Ok Chang Youp, Park Sera, Jang Hye-Ock, Bae Moon-Kyoung, Bae Soo-Kyung
Department of Dental Pharmacology, BK21 PLUS Project, Education and Research Team for Life Science on Dentistry, School of Dentistry, Pusan National University, Yangsan, South Korea.
Periodontal Disease Signaling Network Research Center (MRC), Pusan National University, Yangsan, South Korea.
J Dent Sci. 2023 Jul;18(3):1177-1188. doi: 10.1016/j.jds.2022.10.008. Epub 2022 Oct 21.
/purpose: Dental pulp plays an important role in the maintenance of tooth homeostasis and repair. The aging of dental pulp affects the functional life of the tooth owing to the senescence of dental pulp cells. Toll-like receptor 4 (TLR4) is involved in regulating cellular senescence in dental pulp. We have recently demonstrated that visfatin induces the senescence of human dental pulp cells (hDPCs). Here, we explored the association of TLR4 with visfatin signaling in cellular senescence in hDPCs.
mRNA levels were determined using reverse transcription polymerase chain reaction (PCR) and quantitative real time-PCR. Protein levels were determined using immunofluorescence staining and Western blot analysis. Gene silencing was performed using small interfering RNA. The degree of cellular senescence was measured by senescence-associated-β-galactosidase (SA-β-gal) staining. Oxidative stress was determined by measurement of NADP/NADPH levels and intracellular reactive oxygen species (ROS) levels.
Neutralizing anti-TLR4 antibodies or TLR4 inhibitor markedly blocked visfatin-induced hDPCs senescence, as revealed by an increase in the number of SA-β-gal-positive hDPCs and upregulation of p21 and p53 proteins. Moreover, visfatin-induced senescence was associated with excessive ROS production; NADPH consumption; telomere DNA damage induction; interleukin (IL)-1β, IL-6, IL-8, cyclooxygenase-2, and tumor necrosis factor-α upregulation; and nuclear factor-κB and mitogen-activated protein kinase activation. All of these alterations were attenuated by TLR4 blockade.
Our findings indicate that TLR4 plays an important role in visfatin-induced senescence of hDPCs and suggest that the visfatin/TLR4 signaling axis can be a novel therapeutic target for the treatment of inflammaging-related diseases, including pulpitis.
/目的：牙髓在维持牙齿内环境稳定和修复中起重要作用。牙髓老化会因牙髓细胞衰老而影响牙齿的功能寿命。Toll样受体4（TLR4）参与调节牙髓细胞衰老。我们最近证明内脂素可诱导人牙髓细胞（hDPCs）衰老。在此，我们探讨了TLR4与内脂素信号在hDPCs细胞衰老中的关联。
使用逆转录聚合酶链反应（PCR）和定量实时PCR测定mRNA水平。使用免疫荧光染色和蛋白质印迹分析测定蛋白质水平。使用小干扰RNA进行基因沉默。通过衰老相关β半乳糖苷酶（SA-β-gal）染色测量细胞衰老程度。通过测量NADP/NADPH水平和细胞内活性氧（ROS）水平确定氧化应激。
中和性抗TLR4抗体或TLR4抑制剂显著阻断了内脂素诱导的hDPCs衰老，这通过SA-β-gal阳性hDPCs数量增加以及p21和p53蛋白上调得以体现。此外，内脂素诱导的衰老与过量ROS产生、NADPH消耗、端粒DNA损伤诱导、白细胞介素（IL）-1β、IL-6、IL-8、环氧化酶-2和肿瘤坏死因子-α上调以及核因子-κB和丝裂原活化蛋白激酶激活有关。所有这些改变都因TLR4阻断而减弱。
我们的研究结果表明TLR4在内脂素诱导的hDPCs衰老中起重要作用，并提示内脂素/TLR4信号轴可能是治疗包括牙髓炎在内的炎症衰老相关疾病的新治疗靶点。
