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 Table of Contents  
ORIGINAL RESEARCH
Year : 2022  |  Volume : 14  |  Issue : 4  |  Page : 403-408

The effect of alpha-mangostin on interleukin-10 and collagen 1A1 gene in the inflammation process: An experimental in-vitro study


1 Residency Program of Oral and Maxillofacial Surgery, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
2 Academic Staff of Oral and Maxillofacial Surgery, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
3 Academic Staff of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
4 Staff of Oral and Maxillofacial Surgery Department, Pioneer Dental College and Hospital, Bangladesh

Date of Submission22-Feb-2022
Date of Decision30-May-2022
Date of Acceptance10-Jun-2022
Date of Web Publication29-Aug-2022

Correspondence Address:
Dr. Andra Rizqiawan
Academic Staff of Oral and Maxillofacial Surgery, Faculty of Dental Medicine, Universitas Airlangga, Jl. Prof. Moestopo, 47, Surabaya 60132
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JIOH.JIOH_48_22

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  Abstract 

Aims: Bone resorption after tooth extraction can cause serious problems on subsequent denture implants and restorative dentistry procedures that rely on the bone healing process. The peel of the mangostin fruit is one of the natural components that can help with wound healing. Its constituents, particularly alpha-mangostin, have antifungal, antioxidant, antiviral, antibacterial, and anti-inflammatory properties. The research was carried out because there was an effort to speed up the healing process following tooth extraction and because of the high potential of alpha-mangostin. The aim of this study is to see whether taking alpha-mangostin decreases interleukin-10 (IL-10) and increases collagen 1A1 expression in the inflammation process. Materials and Methods: This study was a post-test-only control group design. An in-vitro experiment was conducted on the following four groups. Then lipopolysaccharide (LPS) and alpha-mangostin were inducted into cell culture. In osteoblast cell culture 7F2, real-time polymerase chain reaction was performed to see markers collagen 1A1 and IL-10. The research was statistically analyzed, using a variance test and one-way analysis of variance. Results: The highest IL-10 gene expression was found in group induction with LPS, but there was no significant difference in IL-10 expression between the groups. The highest collagen 1A1 gene expression was found in a group that received induction with LPS and alpha-mangostin, but there was not a significant difference in collagen 1A1 expression between the groups. Conclusion: Alpha-mangostin induction effectively reduces inflammation and IL-10 expression, while increasing collagen 1A1 expression.

Keywords: Alpha-mangostin, Collagen 1A1, Interleukin-10, Lipopolysaccharide, Medicine, Osteoblast


How to cite this article:
Sayekti M, Rizqiawan A, Soesilawati P, Mira NP, Kamadjaja DB, Rahman MZ. The effect of alpha-mangostin on interleukin-10 and collagen 1A1 gene in the inflammation process: An experimental in-vitro study. J Int Oral Health 2022;14:403-8

How to cite this URL:
Sayekti M, Rizqiawan A, Soesilawati P, Mira NP, Kamadjaja DB, Rahman MZ. The effect of alpha-mangostin on interleukin-10 and collagen 1A1 gene in the inflammation process: An experimental in-vitro study. J Int Oral Health [serial online] 2022 [cited 2022 Oct 5];14:403-8. Available from: https://www.jioh.org/text.asp?2022/14/4/403/355024


  Introduction Top


Tooth extraction is a technique of removing teeth from their sockets which involves bone tissues and soft tissues of the oral cavity. The process that will occur next is the wound healing process. Dental implantation has become an important therapeutic modality in the last decade, especially after the works developed by Brånemark in 1985, in which direct contact between functional bone tissue and titanium biomaterials was termed osseointegration.[1] In the first year after implant placement, bone modeling and remodeling occur slowly, contributing to the implant’s increased resistance to shear stresses. Local mechanical stress influences osteoblast proliferation and differentiation, as well as the bone-healing process, in this continual process of bone remodeling.[2]

Many studies have been conducted on the effects of wound healing using natural ingredients. Materials from nature that can be used for wound healing are the skin of the mangostin fruit (Garcinia mangostana).[3] From the content of xanthones, α-, β-, γ-mangostin, α-mangostin has the best anti-inflammatory potential. Alpha-mangostin had the most significant effect compared with β and γ. In addition, α-mangostin has the most abundant xanthone content found in the mangostin peel extract, which is around 80%.[4] Research conducted by Arundina et al.[5] showed that the addition of mangostin rind to mesenchymal stem cells could increase the proliferation of mesenchymal stem cells to differentiate into osteoblasts.

In this study, the process or inflammatory response in vitro was carried out using lipopolysaccharide (LPS), which can induce the inflammatory response in the cultured osteoblastic cell line 7F2. Researchers used the inflammatory mediator interleukin (IL)-10 because it belongs to a group of cytokines that have been reported to be expressed early in the inflammatory phase.[6] Proinflammatory cytokines induce macrophages to produce IL-10 during the inflammatory process.[1] Lobo et al. in their research stated that at the beginning of the inflammatory phase, an increase in IL-10 production was found. The bone-healing process after the administration of alpha-mangostin can trigger many factors, one of which is an increase in the number of osteoblasts and helps in the process of osteogenesis. The process of osteogenesis involves various osteogenesis markers that regulate the expression of bone marker genes, namely, Osterix, alkaline phosphatase, RUNX2, collagen 1A1, and bone sialoprotein.[2] Expression of differentiation markers such as collagen 1A1, alkaline phosphatase, bone sialoprotein, OPN, and OCN can be used to evaluate the process of osteoblastic differentiation in experimental models.[7] Research has shown that xanthones in mangostin peel extract inhibit intracellular reactive oxygen species (ROS) activity. Inhibition of intracellular ROS activity will inhibit enzyme activity and regulate proinflammatory mediators.[8] These inflammatory cytokines can lead to a loss of collagen attachment of the tooth to the bone, promoting osteoclast formation and bone resorption in periodontal tissue.[9] Thus, it is of utmost importance to find new materials that can inhibit bone resorption and inflammatory response by controlling chemical mediators of inflammation. It is hoped that this research will prove the occurrence of the process of inhibiting bone resorption in the post-tooth extraction inflammatory process.


  Materials and Methods Top


Sampling criteria

This study is a post-test-only control group design using cell line osteoblast 7F2. 7F2 cell culture is a precursor cell of osteoblasts derived from the bone marrow of Mus musculus (mouse C57BL/6), and the number ATCC CRL-12557 was purchased at American Type Culture Collection (Manassas, VA, USA): the material alpha-mangostin M3824 was purchased from Sigma Aldrich, Inc. (St. Louis, MO, USA), and LPS L2630 from Sigma Aldrich, Inc. Medium osteogenic (MO) is a cell culture medium that contains osteogenic ingredients by adding DMEM (11885084, Sigma–Aldrich, Inc.), 10% fetal bovine serum (FBS) (10270106, Life Technologies Limited, Paisley, UK), and 1% penicillin-streptomycin (15140148, Sigma–Aldrich, Inc.), L-ascorbic acid 2-phosphate (AA2P) (A2523-53, Sigma Aldrich, Inc.) and β-glycerophosphate (G9422, Sigma Aldrich, Inc.).

Experimental analysis

7F2 cells were taken in 1 mL of the stock solution, then the thawing process is done and in the culture medium 10 mL of DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin + 250 μM L-ascorbic acid 2-phosphate (AA2P) + 10 μM β-glycerophosphate (Gly) was added. The medium was placed on a 10-cm Petri plate and incubated at a temperature of 37°C in 5% CO2 for 72 h. The cells were cultured until an 80% confluent monolayer was formed. The samples were divided into the following four groups:

  • Group P1: 7F2 cells with MO (24 h)


  • Group P2: 7F2 cells + LPS (24 h)


  • Group P3: 7F2 cells + alpha-mangostin (24 h)


  • Group P4: 7F2 cells + LPS + alpha-mangostin (24 and 24 h)


  • The 7F2 cell culture was treated with LPS (10 µg/mL) for 24 h. Each sample was given 5 µg/mL alpha-mangostin in the treatment group and incubated for 24 h in an incubator at 37ºC. After 24 h, each sample was extracted and the RNA using the Total RNA Purification Kit (Norgen, 17200, Canada) was converted into cDNa with iScript cDNA Synthesis Kit (Bio-Rad, 1708890, USA) using T100 Thermal cycle (Bio-Rad, CA, USA). Real-time PCR using SsoFast EvaGreen Supermix (Bio-Rad, 1725200, USA) was performed on 7F2 osteoblast cell culture to see osteogenesis markers of collagen 1A1 and inflammatory markers of IL-10.

    Real-time PCR analysis

    The real-time PCR machine used in this research is Bio-Rad CFX96 Realtime PCR System, USA. With SsoFast EvaGreen Supermix (Bio-Rad, 1725200, USA), the primer sequence was produced by Macrogen, Singapore [Table 1].
    Table 1: Primary sequence by Macrogen, Singapore

    Click here to view


    Statistical analysis

    Statistical Package for the Social Sciences Software (SPSS) 17.0 edition (Version 26, IBM Corp., NY, USA) was used to analyze the data. The Shapiro–Wilk test for assessing the normality of the data showed a P-value of more than 0.05, indicating a normal distribution. In addition to the normality test, Levene’s test was used to do a homogeneity test. The group differences were determined using a one-way analysis of variance (ANOVA) test. Results with P-value of more than 0.05 were considered not significant.


      Results Top


    The samples in this research were divided into four groups: one control group 7F2 osteoblast cell culture with osteogenic medium and three treatment groups. 7F2 osteoblast cell culture with osteogenic medium added LPS. The addition of alpha-mangostin and lipopolysaccharide and alfa-mangostin incubated at a temperature of 37°C in 5% CO2 for 24 h.

    [Figure 1] shows the results of IL-10 expressions on groups P1, P2, P3, and P4. On the results of real-time PCR IL-10 with the control group osteogenic medium as a control, IL 10 gene expression increased in all groups. The highest expression of the IL-10 gene was found in 7F2 osteoblast cell culture with osteogenic medium added with LPS (P2). According to the one-way ANOVA result [Table 2], the treatment in all groups had no significant differences (P> 0.05).
    Figure 1: Bar chart of interleukin-10 expression

    Click here to view
    Table 2: Average and deviation standard of interleukin-10 expression

    Click here to view


    [Figure 2] shows the results of COL 1A1 expression on groups P1, P2, P3, and P4. On the results of real-time PCR COL 1A1 with the control group osteogenic medium as a control, COL 1A1 gene expression increased in P2 and P4 groups and decreased in 7F2 osteoblast cell culture with osteogenic medium added with an alpha-mangostin group (P3). The highest expression of the COL 1A1 gene was found in 7F2 osteoblast cell culture with osteogenic medium added with LPS and alpha-mangostin (P4). According to the one-way ANOVA result [Table 3], the treatment in all groups had no significant differences (P> 0.05).
    Figure 2: Bar chart of collagen 1A1 expression

    Click here to view
    Table 3: Average and standard deviation of COL 1A1 expression

    Click here to view



      Discussion Top


    The main element of the fruit hull of G. mangostana was alpha-mangostin, a traditional Chinese medicine. More notably, alpha-mangostin was well known for a variety of biological activities and pharmacological effects, including anti-inflammatory, antioxidant, anticancer, and COX-2 inhibition.[10] Moreover, these substances potentially inhibit several anti-inflammatory mediators, such as interleukins, which have previously been known to enhance the differentiation and activity of osteoclasts.[8] Alpha-mangostin protects against LPS-induced cytotoxicity through regulating SIRT-1/NF-B signaling, according to a recent study. Although alpha-mangostin had long been utilized as a treatment for a variety of ailments, the mechanism of its actions remained unknown.[2] Alpha-mangostin has an effect on the balancing cytokines of proinflammatory and anti-inflammatory markers (IFN-γ/IL-10 ratio).[11] LPS contains harmful chemicals produced by Gram-negative bacteria that can harm tissues and cause inflammation.[12] The inflammatory process generated by the administration of LPS in cell line 7F2 culture is the starting point for osteogenesis.

    Evaluation of the inflammatory process and osteogenesis in each treatment was calculated quantitatively by real-time PCR to compare the expression levels of IL-10 and COL 1A1 from osteoblast cell line cultures. The study was conducted on the 3rd day of observation after treatment because the acute inflammatory response reaches its peak in the first 24 h; injury or trauma will trigger the inflammatory response needed for the healing process. The inflammatory stage takes place in the first 72 h after injury.[13] The process of osteogenesis begins with the inflammatory process first to trigger active osteogenesis cells. The physiological increase in IL-10 levels after about 72 h from the start of the inflammatory response leads to a progressive resolution of the inflammatory event, according to the results of the study that the expression of the IL-10 gene was increased. A study conducted by Nugrahaeni et al.[11] showed that the compound alpha-mangostin can increase the number of T cells that are producers of IL-10. According to this study, the expression of the IL-10 gene increased in 7F2 osteoblast cell culture with osteogenic medium added to alpha-mangostin. In the P5 group, there was an induction of LPS and alpha-mangostin, the result showed an increase in the expression of the IL-10 gene but did not increase sharply. The highest result of IL-10 production may over control otherwise protective T-cell response, causing the high microbial load. In this research, we can see the highest result of IL-10 in the P3 group.

    Excessive increase in IL-10 can lead to multiple organ failure.[14] The increase of IL-10 is a sign of an increase in the phagocytosis process. The results of the study by Lobo et al. showed that LPS-induced dendritic cells had an increase in IL-10. This is followed by the results of the P3 group. IL-10 binding initiates an intracellular signaling pathway involving STAT3 as a key translocation core factor that induces activation of genes encoding specifically for anti-inflammatory factors. IL-10 is not only a suppressive agent and inhibits the production of proinflammatory mediators but also increases the production of anti-inflammatory factors including the soluble TNF-α receptor and IL-1RA. Physiological increase in IL-10 levels after approximately 72 h from the onset of the inflammatory response leads to progressive resolution of the inflammatory event.

    The immune system’s fundamental regulator, IL-10, regulates immune response hyperactivation and maintains the so-called physiological inflammation by adjusting its activity. Another anti-inflammatory cytokine that affects bone growth is IL-10. In mice, mechanistic studies revealed that IL-10 influences endochondral bone formation by promoting chondrocyte proliferation and differentiation via the bone morphogenetic protein (BMP) pathway. Bone formation and osteoblastogenesis were decreased in IL-10-deficient animals, resulting in osteopenia and increased bone fragility. However, different doses of IL-10 have varied effects on human bone marrow mesenchymal stromal cell osteogenesis. Low physiological concentrations of IL-10 (0.01–1.0 ng/mL) stimulate osteogenesis by activating the p38/MAPK signaling pathway, but larger dosages of IL-10 (10–100 ng/mL) inhibit osteogenesis by activating NF-κβ. Genetically engineered IL-10 and TGF-overexpressing osteoclasts prevented osteoblast apoptosis and decreased osteoclast development and bone absorption ability in an in-vitro osteoblast–osteoclast coculture paradigm.[6]

    In this study, the osteogenesis marker collagen 1A1 was used because it is a gene that is commonly found in mature osteoblasts. During bone formation, mature osteoblasts synthesize and secrete collagen 1A1 and other non-collagenous proteins such as osteocalcin, osteopontin, and bone sialoproteins. According to the research conducted by Gencheva et al.,[15] the extracellular matrix proteins found in bone tissue are collagen 1A1 and OCN. Both are closely related to bone mineralization. Collagen 1A1 is one of the important markers of the osteoblast differentiation process. The expression of the COL 1A1 gene in 7F2 osteoblast cell culture with osteogenic medium added with alpha-mangostin decreased because alpha-mangostin had not yet been able to give its maximum effect, and the time of administration of alpha-mangostin for 24 h still did not affect osteoblast cells. The study by Gunter et al. mentioned that 7F2 osteoblast cells began to differentiate on day 4, and COL 1A1 gene expression began to appear in the early phase of cell differentiation. The expression of the COL 1A1 gene in 7F2 osteoblast cell culture with osteogenic medium added to alpha-mangostin decreased because osteoblast cells were not induced by LPS so there was no inflammatory process required by the osteogenesis process. Another influencing factor is the concentration of alpha-mangostin after 24 h, and the dose decreased by 4%/h. One of the limitations of the study is that it only uses one type of 7F2 cell line which is a mature osteoblast, so that the expression of osteogenesis that occurs in the early phase cannot be seen. This study has limitations, including limited observation time and limited number of samples. It takes varying concentrations of alpha-mangostin to see the osteogenic potential of alpha-mangostin.


      Conclusion Top


    The induction of alpha-mangostin is effective in reducing inflammation, decreasing the expression of IL-10, an anti-inflammatory cytokine marker, and increasing the expression of collagen 1A1, an osteogenesis marker.

    Acknowledgement

    We express our gratitude to the Faculty of Dental Medicine, Universitas Airlangga, Indonesia, for their invaluable support for the research.

    Financial support and sponsorship

    Nil.

    Conflicts of interest

    The authors have no conflicts of interest regarding this investigation.

    Authors’ contribution

    AR, MS: Concept, design, data acquisition, and statistical analysis, article preparation, editing, and review. PS, NPM, DK, MZR: Concept, design, article preparation, editing, and review. Finally, all authors had given approval for publication.

    Ethical policy and Institutional Review Board statement

    All procedures were in accordance with the ethical standards of the Airlangga Research Center of the Faculty of Dental Medicine, Universitas Airlangga, Indonesia, with approval number 522/HRECC.FODM/XI/2020 (dated November 24, 2020).

    Patient declaration of consent

    Not applicable.

    Data availability statement

    Data will be available on request from Andra Rizqiawan ([email protected]).

     
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        Figures

      [Figure 1], [Figure 2]
     
     
        Tables

      [Table 1], [Table 2], [Table 3]



     

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