Combination of resolvin E1 and lipoxin A4 promotes the resolution of pulpitis by inhibiting NF‐κB activation through upregulating sirtuin 7 in dental pulp fibroblasts

Abstract Objectives To determine whether the combination of resolvin E1 (RvE1) and lipoxin A4 (LXA4) could promote resolution of pulpitis and to investigate the mechanism. Materials and Methods Preliminary screening was first conducted in four specialized pro‐resolving mediators (SPMs). Real‐time quantitative polymerase chain reaction, western blotting, enzyme‐linked immunosorbent assay and double‐immunofluorescence labelling were employed to assess the expression of RelA, SIRT1, SIRT6, SIRT7 and pro‐inflammatory factors. Dental pulp fibroblasts (DPFs) were transfected with siRNA to assess the biological role of SIRT7. A pulpitis model was utilized to evaluate the in vivo curative effect. Results Preliminary results showed that RvE1 and LXA4 reduced the expression of RelA more markedly than other two SPMs. Both RvE1 and LXA4 treatment downregulated nuclear factor kappa B (NF‐κB) activation and increased the expression of SIRT1, SIRT6 and SIRT7, more so in combination than alone. Double‐immunofluorescence labelling showed that SIRT7 co‐localized with p‐p65 and Ac‐p65 in the nucleus. Inhibiting ChemR23 and ALX reversed the expression of RelA mRNA, p‐p65 and Ac‐p65 proteins, pro‐inflammatory factors, SIRT1, SIRT6 and SIRT7. Silencing SIRT7 significantly increased p‐p65 and Ac‐p65 protein levels and decreased SIRT1 and SIRT6 expression. In vivo experiments showed that combined administration of RvE1 and LXA4 promoted pulpitis markedly to resolution. Conclusions Combination of RvE1 and LXA4 effectively inhibited NF‐κB activation by upregulating SIRT7 expression in DPFs, leading to reduced production of pro‐inflammatory factors and promotion of pulpitis resolution.


| INTRODUCTION
Pulpitis is one of the most common inflammatory dental diseases, accounting for approximately 24%-44% of emergency problems observed in oral health clinics. 1,2 By causing irreversible damage of the dental pulp and periapical tissue, pulpitis eventually results in functional loss of the pulp and exfoliation of teeth. 3 An inflammatory response is initiated after infection and/or injury as a protective mechanism to eliminate invading organisms and permit tissue repair.
However, a dysregulated inflammatory response is the fundamental cause of chronic pulpitis and irreversible pulp damage. 4 Hence, attenuation of excessive inflammation has been the most substantial challenge in modern endodontics. Excessive inflammation is due to an imbalance between production and clearance of inflammatory mediators, which causes disruption of tissue homeostasis. 5,6 In contrast to the outdated view of a passive process of inflammatory dilution, resolution of inflammation is considered to be an active, programmed response that is 'turned on' in the body. 7,8 Pro-resolution strategy is a relatively new approach for regulating inflammation, which avoids inhibition of the protective acute inflammatory response and reduces immune tolerance and other side effects of conventional anti-inflammatory treatment. 9,10 The basis of resolution is sequestration of pro-inflammatory cytokines, thereby improving the local inflammatory microenvironment, promoting clearance of apoptotic neutrophils and removal of inflammatory stimulation. 8 Throughout the inflammatory process of pulpitis, an essential source of cytokines is the resident dental pulp fibroblasts (DPFs), which deteriorate the microenvironment, leading to soaring cytokine levels via a signal cascade. [11][12][13] Therefore, regulation of DPFs could reduce the acute inflammatory infiltration and promote resolution.
Inflammatory resolution is mediated by specialized pro-resolving mediators (SPMs), a family of bioactive lipids including, to date, four classes termed lipoxins, resolvins, maresins and protectins. 14 In oral environment, independent evidence supports the protective effect of SPMs in experimental periodontitis. Lee et al. 15 and Hasturk et al. 16 demonstrated that resolvin E1 (RvE1) could decrease the expression of proinflammatory genes and prevent alveolar bone loss by reducing osteoclast density and inflammatory cell infiltration. Besides, resolvin D2 has been shown to exhibit similar effects in murine periodontitis by restraining Th1 immunity. 17 Furthermore, a recent clinical trial showed that methyl esterbenzo-lipoxin A4 (a lipoxin A4 analogue) reduced gingival inflammation and increased the abundance of pro-resolution molecules that prevent periodontitis. 18 These studies exhibit promise with respect to prevention and treatment of oral infectious inflammation. However, studies have rarely focused on the effects of SPMs in endodontics. Dondoni et al. first illustrated the protective role in a rat model of pulpitis. 19 We previously showed that RvE1 could suppress inflammatory infiltration and accelerate pulp repair in pulpitis by reducing activation of nuclear factor kappa B (NF-κB) in lipopolysaccharide (LPS)-induced DPFs to some extent, but not to normal levels. 20,21 This is because SPMs are biosynthesized during resolution in a certain order and act on different stages and aspects of resolution. 22,23 Thus, complete inhibition of inflammatory pathway signalling and resolution cannot be achieved by using RvE1 alone, which is only synthesized in the later stage of resolution. To increase pulpitis resolution, combined application of different SPMs may be more effective. The optimal combination of SPMs and their mechanism of action in resolving pulpitis require further elucidation.
The key to sequestrating pro-inflammatory factors is to inhibit the cascade of inflammatory pathway signalling. 24 Among these pathways, the NF-kB pathway is a major regulator of inflammation due to its ability to cause transcription of numerous genes involved in the inflammatory response. [25][26][27] Therefore, inhibition of NF-κB signalling in DPFs should be continued throughout the resolution process.
Phosphorylation and nuclear localization, as well as steady binding to DNA through acetylation, are key steps in persistent NF-κB activation and the inflammatory cascade. Persistent phosphorylation of NF-κB is tightly regulated by acetylation, which controls the duration of NF-kB activation. 28 Deacetylation of NF-kB has been demonstrated to be an intranuclear molecular switch that could prevent its activation by inhibiting the p65 subunit from entering the nucleus, as well as abrogating binding of activated p65 to DNA, and its translocation back to the cytoplasm. 29 Thus, deacetylation may be a vital means to inhibit the NF-kB pathway and promote resolution of pulpitis. It remains unknown whether combinations of SPMs could inhibit NF-kB activation and promote its degradation by deacetylation.
Deacetylation of NF-κB is directly regulated by sirtuins (SIRTs), a highly conservative family of deacetylases, including seven members in mammals. The enzymatic activities of SIRTs are amenable to regulation by nicotinamide adenine dinucleotide (NAD+), as NAD+ is a cofactor in the deacetylation reaction. SIRT1, SIRT6 and SIRT7 are predominantly expressed in the nucleus. 29 As previous studies reported, SIRT1 and SIRT6 are able to deacetylate the p65 subunit, affecting its transcriptional activity and decreasing expression of pro-inflammatory target genes. 30,31 It was recently suggested that SIRT7 is critical for inflammatory regulation and tissue homeostasis. Wyman et al. found that SIRT7 deficiency in primary pulmonary endothelial cells increased vascular permeability, which is involved in acute lung injury, 32 although the underlying mechanism remains poorly understood. In infectious pulpitis, it also remains unknown whether a combination of SPMs could upregulate SIRT7 expression and promote resolution of inflammation.
Besides, Fukuda et al. have demonstrated that SIRT7 cooperates with SIRT1 to promote OSX deacylation in bone metabolism. 33 No data are available on the relationship of SIRT7 with SIRT1 or SIRT6 in NF-kB deacetylation in an inflammatory environment.
In this study, we sought to clarify the mechanism of pulpitis resolution to provide new ideas and potential treatment strategies for vital pulp preservation. To this end, the inhibition of NF-κB by four representative SPMs were first determined, which indicated the best combination of SPMs for further applications. Thereafter, we evaluated the effect of this combination of SPMs on LPS-induced DPFs to investigate the phosphorylation and acetylation levels of NF-kB and expression of SIRT1, SIRT6 and SIRT7, as well as that of proinflammatory cytokines. Furthermore, the effect of SIRT7 on the RvE1 and lipoxin A4 (LXA4) combination was explored by silencing SIRT7, and the expression of SIRT1 and SIRT6 was observed concurrently to illustrate the relationship between them. Finally, an animal model of pulpitis (rat incisors) was employed to evaluate the therapeutic effect of the combined usage of RvvE1 and LXA4 on pulpitis.

| Cell culture
Ten-third molars with healthy pulp, extracted for orthodontic requirements, were collected at the Affiliated Stomatology Hospital of Tongji University with approval from the Ethics Review Board (No. [2021]-SR-09). Briefly, the dental pulp was cut into fragments and digested by 2 mg/ml collagenase, type 1 (Sangon Biotech, Shanghai, China) for 1 h. After centrifugation at 1000 rpm for 10 min by an SL8 centrifuge (Thermo Scientific), the precipitate was removed into Dulbecco's modified Eagle's medium (Hyclone) with 10% of foetal bovine serum (Gibco), which was renewed every 3 days. Passage 3-5 DFPs were used for this study.
In the preliminary screening experiment, RvE1 alone (

| Real-time quantitative PCR
Total cellular RNA was extracted using a TRIeasy Total RNA Extraction Reagent (Yeasen) for reverse-transcription, using a cDNA Synthesis SuperMix for quantitative polymerase chain reaction (qPCR) with gDNA Eraser (Yeasen). To quantify mRNA levels, real-time qPCR was performed using a qPCR SYBR Green Master Mix (Yeasen) on a Lig-htCycler ® 96 (Roche) device. The primer sequences are listed in Table 1. Data were normalized to the housekeeping gene ACTB (β-actin), and relative expression was evaluated using the 2 ÀΔΔCt method.     Briefly, under general anaesthesia using 1% Pelltobarbitalum Natricum (Sangon Biotech), 2 mm of the crowns of the maxillary incisors were gently ground using a no. 1/2 round burr (Mani Inc), and access to the pulp was gained using a #20 K-file followed by enlargement using with a #40 K-file (Mani). Pulp was exposed to the oral environment for 12 h. Thereafter, 0.9% of sterile saline solution was used for irrigation to remove debris above the pulp. The cavities were dried with sterile cotton pellets and were covered using sponge (Jinling Pharmaceutical Co., Ltd) with 50-ng RvE1 and/or LXA4. A control group was set up with a sham, non-exposure operation. Rats were euthanized to extract samples at days 1, 3 and 7.

| Statistical analysis
Three replicates were performed in all experiments independently, and data were expressed as mean ± standard deviation (SD

| Combined administration of RvE1 and LXA4 downregulates RelA mRNA levels and production of inflammatory cytokines by LPS-induced DPFs
Four representative SPMs were used in LPS-induced DPFs. Preliminary results showed that RvE1 and LXA4 reduced the expression of RelA more markedly than did PD1 and MaR1 ( Figure 1A). Moreover, both RvE1 and LXA4 showed the best inhibitory effect on RelA expression at the concentration of 10 nM ( Figure S1A,B), which was applied in follow-up experiments. Then, a combination of RvE1 and LXA4 was used, which resulted in a significantly greater decrease in RelA expression than that achieved with RvE1 or LXA4 alone ( Figure 1B). Translocation of p65 was detected by western blotting of nuclear and cytosolic protein, respectively. As shown in Figure 2E, the amount of nuclear p65 protein was sharply augmented in LPS-induced DPFs, while cytoplasmic p65 protein only slightly increased, resulting in an increased ratio of nuclear to cytoplasmic p65. After RvE1-or LXA4-only treatment, the amount of nuclear p65 protein decreased, with a small increase in cytoplasmic p65. The combination of RvE1 and LXA4 caused a further decline of nuclear p65 and a slight decrease in cytoplasmic p65. Meanwhile, the phosphorylation and the acetylation of p65 (p-p65 and Ac-p65) were concurrently decreased, and combined usage showed a much greater inhibition than using separately ( Figure 2F).
Double-immunofluorescence labelling was employed to observe the cellular co-localization of SIRT7 and p-p65 ( Figure 2G) or Ac-p65 ( Figure 2H). Images revealed that after LPS stimulation, both p-p65 and Ac-p65 were highly expressed in and around the nucleus, while SIRT7 maintained a low expression in the nucleus. In contrast, increased SIRT7 expression and reduced p-p65 and Ac-p65 levels were observed upon combined administration of RvE1 and LXA4. These effects were more marked for the combined treatment than for either single treatment.

| Silencing SIRT7 weakens suppression of NF-κB by the RvE1 and LXA4 combination
To assess the biological role of SIRT7 in the resolution of pulpitis, siRNA targeting SIRT7 was designed and transfected into DPFs.
Results of qPCR ( Figure 4A) and western blotting ( Figure 4B staining ( Figure 5A) and CD11b immunofluorescence ( Figure 5B) suggested that infiltration of immune cells was significantly less in the RvE1-and/or LXA4-treated group than in the pulpitis tissue at day 1. On day 3, the inflammation continued to spread in the pulpitis tissue but was notably limited in the RvE1-and/or LXA4-treated group ( Figure 5D). In the RvE1-and LXA4-only treatment groups, ectopic mineralization in the pulp cavity, a typical feature of the chronic pulpitis stage, was markedly present, while it was virtually absent in rats treated with the combination of RvE1 and LXA4 ( Figure 5A).
Immunohistochemical staining showed that ectopic mineralization Images of double-immunofluorescence labelling of day 3 samples indicated a similar tendency to the in vitro experiments. In the pulpitis group, both p-p65 and Ac-p65 were expressed at higher levels, while SIRT7 showed a lower expression than in normal pulp. After combined RvE1 and LXA4 treatment, SIRT7 increased and p-p65 and Ac-p65 decreased, with changes that were more significant than with either treatment alone ( Figure S3A,B). Immunohistochemistry staining for NLRP3, caspase-1, IL-1β and IL-18 ( Figure 6A,B) and statistical analysis ( Figure 6C-F) showed the same trend, indicating that use of RvE1 or LXA4 alone could reduce the expression of these inflammatory markers to some extent, but that their levels were still higher than in normal pulp tissue. However, combined administration of RvE1 and LXA4 could promote pulpitis resolution to a greater extent, facilitating recovery of homeostasis in the pulp tissue.

| DISCUSSION
In this study, we investigated whether combined administration of RvE1 and LXA4 could promote resolution of pulpitis better than RvE1 or LXA4 alone. We also investigated the associated mechanism in DPFs and in a rat incisor model of pulpitis. We showed that combined administration of RvE1 and LXA4 could effectively promote pulpitis resolution by inhibiting NF-κB activation via upregulation of SIRT7 expression, reducing expression of pro-inflammatory factors.
Inflammatory regulation is crucial to vital pulp preservation and pulp, including DPFs, stem cells and odontoblasts, contribute substantially to inflammatory resolution and are critical for tissue homeostasis. 37 Horibe et al. found that ALX could be observed in odontoblastic layer during physiological and reparative dentin formation. 38 Our group has previously shown that RvE1 can accelerate repair of injured pulp by regulating inflammation and promoting odontogenic differentiation in dental pulp stem cells. 21 These results highlight the proregeneration function of SPMs in dental pulp, but gaps remain regarding understanding of inflammatory regulation. Our previous study also verified that using RvE1 alone could inhibit inflammatory reactions and NF-κB activation to a certain extent but failed to achieve complete resolution of pulpitis. 20 In our present study, we also found that single usage of RvE1 or LXA4 significantly reduced the expression of pro-inflammatory cytokines ( Figure 1B) but failed to decrease to that of normal pulp. These results were similar to previous findings regarding SPMs in periodontitis 15 and lung injury. 39 SPMs act at different stages of resolution and on diverse inflammatory biomarkers. 22 Therefore, combined administration of SPMs may be an optimal tactic in pro-resolving approach.
SPMs are a family of bioactive lipids derived from ω-3 and ω-6 essential polyunsaturated fatty acids including, to date, four classes named lipoxins, resolvins, maresins and protectins. 14 Considering the classification, metabolic precursors and receptors of SPMs, we selected one from each category for preliminarily screening. We found that LXA4 and RvE1 showed a greater inhibitory effect on NF-κB signalling in LPS-induced DPFs than the two other SPMs ( Figure 1A).
Hence, we applied them in combination in the follow-up experiments.
In this study, we first explored the optimal concentration of RvE1 and LXA4 on LPS-induced DPFs, finding that the best inhibitory effect on both Rel was at 10 nM (either with RvE1 or LXA4, Figure S1A study, separate determination of p65 expression in the nucleus and cytoplasm was examined using western blotting. An increased ratio of nuclear to cytoplasmic p65 was observed in LPS-induced DPFs, and some p65 protein translocated back to the cytoplasm after RvE1-or LXA4-treatment, leading to a decrease in nuclear p65. The combination of RvE1 and LXA4 caused a further decline of nuclear p65 and a slight decrease in cytoplasmic p65 ( Figure 2E); this simultaneous reduction of nuclear and cytoplasmic p65 is due to a significant decrease in p65 transcription ( Figure 1A). Meanwhile, the ratio of nuclear to cytoplasmic p65 was much lower than that with RvE1-or LXA4-only treatment. Moreover, phosphorylation and acetylation of p65, which represent the duration and stable transcription of NF-κB, 44,45 tended to be reduced more with the combination treatment ( Figure 2F). As a result, combined administration exhibited greater suppression of pro-inflammatory cytokines transcribed by NF-κB than either RvE1 or LXA4 alone ( Figure 1C Furthermore, the molecular mechanism of the RvE1 and LXA4 combination on NF-κB suppression was also investigated in this study.
SIRTs are a highly conserved family of deacetylases, of which the enzymatic activities are strictly regulated by NAD+. 52,53 In LPSinduced DPFs in vitro, we found that the expression of SIRT1 and SIRT6, which are located in the nucleus, was significantly reduced.
These results are similar to those of well-established studies in cardiovascular disease and vascular cells. 30,31 Particularly, our results also demonstrated expression of SIRT7 decreased in pulpitis and was consistent with a recent report regarding mastitis, 54 in which activation of NF-κB was increased as well, but in which the relationship of SIRT7 and NF-κB remained unclear. Combined use of RvE1 and LXA4 could strongly increase the NAD+/NADH ratio ( Figure S3), indicating that high expression of SIRT7 may be closely related to NF-κB inactivation. By silencing SIRT7 in DPFs, the pro-resolving effect of RvE1 and LXA4 was largely abrogated, and the RelA mRNA level, ratio of nuclear to cytoplasmic p65 protein, and phosphorylation and acetylation of p65 were increased after combined RvE1 and LXA4 administration ( Figure 4C,G-I). These results suggested that the upregulation of SIRT7 was an indispensable mechanism of the inhibitory effect of NF-κB on RvE1 and LXA4 and may be a potential target for resolution of pulpitis. However, whether SIRT7 binds directly to NF-κB, and the location of its binding site, remain to be studied. Interestingly, the NAD+/NADH ratio and the expression of SIRT1 and SIRT6 were observed to be decreased in combined-treated si-SIRT7 DPFs ( Figure 4D-F), demonstrating that SIRT7 may play a synergistic role with SIRT1 and SIRT6 in NF-κB inhibition and inflammation resolution.
The present study provides some insights regarding the mechanisms for inhibiting NF-κB activation, which has been seen as a central pathway during pulpitis development. However, further preclinical and clinical trials are needed for verification of our findings. Moreover, future studies on the molecular mechanism of RvE1 and LXA4 combination treatment are necessary identify further potential targets for resolution of pulpitis and other infectious inflammatory conditions.
A visual summary of this study is presented in Figure 7. Based on the above findings, we propose that using a combination of SPMs may be a promising therapeutic strategy against pulpitis and that combined use of RvE1 and LXA4 shows great potential. Through in vitro and in vivo experiments, it was confirmed that the combination of RvE1 and LXA4 could effectively inhibit NF-κB activation by dephosphorylation and deacetylation. At a molecular level, we clarified that SIRT7 was involved in resolution mediated by combination of RvE1 and LXA4 and may affect the expression of SIRT1 and SIRT6, indicating that they may synergistically promote inflammation resolution.
F I G U R E 7 The molecular mechanism by which the RvE1 and LXA4 combination resolves pulpitis as proposed in the present study. LXA4, lipoxin A4; RvE1, resolvin E1