| One of the best-characterized members of the Nod-like receptor (NLR) family is pyrindomain containing3(NLRP3). Intracellular NLRP3is the most versatile innate immunereceptor. Upon activation, NLRP3assembles into a multiprotein complex, termed aninflammasome, which regulates the secretion and bioactivity of interleukin-1familycytokines by cleaving pro-caspase-1into its active forms. Caspase-1then processescytokine proforms such as interleukin-1β (IL-1β) to generate the active molecules. TheNLRP3/caspase-1/IL-1β pathway has been found in many tissues. NLRP3has broadspecificity for mediating an immune response to a wide range of microbial stimuli ordanger signals.Recent data have elucidated the role of odontoblasts and pulp fibroblasts in theregulation of dental pulp immune and inflammatory responses to cariogenic bacteria.Odontoblasts provide barrier function by protecting the underlying tissue from the invading bacteria; they are also immunocompetent and capable of orchestrating aninflammatory response. Fibroblasts, a major cell type in the dental pulp, are capable ofproducing pro-inflammatory cytokines. We hypothesize that the NLRP3/caspase-1pathway plays an essential role in the detection of bacterial pathogens and the initiationof inflammation within the dental pulp. The aim of the study is to evaluate the expressionof NLRP3/caspase-1in dental pulp and to observe the relationship between thisinflammasome pathway and mineralization repair.The experiment contains two parts:Part1: The expression of NLRP3/caspase-1in human dental pulp tissues.Pulp tissues were collected from teeth that were freshly extracted for orthodonticreasons. Western blotting was performed to detect the levels of NLRP3protein, whileimmunofluorescence staining was used to determine the distribution of NLRP3in pulptissues. Expression of bone-related ALP, OCN, RUNX-2and NLRP3/caspase-1/IL-1βmRNA in human dental pulp tissues was detected separately by qua ntitative RT-PCR. Itwas shown that NLRP3and caspase-1proteins were principally expressed inodontoblasts and some pulp vascular endothelial cells in normal human dental pulptissues, while in inflamed pulp tissues, most types of pulpoblasts including odontoblasts,fibroblasts and inflammatory cells were positive for NLRP3/caspase-1proteins. NLRP3protein expression in inflamed pulp tissues was higher than that in normal tissues.Caspase-1protein was cleaved into its active forms only in inflamed pulp tissues.RT-qPCR revealed that the expression of NLRP3, caspase-1, and IL-1β genes waselevated in inflamed pulp tissues, while expression of mineral-related genes wasdecreased. The mineralization ability of tissues affected by pulpitis was inhibited greatlyby high IL-1β expression, which in turn was mediated by the NLRP3/caspase-1pathway.The distribution and expression levels of NLRP3suggest that NLRP3-mediated signalingpathways may play an important role in dental immune defense. Additionally, both t hedental pulp repair process and the inflammatory process exist in pulpitis. Increasedinflammation inhibits tissue repair process. Part2: Expression of NLRP3/caspase-1in experimental rat pulpitis.In this part, we established a model of experimental rat pulpal lesions by exposing thedental pulp without other stimulation. Immunofluorescence staining was then used todetermine the distribution of NLRP3in pulp tissues. RT-PCR and Western blotting wereperformed to detect the levels of NLRP3/caspase-1/IL-1β mRNA and protein,respectively. The results suggested that NLRP3/caspase-1/IL-1β was more highlyexpressed in inflamed rat dental pulp tissues than in normal tissue.In summary, these results demonstrate that human dental pulp tissues contain afunctional NLRP3-mediated immune response to invading pathogens and that thisresponse interacts with the pulp repair process. |
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