低能量激光照射对脂多糖介导人牙周膜成纤维细胞炎性损伤的保护作用

doi:10.7518/hxkq.2021.03.003

摘要/Abstract

摘要： 目的

研究低能量激光照射（LLLI）对脂多糖（LPS）介导人牙周膜成纤维细胞（hPDLFs）炎性损伤的影响及机制。

方法

将hPDLFs接种于培养板内，随机分为正常组、LPS组和LPS+LLLI组。正常组细胞行常规培养基培养，LPS组及LPS+LLLI组在加入1 mg·L^-1 LPS培养基中培养24 h，LPS+LLLI组3个亚组分别接受不同强度照射。4 d后检测各组细胞凋亡、活力及细胞内游离Ca²⁺浓度，酶联免疫吸附试验检测各组细胞上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-8、IL-1β及IL-6的含量，逆转录聚合酶链反应（RT-PCR）及Western blot检测各个实验组hPDLFs的基质金属蛋白酶（MMP）-2、MMP-3、MMP-9基因和蛋白的表达。

结果

与正常组比较，LPS组hPDLFs细胞凋亡率及细胞内游离Ca²⁺浓度增加，细胞活力降低（P<0.05），细胞上清液中TNF-α、IL-8、IL-1β及IL-6的含量显著升高（P<0.05），hPDLFs的MMP-2、MMP-3、MMP-9基因和蛋白的表达显著升高（P<0.05）。与LPS组比较，LPS+LLLI组细胞凋亡率及细胞内游离Ca²⁺浓度降低，细胞活力升高（P<0.05），细胞上清液中TNF-α、IL-8、IL-1β及IL-6的含量显著降低（P<0.05），hPDLFs的MMP-2、MMP-3、MMP-9基因和蛋白的表达显著降低（P<0.05）。

结论

LLLI对LPS诱导 hPDLFs炎性损伤有保护作用，照射强度为4 J·cm^-2时效果最明显。

关键词: 低能量激光照射, 人牙周膜成纤维细胞, 脂多糖, 炎性损伤, 影响

Abstract: Objective

To study the effect and mechanism of low-level laser irradiation (LLLI) on lipopolysaccharide (LPS)-induced inflammatory injury of human periodontal ligament fibroblasts (hPDLFs).

Methods

hPDLFs were inoculated into well plates and randomly divided into the normal group, LPS group, and LPS+LLLI group. The cells in the normal group were cultured in conventional medium. The hPDLFs in the LPS and LPS+LLLI groups were cultured in RPMI1640 medium containing 1 mg·L^-1 LPS. The three subgroups of the LPS+LLLI group were exposed to different LLLI. After 4 days, the cell apoptosis, viability, and intracellular free Ca²⁺ concentration of each group were measured. The contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1β, and IL-6 were measured by enzyme linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of matrix metalloproteinase (MMP)-2, MMP-3, and MMP-9 genes and proteins of hPDLFs in each group.

Results

Compared with the normal group, the LPS group showed increased apoptosis rate of hPDLFs and intracellular free Ca²⁺concentration and decreased cell viability (P<0.05). The TNF-α, IL-8, IL-1β, and IL-6 levels were higher in the cell supernatant (P<0.05), and the expression of MMP-2, MMP-3, and MMP-9 genes and proteins of hPDLFs was significantly increased (P<0.05). Compared with the LPS group, the LPS+LLLI group showed significantly decreased apoptosis rate and intracellular free Ca²⁺ concentration and significantly increased cell viability (P<0.05). The TNF-α, IL-8, IL-1β, and IL-6 levels in the supernatant of cells and the expression of MMP-2, MMP-3, and MMP-9 genes and proteins of hPDLFs were significantly decreased (P<0.05).

Conclusion

LLLI has a protective effect on the inflammatory injury of hPDLFs induced by LPS, and the effect is most obvious when the irradiation intensity is 4 J·cm^-2.

Key words: low-level laser irradiation, human periodontal ligament fibroblasts, lipopolysaccharide, inflammatory injury, effect

中图分类号:

Q 786

靳晓兰, 张亚男, 孙成蕊, 邹朝晖. 低能量激光照射对脂多糖介导人牙周膜成纤维细胞炎性损伤的保护作用[J]. 华西口腔医学杂志, 2021, 39(3): 260-266.

Jin Xiaolan, Zhang Yanan, Sun Chengrui, Zou Zhaohui. Protective effect of low-level laser irradiation on lipopolysaccharide-mediated inflammatory injury of human periodontal ligament fibroblasts[J]. West China Journal of Stomatology, 2021, 39(3): 260-266.

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参考文献 14

1	Wang F, Guan M, Wei L, et al. IL-18 promotes the secretion of matrix metalloproteinases in human periodontal ligament fibroblasts by activating NF-κB signaling[J]. Mol Med Rep, 2019, 19(1): 703-710.
2	Zhang L, Chen W, Li Y, et al. Effect of 650-nm low-level laser irradiation on c-Jun, c-Fos, ICAM-1, and CCL2 expression in experimental periodontitis[J]. Lasers Med Sci, 2020, 35(1): 31-40.
3	隋华欣, 吕培军, 王宇光, 等. 低能量激光照射对人脂肪基质细胞增殖分化的影响[J]. 北京大学学报(医学版), 2017, 49(2): 337-343.
	Sui HX, Lü PJ, Wang YG, et al. Effect of low-level laser irradiation on proliferation and osteogenic differentiation of human adipose-derived stromal cells[J]. J Peking Univ (Health Sci), 2017, 49(2): 337-343.
4	朱建国, 张菊香, 闫海燕, 等. 低能量氦-氖激光血管内照射与西比灵联合治疗糖尿病性周围神经病[J]. 中华物理医学与康复杂志, 2005, 27(3): 180-181.
	Zhu JG, Zhang JX, Yan HY, et al. Low-energy helium-neon laser intravascular irradiation combined with Sibelin for the treatment of diabetic peripheral neuropathy[J]. Chin J Phys Med Rehabil, 2005, 27(3): 180-181.
5	赵燕娟, 李荣华, 任刚, 等. 低能量激光联合牙周基础治疗对种植体周围炎龈沟液中IL-8、b-FGF及IL-1β水平的影响[J]. 国际生物医学工程杂志, 2019, 42(2): 130-133.
	Zhao YJ, Li RH, Ren G, et al. Effect of low level laser treatment combined with periodontal initial therapy on IL-8, b-FGF and IL-1β content in gingival crevicular fluid on peri-implantitis[J]. Int Biomed Eng J, 2019, 42(2): 130-133.
6	李凤, 徐华顺. 低能量激光配合牙周基础治疗对糖尿病合并慢性牙周炎患者龈沟液细胞因子及LPS、leptin的影响[J]. 上海口腔医学, 2018, 27(6): 637-640.
	Li F, Xu HS. Effects of low level laser combined with basic periodontal therapy on cytokines and LPS, leptin in gingival crevicular fluid of diabetes mellitus complicated with chronic periodontitis patients[J]. Shanghai J Stomatol, 2018, 27(6): 637-640.
7	Lee JH, Chiang MH, Chen PH, et al. Anti-inflammatory effects of low-level laser therapy on human periodontal ligament cells: in vitro study[J]. Lasers Med Sci, 2018, 33(3): 469-477.
8	Wu X, Zhang G, Feng X, et al. Transcriptome analysis of human periodontal ligament fibroblasts exposed to Porphyromonas gingivalis LPS[J]. Arch Oral Biol, 2020, 110: 104632.
9	Lu WL, Song DZ, Yue JL, et al. NLRP3 inflammasome may regulate inflammatory response of human periodontal ligament fibroblasts in an apoptosis-associated speck-like protein containing a CARD (ASC)-dependent manner[J]. Int Endod J, 2017, 50(10): 967-975.
10	Tanaka K, Miyake Y, Hanioka T, et al. The IL18 promoter polymorphism, rs1946518, is associated with the risk of periodontitis in Japanese women: the Kyushu Okinawa maternal and child health study[J]. Tohoku J Exp Med, 2017, 243(3): 159-164.
11	Almeida-Junior LA, Marques NCT, Prado MTO, et al. Effect of single and multiple doses of low-level laser therapy on viability and proliferation of stem cells from human exfoliated deciduous teeth (SHED)[J]. Lasers Med Sci, 2019, 34(9): 1917-1924.
12	任刚, 刘毅, 李荣华, 等. 低能量激光治疗对临床患者种植体周围炎的疗效研究[J]. 国际生物医学工程杂志, 2018, 41(5): 439-442.
	Ren G, Liu Y, Li RH, et al. Effect of low energy laser therapy on peri-implantitis in clinical patients[J]. Int J Biomed Eng, 2018, 41(5): 439-442.
13	Gürsoy M, Könönen E, Tervahartiala T, et al. Longitudinal study of salivary proteinases during pregnancy and postpartum[J]. J Periodontal Res, 2010, 45(4): 496-503.
14	Tiranathanagul S, Pattamapun K, Yongchaitrakul T, et al. MMP-2 activation by Actinobacillus actinomycetemcomitans supernatant in human PDL cells was corresponded with reduction of TIMP-2[J]. Oral Dis, 2004, 10(6): 383-388.

组别	细胞存活率/%	细胞内Ca²⁺浓度/（nmol·L^-1）
正常组	100*	226.69±19.45*
LPS组	34.52±7.86^#	312.38±28.08^#
2 J·cm^-2组	52.36±11.38^#*^@	257.25±26.32^#*^@
4 J·cm^-2组	72.5±12.25^#*	281.42±24.37^#*
6 J·cm^-2组	55.54±9.46^#*^@	251.24±21.32^#*^@

组别	TNF-α	IL-8	IL-1β	IL-6
正常组	0.79±0.14*	0.92±0.02*	1.03±0.04*	0.84±0.03*
LPS组	5.27±0.08^#	6.38±0.06^#	7.14±0.06^#	3.36±0.05^#
2 J·cm^-2组	3.92±0.05^#*^@	5.06±0.07^#*^@	5.86±0.05^#*^@	2.47±0.04^#*^@
4 J·cm^-2组	2.82±0.02^#*	3.56±0.04^#*	3.75±0.06^#*	1.38±0.02^#*
6 J·cm^-2组	3.48±0.03^#*^@	5.01±0.06^#*^@	5.64±0.03^#*^@	2.35±0.03^#*^@

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