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

doi:10.11958/20240738

Abstract

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

CLC Number:

R781.31，R34

Figures/Tables 10

References 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^＊＊