Study On The Mechanisms Of The Expression, Activation And Action Of The NLRP3 Inflammasome In Human Dental Pulp Fibroblasts

The innate immune system is the first line that protect the host from infection of pathogens. It engages an array of germline-encoded pattern-recognition receptors(PRRs) to detect the danger signals from pathogenic microorganisms as well as the host-derived danger signals, which are termed pathogen-associated molecular patterns and danger-associated molecular patterns, respectively. Currently, four different classes of PRR families have been identified,include the TLRs, the CLRs, the RLRs and the NLRs. Distinct from other PRRs, some NLR family members can regulate caspase-1 activity through inflammasome formation, and the active caspase-1 plays an important role in the regulation of IL-1β’s maturation and secretion. The NLRP3 inflammasome is currently the most fully characterized inflammasome, and is found to play a very critical role during the pathological process of diverse diseases.Former experiment results of our research group show that the NLRP3 inflammasome is expressed in human dental pulp fibroblasts(HDPFs) and have a very close relationship with the innate immunity and healing process of dental pulp. However, the exact function and the mechanisms of the expression regulation and activation of the NLRP3 inflammasome in HDPF remain unclear. To illustrate these questions will contribute to further understand the innate immunity of dental pulp and lay a solid foundation for the pursuit of novel treatments for pulpitis.Objectives:1. To investigate the function of NLRP3 inflammasome in HDPFs and the related mechanisms.2. To investigate the mechanisms of NLRP3 inflammasome activation in HDPFs.3. To investigate the transcriptional regulation mechanisms of NLRP3 in HDPFs.4. To investigate the post-transcriptional regulation mechanisms of NLRP3 in HDPFs.Methods:1. Treat HDPFs with LPS and/or ATP, expression of NLRP3 inflammasome related genes were determined by RT-PCR and western-blot, extracellular IL-1β concentration was detected by ELISA. NLRP3 gene and caspase-1 gene were silenced through the transfection of sh RNA lentivirus, respectvely. And their effects on IL-1β expression and secretion were determined by RT-PCR and western-blot, as well as ELISA.2. Intracellular total ROS and superoxide were labeled with fluorescent dye, and the effect of ATP treatment on the intracellular total ROS concentration in HDPFs was determined by fluorescence/confocal microscopy and flow cytometry. ROS inhibitor was used to blockade the generation of ROS, and its effect on the NLRP3 activity was determined by RT-PCR, western-blot and ELISA.3. CLI-095, Pepinh-MYD and Bay 11-7082 were used to inhibit TLR4, My D88 and NF-κB, repectively. Treat HDPFs with LPS, NLRP3, caspase-1 and IL-1β m RNA expression were determined by RT-PCR and western-blot, and extracellular IL-1β concentration was detected by ELISA.4. Expression of mi R-223 and mi R-22 in HDPFs was detected by RT-PCR. Luciferase reporter detection was used to confirm whether mi R-223 and mi R-22 target the 3’UTR of NLRP3 or not. mi RNA mimics were used to overexpress mi R-223 and mi R-22 in HDPF, and the NLRP3 inflammasome activity was detected by western-blot and ELISA.Results:1. LPS can induce an increase in the expression of NLRP3, caspase-1 and IL-1β. Treat the HDPFs with LPS and ATP simultaneously can induce the secretion fo IL-1β. Both NLRP3 gene silence and caspase-1 gene silence can significantly inhibit the secetion of IL-1β; however, no obvious effect on the expression of IL-1β m RNA was detected.2. ATP can potently induce the production of ROS. ROS inhibitor cause significantly reduction in p20 expression and IL-1β secretion.3. TLR4 inhibitor can significantly reduce the expression of NLRP3, caspase-1 and IL-1β. My D88 and NF-κB inhibitors can significantly reduce the expression of NLRP3 and IL-1β, however, they have no obvious effect on the expression of caspase-1.4. RT-PCR results indicate that both mi R-223 and mi R-22 are expressed in HDPFs, and treatment with LPS result in reduction of their expression. The result of Luciferase reporter detection confirmed that both mi R-223 and mi R-22 target the 3’UTR of NLRP3. Further experiment found that overexpression of mi R-223 or mi R-22 can reduce the protein expression level of NLRP3 and the secretion of IL-1β.Conclusion:1. The NLRP3 inflammasome plays a critical role in the regulation IL-1β secretion in HDPFs;2. ATP activates the NLRP3 inflammasome in a ROS-dependent way;3. The TLR4-My D88-NF-κB signaling pathway is the major pathway which regulate the gene expression of NLRP3;4. mi R-223 和 mi R-22 can inhibit expression of NLRP3 in translation process thus reduce the activity of the NLRP3 inflammasome.