基于转录组测序探讨柚皮素对脂多糖作用下人牙周膜干细胞的抗炎作用及相关机制

doi:10.7518/hxkq.2024.2023453

华西口腔医学杂志 ›› 2024, Vol. 42 ›› Issue (4): 512-520.doi: 10.7518/hxkq.2024.2023453

基于转录组测序探讨柚皮素对脂多糖作用下人牙周膜干细胞的抗炎作用及相关机制

李俊玉¹^,²(), 徐晓梅¹^,², 刘兴玉¹^,², 曾婷¹^,², 张丽¹^,², 郑茜¹^,²()

^1.西南医科大学附属口腔医院正畸科，泸州 646000
^2.口颌面修复重建和再生泸州市重点实验室，泸州 646000

收稿日期:2023-12-26 修回日期:2024-05-28 出版日期:2024-08-01 发布日期:2024-07-17
通讯作者: 郑茜 E-mail:601169913@qq.com;317041990@qq.com
作者简介:李俊玉，医师，硕士，E-mail：601169913@qq.com
基金资助:
四川省科技厅创新专项项目(2022YFS0634);四川省中医药管理局面上项目(2023MS080);泸州市科技计划项目(2022-JYJ-116)

Anti-inflammatory effects and related mechanisms of naringenin in human periodontal ligament stem cells under lipopolysaccharide stimulation based on RNA sequencing

Li Junyu¹^,²(), Xu Xiaomei¹^,², Liu Xingyu¹^,², Zeng Ting¹^,², Zhang Li¹^,², Zheng Qian¹^,²()

^1.Dept. of Orthodontics, The Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, China
^2.Oral ＆ Maxillofacial Reconstruction and Regeneration of Luzhou Key Laboratory, Luzhou 646000, China

Received:2023-12-26 Revised:2024-05-28 Online:2024-08-01 Published:2024-07-17
Contact: Zheng Qian E-mail:601169913@qq.com;317041990@qq.com
Supported by:
Sichuan Provincial Science and Technology Department Innovation Project(2022YFS0634);Sichuan Provincial Administration of Traditional Chinese Medicine Project(2023MS080);Luzhou Science and Technology Plan Pro-ject(2022-JYJ-116)

摘要/Abstract

摘要：

目的联合使用转录组测序（RNA-seq）和生信分析，探究柚皮素（Nar）对脂多糖（LPS）刺激下的人牙周膜干细胞（hPDLSCs）的抗炎作用及机制。方法采用细胞计数试剂盒（CCK-8）、实时荧光定量逆转录聚合酶链反应（qRT-PCR）、酶联免疫吸附试验（ELISA）检测Nar对LPS刺激下hPDLSCs增殖及炎性因子表达情况，筛选出Nar的最佳抗炎浓度。采用RNA-seq，以|log2FC|≥1且P≤0.05为标准筛选出显著差异基因（DEGs）。采用火山图分析、京都基因与基因组百科全书（KEGG）通路富集分析、String数据库及Cytoscape的MCODE模块筛选核心基因。ELISA、qRT-PCR和蛋白印迹实验（Western blot）检测对核因子κB（NF-κB）信号通路的影响。结果适宜浓度的Nar可减轻LPS刺激下hPDLSCs的炎症因子表达，促进其增殖，20 μmol/L Nar抗炎效果最佳。RNA-seq提示炎症相关信号通路显著富集。Nar通过抑制NF-κB信号通路产生抗炎作用，Nar与NF-κB抑制剂BAY 11-7802效果相似。结论 Nar可以通过抑制NF-κB信号通路发挥抗炎作用。Nar可能是辅助治疗牙周炎的一种潜在的药效成分。

关键词: 柚皮素, 人牙周膜干细胞, 抗炎作用, 核因子κB, 转录组测序

Abstract:

Objective RNA sequencing (RNA-seq) and bioinformatic analysis were combined and used to explore the anti-inflammatory effects and mechanisms of naringenin (Nar) in lipopolysaccharide (LPS)-stimulated human periodontal ligament stem cells (hPDLSCs). Methods Cell counting kit-8, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA) were adopted to detect the effects of Nar on the proliferation and expression of inflammatory factors in LPS-stimulated hPDLSCs, screening for the optimal anti-inflammatory concentration of Nar. Differentially expressed genes (DEGs) were screened using |log2FC|≥1 and P≤0.05 as criteria. Volcano plot analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the String database, and the MCODE module of Cytoscape were utilized to select core genes and enriched pathways. The effects on the nuclear factor κB (NF-κB) signaling pathway were verified using ELISA, qRT-PCR, and Western blot. Results Appropriate concentrations of Nar could alleviate the expression of inflammatory factors and promote the proliferation of hPDLSCs stimulated by LPS. The best anti-inflammatory effect was achieved with 20 μmol/L Nar. RNA-seq showed significant enrichment of inflammation-related signaling pathways. The anti-inflammatory effect of Nar was mediated by inhibiting the NF-κB signaling pathway, similar to the effect of the NF-κB inhibitor BAY 11-7802. Conclusion Nar could exert its anti-inflammatory effects by inhibiting the NF-κB signaling pathway, making it a potential therapeutic option for the adjuvant treatment of periodontitis.

Key words: naringenin, human periodontal ligament stem cells, anti-inflammatory effect, nuclear factor κB, RNA sequencing

中图分类号:

Q257

李俊玉, 徐晓梅, 刘兴玉, 曾婷, 张丽, 郑茜. 基于转录组测序探讨柚皮素对脂多糖作用下人牙周膜干细胞的抗炎作用及相关机制[J]. 华西口腔医学杂志, 2024, 42(4): 512-520.

Li Junyu, Xu Xiaomei, Liu Xingyu, Zeng Ting, Zhang Li, Zheng Qian. Anti-inflammatory effects and related mechanisms of naringenin in human periodontal ligament stem cells under lipopolysaccharide stimulation based on RNA sequencing[J]. West China Journal of Stomatology, 2024, 42(4): 512-520.

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参考文献 33

1	Nikniaz S, Vaziri F, Mansouri R. Impact of resveratrol supplementation on clinical parameters and inflammatory markers in patients with chronic periodontitis: a randomized clinical trail[J]. BMC Oral Health, 2023, 23(1): 177.
2	Isola G, Tartaglia GM, Santonocito S, et al. Growth differentiation factor-15 and circulating biomarkers as predictors of periodontal treatment effects in patients with periodontitis: a randomized-controlled clinical trial[J]. BMC Oral Health, 2023, 23(1): 582.
3	Liu S, Xie G, Chen M, et al. Oral microbial dysbiosis in patients with periodontitis and chronic obstructive pulmonary disease[J]. Front Cell Infect Microbiol, 2023, 13: 1121399.
4	Pan S, Li Y, He H, et al. Identification of ferroptosis, necroptosis, and pyroptosis-associated genes in perio-dontitis-affected human periodontal tissue using integrated bioinformatic analysis[J]. Front Pharmacol, 2023, 13: 1098851.
5	Feng Q. Gastrodin attenuates lipopolysaccharide-indu-ced inflammation and oxidative stress, and promotes the osteogenic differentiation of human periodontal ligament stem cells through enhancing sirtuin3 expression[J]. Exp Ther Med, 2022, 23(4): 296.
6	Mohammadian Haftcheshmeh S, Momtazi-Borojeni AA. Berberine as a promising natural compound for the treatment of periodontal disease: a focus on anti-inflammatory properties[J]. J Cell Mol Med, 2021, 25(24): 11333-11337.
7	Bae JY, Lee DS, Cho YK, et al. Daphne jejudoensis attenuates LPS-induced inflammation by inhibiting TNF-α, IL-1β, IL-6, iNOS, and COX-2 expression in perio-dontal ligament cells[J]. Pharmaceuticals (Basel), 2022, 15(4): 387.
8	Yu B, Hu J, Li Q, et al. CircMAP3K11 contributes to proliferation, apoptosis and migration of human perio-dontal ligament stem cells in inflammatory microenvironment by regulating TLR4 via miR-511 sponging[J]. Front Pharmacol, 2021, 12: 633353.
9	Wei Y, Fu J, Wu W, et al. Quercetin prevents oxidative stress-induced injury of periodontal ligament cells and alveolar bone loss in periodontitis[J]. Drug Des Devel Ther, 2021, 15: 3509-3522.
10	Zhang K, Li C, Sun J, et al. PRMT5 inhibition ameliorates inflammation and promotes the osteogenic differentiation of LPS‑induced periodontal stem cells via STAT3/NF-κB signaling[J]. Exp Ther Med, 2023, 25(6): 264.
11	Tan W, Dai F, Yang D, et al. MiR-93-5p promotes granulosa cell apoptosis and ferroptosis by the NF-kB signaling pathway in polycystic ovary syndrome[J]. Front Immunol, 2022, 13: 967151.
12	Huang X, Shen H, Liu Y, et al. Fisetin attenuates periodontitis through FGFR1/TLR4/NLRP3 inflammasome pathway[J]. Int Immunopharmacol, 2021, 95: 107505.
13	Zhang J, Dong X, Yan Q, et al. Galectin-1 inhibited LPS-induced autophagy and apoptosis of human periodontal ligament stem cells[J]. Inflammation, 2021, 44(4): 1302-1314.
14	Shen J, Cheng J, Zhu S, et al. Regulating effect of baicalin on IKK/IKB/NF-kB signaling pathway and apoptosis-related proteins in rats with ulcerative colitis[J]. Int Immunopharmacol, 2019, 73: 193-200.
15	Mohamed EE, Ahmed OM, Zoheir KMA, et al. Naringin-dextrin nanocomposite abates diethylnitrosamine/acetylaminofluorene-induced lung carcinogenesis by modulating oxidative stress, inflammation, apoptosis, and cell proliferation[J]. Cancers (Basel), 2023, 15(20): 5102.
16	Wang Y, Li Z, Wang B, et al. Naringenin attenuates inflammation and apoptosis of osteoarthritic chondrocytes via the TLR4/TRAF6/NF-κB pathway[J]. PeerJ, 2023, 11: e16307.
17	Laurindo LF, Santos AROD, Carvalho ACA, et al. Phytochemicals and regulation of NF-κB in inflammatory bowel diseases: an overview of in vitro and in vivo effects[J]. Metabolites, 2023, 13(1): 96.
18	李胜鸿, 彭世元, 罗小玲, 等. 柚皮素通过基质细胞衍生因子1/趋化因子受体4信号轴对脂多糖作用下人牙周膜干细胞抗炎、成血管和成骨分化能力的影响[J]. 华西口腔医学杂志, 2023, 41(2): 175-184.
	Li SH, Peng SY, Luo XL, et al. Effect of naringenin on the anti-inflammatory, vascularization, and osteogenesis differentiation of human periodontal ligament stem cells via the stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 signaling axis stimulated by lipopolysaccharide[J]. West China J Stomatol, 2023, 41(2): 175-184.
19	Polosukhina D, Singh K, Asim M, et al. CCL11 exacerbates colitis and inflammation-associated colon tumorigenesis[J]. Oncogene, 2021, 40(47): 6540-6546.
20	Hanna DN, Smith PM, Novitskiy SV, et al. SMAD4 suppresses colitis-associated carcinoma through inhibition of CCL20/CCR6-mediated inflammation[J]. Gastroente-rology, 2022, 163(5): 1334-1350.e14.
21	Mathur AN, Zirak B, Boothby IC, et al. Treg-cell control of a CXCL5-IL-17 inflammatory axis promotes hair-follicle-stem-cell differentiation during skin-barrier repair[J]. Immunity, 2019, 50(3): 655-667.e4.
22	Camargo LDN, Righetti RF, de Almeida FM, et al. Modulating asthma-COPD overlap responses with IL-17 inhibition[J]. Front Immunol, 2023, 14: 1271342.
23	Lu Y, Xing C, Zhang C, et al. Promotion of IL‑17/NF‑κB signaling in autoimmune thyroid diseases[J]. Exp Ther Med, 2022, 25(1): 51.
24	Wang N, Hao Y, Fu L. Trimethylamine-N-oxide promotes osteoclast differentiation and bone loss via activating ROS-dependent NF-κB signaling pathway[J]. Nutrients, 2022, 14(19): 3955.
25	Liu X, Lu B, Fu J, et al. Amorphous silica nanoparticles induce inflammation via activation of NLRP3 inflammasome and HMGB1/TLR4/MYD88/NF-κB signaling pathway in HUVEC cells[J]. J Hazard Mater, 2021, 404(Pt B): 124050.
26	Camargo LDN, Dos Santos TM, de Andrade FCP, et al. Bronchial vascular remodeling is attenuated by anti-IL-17 in asthmatic responses exacerbated by LPS[J]. Front Pharmacol, 2020, 11: 1269.
27	Somensi N, Rabelo TK, Guimarães AG, et al. Carvacrol suppresses LPS-induced pro-inflammatory activation in RAW 264.7 macrophages through ERK1/2 and NF-κB pathway[J]. Int Immunopharmacol, 2019, 75: 105743.
28	Zusso M, Lunardi V, Franceschini D, et al. Ciprofloxacin and levofloxacin attenuate microglia inflammatory response via TLR4/NF-κB pathway[J]. J Neuroinflammation, 2019, 16(1): 148.
29	Wang Y, Liu P, Ma G, et al. Mechanism of dioscin ameliorating renal fibrosis through NF‑κB signaling path-way‑mediated inflammatory response[J]. Mol Med Rep, 2023, 27(4): 93.
30	Zhuang L, Zong X, Yang Q, et al. Interleukin-34-NF-κB signaling aggravates myocardial ischemic/reperfusion injury by facilitating macrophage recruitment and polarization[J]. EBioMedicine, 2023, 95: 104744.
31	Bonomini F, Favero G, Petroni A, et al. Melatonin modulates the SIRT1-related pathways via transdermal cryopass-laser administration in prostate tumor xenograft[J]. Cancers (Basel), 2023, 15(20): 4908.
32	Tran HG, Shuayprom A, Kueanjinda P, et al. Oxyresveratrol attenuates inflammation in human keratinocyte via regulating NF-κB signaling and ameliorates eczematous lesion in DNCB-induced dermatitis mice[J]. Pharmaceutics, 2023, 15(6): 1709.
33	Xie YQ, Huang JY, Chen YX, et al. Anti-inflammatory and analgesic effects of Streblus indicus[J]. Front Pharmacol, 2023, 14: 1249234.

基因名称	引物序列（5’-3’）
TNF-α	Forward：CCCAGGGACCTCTCTCTAATC
	Reverse：ATGGGCTACAGGCTTGTCACT
IL-6	Forward：AGGAGACTTGCCTGGTGAAA
	Reverse：GCATTTGTGGTTGGGTCAGG
IL-8	Forward：GTTGTAGCGTAGCTAGATGC
	Reverse：TTCTCGTGTGCAGTATCTACG

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基于转录组测序探讨柚皮素对脂多糖作用下人牙周膜干细胞的抗炎作用及相关机制

Anti-inflammatory effects and related mechanisms of naringenin in human periodontal ligament stem cells under lipopolysaccharide stimulation based on RNA sequencing

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