Mouth gel containing phthalocyanine derivative attenuates TNF-α through NF-κB inhibition in experimental periodontitis

doi:10.21203/rs.3.rs-4181301/v1

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Research Article

Mouth gel containing phthalocyanine derivative attenuates TNF-α through NF-κB inhibition in experimental periodontitis

https://doi.org/10.21203/rs.3.rs-4181301/v1

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Background

Tumor necrosis factor alpha (TNF-α) is a major cytokine involved in periodontal disease pathogenesis due to its ability to modulate inflammatory cytokine production and cell influx in tooth-supporting tissues. Therefore, TNF-α is an excellent target for limiting the inflammatory process and avoiding alveolar bone resorption and tooth loss.

Objective

This study investigated the activity and action mechanism behind the anionic iron phthalocyanine (AI-PhyC) derivative on TNF-α production and the impact on experimental periodontitis.

Methods

RAW 264.7 macrophages were treated with AI-PhyC, and cell viability (MTT) was assessed. Afterward, cells were treated with AI-PhyC, primed with LPS (10ng/mL), and the TNF-α levels were measured, as well as the NF-kB activation and the gene expression of Ikkβ. Subsequently, a mouth gel containing 1% AI-PhyC was topically administered in the gingival tissue of mice with periodontitis ligature-induced. Bone loss and the gene expression of Tnfα, Ikkβ, p65 (NF-κB), and receptor-activating nuclear factor kappa B ligand (Rankl) were quantified in the gingival tissue. Lastly, the systemic toxicity of AI-PhyC was estimated in Galleria mellonella larvae.

Results

In an activated RAW 264.7 macrophage cell culture, AI-PhyC at 100µM reduced TNF-α release, nuclear factor kappa B (NF-kB) activation and mRNA expression of Ikkβ. Regarding the experimental periodontitis, topical application of mouth gel containing 1% AI-PhyC blockage alveolar bone loss. Additionally, 1% AI-PhyC reduced the mRNA expression of Tnfα, Ikkβ, p65 (NF-κB), and Rankl in the gingival tissue. Finally, administering AI-PhyC ranging from 1 to 1000mg/kg did not present acute systemic toxicity (72h) in G. mellonella.

Conclusion

Overall, we demonstrate the potential of mouth gel containing AI-PhyC as a therapeutic strategy for managing osteolytic inflammatory disorders, such as periodontitis.

Inflammation

Periodontitis

Cytokines

phthalocyanine

Galleria mellonella

Tumor necrosis factor alpha (TNF-α) is a cytokine that exhibits pleiotropic effects on myriad cells (Jang et al., 2021). Concerning the inflammatory process, TNF-α plays numerous roles in leukocyte recruitment as induction of inflammatory cytokine production and expression of protein folding and cell adhesion (Yang et al., 2005; Vieira et al., 2009; Jang et al., 2021). Moreover, TNF-α is one of the major cytokines involved in developing inflammatory diseases, and its regulation in cells occurs mainly by nuclear factor kappa B (NF-κB). (Bradley, 2008; Loo and Bertrand, 2023). In periodontal disease, TNF-α is tightly related to gingival tissue inflammation and alveolar bone loss (Usui et al., 2021). In this context, TNF-α inhibition is a promising target to control periodontitis, and modulation of the NF-κB pathway is among the strategies used to attenuate TNF-α production by cells (Israël, 2010; Liu et al., 2017).

Phthalocyanines are synthetic molecules composed of macrocyclic compounds with four nitrogen atoms in their center, surrounded by pyrrole rings (Cao et al., 2013). The biological activities and medical application of phthalocyanines can occur along with photodynamic therapy (Roguin et al., 2019; Li et al., 2019) and present promising anti-inflammatory activity (Ayaz et al., 2019; Yüzer et al., 2019). Ayaz et al. (2019) studied the immunomodulatory activity of several subphthalocyanine derivatives. The authors verified that photoinduction in subphthalocyanines could promote an anti-inflammatory effect by decreasing the TNF-α and IL-6 release from activated macrophages. In another study, the authors demonstrated that phthalocyanine derivatives after 10-minute photoinduction were able to exhibit anti-inflammatory activity by reducing macrophage to release inflammatory cytokines and regulating intracellular p38 mitogen-activated protein kinases (p38) phosphorylation (Yüzer et al., 2019). In this context, phthalocyanine derivate presents as an anionic iron phthalocyanine (AI-PhyC), of synthetic origin, with self-activating capacity and, has shown promising antiviral and antimicrobial activity (Santos et al., 2022; Santos et al., 2023). However, its anti-inflammatory activity needs to be further investigated.

In the present study, we investigate the effect and mechanism of action of AI-PhyC on TNF-α production and the impact on experimental periodontitis. Overall, our results demonstrated that AI-PhyC inhibited the release of TNF-α in activated macrophage, which is associated with the blockage of NF-kB activation and Ikkβ expression. Besides, the mouth gel formulation containing 1% AI-PhyC prevented bone loss and inflammation in the periodontium of animals induced with experimental periodontitis. Lastly, dose titration of AI-PhyC presented a safe profile assessed by the Galleria mellonella toxicity model.

2.1 Obtaining AI-PhyC and making a mouth gel

TRIALS (Oral Health & Technologies) supplied AI-PhyC (PHTALOX®) dissolved in 1x Phosphate-Buffered Saline (PBS). The mouth gel containing water, glycerin, sorbitol, hydrated silica, PVP, cellulose gum, AI-PhyC, aroma, sodium benzoate, sucralose, phosphoric acid, sodium hydroxide, and AI-PhyC 1% was assembled for this study by TRIALS and the negative control formulation without the active ingredient (Fig. 1).

2.2 In vitro

2.2.1 Cell culture

The RAW 264.7 macrophages (ATCC TIB-71™) were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 2 mM glutamine at 37°C in a 5% CO₂/95% atmosphere.

2.2.2 Cell viability assay

The RAW 264.7 macrophages were cultured in 96-well plates (2x10⁵ cells/well) for 24 hours at 37°C with 5% CO₂. After the adhesion period, the cells were treated with AI-PhyC dissolved in PBS1x at 1, 10, 100, 1000 and 10000µM concentrations. After 24 hours, the supernatant was removed, RPMI with MTT (0.3 mg/mL) was added, and the plate was incubated for 3 hours. Subsequently, the supernatant was removed, and the cells were resuspended in 200 µL of dimethylsulfoxide (DMSO). The absorbance was measured at 540nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) (Mosmann et al., 1983).

2.2.3 Quantification of TNF-α

The macrophages were cultured in 96-well plates (2 x 10⁵ cells/well) for 24 hours at 37°C with 5% CO₂. After the adhesion period, the cells were treated with AI-PhyC dissolved in PBS 1x at 10, 30, and 100µM concentrations for 30 minutes and were stimulated with lipopolysaccharide (LPS) 10ng/mL for 4 hours. After this period, the supernatant was collected, and the TNF-α cytokine was quantified by the Enzyme-Linked Immunosorbent Assay (ELISA) method.

2.2.4 Activation of NF-κB

The RAW 264.7 macrophages transfected with the NF-κB-pLUC gene (CQB no. 022/97) were cultured in 24-well plates (3 x 10⁵ cells/well) for 24 hours at 37°C with 5% CO2. After the adhesion period, the cells were treated with AI-PhyC dissolved in PBS 1x at a concentration of 100µM for 30 minutes and were stimulated with LPS 10 ng/mL for 4 hours. Afterward, the cells were lysed with 50 µL of Tris-NaCl-Tween buffer, and an aliquot of the suspension was added along with 25 µL of luciferase reagent containing luciferin (Promega Corporation, Madison, WI, USA). A microplate reader (FlexStation 3 Molecular Devices multimode microplate) was used to quantify luminescence.

2.2.5 Ikkβ expression

The macrophages were cultured in 6-well plates (1 x 10⁶ cells/well) for 24 hours at 37°C with 5% CO₂. After the adhesion period, the cells were treated with AI-PhyC dissolved in PBS 1x at 100µM concentrations for 30 minutes and were stimulated with lipopolysaccharide (LPS) 10ng/mL for 30 minutes. After this period, the cell was collected, and the RNA was extracted using Illustra RNAspin Mini Kit (GE Healthcare, Milwaukee, WI, USA) and converted to cDNA using the high-throughput cDNA reverse transcription kit (Applied Biosystems), following the manufacturer's instructions. Subsequently, a cDNA strand was made from 1 µg of total RNA. This procedure was carried out in the Veriti® Thermal Cycler (Applied Biosystems) using the reverse transcriptase enzyme for a final 40 ng of cDNA, using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). The SYBR Green kit (Life Technologies) and the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) were used for quantitative real-time PCR. The following primer was used: Ikkβ, F − 5’-GAAGCGGCAGAAGGAACTCT-3’/ R − 5’-CACTGGAAGGCTGGGACATT-3’. All data were normalized to ACTB (F − 5'-AATTCTCAACCACAGTGCATTGTATGT-3'/ R − 5'-CTTTGGTGAATAGCTGAGTCATTTTCA-3') values. The data are presented as a fold-change of relative quantity using the 2-ΔΔCt method.

2.3 In vivo

2.3.1 Animals

Male C57BL/6 mice weighing 20-22g were used. The animals were obtained from the Central Animal Facility of the Federal University of Alfenas (UNIFAL) in Minas Gerais and randomly

divided into groups (6 mice/groups). During the experiments, the animals were housed in the Animal Facility of the Federal University of Alfenas in a temperature-controlled room (22–25 ºC), 12 h light/ 12 h dark cycle, humidity 40–60% with water and feed ad libitum and 6 mice per cage. The project was submitted and approved by the Ethics Committee on Animal Use of the Federal University of Alfenas CEUA/UNIFAL, protocol nº 0008/2022). The animal procedures followed the ARRIVE guidelines (Sert et al., 2020)..

2.3.2 Ligature-induced periodontitis model in mice (experimental periodontitis)

For experimental periodontitis induction, animals were previously anesthetized with an intraperitoneal injection (90 mg/Kg of Ketamine with 10 mg/Kg of Xylazine). With the assistance of modified forceps, the ligature was made (4 − 0 cotton) around the left lower first molar, kept in position by surgical knots (Abe et al., 2013). It is essential to mention that the knots were tied enough to hold in the position and floppy enough not to induce gingival ischemia. The mice were randomly divided into the following experimental groups and order of treatments (n = 6 per group) using https://www.graphpad.com/quickcalcs/randomize1/: Group 1 - mice without the ligature and treatment; Group 2 - mice received the ligature and topical administration in gingival tissue of the mouth gel vehicle (water, preservative, sweetener, and flavoring) not containing AI-PhyC (50µL/3x/day for 7 days); Group 3 - mice received the ligature and topical administration in gingival tissue of the mouth gel containing AI-PhyC 1% (50µL/3x/day for 7 days).

2.3.3 Bone loss quantification

By the end of the experimental timeline, animals were euthanized by anesthetic overdose, and the mandibles were removed. For bone loss quantification, the mandibles were kept overnight in 3% hydrogen peroxide and then mechanically dissected. The mandibles were stained with 0.5% methylene blue to distinguish the cementoenamel junction. The lingual surfaces were photographed with a digital camera (Canon E0S 1000D) at 2.5x magnification with a stereo microscope (ZEISS CL 1500 ECO). The images were analyzed using Image J software. For quantitative analysis, measurements from the distomesial area (mm2) between the cementoenamel junction and the alveolar bone crest on the lingual side of the first molar were used, according to Prates et al. (2014).

2.3.4 Real-time PCR

Total RNA was extracted from gingival tissue samples using Trizol reagent (Sigma-Aldrich), and the experiment was carried out as previously described. The following primers were used: Tnfα, F – 5’-CCCCCATTACTCTGACCCCT-3’/ R − 5’-CCCAGAGCCACAATTCCCTT-3’; p65, F − 5’-TGAACTTGTGGGGAAGGACTG-3’/ R − 5’-TGGTCTGGATTCGCTGGCTA-3’; Rankl, F − 5'-CCTGAGGCCCAGCCATTT-3'/R − 5'-AGCCTCGATCGTGGTACCAA-3'; Opg, F − 5'-TCCCGAGGACCACAATGAAC-3'/ R − 5'-AAGTACTTTGGAGGAAGGGTTTCC-3'. All data were normalized to ACTB values. Os dados são apresentados como uma variação da quantidade relativa usando o método 2-ΔΔCt.

2.3.5 In vivo toxicity in G. Mellonella

The larvae were raised in the Bioprocesses Laboratory at UNIFAL/MG, following the protocol created by Jorjão et al. (2018), with modifications. The diet was based on an artificial diet consisting of beeswax, honey, powdered milk, roasted soy flour, brewer's yeast and cornmeal. The larvae are kept in the BOD (Biochemical Oxygen Demand) incubator at a controlled temperature of 27°C until testing. The acute toxicity effect was evaluated as described by Megaw et al., (2015) and Rochelle et al., (2016), with modifications. Four increasing doses of AI-PhyC (1, 10, 100 and 1000 mg/kg) were tested to determine the minimum dose capable of killing the larvae over time (72h). For each group, 15 larvae were randomly selected, weighing between 0.25g-0.3g, without signs of melanization. Ten microliters of the sample or control (sterile distilled water) was injected into the hemocoel of each larva through the last proleg using a 10 µL Hamilton Syringe. Larvae were incubated at 27°C and analyzed at intervals of 12, 24, 48 and 72 hours.

2.4 Statistical analysis

Results were expressed as mean and standard deviation (mean ± SEM). Data were tested for normality by the Shapiro-Wilk test. Differences between groups were determined by one-way analysis of variance (ANOVA), followed by Tukey's test (GraphPad Prism, version 9.2) and were considered significant at p < 0.05. There was no exclusion of mice in the experimental groups. The percentage survival of G. mellonella larvae was from the Log-rank test (Mantel-Cox).

3.1 Quantification of TNF-α and evaluation of NF-Κb activation in cell culture

We initially evaluated the toxicity of AI-PhyC in RAW 264.7 macrophage cultures incubated for 24h (Fig. 2A). According to the results, we found that AI-PhyC at 1000µM and 10000µM concentrations reduced macrophage viability, compared to the vehicle group (V) (p < 0.05). On the other hand, AI-PhyC at concentrations of 1, 10, and 100 showed no cytotoxic effect on the cells (p > 0.05). The present study evaluated the anti-inflammatory activity and action mechanism of AI-PhyC in vitro. Initially, the anti-inflammatory effect of AI-PhyC on TNF-α cytokine release in macrophages stimulated with 10ng/mL LPS was evaluated. Figure 2 displays that the group of macrophages that received LPS stimulation (-) significantly increased TNF-α cytokine release (Fig. 2B), compared respectively to the vehicle group (V) without LPS stimulation (p < 0.05). On the other hand, pretreatment with AI-PhyC in RAW 264.7 macrophage culture at concentrations of 30µM and 100µM was able to reduce the release of TNF-α when compared to the control groups activated only with LPS (-) (p < 0.05). Subsequently, we checked the activity of AI-PhyC on transcription factor NF-κB activation (Fig. 2C) and expression of Ikkβ (Fig. 2D). According to the results, pretreatment with AI-PhyC at 100µM was able to reduce NF-kB activation and mRNA expression of Ikkβ in macrophage culture compared to the group that received only stimulation with LPS (-) (p < 0.05).

3.2 Induced periodontal disease in mice and toxicity in G. mellonella

Thus, based on the in vitro anti-inflammatory results, an in vivo study was conducted to prove the activity of AI-PhyC in modulating the inflammatory process and bone loss. This objective was achieved by formulating a mouth gel containing AI-PhyC 1%, which was applied to the gingival tissue of mice with ligature-induced periodontal disease. According to our results (Fig. 3A and 3B), the topical administration of the AI-PhyC 1% mouth gel 3x/day reduced alveolar bone loss compared to the group of mice that received the ligation and treatment with the mouth gel vehicle (V) (p < 0.05).

C57BL/6 mice. Different letters indicate statistically significant differences in intragroup.

To confirm the activity of AI-PhyC in modulating the inflammatory process and bone loss in mice with experimental periodontitis, the gene expression of Tnfα, IKKβ, p65 (NF-κB), receptor activator of nuclear factor-kappa B ligand (Rankl), and osteoprotegerin (Opg) was investigated by real-time PCR in the gingival tissue. According to our results, the topical administration of AI-PhyC 1% reduced the expression of Tnfα (Fig. 4A), IKKβ (Fig. 4B), p65 (Fig. 4C), and Rankl (Fig. 4D) genes, compared to the group of mice that received the ligature and treatment with the mouth gel vehicle (p < 0.05). On the other hand, there was no reduction or increase in Opg gene expression (Fig. 4E) compared to the mouth gel vehicle group (p > 0.05). Finally, concerning gene ratio of Rankl/Opg (Fig. 4F), there was a reduction in the group treated with AI-PhyC 1% mouth gel (p < 0.05).

Finally, the systemic toxicity of AI-PhyC was evaluated in the G. mellonella model. AI-PhyC was administered at doses of 1, 10, 100, and 1000 mg/kg into the hemocoel, and as seen in Fig. 5, AI-PhyC did not present systemic toxicity in G. mellonella compared to the control groups that received distilled water or PBS 1X (p > 0.05).

The present study found that treatment with AI-PhyC (100 µM) mitigates the mRNA expression of Ikkβ, NF-kB activation and, consequently, TNF-α release in LPS-stimulated macrophage culture. Finally, in experimental periodontitis, AI-PhyC avoids the bone resorption induced by experimental periodontitis and diminishes the inflammatory osteolytic reaction.

Activation of TNF-α, via Toll-like receptor (TLR) and LPS binding, involves a Myd88/IRAK/TRAF6/TAK-1/IKK signaling cascade reaction, followed by degradation of IκBα and activation of NF-κB (Muroi and Tanamoto, 2012). Scientific studies have shown that the chronic or augmented activation of NF-κB and TNF-α is directly related to the development of inflammatory diseases such as rheumatoid arthritis, psoriasis, multiple sclerosis, and periodontal disease (Liu et a., 2017). Our results demonstrated that the modulation of NF-κB and reduction of TNF-α by AI-PhyC occurs in part by inhibiting IKKβ, and the ones obtained with AI-PhyC reinforce findings in the literature that investigate IKKβ and NF-κB as promising targets to develop new drugs (Freitas and Fraga, 2018). A study with molecules of natural origin (Pentacyclic Triterpenoids) demonstrated that inhibition of IKKα/β phosphorylation could attenuate NF-κB expression in cell culture (Patil et al., 2015). In another study, a virtual screening technique applied to numerous compounds identified a new one (VH01) with significant inhibition for IKKβ (Nagarajan et al., 2010). Franchin et al. (2016) found that a new coumarin could reduce TNF-α release in stimulated macrophage culture by inhibiting the NF-κB pathway. Therefore, our results put AI-PhyC in evidence as a new drug to block TNF-α (NF-κB pathway) and the inflammatory process.

The in vitro suppressive effects of AI-PhyC on TNF-α release led us to investigate its anti-inflammatory activity on an in vivo model of periodontal disease. Experimental models for rodent periodontitis have been widely used and have great relevance in reproducing the biological hypotheses and characteristics of human periodontitis (Anbinder et al., 2016). In addition, animal models used in periodontal disease allow the study of cellular and molecular mechanisms, elucidating the effect of inflammatory cytokines on disease progression and host-microbial interactions in the inflammatory process (Cavagni et al., 2016; Mascarenhas et al., 2005).

Our results demonstrated that topical treatment with a mouth gel containing AI-PhyC 1% reduced alveolar bone loss and inflammation in mice with ligature-induced periodontitis over 7 days. Studies on periodontal diseases have shown that pro-inflammatory cytokines (such as TNF-α) are likely to play several roles, such as osteoclastic activity and alveolar bone loss (Ramadan et al., 2020). The lipopolysaccharide, associated with other virulent factors of periodontal pathogens, stimulates macrophages, dendritic cells, and other cells, culminating in the production of cytokines, such as TNF-α (Ramadan et al., 2020). Those pro-inflammatory cytokines can stimulate the production of matrix metalloproteinases (MMPs) by macrophages, keratinocytes, and fibroblasts (Ramadan et al., 2020). These cellular events drive osteoclastogenesis activation and bone destruction through direct osteoclast stimulation and releasing tissue-destroying enzymes by inflammatory cells (Ramadan et al., 2020; Usui et al., 2021). Luo et al. (2018) found that TNF-α and RANKL jointly play a crucial role in inducing osteoclast differentiation, and the NF-κB pathway may play a role in this process. Furthermore, TNF-α released in periodontal disease is associated with the increased neutrophil influx into the periodontal pocket, promoting tissue damage (Scott and Krauss, 2012).

Our in vivo results showed that the inhibitory effect of AI-PhyC on the inflammatory process and bone loss in mice periodontal disease occurs by blocking TNF-α and NF-kB pathways. These data corroborate with other studies that evaluated the anti-inflammatory properties of phthalocyanine in experimental periodontal disease (Moraes et al., 2017; Al-Kheraif et al., 2022). For instance, Moraes and collaborators demonstrated that rats with ligature-induced periodontitis and treated with low-intensity laser associated with chloro-aluminum phthalocyanine had decreased inflammatory response (reduction in TNF-α expression) and tissue destruction in gingival tissue (Moraes et al., 2017). In another study, it was found that photodynamic therapy mediated by chloro-aluminum phthalocyanine reduced IL-1β and TNF-α in crevicular fluid and provided an improvement in the results of non-surgical periodontal therapy among patients with stage II chronic periodontitis (smokers and non-smokers). (Al-Kheraif et al., 2022).

In conclusion, this study demonstrated that AI-PhyC inhibited the expression Ikkβ gene, activation of the transcription factor NF-kB, and TNF-α release in RAW 264.7 macrophage culture. In addition, the mouth gel formulation containing 1% AI-PhyC prevented alveolar bone loss in mice with ligature-induced periodontitis, as well as reduced expression of Tnfα, Ikkβ, p65 (NF-κB) and Rankl genes in gingival tissue. Finally, AI-PhyC did not present acute systemic toxicity in Galleria mellonella larvae. Therefore, our results suggest that AI-PhyC is a possible new therapeutic strategy for the adjuvant treatment of pathologies associated with inflammation and bone loss, such as periodontitis.

Funding statement

This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) under grant agreements # 403641/2020-9 (MAI/DAI), 130365/2021-1 and 306673/2019-3.

Author Contributions

Conceived and designed the experiments: IB, RFDB, PLR, FVV, HBA, SMA, TMC, MI, LAF, MF. Performed the experiments in vitro: IB, RFDB, LPA, HBA, JGL, BBS, IMP, LAF, and MF. Performed the experiments in vivo: IB, MF and HBA. Analyzed the data: IB, LPA, HBA, JGL, and MF.

Corresponding authors

Correspondence to Marcelo Franchin

Competing interests

The authors declare no competing interests.

Conflict of interest

All authors report no potential conflicts of interest.

Ethical approval

The procedures were reviewed and approved by the institutional ethics committee on animal care (CEUA/UNIFAL, protocol nº 0008/2022).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

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Mouth gel containing phthalocyanine derivative attenuates TNF-α through NF-κB inhibition in experimental periodontitis

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Abstract

Background

Objective

Methods

Results

Conclusion

Figures

1 INTRODUCTION

2 MATERIAL AND METHODS

2.1 Obtaining AI-PhyC and making a mouth gel

2.2 In vitro

2.2.1 Cell culture

2.2.2 Cell viability assay

2.2.3 Quantification of TNF-α

2.2.4 Activation of NF-κB

2.2.5 Ikkβ expression

2.3 In vivo

2.3.1 Animals

2.3.2 Ligature-induced periodontitis model in mice (experimental periodontitis)

2.3.3 Bone loss quantification

2.3.4 Real-time PCR

2.3.5 In vivo toxicity in G. Mellonella

2.4 Statistical analysis

3 RESULTS

3.1 Quantification of TNF-α and evaluation of NF-Κb activation in cell culture

3.2 Induced periodontal disease in mice and toxicity in G. mellonella

4 DISCUSSION

5 CONCLUSION

Declarations

References

Additional Declarations

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