Stomatology Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Department of Interventional Neuroradiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Int Endod J. 2021 Jun;54(6):916-925. doi: 10.1111/iej.13469. Epub 2021 Jan 20.
To explore the presence and function of NLRP6-caspase 4 inflammasome in human pulp tissue and human dental pulp cells (HDPCs).
Pulp tissue was collected from freshly extracted human caries-free third molars and third molars with irreversible pulpitis. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were performed to assess the expression of NLRP6-caspase 4 inflammasome. HDPCs were prepared from normal human pulp tissues and challenged with Porphyromonas gingivalis LPS. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were performed to assess if LPS can upregulate NLRP6 and caspase-4. HDPCs were further challenged with LPS followed with cytosolic Streptococcus mutans lipoteichoic acid (LTA). SiRNA targeting NLRP6 and Casp4 and pharmacology inhibitor Ac-FLTD-CMK and MCC950 were used to assess if Streptococcus mutans LTA can activate the NLRP6 but not the NLRP3 inflammasome. Western blot and ELISA were performed to evaluate inflammasome activation. The Student's t-test and one-way anova were used for statistical analysis.
NLRP6-caspase 4 inflammasome was upregulated and activated in inflamed human dental pulp tissue. In HDPCs, Porphyromonas gingivalis LPS upregulated the expression of NLRP6, CASP1 and CASP4 in a type I interferon dependent manner. After LPS priming, cytosolic Streptococcus mutans LTA triggered NLRP6-caspase 4 inflammasome activation. Knockdown of NLRP6 or CASP4 using siRNA or using pharmacology inhibitor Ac-FLTD-CMK but not MCC950 efficiently suppressed inflammasome activation by cytosolic LTA.
NLRP6-caspase 4 inflammasome may play an important role in pulp inflammation and immune defence. Inflammatory caspases represent a pharmacological target to restrain pulpal inflammation.
探讨 NLRP6-caspase 4 炎性小体在人牙髓组织和人牙髓细胞（HDPCs）中的存在和功能。
从新鲜拔出的无龋第三磨牙和不可复性牙髓炎的第三磨牙中收集牙髓组织。采用定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹法检测 NLRP6-caspase 4 炎性小体的表达。从正常牙髓组织中分离 HDPCs，并与牙龈卟啉单胞菌 LPS 孵育。采用酶联免疫吸附试验（ELISA）和 qRT-PCR 检测 LPS 是否能上调 NLRP6 和 caspase-4。HDPCs 先用 LPS 孵育，再用细胞质变形链球菌脂磷壁酸（LTA）孵育。用靶向 NLRP6 和 Casp4 的 siRNA 以及药理学抑制剂 Ac-FLTD-CMK 和 MCC950 来评估变形链球菌 LTA 是否能激活 NLRP6 而不是 NLRP3 炎性小体。采用蛋白质印迹法和 ELISA 评估炎性小体的激活。采用 Student's t 检验和单因素方差分析进行统计学分析。
炎性牙髓组织中 NLRP6-caspase 4 炎性小体上调和激活。在 HDPCs 中，牙龈卟啉单胞菌 LPS 以Ⅰ型干扰素依赖的方式上调 NLRP6、CASP1 和 CASP4 的表达。LPS 预处理后，细胞质变形链球菌 LTA 触发 NLRP6-caspase 4 炎性小体激活。用 siRNA 或药理学抑制剂 Ac-FLTD-CMK 而非 MCC950 敲低 NLRP6 或 CASP4 可有效抑制细胞质 LTA 引发的炎性小体激活。
NLRP6-caspase 4 炎性小体可能在牙髓炎症和免疫防御中起重要作用。炎性半胱天冬酶代表了抑制牙髓炎症的药理学靶点。
