金合欢素调节HMGB1/TLR4信号通路对脂多糖诱导牙髓细胞凋亡的影响

doi:10.11958/20240738

天津医药 ›› 2024, Vol. 52 ›› Issue (12): 1238-1243.doi: 10.11958/20240738

金合欢素调节HMGB1/TLR4信号通路对脂多糖诱导牙髓细胞凋亡的影响

林瑶¹(), 刘从娜², 王世霞², 张志勇²^,^△()

1 唐山市妇幼保健院口腔科（邮编063000）
2 河北医科大学第二医院口腔内科

收稿日期:2024-06-06 修回日期:2024-07-26 出版日期:2024-12-15 发布日期:2024-12-17
通讯作者: △E-mail：p39cck@163.com
作者简介:林瑶（1987），女，主治医师，主要从事口腔修复、种植相关研究。E-mail：wbyw23@163.com
基金资助:
河北省医学科学研究重点课题计划项目(20231762)

Effect of acacetin on lipopolysaccharide induced apoptosis of dental pulp cells by regulating the HMGB1/TLR4 signaling pathway

LIN Yao¹(), LIU Congna², WANG Shixia², ZHANG Zhiyong²^,^△()

1 Department of Stomatology, Tangshan Maternal and Child Health Hospital, Tangshan 063000, China
2 Department of Stomatology, Second Hospital of Hebei Medical University

Received:2024-06-06 Revised:2024-07-26 Published:2024-12-15 Online:2024-12-17
Contact: △E-mail：p39cck@163.com

林瑶, 刘从娜, 王世霞, 张志勇. 金合欢素调节HMGB1/TLR4信号通路对脂多糖诱导牙髓细胞凋亡的影响[J]. 天津医药, 2024, 52(12): 1238-1243.

LIN Yao, LIU Congna, WANG Shixia, ZHANG Zhiyong. Effect of acacetin on lipopolysaccharide induced apoptosis of dental pulp cells by regulating the HMGB1/TLR4 signaling pathway[J]. Tianjin Medical Journal, 2024, 52(12): 1238-1243.

摘要/Abstract

摘要：

目的探究金合欢素（ACE）通过调节高迁移率族蛋白1（HMGB1）/Toll样受体4（TLR4）信号通路对脂多糖（LPS）诱导牙髓细胞凋亡的影响。方法从5例正畸、阻生患者拔除的健康牙齿的牙髓中分离、克隆、纯化第三代牙髓细胞，观察细胞形态并通过免疫荧光鉴定基质细胞表面抗原1（STRO1）；流式细胞术鉴定表皮抗原CD90、CD105、CD34、CD45；MTT鉴定ACE对原代牙髓细胞（DPSCs）活性影响。将DPSCs分为Control组（0 μmol/L ACE培养）、LPS组（1 ng/L LPS处理）、L-ACE组（LPS组基础上加20 μmol/L ACE）、H-ACE组（LPS组基础上加40 μmol/L ACE）、pcDNA-NC组（H-ACE组基础上转染pcDNA-NC质粒），HMGB组（H-ACE组基础上转染pcDNA-HMGB）。CCK-8检测细胞增殖；克隆形成实验检测克隆形成数；Western blot实验检测HMGB1/TLR4通路、凋亡、炎症相关蛋白的表达；酶联免疫吸附试验检测细胞上清液中白细胞介素（IL）-1β、IL-4、肿瘤坏死因子α（TNF-α）的含量。结果从细胞形态、STRO1阳性表达以及CD90、CD105呈阳性，CD34、CD45呈阴性鉴定成功分离DPSCs。与Control组比较，LPS组细胞增殖活力、克隆细胞数、B细胞淋巴瘤-2（Bcl-2）、IL-4水平减少，细胞凋亡率、通路相关蛋白HMGB1、TLR4，凋亡相关蛋白胱天蛋白酶（Caspase）-3、Caspase-7、Bcl-2相关X蛋白（Bax）以及炎症因子IL-1β、TNF-α水平升高（P<0.05）；与LPS组比较，L-ACE组、H-ACE组细胞增殖活力、Bcl-2、IL-4依次升高，细胞凋亡率、HMGB1、TLR4、Caspase-3、Caspase-7、Bax以及IL-1β、TNF-α水平依次降低（P<0.05）；过表达HMGB逆转了H-ACE对细胞增殖、凋亡、相关蛋白及炎症因子表达的影响（P<0.05）。结论 ACE通过影响HMGB1/TLR4信号通路活化，实现抑制LPS诱导的DPSCs凋亡和炎症反应的作用。

关键词: HMGB1蛋白质, Toll样受体4, 金合欢属, 脂多糖类, 牙髓炎, 细胞凋亡, 细胞增殖, 牙髓细胞

Abstract:

Objective To investigate the effect of acacetin (ACE) on the apoptosis of dental pulp cells induced by lipopolysaccharide (LPS) by regulating the high mobility histone 1 (HMGB1)/Toll-like receptor 4 (TLR4) signaling pathway. Methods Third generation dental pulp cells were isolated, cloned and purified from pulp of five patients with healthy teeth extracted from orthodontics and impaction. Cell morphology was observed and identification of stromal cell surface antigen1(STRO1) by immunofluorescence, and epidermal antigens CD90, CD105, CD34 and CD45 were identified by flow cytometry. MTT was applied to identify the effect of ACE on the activity of primary dental pulp cells (DPSCs). DPSCs were divided into the control group (0 μmol/L ACE culture), the LPS group (1 ng/L LPS treatment), the L-ACE group (added 20 μmol/L ACE on the basis of LPS group), the H-ACE group (added 40 μmol/L ACE on the basis of LPS group), the pcDNA-NC group (transfected pcDNA-NC plasmid on the basis of H-ACE group) and the HMGB group (transfected pcDNA-HMGB on the basis of H-ACE group). Cell proliferation was detected by CCK-8. Clone formation number was detected by clone formation assay. Western blot experiments were applied to detect the expression of HMGB1/TLR4 pathway, apoptosis and inflammation related proteins. ELISA assay was applied to detect levels of interleukin (IL-)-1β, IL-4 and tumor necrosis factor-α (TNF-α) in cell supernatant. Results DPSCs were successfully isolated and identified based on cell morphology, positive expression of STRO1, positive expression of CD90 and CD105, and negative identification of CD34 and CD45. Compared with the control group, cell proliferation activity, clonal cell count, B-cell lymphoma-2 (Bcl-2) and IL-4 levels were decreased in the LPS group, while apoptosis rate, the pathway related proteins HMGB1, TLR4, apoptosis related proteins aspartate-specific cysteine protease (Caspase) -3, Caspase-7, Bcl-2 associated X protein (Bax), and inflammatory factors IL-1β, TNF-α were increased in the LPS group (P<0.05). Compared with the LPS group, cell proliferation activity, Bcl-2 and IL-4 were increased successively in the L-ACE group and the H-ACE group, and apoptosis rate, pathway-related proteins HMGB1, TLR4, Caspase-3, Caspase-7 and Bax, and inflammatory factors IL-1β, TNF-α were reduced successively (P<0.05). Overexpression of HMGB reversed the effects of H-ACE on cell proliferation, apoptosis, expression of related proteins and inflammatory factors (P<0.05). Conclusion ACE inhibits LPS induced apoptosis of dental pulp cells, and it may be achieved by inhibiting the HMGB1/TLR4 signaling pathway.

Key words: HMGB1 protein, Toll-like receptor 4, acacia, lipopolysaccharides, pulpitis, apoptosis, cell proliferation, pulp cell

中图分类号:

R781.31，R34

图/表 10

参考文献 20

[1]	CHEN J, XU H, XIA K, et al. Resolvin E1 accelerates pulp repair by regulating inflammation and stimulating dentin regeneration in dental pulp stem cells[J]. Stem Cell Res Ther, 2021, 12(1):75. doi:10.1186/s13287-021-02141-y.
[2]	GALLER K M, WEBER M, KORKMAZ Y, et al. Inflammatory response mechanisms of the dentine-pulp complex and the periapical tissues[J]. Int J Mol Sci, 2021, 22(3):1480. doi:10.3390/ijms22031480.
[3]	IACULLI F, RODRíGUEZ-LOZANO F J, BRISEÑO-MARROQUÍN B, et al. Vital pulp therapy of permanent teeth with reversible or irreversible pulpitis: an overview of the literature[J]. J Clin Med, 2022, 11(14):4016. doi:10.3390/jcm11144016.
[4]	ZHANG P, CUI Z, LI S. The protective effects of S14G-humanin (HNG) against lipopolysaccharide (LPS)- induced inflammatory response in human dental pulp cells (hDPCs) mediated by the TLR4/MyD88/NF-κB pathway[J]. Bioengineered, 2021, 12(1):7552-7562. doi:10.1080/21655979.2021.1979914.
[5]	VASEENON S, WEEKATE K, SRISUWAN T, et al. Observation of inflammation,oxidative stress,mitochondrial dynamics,and apoptosis in dental pulp following a diagnosis of irreversible pulpitis[J]. Eur Endod J, 2023, 8(2):148-155. doi:10.14744/eej.2022.74745.
[6]	BU J, MAHAN Y, ZHANG S, et al. Acacetin inhibits inflammation by blocking MAPK/NF-κB pathways and NLRP3 inflammasome activation[J]. Front Pharmacol, 2024, 15:1286546. doi:10.3389/fphar.2024.1286546.
[7]	WU C, CHEN R L, WANG Y, et al. Acacetin alleviates myocardial ischaemia/reperfusion injury by inhibiting oxidative stress and apoptosis via the Nrf-2/HO-1 pathway[J]. Pharm Biol, 2022, 60(1):553-561. doi:10.1080/13880209.2022.2041675.
[8]	SHANG J, LIU W, YIN C, et al. Cucurbitacin E ameliorates lipopolysaccharide-evoked injury, inflammation and MUC5AC expression in bronchial epithelial cells by restraining the HMGB1-TLR4-NF-κB signaling[J]. Mol Immunol, 2019, 114:571-577. doi:10.1016/j.molimm.2019.09.008.
[9]	WANG D R, WANG Y H, PAN J, et al. Neurotrophic effects of dental pulp stem cells in repair of peripheral nerve after crush injury[J]. World J Stem Cells, 2020, 12(10):1196-1213. doi:10.4252/wjsc.v12.i10.1196.
[10]	杜暘, 金鼎, 高秀秋. 紫草素通过TLR4/NF-κB信号通路抑制由LPS诱导的人牙髓细胞炎症研究[J]. 实用口腔医学杂志, 2020, 36(1):15-19.
	DU Y, JING D, GAO X Q. The effects of Shikonin on LPS-induced cell inflammation of human dental pulp cells through TLR4/NF-κB signaling pathway[J]. Journal of Practical Stomatology, 2020, 36(1):15-19. doi:10.3969/j.issn.1001-3733.2020.01.003.
[11]	彭佳欣, 张自辉, 洪莉. 金合欢素逆转卵巢癌铂耐药的机制研究[J]. 中国实用妇科与产科杂志, 2023, 39(8):842-848.
	PENG J X, ZHANG Z H, HONG L. Study on the mechanism of acacetin reversing cisplatin resistance in ovarian cancer[J]. Chinese Journal of Practical Gynecology and Obstetrics, 2023, 39(8):842-848. doi:10.19538/j.fk2023080116.
[12]	WANG H S, YANG F H, WANG Y J, et al. Odontoblastic exosomes attenuate apoptosis in neighboring cells[J]. J Dent Res, 2019, 98(11):1271-1278. doi:10.1177/0022034519869580.
[13]	WANG X, SUN H, HU Z, et al. NUTM2A-AS1 silencing alleviates LPS-induced apoptosis and inflammation in dental pulp cells through targeting let-7c-5p/HMGB1 axis[J]. Int Immunopharmacol, 2021, 96:107497. doi:10.1016/j.intimp.2021.107497.
[14]	WANG H, JIANG Z, PANG Z, et al. Acacetin alleviates inflammation and matrix degradation in nucleus pulposus cells and ameliorates intervertebral disc degeneration in vivo[J]. Drug Des Devel Ther, 2020, 14:4801-4813. doi:10.2147/DDDT.S274812.
[15]	吴琼, 李锦源, 黄文涛, 等. 金合欢素对肝癌HepG2细胞增殖、凋亡和迁移的影响及机制研究[J]. 天津医药, 2023, 51(3):235-239.
	WU Q, LI J Y, HUANG W T, et al. Effects of acacetin on proliferation, apoptosis and migration of hepatocellular carcinoma HepG2 cells and its mechanism[J]. Tianjin Med J, 2023, 51(3):235-239. doi:10.11958/20221115.
[16]	REN J, YUE B, WANG H, et al. Acacetin ameliorates experimental colitis in mice via inhibiting macrophage inflammatory response and regulating the composition of gut microbiota[J]. Front Physiol, 2020, 11:577237. doi:10.3389/fphys.2020.577237.
[17]	YANG H, WANG H, ANDERSSON U. Targeting inflammation driven by HMGB1[J]. Front Immunol, 2020, 11:484. doi:10.3389/fimmu.2020.00484.
[18]	LI J, XIA Y, SUN B, et al. Neutrophil extracellular traps induced by the hypoxic microenvironment in gastric cancer augment tumour growth[J]. Cell Commun Signal, 2023, 21(1):86. doi:10.1186/s12964-023-01112-5.
[19]	ZHENZHEN Z, FENGHAO L, MEINA M, et al. Targeting HMGB1-TLR4 signaling by miR-216a-5p elevation alleviates the inflammatory behavioral hypersensitivity[J]. Neurosci Lett, 2021, 759:136043. doi:10.1016/j.neulet.2021.136043.
[20]	LI Q, XU M, LI Z, et al. Maslinic acid attenuates ischemia/reperfusion injury-induced myocardial inflammation and apoptosis by regulating HMGB1-TLR4 axis[J]. Front Cardiovasc Med, 2021, 8(1):e768947. doi:10.3389/fcvm.2021.768947.

组别	细胞增殖活力（OD值）			克隆细胞数/（个／视野）
组别	24 h	48 h	72 h	克隆细胞数/（个／视野）
Control组	0.65±0.03	0.67±0.01	0.73±0.05	118.32±4.03
LPS组	0.38±0.02^a	0.42±0.03^a	0.48±0.03^a	71.43±4.34^a
L-ACE组	0.42±0.01^b	0.49±0.03^b	0.51±0.03^b	83.27±5.14^b
H-ACE组	0.56±0.03^bc	0.57±0.04^bc	0.63±0.05^bc	94.95±4.32^bc
pcDNA-NC组	0.55±0.05	0.56±0.06	0.63±0.07	94.33±5.82
HMGB组	0.40±0.05^de	0.46±0.04^de	0.48±0.04^de	81.58±4.05^de
F	58.323^＊＊	33.669^＊＊	28.331^＊＊	71.970^＊＊

组别	细胞增殖活力（OD值）			克隆细胞数/（个／视野）
组别	24 h	48 h	72 h	克隆细胞数/（个／视野）
Control组	0.65±0.03	0.67±0.01	0.73±0.05	118.32±4.03
LPS组	0.38±0.02^a	0.42±0.03^a	0.48±0.03^a	71.43±4.34^a
L-ACE组	0.42±0.01^b	0.49±0.03^b	0.51±0.03^b	83.27±5.14^b
H-ACE组	0.56±0.03^bc	0.57±0.04^bc	0.63±0.05^bc	94.95±4.32^bc
pcDNA-NC组	0.55±0.05	0.56±0.06	0.63±0.07	94.33±5.82
HMGB组	0.40±0.05^de	0.46±0.04^de	0.48±0.04^de	81.58±4.05^de
F	58.323^＊＊	33.669^＊＊	28.331^＊＊	71.970^＊＊

组别	HMGB1	TLR4	Bcl-2
Control组	1.05±0.06	1.08±0.03	1.01±0.04
LPS组	1.54±0.03^a	1.34±0.04^a	0.75±0.03^a
L-ACE组	0.94±0.03^b	0.87±0.04^b	1.24±0.05^b
H-ACE组	0.82±0.03^bc	0.71±0.05^bc	1.56±0.03^bc
pcDNA-NC组	0.83±0.07	0.70±0.09	1.55±0.06
HMGB组	1.24±0.03^de	1.16±0.07^de	1.21±0.03^de
F	230.400^＊＊	123.183^＊＊	339.231^＊＊
组别	Bax	Caspase-3	Caspase-7
Control组	1.03±0.04	1.07±0.03	1.02±0.05
LPS组	1.52±0.04^a	1.78±0.03^a	1.52±0.03^a
L-ACE组	0.77±0.04^b	1.26±0.05^b	1.36±0.03^b
H-ACE组	0.62±0.04^bc	0.88±0.04^bc	0.75±0.02^bc
pcDNA-NC组	0.61±0.05	0.87±0.05	0.74±0.03
HMGB组	0.82±0.05^de	1.17±0.04^de	1.22±0.03^de
F	370.705^＊＊	406.284^＊＊	572.548^＊＊

组别	HMGB1	TLR4	Bcl-2
Control组	1.05±0.06	1.08±0.03	1.01±0.04
LPS组	1.54±0.03^a	1.34±0.04^a	0.75±0.03^a
L-ACE组	0.94±0.03^b	0.87±0.04^b	1.24±0.05^b
H-ACE组	0.82±0.03^bc	0.71±0.05^bc	1.56±0.03^bc
pcDNA-NC组	0.83±0.07	0.70±0.09	1.55±0.06
HMGB组	1.24±0.03^de	1.16±0.07^de	1.21±0.03^de
F	230.400^＊＊	123.183^＊＊	339.231^＊＊
组别	Bax	Caspase-3	Caspase-7
Control组	1.03±0.04	1.07±0.03	1.02±0.05
LPS组	1.52±0.04^a	1.78±0.03^a	1.52±0.03^a
L-ACE组	0.77±0.04^b	1.26±0.05^b	1.36±0.03^b
H-ACE组	0.62±0.04^bc	0.88±0.04^bc	0.75±0.02^bc
pcDNA-NC组	0.61±0.05	0.87±0.05	0.74±0.03
HMGB组	0.82±0.05^de	1.17±0.04^de	1.22±0.03^de
F	370.705^＊＊	406.284^＊＊	572.548^＊＊

组别	IL-1β	IL-4	TNF-α
Control组	80.23±5.48	24.50±4.71	113.22±7.13
LPS组	94.62±4.57^a	16.41±4.28^a	129.41±7.24^a
L-ACE组	70.18±5.42^b	28.77±4.34^b	98.62±8.47^b
H-ACE组	61.47±4.59^bc	37.75±4.02^bc	75.18±6.73^bc
pcDNA-NC组	61.51±4.68	37.77±5.10	75.20±6.46
HMGB组	73.59±4.44^de	29.54±4.83^de	95.89±6.24^de
F	39.823^＊＊	19.176^＊＊	54.154^＊＊

金合欢素调节HMGB1/TLR4信号通路对脂多糖诱导牙髓细胞凋亡的影响

Effect of acacetin on lipopolysaccharide induced apoptosis of dental pulp cells by regulating the HMGB1/TLR4 signaling pathway

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[15]	郝凯凯, 王晓敏, 刘峥, 刘东洋, 李静. 藁本内酯调节RhoA/ROCK信号通路对食管癌细胞生物学行为的影响[J]. 天津医药, 2024, 52(11): 1164-1170.