Treatment Of Early Pulpitis With An Antisense Oligonucleotide Targeting NF-κB P65 MRNA

Objectives: Our research group designed the ASOs targeting NF-κB p65 mRNA.After screening in vitro model,the effect of antisense oligonucleotide on NF-κB p65 expression and its therapeutic effect on early pulpitis in rat model were observed,which may provide a new idea for clinical inhibition of inflammation and preservation of vital pulp.Methods: 1.Design of antisense oligonucleotide targeting NF-κB p65 mRNA: First,using NCBI database,we obtained human and rat NF-κB p65 mRNA sequences.Then,the BLAST database was used to screen the target regions.In primer software,candidate ASOs complementary to the target regions were obtained.Then,the possibility of candidate ASOs binding to the target region was evaluated using Sfold online analysis software and RNAfold web server online analysis software.In addition,Nucleotide BLAST database was used to analyze the specificity of the selected ASOs.2.Screening of PMOs in vitro: We extracted h DPCs and r DPCs by enzymatic digestion and tissue block method,and completed the identification of DPCs by observation of light microscopy and immunofluorescence experiments.h DPCs and r DPCs were stimulated with 1 μg/ml LPS,and the optimal time point of NF-κB inhibition was determined by RT-q PCR.Then,the PMOs labeled with Cy3+ fluorescence were co-cultured with the cells,and the positional relationship between the PMOs and the cells was observed using laser confocal microscopy.The experimental groups were divided into blank group,LPS group,LPS+PMO1 group,LPS+PMO2 group and LPS+PMO3 group.The experimental groups were divided into blank group,LPS group,LPS+PMO1 group,LPS+PMO2 group and LPS+PMO3 group.RT-q PCR and WB experiments were used to observe the inhibitory effects of PMO1,PMO2 and PMO3 on the mRNA and protein expression of NF-κB.The PMO with the best inhibitory effect was selected for further in vitro study.3.Therapeutic effect of PMO3 on early pulpitis: Eighteen male Wistar rats（8 weeks old,weight 220g±20g）were randomly divided into three experimental groups,with 6 rats in each group.Blank group: rats were not treated.In LPS group,the bilateral maxillary first molars of rats were pulposed,and a gel sponge adsorbing 5 μl LPS（10 μg/ml）was placed on the exposed pulp tissue,and the cavities were temporarily sealed with glass ions.In LPS+PMO3 group,the bilateral maxillary first molars of rats were pulpotted,and a gel sponge adsorbing 5 μl LPS（10 μg/ml）and PMO3（10 μg/ml）was placed on the exposed pulp tissue,and the cavities were temporarily sealed with glass ions.After 24 h,the rats were sacrificed for sampling.HE staining was used to observe the pathological changes of the dental pulp tissue,including the extent of inflammation and the number of infiltrating inflammatory cells.IHC and WB were used to detect the protein expression levels of NF-κB p65 and its downstream inflammatory factors IL-1β and TNF-α in dental pulp tissues.RT-q PCR was used to detect the mRNA expression levels of NF-κB p65 and its downstream inflammatory factors IL-1β and TNF-α in dental pulp tissues.The heart,liver,spleen,lung and kidney were observed by HE staining to analyze whether PMO had drug toxicity.Results: 1.Using the NCBI network database,the NF-κB p65 mRNA sequences of human and rat were obtained,and the numbers were NM₀01145138.2 and NM₁99267.2,respectively.Then,12 conserved sequences with length ≥25 bp in the common conserved sequence of human and rat NF-κB p65 mRNA were screened by BLAST website.In primer software,138 candidate ASOs complementary to the target regions were obtained.Then,Sfold online analysis software and RNAfold web server online analysis software were used to evaluate the possibility of candidate ASOs binding to the target region,and the 29,30 and 72 candidate ASOs were screened out.Finally,the Nucleotide BLAST network database comparative analysis showed that the selected ASOs were specific.2.Optical microscopy showed that both h DPCs and r DPCs were fusiform or flat star shaped,but r DPCs were shorter than h DPCs.Both kinds of cells grew in a certain direction and arranged in a palisade pattern after covering the bottom of the bottle.There was no multilayer growth and contact inhibition phenomenon.Immunofluorescence staining showed that the cells were positive for vimentin and negative for keratin.The results of RT-q PCR showed that the expression of NF-κB p65 mRNA in h DPCs was the highest at 10 h of LPS stimulation.In r DPCs,the highest expression of NF-κB p65 mRNA was observed at 8h after LPS stimulation.Immunofluorescence assay showed that PMO1,PMO2,and PMO3 could enter h DPCs and r DPCs.The results of RT-q PCR showed that the mRNA expressions of NF-κB p65,IL-1β and TNF-α in the LPS group were higher than those in the blank group.However,the mRNA expression level of NF-κB p65 was decreased in the LPS+PMO group,and the mRNA expression levels of the downstream inflammatory factors IL-1β and TNF-α were reduced more significantly.WB results showed that the protein expressions of NF-κB p65,p-p65,IL-1β and TNF-α in the LPS group were higher than those in the blank group.However,the protein expression levels of NF-κB p65,p-p65,IL-1β and TNF-α were decreased in the LPS+PMO group.PMO3 has the best anti-inflammatory effect in vitro,and can be further used for the study of rat pulpitis in vivo.3.HE staining of rat dental pulp tissue showed that compared with the blank group,the pulp inflammation in the LPS group was concentrated in the coronal pulp,with a large number of inflammatory cells infiltrating.Compared with the LPS group,the extent of inflammation and the infiltration of inflammatory cells were reduced in the LPS+PMO3 group.The results of IHC staining and WB experiments of rat dental pulp tissues were howed that compared with the control group,the protein expression levels of NF-κB p65 and p-p65 in the dental pulp tissue of the LPS group increased,and the protein expression levels of its downstream inflammatory factors IL-1β and TNF-α increased more significantly.Compared with the LPS group,the LPS+PMO3 group had significant reductions in the protein expression levels of NF-κB p65 and p-p65,and the protein expression levels of its downstream inflammatory factors IL-1β and TNF-α.RT-q PCR results of rat dental pulp tissue showed that the mRNA expression levels of NF-κB p65 and its downstream inflammatory factors IL-1β and TNF-α in the dental pulp tissues of the LPS group were significantly higher than those of the blank group.Compared with the LPS group,the LPS+PMO3 group had a significant reduction in the mRNA expression of NF-κB p65 and its downstream inflammatory factors IL-1β and TNF-α.HE staining results of the important organs of rats: compared with the control group,the LPS+PMO3 group had no abnormal cell morphology in each organ.Conclusion: 1.The design and synthesis of antisense oligonucleotides targeting NF-κB p65 mRNA were completed 2.Cell and animal models of pulpitis were successfully established.3.PMOs could enter into dental pulp cells and successfully inhibit the expression of NF-κB p65 and its downstream inflammatory factors in the pulpitis model.4.PMO3 can treat LPS-induced pulpitis with biological safety.