Identification and validation of novel biomarkers and therapeutics for pulpitis using connectivity mapping

Abstract

Aim To create an irreversible pulpitis gene signature from microarray data of healthy and inflamed dental pulps, followed by a bioinformatics approach using connectivity mapping to identify therapeutic compounds that could potentially treat pulpitis. Methodology The Gene Expression Omnibus (GEO) database, an international public repository of genomics data sets, was searched for human microarray datasets assessing pulpitis. An irreversible pulpitis gene expression signature was generated by differential expression analysis. The statistically significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. qPCR was used to validate novel pulpitis genes. An ex vivo pulpitis model was used to test the effects of the compounds identified, and the level of inflammatory cytokines was measured with qPCR, ELISA and multiplex array. Means were compared using the t‐test or ANOVA with the level of significance set at p ≤ .05. Results Pulpitis gene signatures were created using differential gene expression analysis at cutoff points p = .0001 and .000018. Top upregulated genes were selected as potential pulpitis biomarkers. Among these, IL8, IL6 and MMP9 were previously identified as pulpitis biomarkers. Novel upregulated genes, chemokine (C‐C motif) ligand 21 (CCL21), metallothionein 1H (MT1H) and aquaporin 9 (AQP9) were validated in the pulp tissue of teeth clinically diagnosed with irreversible pulpitis using qPCR. ssCMap analysis identified fluvastatin (Statin) and dequalinium chloride (Quaternary ammonium) as compounds with the strongest correlation to the gene signatures (p = .0001). Fluvastatin reduced IL8, IL6, CCL21, AQP9 (p < .001) and MMP9 (p < .05) in the ex vivo pulpitis model, while dequalinium chloride reduced AQP9 (p < .001) but had no significant effect on the other biomarkers. Conclusions AQP9, MT1H and CCL21 were identified and validated as novel biomarkers for pulpitis. Fluvastatin and dequalinium chloride identified by the ssCMap as potential therapeutics for pulpitis reduced selected pulpitis biomarkers in an ex vivo pulpitis model. In vivo testing of these licenced drugs is warranted.

A critical analysis of research methods and experimental models to study pulpitis

“…Used in the context of the Chromosome‐centric Human Proteome Project, N‐terminomics led to the identification of 17 missing protein products in the healthy pulp, opening the path to the identification of pulp‐specific proteins that could be novel biomarkers of pulpitis progression (Eckhard et al, 2015). Recently, a gene signature of irreversible pulpitis was created from microarray data of healthy and inflamed pulps, and AQP9, MT1H and CCL21 genes were identified and validated as pulpitis biomarkers (Al‐Natour et al, 2021). All these data constitute cues for the future development of individual diagnostic tests valuable to determine pulpitis progression and that could guide the researcher to create more accurate pulpitis experimental models and the practitioner toward personalised pulpitis treatments (Figure 2).…”

“…A bioinformatics study using connectivity mapping (ssCMap) allowed identifying compounds that could potentially help treat pulpitis‐affected teeth. Indeed, fluvastatin and dequalinium chloride identified by ssCMap reduced pulpitis biomarkers and the authors proposed these molecules as potential therapeutic tools (Al‐Natour et al, 2021). A limitation of this study is that the validation of the ssCMap results was only performed in an ex vivo model of 2D co‐culture of DPCs and macrophages (Rankin et al, 2020).…”

Delayed COVID‐19‐induced cytokine storm after root canal therapy with favorable response to plasmapheresis, tocilizumab, and methylprednisolone pulses therapy: A case report

“… 24 IL8, IL6, MMP9, and chemokines were also introduced as pulpitis biomarkers. 25 In cases of endodontic failure, persisting microorganisms in the root canal, IL‐17A, IL‐1β, TNF‐α, and IFN‐γ could be increased. 26 Not performing proper endodontics protocol in filing and flaring, irrigation and obscuration is a source of inflammation production.…”

Protective effects of budesonide on LPS‑induced podocyte injury by modulating macrophage M1/M2 polarization: Evidence from in vitro and in silico studies

“…GEO (http://www. ncbi.nlm.nih.gov/geo/) is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics datasets (11). A gene expression dataset, namely, GSE83233 (https://www.ncbi.nlm.…”