Investigation of the Potential Targets and Mechanism Actions of Berberine in the Treatment of Pulpitis Based on Bioinformatics Analysis

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Introduction
Pulp infammation (pulpitis) is one of the common oral diseases, which is accompanied by spontaneous or provoked pain evoked by stimulating nerve cells of the dental pulp [1,2].Pulpitis is caused by a sustained and severely stimulated response that may lead to infammatory damage and apoptosis of pulp cells, resulting in long-term tissue impairments [3][4][5].It has been demonstrated that pathogenic microorganism infection is closely related to the initiation and progression of pulpitis [6].Immune responses are one of the primary factors implicated in infammatory responses in infamed pulp tissue.Te balance between repair processes and infammation in the host defense response could afect the degree of injury in pulpitis, in which multiple signaling pathways have participated, including toll-like receptors, nuclear factor-kappa B, and NLR family pyrin domain containing 3 (NLRP3) infammasomes' signaling pathways [7,8].Nonimmune and immune cells including endothelial cells, odontoblasts, and macrophages could identify pathogen-related molecules and trigger immune responses via the expression of toll-like receptors in pulp tissue [9][10][11].Te previous report indicated that nitric oxide-induced infammatory cytokines expression is involved in nuclear factor-kappa B activation in primary cultured pulp cells [12].Besides, astrocyte-elevated gene-1 could initiate the generation of proinfammatory cytokines via the nuclear factorkappa B pathway in pulp cells [13].It has revealed that lipopolysaccharide (LPS) stimulation activated the NLRP3 infammasome pathway, thereby leading to the secretion of proinfammatory cytokines, which contributes to the immune responses in human dental pulp tissue [14].Currently, clinical interventions for the prevention and treatment of pulpitis are limited due to the lack of understanding of the specifc molecular mechanisms underlying pulpitis.Tus, clarifying potential molecular mechanisms involved in the progression of pulpitis and infammatory responses might be useful for the prevention and treatment of this oral disease.
Berberine is an isoquinoline alkaloid extracted from popular medicinal plants, such as Chelidonium majus, Xanthoriza simplicissima, and Xanthoriza simplicissima.Recently, studies have indicated that berberine exerts a wide range of pharmacological efects, such as antiviral, antitumor, anti-infammatory, hypolipidemic, anti-infective, neuroprotective, and hepatoprotective efects [15,16].Berberine has been found to inhibit interleukin 6 (IL-6) and C-C motif chemokine ligand 11 (CCL11) production via regulation of the signal transducer and activator of transcription 6 (STAT6) pathway in proinfammatory cytokineactivated human bronchial epithelial cells [17].Berberine reportedly alleviates streptozotocin (STZ)-induced diabetic nephropathy by inhibiting the TLR4/NF-lB signaling pathway [18].Berberine has been reported to protect the intestinal mucosal barrier via the regulation of the toll-like receptor pathway [19].Importantly, berberine promotes odontoblast diferentiation via activating the Wnt/β-catenin pathway, and it could promote human dental pulp cell osteogenic diferentiation via EGFR-MAPK-Runx2 signaling pathway activation, suggesting that berberine is a potential therapeutic agent for the treatment of pulpitis [20,21].Moreover, our previous study revealed that berberine regulates LPS-induced infammation in human dental pulp fbroblast via the miR-21/KBTBD7 axis [22].A mechanistic randomized controlled trial demonstrated that berberine efectively reduces total cholesterol levels and potentially lowers LDL-c, while maintaining a good safety profle [23].A comprehensive meta-analysis of randomized controlled trials revealed that the utilization of berberine in individuals sufering from metabolic syndrome and associated disorders exhibited a notable reduction in infammatory markers, such as CRP, TNF-α, and IL-6 [24].Based on these reports, we speculated that berberine might be a potential therapeutic drug for pulp infammation via the inactivation of infammatory-related pathways.
Network pharmacology is a novel method for drug development and pharmacological mechanistic research, which combines multidirection pharmacology, bioinformatics, system biology, and interdisciplinary theories to screen therapeutic targets quickly, economically, and efciently [25].In recent years, molecular docking is a wellestablished computational method based on the structure of molecules, which is extensively utilized in the feld of drug discovery [26,27].In the present study, computational tools and public databases were used to screen relevant therapeutic targets of berberine in the treatment of pulpitis.Subsequently, molecular docking was performed to validate whether berberine exerts an antipulpitis efect by binding to PTGS2 protein.Finally, the potential mechanisms were systematically predicted based on the results of enrichment analyses.Te fowchart of the present study is presented in Figure 1.We aimed to investigate the potential targets and mechanism of berberine against pulpitis, and our fndings provided the theoretical basis for the investigation of molecular mechanisms of berberine in the treatment of pulpitis.

Collection of Putative Targets of Pulpitis and Berberine.
We collected the microarray dataset (GSE77459) from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/) with the keyword "pulpitis."Te selection criteria for the datasets involved in the present study are as follows: the species is "Homo sapiens," the data type is "expression profle microarray," and GEO2R analysis can be performed.Te GSE77459 dataset contains 6 infamed pulps from patients with irreversible pulpitis and 6 pulp tissue samples from healthy donors.Te raw expression data were processed using Bioconductor Packages, which included steps such as creating an expression matrix and matching probes to gene symbols.To ensure accuracy, a manufacturer-provided annotation fle was used to convert probes to gene symbols.Additionally, duplicate probe sets were eliminated by calculating the median expression value of all corresponding probes.Probes that did not have corresponding gene symbols were also excluded.Te microarray datasets were preprocessed and normalized using the afy package in the R environment.Subsequently, the R package was used to screen diferentially expressed genes (DEGs) between the healthy donors and infamed pulps.|log fold change|≥ 1 and p < 0.05 as the cutof criteria were considered statistically signifcant.Te chemical structure of berberine was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and inputted into the SwissTargetPrediction database (https://www.swisstargetprediction.ch/) to obtain berberine-related genes [28].Moreover, we searched the keyword "berberine" in the comparative toxicogenomics database (CTD) (https://ctdbase.org/) to collect berberine-related genes.Ten, the berberineassociated targets were obtained by combining these targets derived from the two databases.We used a Venny online tool to visualize the intersection targets of berberine-related targets and DEGs.Te volcano map and heat map of DEGs were visualized using a bioinformatics platform.In addition, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of berberine were obtained from the pkCSM (https:// biosig.unimelb.edu.au/).

Construction of the Integrated Network in Antipulpitis
Targets of Berberine.Te shared targets of berberine and pulpitis were imported to the String database (https://www.string-db.org/)with a confdence score greater than 0.9 to generate the protein-protein interaction (PPI) network construction [29].Ten, the PPI network was visualized using Cytoscape software, and the CytoHubba plugin was used to screen hub genes based on 10 algorithms [30].Besides, the common genes of berberine and DEGs were grouped into two clusters using the molecular complex detection (MCODE) algorithm in Metascape [31].

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Molecular Docking Validation.
We performed molecular docking to interpret the potential binding mode of berberine to a macromolecular receptor.Te hub gene (PTGS2) was selected for molecular docking analysis.Te 3D conformations of proteins with a crystal resolution <3 Å, as measured by X crystal difraction, were chosen.Te 3D structure of the PTGS2 protein (PDB ID: 5IKR with resolution 2.342 Å) was downloaded from the PDB (https:// www.rcsb.org)database [32].Te PyMOL software package was applied to remove the solvent and organic of the protein conformations.For the processing of ligand molecules, we applied ChemDraw In this study, a total of 100 GA runs were conducted while keeping other docking criteria fxed at default settings.Te result of molecular docking was visualized by using PyMOL software.

Enrichment Analysis of Crucial Targets. Te Metascape online platform was used to perform (gene ontology) GO enrichment analyses including biological process (BP), cellular component (CC), and molecular function (MF) and
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses.Based on p < 0.05 and a high count, the top 10 BP, CC, MF, and KEGG pathways were chosen to visualize circle, bubble, and heat graphs using the bioinformatics platform (https://www.bioinformatics.com.cn/).Besides, the Gene Set Enrichment Analysis (GSEA) was used to identify the enriched pathways in patients with pulpitis; p.adjust <0.05 and false discovery rate (FDR) <0.25 were considered as the inclusion criteria.

Human Dental Pulp Stem Cell Culture and Treatment.
Human dental pulp stem cells were incubated in alpha modifcation of Eagle's medium containing 10 U/mL penicillin, 10 mg/mL streptomycin, and 20% fetal bovine serum and maintained in an incubator supplemented with 5% CO 2 at 37 °C.Berberine (purity >98%) and LPS (Escherichia coli) were obtained from Sigma-Aldrich and dissolved in DMSO.Cells from the sixth passage were used in the next experiments.Human dental pulp stem cells were treated or not treated with berberine (10 or 20 μM) in the presence of LPS (1 μg/ml) for 24 h.Te pulpitis model induced by LPS was established based on a previous study [33].

Determination of Proinfammatory Cytokines.
Te cell culture media were collected and centrifuged at 1000 × g for 20 min.Ten, the supernatant was collected, and the level of IL-6 was measured using ELISA kits as recommended by the manufacturer's protocols (Elabscience Biotechnology, Wuhan, China).

Quantitative Polymerase Chain Reaction (qPCR).
Te total RNA was extracted from human dental pulp stem cells using TRIzol (Invitrogen, USA) based on the protocols recommended by the manufacturer.1 μg of total RNA was reverse-transcribed by a First Strand cDNA Synthesis Kit (Invitrogen, USA).qPCR was carried out using the SYBR Green PCR master mix reagent (Takara) in a thermocycler instrument (Applied Biosystems, USA).Relative quantifcation of target gene expression was measured by the 2 −ΔΔCt method, and GAPDH was used as the reference gene.

Statistical Analysis.
Te means ± SD were used to present all data.GraphPad Prism software was employed for conducting all statistical analyses.One-way analysis of variance followed by Dunnett's multiple comparison test was utilized to compare the statistical diferences between two groups.Te statistical diference was considered signifcant at a p value of less than 0.05.

Prediction of ADMET Properties of Berberine.
In the present study, we used pkCSM to estimate the ADMET properties of berberine.As shown in Table 1, the human intestinal absorption of berberine is 97.14, indicating that it showed good absorption.Te blood-brain barrier (BBB) permeability (LogBB) was used to assess the brain distribution of berberine.Te result of BBB permeability is 0.198, indicating that berberine could permeate the BBB.In addition, berberine may not inhibit the activity of CYP2C19 and CYP2C9 enzymes, indicating that it may not impact the clearance and metabolism of the drug substrates of CYP2C19 and CYP2C9.Te renal total clearance is 1.27, and berberine is not likely to be organic cation transporter 2 (OCT2) substrate.Furthermore, the toxicity data indicated that berberine exhibited a promising safety profle.

Prediction of Potential Targets Involved in the Antipulpitis
Efect of Berberine.As shown in Figure 2(a), we identifed 350 berberine-related genes from CTD and Swis-sTargetPrediction databases.A total of 1104 pulpitis-related DGEs were collected from the GSE77459 dataset, which included 325 downregulated genes and 779 upregulated genes, and the volcano plot of DEGs is presented in

Identifcation of Hub Genes and Molecular Docking Study.
PTGS2 was identifed as a hub gene based on the overlapped parameters of the top 10 genes in 10 algorithms (including stress, MNC, MCC, EPC, radiality, DMNC, degree, closeness, bottleneck, and betweenness) (Figure 3(a)).Ten, molecular docking analysis was performed on berberine with the PTGS2 gene to validate the potential target of berberine against pulpitis.Te results showed that the berberine binds to PTGS2 with a binding energy of −9.0 kcal/mol.Eicosapentaenoic acid (EA) is a typical PTGS2 enzyme inhibitor, so it was used as a control compound in the present study.EA binds to PTGS2 with a binding energy of −5.79 kcal/mol.Berberine formed hydrophobic interactions with TRP-387 and ASN-382 residues; EA formed hydrophobic interactions with GLN-278 residue.Tese fndings implied that berberine showed a strong binding afnity for PTGS2 protein.Te binding modes and interactions between the PTGS2 receptor and small molecule ligands are presented in Figures 3(b) and 3(c).

PPI Network from 30 Overlapping
Genes.In the present study, 30 overlapping genes were inputted into the Metascape platform to construct the PPI network.As shown in Figure 4(a), this network contained 24 nodes and 63 edges.All targets were ranked in descending order of degree value, and the top 10 genes are presented in Table 1.Besides, the PPI network was grouped into two clusters using the MCODE algorithm.As shown in Figures 4(b) and 4(c), cluster 1 contained several infammation-related genes, such as CXCL8, TNF, IL1B, and IL-6.Cluster 2 contained three genes (TGM2, H3C2, and H3C8).

Enrichment Analyses of Crucial Targets from Cluster 1.
Te bioinformatics platform was used for generating enriched terms in the BP, CC, and MF categories.Te top 10 biological processes are shown in Figure 5 and Table 2. GO-BP enrichment analysis revealed that the genes of  cluster 1 were implicated in multiple biological processes, including regulation of neuroinfammatory response, response to lipopolysaccharide, response to molecule of bacterial origin, neuroinfammatory response, and positive regulation of smooth muscle cell proliferation.CC enrichment analysis indicated that these crucial targets were mainly related to the endoplasmic reticulum lumen, membrane raft, membrane microdomain, and membrane region (Figure 6).MF enrichment analysis revealed that these crucial targets were mainly enriched in cytokine   3. KEGG enrichment analysis revealed that these crucial genes were involved in multiple pathways, including rheumatoid arthritis, toll-like receptor signaling pathway (Figure 8), Chagas disease (American trypanosomiasis), malaria, NOD-like receptor signaling pathway (Figure 9), and leishmaniasis.
3.6.GSEA.GSEA of DEGs was carried out to further assess the related pathways in the development of pulpitis.As shown in Figure 10, our results showed that the DEGs were signifcantly enriched in the toll-like receptor signaling pathway and regulation of the toll-like receptor signaling pathway, which is consistent with the KEGG results.

Validation of Network Pharmacology Results by Cell
Experiments.As shown in Figure 11(a), compared with the control group, the level of IL-6 was signifcantly increased, which was repressed by treatment with berberine.In addition, compared with the control group, the expression level of PTGS2 was signifcantly upregulated, which was downregulated by treatment with berberine (Figure 11(b)).Tese fndings revealed that the activated infammatory response in LPS stimulation of dental pulp stem cells was mitigated by berberine.

Discussion
Pulpitis is an oral disease mainly caused by a pathogenic microorganism infection of the pulp space, but its exact pathologic mechanisms remain unknown [6].Berberine has been reported to promote odontoblast diferentiation in human dental pulp cells, suggesting its potential therapeutic efects against pulpitis [21].However, the therapeutic mechanism of berberine in the treatment of pulpitis remains unknown.In the feld of natural product development and utilization, network pharmacology is increasingly being applied to repurpose approved drugs and to develop new therapeutic drugs [34].In the present study, network pharmacology was applied to identify the pharmacological mechanism of berberine against pulpitis.
First, we identifed 30 potential targets of berberine by combining several databases' predication.Subsequently, a core subnetwork including 8 potential targets was identifed by MCODE analysis.GO and KEGG enrichment analyses indicated that berberine exerts an antipulpitis efect via regulating multiple pathways, including regulation of neuroinfammatory response, toll-like receptor signaling pathway, and NOD-like receptor signaling pathway.In addition, the fndings of molecular docking revealed that there existed hydrogen bondings between the berberine and the PTGS2 protein, which further confrmed our results from bioinformatics analysis.
Infammation is one of the main drivers of various diseases [35].Pulpitis is a chronic infammatory disease, characterized by the aggregation of proinfammatory mediators, including tumor necrosis factor-α (TNF-α), IL-6, C-X-C motif chemokine ligand 8 (CXCL8), IL-1, and IL-1β [36,37].It has been demonstrated that the overexpression of proinfammatory cytokines could afect the diferentiation of odontoblasts, thus infuencing the formation of restorative dentin [8,38].Besides, these cytokines were considered diagnostic markers of irreversible pulpitis [39].Te activation of the toll-like receptor 2 (TLR2) signaling pathway is involved in the upregulation of IL-6, CXCL8, and TLR2 in odontoblast-like cells stimulated with lipoteichoic acid [40].Berberine has been revealed to exert its antimicrobial and anti-infammatory activities in the treatment of chronic diseases [41].Importantly, berberine was reported to promote odontoblast diferentiation via activating the Wnt/ β-catenin pathway [20].Interestingly, these reports supported our putative therapeutic efects that berberine could regulate infammatory responses.
Te molecular docking method is employed to examine the molecular interactions between small molecules and the binding site of the target protein.Molecular docking was performed to assess the binding modes and interactions between the core target protein (PTGS2) and berberine.Te binding energy was −9.0 kcal/mol, and berberine formed hydrophobic interactions with TRP-387 and ASN-382    Journal of Clinical Pharmacy and Terapeutics residues.Tese fndings implied that berberine showed a strong binding afnity for PTGS2 protein.PTGS2, also known as COX-2, plays a vital role in the regulation of infammation [42].Te positioning of the p-methoxy group in naproxen is directed towards the apex of the COX active site, establishing van der Waals interactions with TRP-387 and TYR-385 [43].A recent study showed that berberine had anti-ischemia-reperfusion efects via inhibiting the PTGS2/ MAPK pathway [44].Berberine exerted the antiangiogenic efect via the inhibition of COX-2 expression and proinfammatory cytokines [45].In line with those previous results, our fndings suggested that berberine might contribute to the therapeutic efect against pulpitis by inhibiting the activity of PTGS2.However, cell or animal studies are needed to confrm the results.Toll-like receptors (TLRs) are important receptors regulating the innate immune response.Activation of TLRs is mainly related to the induction of cell-mediated immune responses and infammatory processes via the generation of immune messenger molecules [46].It has been demonstrated that the expression levels of toll-like receptor 4 (TLR4) and TLR2 are related to the early stage of pulpitis induced by pathogenic microorganism infection [11].Activation of a TLR4 signaling pathway in trigeminal ganglion has been reported to be involved in the nociception associated with acute pulpitis in the rat [10].Te neural TLR2    Journal of Clinical Pharmacy and Terapeutics plays an important role in pulpitis and suggesting inactivation of the TLRs signaling pathway may be a promising therapy in the treatment of pulpal infammation [47,48].Te anti-infammatory efects of S14G-humanin were reported to protect LPS-stimulated human dental pulp cells via inactivating the TLR4/MyD88/NF-κB signaling pathway [49].Phoenixin-20 was shown to inhibit LPS-induced infammation via downregulation of TLR4 expression in dental pulp cells [50].NOD-like receptors represent a wide range of complex signal regulators that are implicated in cellular senescence, tumorigenesis, and infammatory responses [51,52].NLRP3 is one of the most characteristic members of the NOD-like receptor family.Terefore, TLRs and NOD-like receptors might be potential therapeutic targets for the prevention and treatment of oral diseases, such as pulpitis [53].Berberine treatment downregulated TLR4 expression in necrotizing enterocolitis [54].Berberine attenuated intestinal mucosal barrier injury through the TLRs pathway [19].Berberine exerted an inhibitory efect on NLRP3 infammasome activation in nonalcoholic steatohepatitis [55].Berberine exhibited potential therapeutic efects via targeting the endothelial NLRP3 infammasome in infammatory vascular damage [56].In line with these reports, our results of bioinformatics analysis indicated that berberine may exert a therapeutic efect on pulpitis via the inactivation of TLRs and NOD-like receptor signaling pathways.
Te main limitation of the current study is that the results of the analyses were based on existing public databases.Terefore, our conclusions need to be verifed using cell and   Journal of Clinical Pharmacy and Terapeutics animal experiments.In future research, we will explore the following topics: the cellular and molecular mechanism of berberine against pulpitis, the appropriate dose of berberine to improve pulpitis, and whether it is suitable for long-term maintenance treatment of pulpitis.We hope that our fndings will shed light on the potential mechanism of action of berberine against pulpitis and provide a theoretical basis for the application of berberine in clinical practice.

Conclusion
In summary, this study employed bioinformatics analysis to explore the potential mechanism of berberine in the treatment of pulpitis.Our fndings showed that berberine may play a signifcant role in the treatment of pulpitis by infuencing key biological targets, such as PTGS2.Additionally, our molecular docking analysis indicated that berberine has the potential to efectively interact with this target.Berberine also could exert antipulpitis efects via the regulation of TLRs and NOD-like receptor signaling pathways.Tese results provided initial insights into the working of berberine and opened up new possibilities for future research on its mechanism in pulpitis treatment.Moreover, these fndings lay the groundwork for the clinical application of berberine in pulpitis treatment.However, further pharmacological experiments are required in subsequent stages to validate the primary regulatory target of berberine in pulpitis.
19.0 software to draw the molecular structure of the small molecule compound, imported the format fles into Chem3D 19.0 software, performed the program to optimise the energy, and saved them in the PDB format.Te PDB format fle was then imported into AutoDockTools 1.5.6 software, which was applied to adjust the charge of the ligand and saved as a PDBQT fle.Ten, AutoDock software (1.5.6) was used to perform molecular docking analysis.Te detailed procedure for molecular docking is as follows: the PDBQT fle of the receptor macromolecule and ligand was opened in Auto-DockTools 1.5.6 software.To identify the active binding site for molecular docking, we utilized the coordinates of the cocrystallized ligand.Based on the active pocket of the target protein, we then set the grid-box coordinates and size.Te grid-box parameters were set as follows: spacing of 0.503 Å, x-dimension of 80 points, y-dimension of 80 points, and zdimension of 80 points.Docking was performed with the Vina force feld, using the Lamarckian GA (4.2) algorithm.

Figure 1 :
Figure 1: Flow diagram designed for the present study to investigate the therapeutic mechanisms of berberine against pulpitis via a network pharmacology approach.

Figure 2 (
Figure 2(b).Ten, 30 intersection genes were chosen as potential targets of berberine in the treatment of pulpitis.Te heat diagram of 30 overlapping targets is presented in Figure 2(c).

Figure 2 :
Figure 2: Identifcation of potential genes involved in the antipulpitis efects of berberine.(a) Venn diagram of shared genes between pulpitis and berberine.(b) Volcano plot of DEGs in the GSE77459 dataset.Blue and red dots indicate downregulated and upregulated genes, respectively.Te DEGs were screened based on the criteria p value <0.05 and |log2(fold change)| > 1. (c) Heat map of the shared genes between pulpitis and berberine.

Figure 3 :
Figure 3: Identifcation and validation of the hub gene.(a) PTGS2 was identifed by ten algorithms.(b) Molecular docking analysis of berberine binding to PTGS2.(c) Molecular docking analysis of eicosapentaenoic acid binding to PTGS2.Te left side presents the 3D surface structure of the PTGS2 receptor and small molecular ligand.Te right side presents the binding pattern of the small molecular ligand to the PTGS2 protein.

Figure 4 :
Figure 4: Protein-protein interaction (PPI) network for berberine against pulpitis.(a) Te PPI network of intersection genes between pulpitis and berberine.(b) Cluster 1 of the PPI network.(c) Cluster 2 of the PPI network.
(-log10 (pvalue)) response to lipopolysaccharide response to molecule of bacterial origin positive regulation of smooth muscle cell proliferation response to glucocorticoid positive regulation of nitric oxide biosynthetic process positive regulation of nitric oxide metabolic process acute-phase response regulation of neuroinflammatory response neuroinflammatory response positive regulation of acute inflammatory response Biological Process (b)

Figure 5 :
Figure 5: Te GO-BP enrichment analysis of cluster 1. Te results of GO-BP were presented by circle (a) and bubble (b) graphs.

Figure 7 :
Figure 7: Te KEGG enrichment analysis of cluster 1. Te results of KEGG were presented by circle (a) and bubble (b) charts.

Figure 9 :
Figure 9: Terapeutic efects of berberine against pulpitis involved in the NOD-like receptor signaling pathway.Te red rectangle indicates the genes associated with the PPI network.

Figure 8 :Figure 10 :Figure 11 :
Figure 8: Terapeutic efects of berberine against pulpitis involved in the toll-like receptor signaling pathway.Te red rectangle indicates the genes associated with the PPI network.