The Effect Of Melatonin On Pulpitis TLR4、NF-κB Regulation Mechanism

Microbes or their products of dental caries may enter the pulp tissues and cause immune responses through the dentinal tubules.The immune responses in pulpitis involve various immune cells,especially macrophages and dendritic cells.Recently,studies have shown that pulp fibroblasts also play an important role in the genesis and development of pulpitis.The regulatory role of Toll-like receptors(TLRs) 2,3,4and5 in HDPCs and their inflammatory pathways has drawn widespread attention.Toll like receptor(Toll-like receptor,TLR) is one of the most common molecules of the pathogen associated molecular patterns(Pathogen Associated Molecular Patterns invasion,PAMPs)and dangerous mode(Damage Associated Molecular Patterns,DAMPs), initiating and regulating inflammation development and prognosis.TLR4 is an important recognition molecule of endotoxin receptor and is involved in inflammation development. Our previous study found that in human dental pulp fibroblast cells stimulated with LPS,TLR4, My D88,NF-κ B signaling pathway has a role in transcriptional regulation on NLRP3 and IL-1β,the mechanism of which remains unkown.With the accelerating pace of life,sleep disorders increase the incidencs of a variety of inflammatory related diseases.Melatonin plays an important role in regulating sleep and immune system.It is reported that melatonin can reduce the expression of TLR4、NF-κ B signaling pathway in macrophages and other immune cells.However,the effects of melatonin on pulpitis remains unkown at present.Thus,in this study,we established pulpitis models both in vitro and in vivo and treatied with melaonin to elucidate the changes of TLR4、NF-κB signaling pathway.We aimed to clarify the relationship among melatonin,TLR4、NF-κB signaling pathway and pulp innate immunity.The main contents are as follows:1.Establishment of rat pulpitis model and researches on signaling pathway TLR4、NF-κB activation mechanism as well as related inflammation factors IL-1β,TNF-α.2.The influence of melatonin TLR4、NF-κB signaling pathway and inflammatory factors IL-1β,TNF-α in experimental rat pulpitis.3.The expression of mlatonin recepors in HDPCs and their role in the genesis and development of pulpitis.Method:1.The rat pulpitis model was established by pulp exposure in first molars.After the sample was prepared,morphological examinations were conducted to evaluate pulpitis severity;real time PCR were also performed to examine the expression levels of relative genes in TLR4 、 NF-κB signaling pathway;immunohistochemical examinations were applied to analyze TLR4、NF-κB;ELISA was used to detect serum levels of IL-1β 、IL-18、TNF-α and melaton as well as TNF-α and IL-1β levels in pulp.2.The rat pulpitis model were treated with extrinsic melatonin while pulpitis was induced by LPS in HDPCs with melatonin added.Real time RRRRRREEWQPCR were performed to examine the expression levels of relative genes in TLR4、NF-κB signaling pathway as well as IL-1β and TNF-α;western botting was applied to analyze TLR4、NF-κB;ELISA was used to detect TNF-α and IL-1β levels in serum and HDPCs.3.RT-PCR and immunohistochemical examinations were applied to detect the expression the melatonin receptor MT1 and MT2 in HDPCs.Lentivirus mediated RNA interference was performed to specifically knoc down expression levels of melatonin.After LPS stimulation and melatonin treatment,RRRRRREEWQReal time PCR were performed to examine the expression levels of relative genes in TLR4、NF-κB signaling pathway as well as IL-1β and TNF-α;western botting was applied to analyze TLR4、NF-κB;ELISA was used to detect TNF-α and IL-1β levels in serum and HDPCs.Results:1.Rat pulpitis model was successfully established by molar pulp exposure.After pulpitis was established IL-1β,IL-18 and TNF-α levels in serum increased while IL-1β,TNF-α levels elevated in pulps.The melatonin concentrations in serum first increased and then decreased.Immunohistochemistry showed the expression of TLR4、NF-κB were highest at the site of inflammation.The expression levels of TLR4、NF-κB protein increased in dental pulp tissue.2.Compared with non-melatonin-treated rats,melatonin-treated rats showed a significant decrease in IL-1β and TNF-α levels;the expression levels of TLR4、NF-κB in inflammatory sites significantly reduced;m RNA expression levels of TLR4 、 NF-κB lowered significantly.Compared with non-melatonin-treated HDPCs,melatonin-treated HDPCs presented reduced levels of IL-1β and TNF-α;both TLR4、NF-κB m RNA and protein levels decreased significantly.3.Positive expression of MT1 was detected in HDPCs while MT2 was rarely detected.After MT1 was specifically knocked down,the inhibitory effects of melatonin in inflammation of HDPCs dereased significantly.Compared with MT1-NC-sh RNAHDPCs,MT1-sh RNA-HDPCs presented increased levels of IL-1 β and TNF- α;both TLR4、NF-κB m RNA and protein levels rose significantly.Conclusion:1.After pulpitis was established,IL-1β,IL-18 and TNF-α levels in serum increased while IL-1β,TNF-α levels elevated in pulps.The melatonin concentrations in serum first increased and then decreased.2.Melatonin could decrease the expression levels of TLR4、NF-κB,IL-1β,TNF-α in rat pulpitis.3.Melatonin might exert inhibitory effects in inflammation of human dental pulp cells via MT1.