Evaluating the effect of LPS from periodontal pathogenic bacteria on the expression of senescence‐related genes in human dental pulp stem cells

Abstract The human dental pulp stem cells (hDPSCs) are one of the readily available sources of multipotent mesenchymal stem cells (MSCs) and can be considered as a type of tool cells for cell‐based therapies. However, the main limitation in the clinical use of these cells is DPSC senescence, which can be induced by lipopolysaccharide (LPS) of oral pathogenic bacteria. Up to now, far little attention has been paid to exploring the molecular mechanisms of senescence in DPSCs. So, the current study aimed to investigate the underlying molecular mechanism of senescence in hDPSCs stimulated with Porphyromonas gingivalis (P. gingivalis) and Escherichia coli (E. coli)‐derived LPSs, by evaluating both mRNA and protein expression of four important senescence‐related genes, including TP53, CDKN1A, CDKN2A and SIRT1. To this purpose, hDPSCs were stimulated with different LPSs for 6, 24 and 48 h and then the gene expression was evaluated using quantitative real‐time polymerase chain reaction (qPCR) and western blotting. Following stimulation with P. gingivalis and E. coli‐derived LPSs, the relative mRNA and protein expression of all genes were significantly up‐regulated in a time‐dependent manner, as compared with unstimulated hDPSCs. Moreover, the hDPSCs stimulated with P. gingivalis LPS for 6 and 24 h had the highest mRNA expression of CDKN1A and SIRT1, respectively (p < 0.0001), whereas the highest mRNA expression of CDKN2A and TP53 was seen in hDPSCs stimulated with E. coli LPS for 48 h (p < 0.0001). In summary, because DPSCs have been reported to have therapeutic potential for several cell‐based therapies, targeting molecular mechanisms aiming at preventing DPSC senescence could be considered a valuable strategy.


| INTRODUC TI ON
Human dental pulp stem cells (hDPSCs) are one of the readily available sources of multipotent mesenchymal stem cells (MSCs), characterized by the clonogenic, plastic-adherent, highly proliferative cells capable of self-renewal and differentiation potential into several cell lineages, such as osteo/odontogenic, adipogenic, chondrogenic, myogenic and neurogenic lineages. 1,2 The hDPSCs express cell surface markers specific for MSCs, such as CD105, CD73 and CD90.
However, these cells are negative for surface expression of haematopoietic molecules, including CD14, CD34 and CD45. 1,3 In recent years, there has been an increasing interest in the use of hDPSCs in cell-based therapies, as these cells have important features including ease to access, cost-benefit, low immunogenicity, simplicity and convenience of isolation and minimum ethical issues. [4][5][6] However, before clinical usage of hDPSCs in cell-based therapies, there is an urgent need to address the biological properties of these cells in response to intrinsic and extrinsic stimuli. The dental pulp tissues, which are the source of DPSCs, are often repeatedly encountered with the various types of stimuli, especially infectious agents, such as Gram-negative bacteria. In this regard, one of the main stimulators that binds to the toll-like receptor 4 (TLR4) on the surface of DPSCs and exerts its immunopathological effects is LPS. LPS (a major component of G − bacteria) plays a crucial role in the induction and promotion of inflammation in the oral cavity by inducing the production of several pro-inflammatory mediators such as tumour necrosis factorα (TNFα), interleukin-1 (IL-1), IL-6 and CCL8. 7,8 Increasing research evidence has indicated that inflammatory responses can induce senescence (aging) in MSCs. [9][10][11][12] In this respect, recent studies by Feng et al. have revealed that stimulation with LPS, to imitate an inflammatory microenvironment, promotes senescence of DPSCs. 13,14 Although senescence plays physiological roles in the human body, this phenomenon can exert pathological roles in MSCs, which is characterized by the reduced capacity of proliferation and differentiation, and functional disorders. 15,16 Therefore, the senescence of DPSCs is one of the main challenges faced by therapies targeting tissue regeneration. Up to now, few studies have investigated the molecular mechanism of DPSC senescence induced by LPS. Hence, the current study aimed to investigate the underlying molecular mechanism of senescence in LPS-stimulated DPSCs by evaluating the expression of four important genes (CDKN1A, CDKN2A, TP53 and SIRT1), which are involved in the process of aging, at the mRNA and protein levels. [17][18][19][20][21] To this purpose, the current study evaluates and compares the impact of LPS derived from P. gingivalis and E. coli on hDPSCs. The hDPSCs were isolated from impacted third molars by disinfecting the tooth surface using 70% ethanol, cutting around the cementoenamel junction using sterilized dental fissure and then removing coronal pulpal tissue using sterile dental excavator burs.

| Cell cultures
Then, the digestion of the minced dental pulp tissues was performed for 1 h at 37°C using a solution of 3 mg/ml collagenase type I (Sigma-Aldrich, St. Louis, MO, USA). Then, digested dental pulp tissues were filtered using a 70μm cell strainer and centrifuged at 12000 RPM for 5 min. Finally, cell pellets were resuspended in Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F12 (Biosera, England) medium containing 12% fetal bovine serum (FBS) (Gibco, UK), 100 U/ml penicillin and 100 μg/ml streptomycin (Biosera, England) and cultured at 37°C in an incubator containing 5% CO 2 . To obtain precise analysis, all tests were done using the third passage of cells (passage 3).

| Cell number determination
The hDPSCs at a density of 0.5 × 10 4 cells/well were seeded into 24-well plates in triplicate. The hDPSCs were collected after plating and dissociated and the total cell numbers were counted as follows:

| Differentiation assays
To evaluate the ability of hDPSCs to differentiate into osteogenic and adipogenic cells, third passage cells were used. Briefly, hDPSCs at the density of 3 × 10 4 cells/well were seeded into 24-well plates

| LPS stimulation
The hDPSCs at the density of 3 × 10 4 cells/well were seeded into 24-well plates and incubated overnight. After incubation, the culture medium received either 1 μg/ml P. gingivalis LPS (InvivoGen), 1 μg/ ml E. coli LPS (Sigma), or normal saline (as a control) once for 6, 24 and 48 h.

| Quantitative real-time PCR
Specific primers (forward and reverse primers) for GAPDH (as a housekeeping gene), TP53, CDKN1A, CDKN2A and SIRT1 were designed using AlleleID® Software (version 7.0, Premier Biosoft International) ( Table 1) ing) and 10 s 72°C (extension), which was followed by a final melting curve analysis at 65-92°C for 15 seconds. The relative expression of target genes was normalized to the GAPDH gene, internal control and then measured using the 2 −Δct method. 22,23 All experiments were done three times.

| Statistical analysis
Data analysis was performed using GraphPad Prism software (Version 8.0.2, San Diego, California). Comparisons between groups were performed using one-way analysis of variance (anova) followed by Tukey's multiple comparison tests. Descriptive data were generated for all variables. Significance levels were set at <5%.

| Differentiation of hDPSCs
As shown in Figure 2B,

| DISCUSS ION
The key question of the current study was whether treatment with mitogen-activated protein kinases (MAPKs) and myeloid differentiation factor 88 (MyD88) signalling pathways. 24 As a consequence of these events, several inflammatory responses are promoted, which in turn may stimulate senescence in DPSCs. 13,14 In this regard, several important genes such as TP53, CDKN1A, CDKN2A and SIRT1 play key regulatory roles in the DPSC senescence.
TP53, a 53-kDa protein located at position 17p13.1, is one of the first transcription factors that play crucial regulatory roles in several cellular events such as self-renewal, genome stability, cell-cycle arrest and apoptosis. 25,26 Importantly, several triggers such as DNA damage, oxidative stress and inflammation can initiate the p53 signalling cascade, which eventually leads to cellular senescence. 25,26 In the downstream of p53 signalling pathway, CDKN1A gene as the first identified senescence-associated target gene of p53 has been located, which encodes the cyclin-dependent kinase (CDK) inhibitor p21. 25 Several research reports have revealed that p21 effectively is capable of inducing cellular senescence in vitro. [27][28][29][30][31] Indeed, p21 has been defined as a pivotal mediator of p53-regulated cellular senescence in response to various stimuli such as DNA damage and inflammation. 31,32 In this respect, our in vitro study showed that   as the important inducer of DPSC senescence. [33][34][35] In this regard, the current study found that P. gingivalis-derived LPS can markedly up-regulate the gene expression of TP53 and CDKN1A in hDPSCs.

Mean ± SD Mean ± SD
Meanwhile, P. gingivalis LPS also has a high capacity to increase the mRNA and protein expression of CDKN1A, over E. coli LPS.
One of the most important findings in the current study was that hDPSCs treated with P. gingivalis and E. coli -derived LPSs had a high level of CDKN2A, compared with unstimulated hDPSCs, in a time-dependent manner. CDKN2A is one of the main regulatory proteins, which encodes by the p16 gene, participating in the G1/S cell cycle checkpoint. 36 This protein was reported to be a unique marker for cell senescence in vitro and in vivo. 37  F I G U R E 6 mRNA and protein expression of SIRT1 gene. The hDPSCs at the density of 3 × 10 4 cells/well were seeded into 24-well plates and then treated with either 1 μg/ml Porphyromonas gingivalis LPS, 1 μg/ml Escherichia coli LPS, or normal saline (as a control or untreated) once for 6, 24 and 48 h. Both mRNA (A) and protein levels (B) of SIRT1 significantly up-regulated following treatment with different LPSs, in a time-dependent manner, as compared to unstimulated hDPSCs. The results are expressed as mean ± SD (n = 3). SIRT1: 81 kDa, GAPDH: 37 kDa. Cnt: Control, P. G: P. gingivalis, E.C: E. coli. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.