INTRODUCTION:

Periodontitis is an infectious disease characterized by inflammation of the periodontal tissue, attachment loss, and alveolar bone destruction and is an oral disease that ranks first in the 2001 world record book as the most common disease experienced by humans.^1,2 According to WHO, inflammatory diseases are ranked as the biggest threat to the health of the world's population.^3,4 The presence of an inflammatory process in the periodontal tissue as a response to the induction of plaque bacteria and its products causes damage to all components of the periodontal tissue, resulting in tooth loss.⁵

In principle, periodontitis treatment is aimed at achieving periodontal tissue regeneration, namely the formation of cementum and new alveolar bone, functional attachment of periodontal ligament fibers, and normal gingiva.^6,7 In addition, the success of periodontitis therapy is not only demonstrated by improving clinical parameters, but also by preventing the possibility of recurrence of inflammation. Therefore, a treatment is needed that is not only able to regenerate tissue but also prevents inflammation from occurring.^5,8

Stem cell-based tissue engineering therapy is rapidly emerging as a potential strategy for periodontal tissue regeneration due to its self-renewal ability and multilineage differentiation capacity.,^7,9-11 Several sources of confirmed stem cells, namely mesenchymal stem cells from dental pulp tissue or what are known as Dental Pulp Stem Cells (DPSC), have various advantages. Based on their available sources, DPSC can be easily collected from extracted permanent teeth so that they are obtained in a minimally invasive and relatively safe way. Several studies have confirmed that DPSCs have higher angiogenic, neurogenic, and regenerative potential compared to bone marrow and adipose stem cells, thus making DPSCs a flexible alternative stem cell source for cellular therapy.^12,13

In vivo studies reported that DPSC has the potential forperiodontal regenerative therapy by providing optimal results of bone repair, cementum formation, and periodontal ligament around the bone defects. DPSC wasalso effective in increasing bone regeneration in the treatment ofintrabony defects and socket preservation.^14,15 However, the presence of bacterial toxins, such as Lipopolysaccharide (LPS) of P. gingivalis in the periodontal tissue can affect the success of DPSC in regenerating periodontal tissue. Several studies reported that LPS of P. gingivaliscan stimulate the secretion of proinflammatory cytokines such as Interleukin 1 Beta (IL-1β) and Tumor Necroting Factors Alfa (TNF-α) in excess, thereby inhibiting the proliferation and differentiation of osteogenic stem cells. This has an impact on delaying the success of periodontal treatment.^16,17

Besides the advantages of stem cells as a periodontal regenerative therapy, this therapy must also be able to control the possibility of excessive inflammation that occurs and prevent disease severity and progression. It is because stem cell therapy will be used in patients with periodontitis, which is a chronic inflammatory disease. Therefore, the addition of a therapeutic substance to help DPSC against the excessive inflammation potential in periodontal treatment needs to be explored further so that it can increase the success of periodontal regeneration therapy.^18,19

Chlorogenic acid is well known as anti-inflammatory, antioxidant, antimicrobial, antifungal, and antiviral.^20,21 In vitro and in vivo experiments revealed that chlorogenic acid is an effective anti-inflammatory, antipyretic, and analgesic agent.^22-25 Chlorogenic acid is found in seeds and nuts.However, the highest content of chlorogenic acid can be found in robustagreen beans, which is 6.1-11.3%. This amount is the largest content compared to other sources.²⁶

Robusta green beans also contain several anti-inflammatory compounds, including caffeine, caffeic acid, and trigonelline. These chemical components are a good source of anti-inflammatory for the body.²⁷ However, the highest content of anti-inflammatory compounds in robusta green beans is chlorogenic acid.^26,28,29 In addition to the high anti-inflammatory compounds in robustagreen beans, robusta green beansare the main and largest commodity in Indonesia. Its availability is very abundant in Indonesia, making this type of source easy to obtain.³⁰

The high anti-inflammatory content and the abundant availability of ingredients are the backgrounds of researchers in utilizing robustagreen bean extract to increase the anti-inflammatory power of DPSC. Its use to increase the anti-inflammatory power of stem cells is still not well understood. Therefore, this study aims to provide an overview of the anti-inflammatory effect of robustagreen bean extract on DPSC induced by LPS P. gingivalis. LPS P. gingivalisinduction aims to create an inflammatory microenvironment. The anti-inflammatory effect was seen by measuring the levels of IL-1β cytokines at 24, 48, and 72 hours of observation.

MATERIALS AND METHODS:

Material:

Robusta green bean extract was obtained from the Coffee and Cocoa Research Center, Jember-Indonesia which was processed untilthe content of chlorogenic acid was 30%. This extract was then made at a concentration of 0.5%; 0.25%; 0.125%; 0.0625%. LPS from P.gingivalis (#05H23-SV) (InvivoGen, San Diego, CA) at a concentration of 0.5µg/ml; 1µg/ml; 5µg/ml; 10 µg/ml. DPSC was obtained from the pulp of the 1st or 2nd premolars that were extracted due to indications for orthodontic treatment. The teeth that can be used are healthy, no caries, and perfectly closed tooth roots, donors aged between 19-29 years, healthy, and no infectious diseases.

DPSC Culture and Isolation:

The pulp tissue was chopped and given 3ml of collagenase type 1, then incubated in a 5% CO₂ incubator, at 37^oC for 30 minutes. After incubation, 3ml of 25% αMEM solution was added and incubated again. The pulp and media were transferred to a 15ml conical tube, then centrifuged at 3500rpm for 5 minutes at 28^oC. After being centrifuged, the supernatant was discarded and the pellet was planted in a petri dish containing 3 ml of 25% αMEM solution media and incubate. After the cell culture was 80% confluent, the culture was harvested and reseeded up to 2-3 times (passage). This experiment used cell culture passage 4.³¹

Identify the characteristics of DPSC:

DPSC characteristics are identified by observing the morphology and surface proteins of the cells. The identification of cells morphologywas carried out using an inverted light microscope. While the identification of cell surface proteins using flow cytometry (FCM) test with antibody markers. The antibody markers used as positive controls were CD90 and CD105 as markers of mesenchymal stem cell lineage. Meanwhile, the markers used as negative controls were CD34 and CD45, which are markers of hematopoietic stem cell lineage.

DPSC viability analysis:

Cells were cultured on 96 well plates and incubated for 24hours. After 24hours, all well plates were given various concentrations of LPS P. gingivalisand Robusta green bean extract. Robusta green bean extract each concentration of 0.5%; 0.25%; 0.125%; 0.0625% givenas much as 200µl/well plate. While the LPS given were 0.5µl, 1µl, 5µl, 10µl/well plate. Each well plate was incubated for 24, 48, and 72hours. After incubation, 25µl of MTT (3-4-5-dimethylthiazol-2YL-2,5-dibromurodiphenyltetrazolium) was added to each well. Then incubated for 4 hours at 37°C. The color change was observed with Elisa Reader at a wavelength of 595nm.³²

Measurement of IL-1β levels:

DPSC were cultured on 96 well plates. Robusta green bean extract concentration 0.125% and 0.0625% was given to the treatment group 30 minutes before LPS 10 µg/ml was given. Each well plate was incubated for 24, 48, and 72hours. After incubation, the supernatant was extracted and placed in a labeled 96 well plate. The supernatant volume given is at least 50µl/well. Then the levels of IL-1β were analyzed using the ELISA test.

Data analysis and statistics:

The data were analyzed statistically with a significance level (α= 0.05) using SPSS 20. The normality of the data was tested with Shapiro – Wilk. The homogeneity of the data was tested by the Levene test. Differences between groups were tested with OnewayAnova to see if there were significant differences between the treatment groups. If there is a significant difference (significance value <0.05), then proceed with the post hoc Tukey HSD test to find out which group has a significant difference.

RESULTS:

Identify the characteristics of DPSC:

On observation, the morphology of DPSC was a fusiform shape with a tapered tip, a cell nucleus in the middle, and a large cytoplasm, which can be seen in Figure 1.

Figure 1: Morphology of DPSC (passage 4).

A fibroblast-like morphology appears, which is a fusiform shape with a tapered tip (red arrow), a cell nucleus in the middle, and a large cytoplasm (blue arrow). Observations under a microscope with a magnification of 100x.

Observation of DPSC surface expression markers was carried out using the FCM test. This observation was performedto analyze the surface characteristics of stem cells used in this study. The FCM test shows the results according to Figure 2, based on the histograms, DPSCs express the cell surface protein markers CD90 92.16% and CD105 89.72%. While DPSC express the CD34 0.68% and CD45 1.71%.

Figure 2: DPSC FCM test histogram graph.

(A, B) DPSCs express a very large number of cell surface protein markers CD90 and CD105 which are shown in the green and red diagrams on the right side of the histogram. (C, D) While DPSC expresses very low cell surface protein markers CD34 and CD45 as indicated by the slight presence of green and red diagrams on the right side of the histogram.

DPSC viability analysis:

The results of the viability test showed that the administration of LPS P. gingivalis in various concentrations did not cause toxicity to the DPSC. This was indicated by the percentage of DPSC living cells >80% after administration of LPS P. gingivalis at various concentrations, as shown in Figure 3.

Therefore, all concentrations can be used for further research. However, the next study used LPS P. gingivalis with a concentration of 10g/ml to induce inflammation in DPSC because this concentration was more able to suppress cell proliferation. These results are in line with several in vitro studies, in which the higher the LPS concentration of P. gingivalis, the more it will suppress cell proliferation and further increase the production of proinflammatory cytokines.^33–35

Figure 3: Graph of DPSC Viability Test after LPS administration of P. gingivalis.

All concentrations of P. gingivalis LPS did not cause toxicity to DPSC either after administration for 24 hours, 48 hours,or 72 hours.

The results of the DPSC viability test after administration of Robusta green bean extract at various concentrations showed results as shown in Figure 4. Robusta green bean extract concentrations of 0.5% and 0.25% caused toxicity to DPSC at all observation times. These were indicated by the percentage of DPSC living cells <80% after the concentration was given. Therefore, only robustagreen bean extract concentrations of 0.125% and 0.0625% can be used for further research.

Figure 4: Graph of DPSC Viability Test after Robusta green bean extract was administered.

Concentrations 0.125% and 0.0625% are not toxic to DPSC.

Robusta green bean extract decrease IL-1B levels in DPSC:

Statistical analysis of the effect of Robusta green bean extract on the average level of IL-1β aims to see which treatment group most significantly decrease IL-1β levels at one time, at 24 hours, 48 hours, and 72 hours, respectively. The mean IL-1β in all treatment groups at each observation time showed normal data distribution and homogeneous data variance (p>0.05) (Table 1).

Inferential statistical test using One-way ANOVA showed a significant difference between all treatment groups at each observation time (p<0.05) (Table 1). The Post Hoc Tukey HSD test was then carried out to analyze the treatment group that had the highest significance in suppressing IL-1β levels (Figure 5).

Based on the results of post hoc Tukey HSD analysis, the treatment had a significant effect on IL-β levels at each observation time. Observation of the effectiveness of the addition of coffee extract in suppressing IL-1β levels is by looking at the comparison between the LPS treatment group given Robusta green bean extract and the LPS treatment group without Robusta green bean extract. In observations based on treatment, the addition of Robusta green bean extract which most significantly suppressed IL-1β was a concentration of 0.125% both at 24 hours, 48 hours, and 72 hours.

DISCUSSION:

Observations of morphological characteristics confirmed that DPSCs are fusiform shape with a tapered tip, a cell nucleus in the middle, and a large cytoplasm, which is often referred to as a fibroblast-like spindle formation. These results are following the previous theory, which confirmed that DPSCs as mesenchymal stem cells have a morphology similar to fibroblast cells.^36,37 In this study, DPSCs can also grow and attach to plastic substrates and can colonize. These results are in line with the in vitro research of Martens et al³⁸ who investigated the characteristics and surface expression of DPSC. His research explains that in the first 24 hours of culture, cells can grow and migrate out of the cultured pulp tissue and stick to the bottom of the plastic media container which then forms colonies.

Observation of other characteristics by analyzing DPSC surface expression markers was performed using the FCM test. The results of the FCM test showed a large numberof expressions of CD90 and CD105, while CD34 and CD45 were slightly expressed. DPSCs express the cell surface protein markers CD90 92.16% and CD105 89.72%. While DPSC express the CD34 0.68% and CD45 1.71%. Specific antibodies CD90 and CD105 are markers of mesenchymal stem cell lineage. While the specific antibodies CD34 and CD45 are markers of hematopoietic stem cell lineage. The results of this study are following pre-existing theories.³⁹ Martens et al³⁸in theirin vitro research also showed the same results. Other studies also explained that a cell is classified as a mesenchymal stem cell if the cellscan stick to plastic medium containers when cultured; expresses CD105, CD90, CD73, and CD44, and lacks expression of CD45, CD34, CD14 or CD11b, CD79 or CD19, and HLA-DR; and also can differentiate into osteoblasts, adipocytes, and chondroblasts in vitro.⁴⁰

The highest level of IL-1β at 24-hour observation was found in the DPSC group induced by LPS P. gingivalis, which was an average of 953.40 ng/ml. This value was significantly different compared to the control group at 24 hours of observation. This indicated that LPS P. gingivalis 10 µg/ml was able to significantly increase the expression of proinflammatory cytokines in DPSC. There was a drastic increase in IL-1β levels in the LPS-induced P. gingivalis DPSC group in line with previous studies. Albiero et al³³ in their research explained that LPS P. gingivalis 10 µg/ml was able to induce an increase in IL-1β mRNA expression in periodontal ligament stem cells (PDLSC). The same result was also explained by Kato et al¹⁶and Tang et al¹⁷.

The results of observations after administration of LPS P. gingivalis for 48 hours and 72 hours in this study also showed the same results as 24 hours, that the group induced by LPS P. gingivalis had significantly higher levels of IL-1β compared to other groups in each of the groups. that time. This indicates that DPSC can capture the pathogen signal well. DPSC can recognize the induction of a pathogen through cell surface receptors. LPS from P. gingivalis bacteria will cause cell membrane surface receptors to be activated. These surface receptors are known as Toll-like receptors (TLRs). This TLR is the initial key in the process of immunomodulating a cell. Some literature mentions that TLRs that can recognize the presence of LPS induced P. gingivalis are TLR 4 and TLR 2.^41-43 In vitro studies have proven that TLR 2 and TLR 4 are transmembrane receptors that can be activated when exposed to LPS P. gingivalis.^44,45

Activated TLR2 and TLR4 then signal and cause Myeloid Differentiation Factor 88 (MyD88) and IL-1R-associated kination 4 (IRAK-4) to be activated. MyD88 and IRAK-4 are media adapter molecules for signal transduction of the TLR/IL-1R superfamily. The activation of IRAK-4 and MyD88 will then activate TRAF-6 which will then forward the signal through the TAK-1 pathway. TAK-1 will then activate the Inhibitor of Nuclear Factor kinase (IKK) complex, namely IKKα and IKKβ. IKK will phosphorylate Inhibitor of Nuclear Factor (IKβ) so that IKβ which binds to Nuclear Factor (NFκβ) will be degraded. NFκβ which is in its inactive form binds to IKβ which is an inhibitory protein of NF. If there is a stimulus in IKβ, it will result in phosphorylation, ubiquitanation, and degradation of IKβ. This process causes inactive NFκβ in the cytoplasm to translocate into the cell nucleus and become active. Activated NFκβ will activate cell DNA which in turn produceproinflammatory cytokines such as IL-1β, TNF, and IL-6.⁴² The pro-IL-1β cytokine produced is inactive. The caspase-1 process will then break the pro-IL-1β bond so that it becomes active and is secreted through the gaps between epithelial cells and lysosomal secretion.⁴¹

The administration of Robusta green bean extract to DPSC induced by high dose LPS P. gingivalis (10 µg/ml) showed a significant decrease in IL-1β expression. The levels of IL-1β in the DPSC group which was induced by LPS P. gingivalis and Robusta green bean extract 0.125% and 0.0625% after 24 hours were significantly lower than the DPSC group which was only induced by LPS P. gingivalis. The addition of Robusta green bean extract with a concentration of 0.125% was more effective in suppressing IL-1β levels when compared to a concentration of 0.0625%. The effectiveness of reducing IL-1β levels was more optimum after 48 hours and 72 hours of administration of Robusta green bean extract on DPSC induced by LPS P. gingivalis. This indicated that the administration of Robusta green bean extract was able to suppress IL-1β levels in inflamed DPSCs. These results are in line with several studies examining the role of the active compounds in the Robusta green bean, especially phenol components as antioxidants and anti-inflammatory.⁴³

Robusta green beans contain a wide variety of bioactive compounds that have anti-inflammatory properties. This anti-inflammatory activity was obtained due to the presence of phenolic compounds in the Robusta green bean. The main component of phenolic compounds contained in Robusta green bean is chlorogenic acid. Apart from being anti-inflammatory, chlorogenic acid also acts as an anti-oxidant and anti-cancer.²⁰

Several studies have confirmed the anti-inflammatory activity of chlorogenic acid. In vitro, administration of 20 M chlorogenic acid for 24 hours to murine RAW 264.7 macrophage cells that had been induced by LPS E. coli 1 µg/ml was able to reduce the expression of IL-1β and TNF-α.²³ Gao et al²² in their research also proved that the administration of chlorogenic acid with a concentration of 12.5; 25 and 50 µg/ml in LPS-induced mammary epithelial cells can significantly reduce the secretion of IL-1β, TNF-α, IL-6. Another coffee phenolic content, namely caffeic acid, also shows anti-inflammatory activity by inhibiting the production of IL-1β.⁴⁶

The results of previous studies are in line with the results of our research. Robusta green bean extract can reduce the expression of IL-1β in inflamed DPSC, possibly due to the presence of polyphenolic compounds such as chlorogenic acid, caffeic acid, and caffeine which are known to have anti-inflammatory properties. The ability of Robusta green bean extract to reduce IL-1β levels proves that Robusta green bean extract can be used as a therapeutic agent to prevent cells from being damaged due to excessive inflammation. Although this study did not confirm the mechanism of how Robusta green bean extract can reduce IL-1β levels in DPSC, other studies have confirmed the mechanism of the effect of chlorogenic acid in reducing proinflammatory cytokines.

The mechanism of decreased secretion of pro-inflammatory cytokines was confirmed by decreased phosphorylation of Nf-κB p65. Prevention of Nf-κB phosphorylation causes cells to stop the production of proinflammatory cytokines.²² Further research by Liu et al²⁴ confirmed that chlorogenic acid was not only able to significantly inhibit the phosphorylation of Nf-κB but also was able to increase the expression of IkB-α which is an inhibitory protein of Nf-κB.

CONCLUSION:

Robusta green bean extract concentrations of 0.125% and 0.0625% can significantly reduce IL-1β levels in DPSC induced by LPS P. gingivalis. The most effective concentration at reducing IL-1β levels was 0.125% at 24, 48, and 72 hours of observation.

ACKNOWLEDGEMENT:

The authors would like to thank Prof. Dr. Ernie Maduratna S., drg., M.Kes., Sp.Perio(K) as a chairman of the Periodontology Department in the Faculty of Dental Medicine, Universitas Airlangga for providing us with a good environment and facilities to complete this project.

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Received on 07.12.2021 Modified on 19.08.2022

Research J. Pharm. and Tech 2023; 16(8):3638-3644.

DOI: 10.52711/0974-360X.2023.00599

Observation Time	Groups	N	Shapiro-wilk	Levene Test	Oneway ANOVA
24 Hours	DPSC (control)	5	0.980*	0.823*	0.000**
	DPSC+LPS	5	0.677*
	LPS+Robusta 0.125%	5	0.419*
	LPS+Robusta 0.0625%	5	0.794*
48 Hours	DPSC (control)	5	0.794*	0.803*	0.000**
	DPSC+LPS	5	0.980*
	LPS+Robusta 0.125%	5	0.884*
	LPS+Robusta 0.0625%	5	0.419*
72 Hours	DPSC (control)	5	0.940*	0.566*	0.000**
	DPSC+LPS	5	0.955*
	LPS+Robusta 0,125%	5	0.737*
	LPS+Robusta 0,0625%	5	0.390*