The Preliminary Study Of The NLRP3/Caspase-1Inflammation Pathway In Human Dental Pulp Fibroblasts

Dental caries have emerged as the major disease in oral medicine, and their incidenceand prevalence have been increasing in the last decade. The invasion of cary-relatedbacteria is a main cause of dental caries, which can lead to demineralization of enamel anddentin and subsequent pulp tissue injury. It has been reported that the bacterial invasionaccompanying the advance of dental caries subsequently initiates immune reactions.Unlike the widely studied immune cells, little is known about the role of human dentalpulp fibroblasts (HDPFs), the main cells in dental pulp, in the innate immune responseagainst bacterial infection. Although the inflammasome NLRP3/caspase-1pathway playsan important role in the immune cell defense against bacterial infection, whether it existsin HDPFs (considered a non-immune cell) remains poorly understood. Moreover, there arefew studies indicating how the inflammasome NLRP3/caspase-1pathway can be induced in HDPFs. In this study, we investigated the role of the inflammasome NLRP3/caspase-1pathway in HDPFs to better understand the inflammatory reaction in pulpitis so thattreatments aimed at controlling inflammation and saving the pulp can be developed.The results are as follows:1.Separation, culture and identification of HDPFs.We used enzymatic digestion to separate HDPFs from fresh and healthy dental pulptissue and expanded the cells in in vitro culture. Flow cytometry andimmunocytochemistry were used to identify the surface markers of HDPFs to ensure thatthe cells were mesenchyma-derived.2.The detection of gene and protein expression of P2X7, NLRP3, caspase-1, and IL-1βin normal HDPFs.The protein expression of NLRP3and caspase-1in HDPFs was detected byimmunocytochemistry. The gene expression of NLRP3, caspase-1, P2X7, and IL-1β wasdetected by semi-quantitative RT-PCR. The results showed that the expression of NLRP3and caspase-1proteins and P2X7, NLRP3, caspase-1, and IL-1β genes were observed inHDPFs.3.The gene expression of P2X7, NLRP3, caspase-1, and IL-1β in HDPFs stimulatedby ATP and Streptococcus mutans.After stimulation with ATP and Streptococcus mutans, the gene expression of NLRP3,caspase-1, P2X7, and IL-1β was detected by quantitative real-time reverse-transcriptasepolymerase chain reaction. The results show that compared with the control group, theexpression of NLRP3, caspase-1, P2X7, and IL-1β mRNA that was detected in HDPFsafter stimulation with ATP and Streptococcus mutans was remarkably elevated.4. In HDPFs, the gene and protein expression of P2X7, NLRP3, caspase-1, and IL-1β,as well as the regulation of the inflammasome NLRP3/caspase-1pathway, isstimulated by ATP and LPS.First, we stimulated HDPFs with ATP and LPS and determined the gene and proteinexpression levels of NLRP3, caspase-1, P2X7, and IL-1β using quantitative real-timereverse-transcriptase polymerase chain reaction, Western blot and ELISA. The results showed that exposure to LPS could not only induce the activation of the inflammasomeNLRP3/caspase-1pathway, but could also lead to the release of IL-1β; on the other hand,ATP stimulation alone could partially activate NLRP3inflammation and caspase-1protein,but was not able to induce the release of IL-1β. However, when pulsed with ATP followedby LPS exposure, HDPFs showed induction of P2X7and activation of the inflammasomeNLRP3/caspase-1pathway, resulting in the production of IL-1β, which is involved in theinnate immune defense reaction.Second, we used quantitative real-time reverse-transcriptase polymerase chain reactionand Western blotting to detect the effect of ATP plus ultra-pure LPS on the expression ofP2X7,NLRP3, caspase-1and IL-1β in HDPFs after exposure at different time points. Theresults showed that maximal induction of P2X7,NLRP3, caspase-1and IL-1β messengerRNA expression was observed after6hours of exposure to10μg/mL LPS and5mM ATPand that the increases in NLRP3and caspase-1protein production correlated directly withincreased NLRP3and caspase-1messenger RNA levels.Third, we investigated whether the purogenic P2X7ATP-gated ion channel wasinvolved in LPS–induced IL-1β expression in HDPFs. We therefore use a highextracellular potassium concentration and glibenclamide, a selective inhibitor ofATP-dependent potassium channels, to block potassium efflux. Quantitative real-timereverse-transcriptase polymerase chain reaction and ELISA were used to detect theamount of secreted IL-1β messenger RNA and protein, respectively. The results showedthat both a high extracellular potassium concentration and glibenclamide couldsignificantly diminish the secretion of IL-1β triggered by exposure to ATP plus LPS.Forth, we investigated whether reactive oxygen species (ROS) were involved inLPS–induced IL-1β expression in HDPFs. We therefore used N-acetyl cysteine (NAC), ananti-oxidant, to neutralize ROS. Quantitative real-time reverse-transcriptase polymerasechain reaction and ELISA were used to detect the amount of secreted IL-1β messengerRNA and protein, respectively. The results showed that NAC could significantly diminishthe secretion of IL-1β triggered by exposure to ATP plus LPS.In conclusion, we conducted a preliminary exploration of the inflammatory pathway of NLRP3/caspase-1in HDPFs and found that the NLRP3/caspase-1inflammation pathwayplayed an important role in HDPFs involved in the innate immune process. This will helpus further study the inflammatory pathway in order to better regulate this pathway in thepulp and avoid pulp tissue necrosis.