350.
8	吴洁, 崔占琴, 韩瑜, 等. 持续静压力作用下牙周膜细胞骨保护素、核因子-κB受体活化因子配体表达的增龄性变化[J]. 华西口腔医学杂志, 2022, 40(6): 654-661.
	Wu J, Cui ZQ, Han Y, et al. Aging effect of osteoprotegerin and receptor activator of nuclear factor-κB ligand expression in human periodontal ligament cells under continuous static pressure[J]. West China J Stomatol, 2022, 40(6): 654-661.
9	Baud'huin M, Lamoureux F, Duplomb L, et al. RANKL, RANK, osteoprotegerin: key partners of osteoimmunology and vascular diseases[J]. Cell Mol Life Sci, 2007, 64(18): 2334-2350.
10	Trouvin AP, Goëb V. Receptor activator of nuclear factor‑κB ligand and osteoprotegerin: maintaining the balance to prevent bone loss[J]. Clin Interv Aging, 2010, 5: 345-354.
11	Mizuno A, Amizuka N, Irie K, et al. Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin[J]. Biochem Biophys Res Commun, 1998, 247(3): 610-615.
12	Pan S, Hu B, Sun J, et al. Identification of cross-talk pathways and ferroptosis-related genes in periodontitis and type 2 diabetes mellitus by bioinformatics analysis and experimental validation[J]. Front Immunol, 2022, 13: 1015491.
13	Li Y, Zhao Y, Song L, et al. High glucose levels contribute to vascular fibrosis via the activation of the endothelial-to-mesenchymal transition in periodontitis[J]. J Periodontal Res, 2023, 58(2): 225-236.
14	Banjar A, Alyafi R, AlGhamdi A, et al. The relationship between glycated hemoglobin level and the stage of pe-riodontitis in individuals without diabetes[J]. PLoS One, 2023, 18(1): e0279755.
15	Dong L, Xu P. Danzhi Jiangtang capsule alleviate hyperglycemia and periodontitis via Wnt/β-catenin signaling in diabetic rat[J]. J Tradit Chin Med, 2021, 41(4): 608-616.
16	Zhou M, Graves DT. Impact of the host response and osteoblast lineage cells on periodontal disease[J]. Front Immunol, 2022, 13: 998244.
17	Graves DT, Ding Z, Yang Y. The impact of diabetes on periodontal diseases[J]. Periodontol 2000, 2020, 82(1): 214-224.
18	Miranda TS, Napimoga MH, Feres M, et al. Antagonists of Wnt/β-catenin signalling in the periodontitis associa-ted with type 2 diabetes and smoking[J]. J Clin Perio-dontol, 2018, 45(3): 293-302.
19	Song X, Yue Z, Fan L, et al. Relationship between circulating senescence-associated secretory phenotype levels and severity of type 2 diabetes-associated periodontitis: a cross-sectional study[J]. J Periodontol, 2023. doi: 10. 1002/JPER.22-0445 .
20	Irwin CR, Myrillas TT. The role of IL-6 in the pathoge-nesis of periodontal disease[J]. Oral Dis, 1998, 4(1): 43-47.
21	Yakovlev E, Kalichman I, Pisanti S, et al. Levels of cytokines and collagen typeⅠand type Ⅲ as a function of age in human gingivitis[J]. J Periodontol, 1996, 67(8): 788-793.
22	Takeichi O, Saito I, Tsurumachi T, et al. Expression of inflammatory cytokine genes in vivo by human alveolar bone-derived polymorphonuclear leukocytes isolated from chronically inflamed sites of bone resorption[J]. Calcif Tissue Int, 1996, 58(4): 244-248.
23	Gaspersic R, Stiblar-Martincic D, Osredkar J, et al. In-fluence of subcutaneous administration of recombinant TNF-alpha on ligature-induced periodontitis in rats[J]. J Periodontal Res, 2003, 38(2): 198-203.
24	Lee HJ, Kang IK, Chung CP, et al. The subgingival microflora and gingival crevicular fluid cytokines in refractory periodontitis[J]. J Clin Periodontol, 1995, 22(11): 885-890.
25	Huang YY, Sharma SK, Carroll J, et al. Biphasic dose response in low level light therapy-an update[J]. Dose Response, 2011, 9(4): 602-618.
26	Soi S, Bains VK, Srivastava R, et al. Comparative eva-luation of improvement in periodontal and glycemic health status of type 2 diabetes mellitus patients after scaling and root planing with or without adjunctive use of diode laser[J]. Lasers Med Sci, 2021, 36(6): 1307-1315.
27	张建兴, 黄生高, 钟孝欢, 等. 人牙周膜细胞体外培养和机械压力模型构建[J]. 口腔医学研究, 2006, 22(1): 38-42.
	Zhang JX, Huang SG, Zhong XH, et al. Culture of human periodontal ligament cells and establishment of mechanical pressure model in vitro [J]. J Oral Sci Res, 2006, 22(1): 38-42.
28	Nie F, Hao S, Ji Y, et al. Biphasic dose response in the anti-inflammation experiment of PBM[J]. Lasers Med Sci, 2023, 38(1): 66.
29	Prado TP, Zanchetta FC, Barbieri B, et al. Photobiomo-dulation with blue light on wound healing: a scoping review[J]. Life (Basel), 2023, 13(2): 575.
30	Tetè G, Ferrante L, Moretto M, et al. The efficacy of the diode laser at 800-980 nm as an aid to non-surgical the-rapy in periodontitis treatment: a review of literature[J]. J Biol Regul Homeost Agents, 2021, 35(4 suppl. 1): 51-63.
31	Cruz DR, Kohara EK, Ribeiro MS, et al. Effects of low-intensity laser therapy on the orthodontic movement velocity of human teeth: a preliminary study[J]. Lasers Surg Med, 2004, 35(2): 117-120.
32	Rigi-Ladez MA, Hendi SS, Mirzaei A, et al. Near infrared laser photobiomodulation of periodontal ligament stem cells[J]. Chin J Dent Res, 2022, 25(1): 57-65.
33	张瑞林, 孙瑶. 正畸牙槽骨改建中RANKL/OPG表达动态变化特征及牙周组织细胞凋亡观察[J]. 口腔医学, 2019, 39(6): 488-493.
	Zhang RL, Sun Y. Observation of RANKL/OPG temporal expression and apoptosis of periodontal tissue during alveolar bone remodeling induced by orthodontic force[J]. Stomatology, 2019, 39(6): 488-493.
34	柏思羽, 陈悦, 戴红卫, 等. 机械压应力下骨硬化蛋白对成牙骨质细胞功能影响及机制的体外研究[J]. 华西口腔医学杂志, 2019, 37(2): 162-167.
	Bai SY, Chen Y, Dai HW, et al. Effect of sclerostin on the functions and related mechanisms of cementoblasts under mechanical stress[J]. West China J Stomatol, 2019, 37(2): 162-167.
35	刘长庚, 赵殿才, 程自力, 等. 正畸对牙周膜细胞OPG、RANKL表达的影响[J]. 临床口腔医学杂志, 2018, 34(3): 138-140.
	Liu CG, Zhao DC, Cheng ZL, et al. The effects of or-thodontic force on the expression of OPG and RANKL in human periodontal ligament cells during orthodontic tooth movement[J]. J Clin Stomatol, 2018, 34(3): 138-140.

组别	0 h	12 h	24 h	48 h	72 h
A	19.912 1±2.526 1^cef	25.194 2±3.367 7^ef	27.215 4±0.924 3^ef	34.751 1±1.404 7	35.603 6±1.449 4
B	21.994 4±2.689 1^cdef	30.538 8±2.538 1^aef	37.885 6±1.792 2^ae	42.807 6±1.433 9^a	47.730 7±4.998 5^a
C1	21.593 5±2.710 5^def	23.679 7±2.230 3^aef	25.876 1±2.431 9^aef	31.616 6±0.583 1^a	34.125 2±1.733 9^a
C2	22.090 6±0.895 9^cf	24.142 0±0.749 3^af	24.234 4±1.737 3^ae	29.156 0±2.683 6^a	31.390 3±2.359 6^a
D1	20.595 9±3.024 3^ef	23.930 9±5.488 3^g	30.100 2±2.030 4^efg	35.926 1±1.602 0^g	41.902 2±3.158 6^g
D2	21.382 6±2.623 2^df	25.106 3±7.507 0^defg	28.040 7±1.121 9^fg	35.011 5±4.345 6^g	39.362 2±0.990 3^g

组别	0 h	12 h	24 h	48 h	72 h
A	25.727 5±11.556 2^df	45.080 8±1.653 1^ef	52.901 8±5.881 0	54.180 3±1.974 7^f	64.265 6±2.689 0
B	28.412 1±5.603 2^cdef	50.983 6±3.493 7^adef	59.516 8±4.767 8^af	72.285 2±2.387 5^af	80.907 7±3.394 2^a
C1	27.553 5±4.803 2^ef	35.299 5±8.727 8^adef	45.925 4±9.939 2^af	53.081 2±4.778 2^af	59.265 5±6.594 9^a
C2	23.846 7±1.273 4^cdef	36.232 1±4.080 9^aef	44.258 6±0.809 2^af	50.383 1±6.093 9^a	62.203 1±2.891 9^a
D1	27.026 6±3.701 9^cdef	37.887 1±6.519 2^efg	44.613 9±0.315 4^efg	57.526 1±1.456 2^fg	64.300 8±1.192 3^g
D2	26.103 8±2.854 8^cdef	44.425 9±4.867 5^efg	51.737 0±1.251 4^efg	62.782 9±1.480 6^fg	71.818 0±2.939 5^g

组别	0 h	12 h	24 h	48 h	72 h
A	7.121 5±0.508 1^cd	8.415 0±0.113 3^d	9.015 8±0.217 4^ef	8.280 7±0.008 3^f	7.761 6±0.208 4
B	6.823 3±0.271 8^cde	7.840 5±0.033 3^adef	8.197 1±0.052 1^aef	7.976 5±0.020 0^af	7.007 2±0.034 4^a
C1	7.000 3±0.581 4^cde	8.794 4±0.161 3^ad	9.857 2±0.533 3^a	9.251 7±0.512 3^a	8.451 3±0.206 7^a
C2	7.116 7±0.063 2^cdef	8.762 6±0.070 1^ade	10.068 5±0.220 2^af	9.710 7±0.121 5^af	8.904 5±0.378 3^a
D1	6.809 1±0.146 0^df	8.365 0±0.746 1^b	8.909 6±0.079 5^bf	8.507 8±0.925 9^b	8.054 5±0.253 8^b
D2	7.216 1±0.110 0^cdef	8.103 7±0.065 9^bdf	9.265 4±0.257 9^bf	8.349 2±0.193 4^b	8.334 4±0.008 8^b

组别	0 h	12 h	24 h	48 h	72 h
A	0.408 3±0.000 7^cdef	0.609 8±0.007 8^def	1.075 5±0.047 6^ef	2.123 1±0.109 8^f	3.257 9±0.015 5
B	0.392 5±0.009 1^cdef	0.938 0±0.042 5^adef	2.419 1±0.059 2^aef	3.734 5±0.070 4^af	4.959 5±0.227 9^a
C1	0.395 2±0.014 8^cdef	0.641 6±0.027 5^defg	1.421 5±0.168 5^efg	2.753 4±0.039 3^fg	3.930 9±0.146 1^g
C2	0.396 7±0.009 2^def	0.615 2±0.028 2^defg	1.517 5±0.325 4^efg	2.976 3±0.182 7^fg	4.212 6±0.285 2^g
D1	0.397 4±0.017 5^cdef	0.765 4±0.039 5^bdef	1.806 6±0.040 3^bef	3.145 4±0.110 8^b	4.169 4±0.425 3^b
D2	0.393 1±0.006 3^cdef	0.725 5±0.015 5^bdef	1.898 5±0.046 9^bef	3.090 6±0.175 1^bf	4.681 0±0.172 2^b

组别	0 h	12 h	24 h	48 h	72 h
A	17.440 3±1.241 1^cdef	13.801 6±0.234 9^def	8.394 7±0.446 1^ef	3.907 4±0.203 7^f	2.382 5±0.068 5
B	17.398 6±1.030 6^cdef	8.370 7±0.397 5^adef	3.389 6±0.076 8^aef	2.136 3±0.036 0^af	1.414 6±0.057 6^a
C1	17.759 0±2.008 8^def	13.716 8±0.347 5^defg	6.968 4±0.427 9^efg	3.362 3±0.232 1^fg	2.160 6±0.186 0 ^g
C2	17.946 1±0.521 5^def	14.260 9±0.447 2^defg	6.803 5±1.180 3^efg	3.272 7±0.244 1^g	2.147 8±0.295 5^g
D1	17.149 9±0.627 1^cdef	10.976 9±1.510 5^bdef	4.932 7±0.066 7^bef	2.710 4±0.346 2^b	1.945 0±0.205 3^b
D2	18.361 9±0.511 9^cdef	11.172 0±0.292 1^bdef	4.880 7±0.099 3^bef	2.705 2±0.102 7^bf	1.781 9±0.058 9^b