Zhai Shafei, Zhang Lihui, Li Xue, Yu Qi, Liu Changkui
Department of Stomatology, Xi'an Medical University, Xi'an, 710075, Shaanxi Province, China.
Shaanxi Key Laboratory of Ischemic Cardiovascular Disease, Institute of Basic and Translational Medicine, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, China.
Regen Ther. 2024 Mar 7;27:12-20. doi: 10.1016/j.reth.2024.02.010. eCollection 2024 Dec.
The objective of the present study was to investigate whether NOD-like receptor family pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes pathways were involved in an experimental model of fibroblast activation named nemosis, which was used to mimic circumstances without bacteria stimulation.
Nemosis of human dental pulp fibroblast (DPFs) was induced by three-dimensional culture in U-shaped 96-well plates and investigated by scanning electron microscopy (SEM). DPFs monolayers were used as control. Annexin V-FITC/7-AAD apoptosis assay was performed on the DPFs spheroids by flowcytometry. Caspase-1 activity detection assay was conducted on the DPFs spheroids. Quantitative real-time polymerase chain reaction (qRT-PCR), cytokine measurements, Western blot and the effect of COX-2 inhibitor on spheroids was studied.
SEM study observed human dental pulp fibroblast clusters and cell membranes damage on the surface of DPFs spheroids. The percentages of necrotic cells from DPFs spheroids gradually increased as the incubation time increased. A statistically significant increase in caspase-1 activity was observed after DPFs spheroids formation. DPFs spheroids displayed significant amounts of NLRP3, AIM2 mRNA and protein expression, caspase-1 mRNA expression and cleaved Caspase-1 protein expression and high IL-1β concentrations ( < 0.05) than DPFs monolayers. Specific COX-2 inhibitor (NS-398) decreased NLRP3 mRNA and protein expression, cleaved Caspase-1 protein expression, Caspase-1 activity and IL-1β mRNA expression and IL-1β concentrations ( < 0.05). However, Specific COX-2 inhibitor had no impact on AIM2 mRNA and protein expression, caspase-1 mRNA expression and pro-Caspase-1 protein expression.
In conclusion, clustering human DPFs spontaneously activated NLRP3 and AIM2 inflammasomes and induced IL-1β secretion which could be partially attenuated by COX-2 inhibitor. Thus, nemosis could become a powerful model for studying mechanisms underlying aseptic pulpitis.
本研究的目的是调查含NOD样受体家族pyrin结构域蛋白3（NLRP3）和黑色素瘤缺乏因子2（AIM2）炎性小体途径是否参与一种名为nemosis的成纤维细胞激活实验模型，该模型用于模拟无细菌刺激的情况。
通过在U形96孔板中进行三维培养诱导人牙髓成纤维细胞（DPFs）发生nemosis，并通过扫描电子显微镜（SEM）进行观察。将DPFs单层用作对照。通过流式细胞术对DPFs球体进行膜联蛋白V-FITC/7-AAD凋亡检测。对DPFs球体进行半胱天冬酶-1活性检测。研究了定量实时聚合酶链反应（qRT-PCR）、细胞因子测量、蛋白质免疫印迹以及COX-2抑制剂对球体的影响。
SEM研究观察到DPFs球体表面有人牙髓成纤维细胞簇和细胞膜损伤。随着孵育时间增加，DPFs球体中坏死细胞的百分比逐渐升高。DPFs球体形成后，观察到半胱天冬酶-1活性有统计学意义的增加。与DPFs单层相比，DPFs球体显示出大量的NLRP3、AIM2 mRNA和蛋白表达、半胱天冬酶-1 mRNA表达和裂解的半胱天冬酶-1蛋白表达以及高浓度的IL-1β（P<0.05）。特异性COX-2抑制剂（NS-398）降低了NLRP3 mRNA和蛋白表达、裂解的半胱天冬酶-1蛋白表达、半胱天冬酶-1活性以及IL-1β mRNA表达和IL-1β浓度（P<0.05）。然而，特异性COX-2抑制剂对AIM2 mRNA和蛋白表达、半胱天冬酶-1 mRNA表达和前半胱天冬酶-1蛋白表达没有影响。
总之，人DPFs聚集可自发激活NLRP3和AIM2炎性小体并诱导IL-1β分泌，COX-2抑制剂可部分减弱这种分泌。因此，nemosis可能成为研究无菌性牙髓炎潜在机制的有力模型。
