低能量激光对人牙周膜细胞白细胞介素-6、肿瘤坏死因子-α、骨保护素、核因子-κB受体活化因子配体表达的影响

doi:10.7518/hxkq.2023.2023133

华西口腔医学杂志 ›› 2023, Vol. 41 ›› Issue (5): 521-532.doi: 10.7518/hxkq.2023.2023133

低能量激光对人牙周膜细胞白细胞介素-6、肿瘤坏死因子-α、骨保护素、核因子-κB受体活化因子配体表达的影响

汤盟¹(), 崔占琴¹, 王阳阳¹, 陈增国², 李文静²(), 张翠萍³

^1.河北医科大学第二医院口腔正畸科，石家庄 050000
^2.河北医科大学第二医院口腔内科，石家庄 050000
^3.福建省龙岩市永定区医院口腔科，龙岩 364100

收稿日期:2023-04-24 修回日期:2023-07-26 出版日期:2023-10-01 发布日期:2023-10-08
通讯作者: 李文静 E-mail:369849317@qq.com;lwjkq@163.com
作者简介:汤盟，医师，硕士，E-mail：369849317@qq.com
基金资助:
2022年河北省政府资助临床医学优秀人才项目(冀财预复（2022）180号)

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

Tang Meng¹(), Cui Zhan-qin¹, Wang Yangyang¹, Chen Zengguo², Li Wenjing²(), Zhang Cuiping³

^1.Dept. of Orthodontics, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, China
^2.Dept. of Oral Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, China
^3.Dept. of Stomatology, Yongding District Hospital of Longyan City, Fujian Province, Longyan 364100, China

Received:2023-04-24 Revised:2023-07-26 Online:2023-10-01 Published:2023-10-08
Contact: Li Wenjing E-mail:369849317@qq.com;lwjkq@163.com
Supported by:
Hebei Provincial Government Funded the Clinical Medicine Outstanding Talents Project in 2022(JCYF[2022]180)

摘要/Abstract

摘要：

目的通过观察高糖环境下低能量激光（LLL）对受静压力刺激的人牙周膜细胞（HPDLC）白细胞介素-6（IL-6）、肿瘤坏死因子（TNF）-α、骨保护素（OPG）、核因子-κB受体活化因子配体（RANKL）表达的影响，以期探究LLL对糖尿病患者牙周组织炎症及正畸治疗过程中骨改建调控的分子生物学机制。方法体外培养HPDLC，模拟正畸加力，结合LLL照射，将所培养细胞随机分为4组：低糖型杜氏改良Eagle培养基（DMEM）+压力刺激（A组），高糖型DMEM+压力刺激（B组），低糖型DMEM+LLL照射+压力刺激（C组），高糖型DMEM+LLL照射+压力刺激（D组），其中C、D组根据给予的激光能量密度值不同又分C1、D1组（能量密度值：3.75 J/cm²）和C2、D2组（能量密度值：5.625 J/cm²）。根据已有分组，对A、B、C、D组细胞进行加力，对C、D组细胞在细胞加力前进行LLL照射。分别观察0、12、24、48、72 h，定时提取各组细胞培养上清液，采用酶联免疫吸附（ELISA）法检测各组不同时段IL-6、TNF-α、OPG、RANKL的蛋白表达。结果 1）在持续静压力刺激下，HPDLC分泌的IL-6、TNF-α浓度随时间逐渐上升；12 h后IL-6、TNF-α的浓度在A组与B、C1、C2组组间两两比较的差异均有统计学意义（P<0.05），B组与D1、D2组组间两两比较的差异也均有统计学意义（P<0.01）。2）OPG蛋白浓度在24 h之前呈现出上升趋势，24 h后随时间出现下降趋势；RANKL蛋白浓度随时间呈上升趋势；OPG/RANKL比值随时间呈下降趋势；持续静压力刺激12 h后，A组与B、C1、C2组，B组与D1、D2组组间两两比较的OPG、RANKL及OPG/RANKL的比值的差异均具有统计学意义（P<0.05）。结论 1）在高糖环境持续静压力刺激下，HPDLC分泌的IL-6、TNF-α浓度随时间呈现上升趋势；OPG的表达水平降低，RANKL的表达水平增加，OPG/RANKL的比值减小，提示高糖环境会促进骨吸收反应的发生；给予LLL照射干预后发现，IL-6、TNF-α的浓度出现下降，表明其可拮抗高糖环境所致的炎症因子水平的升高，并且能上调人HPDLC中OPG的表达，下调HPDLC中RANKL的表达，从而上调OPG/RANKL的比值，逆转高糖所致的骨代谢失衡。2）在3.75~5.625 J/cm²这一能量密度范围内，随着给予的激光能量密度值的增大，其表现出的降低炎症因子及对HPDLC骨代谢的调控作用增强，提示在一定范围内，LLL调节骨改建的能力随剂量增加而增强。

关键词: 高糖环境, 白细胞介素-6, 肿瘤坏死因子-α, 骨保护素, 核因子-κB 受体活化因子配体, 人牙周膜细胞, 低能量激光

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

中图分类号:

R 783.5

汤盟, 崔占琴, 王阳阳, 陈增国, 李文静, 张翠萍. 低能量激光对人牙周膜细胞白细胞介素-6、肿瘤坏死因子-α、骨保护素、核因子-κB受体活化因子配体表达的影响[J]. 华西口腔医学杂志, 2023, 41(5): 521-532.

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.

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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

低能量激光对人牙周膜细胞白细胞介素-6、肿瘤坏死因子-α、骨保护素、核因子-κB受体活化因子配体表达的影响

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

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