Distilled liquid smoke coconut shell attenuates the cytokine profile of macrophages in oral ulcer in experimental model of diabetes mellitus

Meircurius Dwi Condro Surboyo1 , Diah Savitri Ernawati1* , Desiana Radithia1 , Bagus Soebadi1 , Fatma Yasmin Mahdani1 , Nurina Febriyanti Ayuningtyas1 , Fiona Cherrilia Adji2, Novia Ambar Larasati2 1Department of Oral Medicine. Faculty of Dental Medicine, Universitas Airlangga. Surabaya. Indonesia. 2Graduated Student of Doctor Dental Surgery. Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.


INTRODUCTION
The prevalence of oral mucosal lesions in diabetes mellitus type 1 and 2 patients is higher than in normal persons, and the most common lesions are ulcerative, such as traumatic and aphthous ulcers, with a prevalence of 24.6% (González-Serrano et al., 2016;Silva et al., 2015). Oral ulcers can cause pain, disturb nutrition intake and oral hygiene, and increase the risk of infection (Lim et al., 2016). One of the complications found with diabetes mellitus patients' oral ulcers is delayed healing (Selimovic et al., 2010;Suresh et al., 2014). Delayed healing is caused by a prolonged inflammation phase, resulting from the excessive secretion of cytokines of macrophages, such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Another factor is a decrease in antioxidant capacity with the decrease in nuclear factor erythroid-2-related factor 2 (Nrf2). The decrease in Nafr2 increases the migration of macrophages and the level of TNF-α, IL-6, and IL-1β (Tian et al., 2018). The oral ulcer, with pain as the chief complaint, is treated with an antiseptic and anti-inflammation drug in the form of a mouthwash (Tripathi and Tripathi, 2015). Sometimes, oral ulcers in diabetes mellitus patients do not respond to medication and an alternative substance is needed (Baltzis et al., 2014;Donath and Shoelson, 2011;Guo and DiPietro, 2010;Panchbhai, 2012;Tsourdi et al., 2013).
Coconut is one of the most common fruits in Indonesia, with consumption reaching 15.5 billion/year. The coconut industry only uses the flesh, and the shells are usually thrown out. Coconut shells are a waste that can be reused because of their contents, such as cellulose, pentosans, lignin, ash, solvents, anhydrous urinate, nitrogen, and water, which have a high potential for antiinflammation and as antioxidants (Surboyo et al., 2012).
Distilled liquid smoke coconut shell (DLS-CS) is a complex liquid compound produced with the pyrolysis of coconut shells at 400°C (Budijanto et al., 2008;Wagiman et al, 2014). Coconut shells contain cellulose, hemicellulose, and lignin. The latter substance forms phenolic compounds after going through pyrolysis, which has been proven to have analgesic, antiinflammatory, antiseptic, and antioxidant effects (Wagiman et al, 2014). Phenolic compounds are known to be able to decrease secretion and profiles of proinflammation cytokines, that is, TNF-α, IL-6, and IL-1β (Ambriz-Pérez et al., 2016;Kang et al., 2017;Lee and Yang, 2012).
TNF-α, IL-6, and IL-1β inhibitions are essential for the healing of diabetes mellitus patient's oral ulcers. The cytokine profiles of macrophage (TNF-α, IL-6, and IL-1β) inhibition in diabetes mellitus induct transformation of the macrophage phenotype, which is associated with an increased wound growth factor (Mirza et al., 2013). TNF-α, IL-6, and IL-1β are also known to decrease collagen synthesis, which is useful in the proliferation phase of wound healing (Adams et al., 1996;Mirza et al., 2013).
DLS-CS is expected to help accelerate the healing of oral ulcers with delayed healing in diabetes mellitus by decreasing the profiles of the proinflammation cytokines of macrophages, such as TNF-α, IL-6, and IL-1β, increasing Nrf2 and promoting macrophage migration. This research was conducted to discover the Nrf2, TNF-α, IL-6, and IL-1β profiles and macrophage count in oral ulcers with diabetes mellitus as an anti-inflammation indicator for the application of DLS-CS.

Distilled liquid smoke coconut shell (DLS-CS) production
The DLS-CS used in this research is made of (Surboyo et al., 2019a) coconut shells (Cocos nucifera L.) that come from 8-to 10-month-old coconuts. Five kilograms of coconut shells is pyrolyzed at a temperature of 400°C, with the heating rate of 3.3°C, for 4 hours and 30 minutes (Surboyo et al., 2019a).

Diabetic animals model
The experimental animals used were 36 male Wistar rats (Rattus norvegicus) with the following criteria: 2 months old, 120-160 g, and healthy (active movements and shiny fur). The rats were injected with 150 mg/kg body weight of alloxan (Alloxan monohydrate A7413, Sigma Aldrich., St. Louis, MO) intraperitoneally to induce diabetes mellitus after being fasted for 12 hours. Diabetes mellitus confirmation was carried out after 72 hours by measuring their fasting glucose level, with a minimum of 200 mg/dl, using GlucoDR (AGM-2100, All Medicus, Korea) (Ajiboye et al., 2018;Ernawati et al., 2019).
This study was carried out in strict accordance with the Guide for the Care and Use of Laboratory

Oral ulcer and treatment
Rats confirmed with diabetes were given a 10-mm oral ulcer using a round stainless steel blade in the inferior labial fornix region after anesthesia (ketamine/xylazine cocktail, intraperitoneal, 1 ml) to reduce pain. Ulceration confirmation was carried out after 24 hours by observing clinical signs, a white-yellowish lesion with a reddish edge (Hitomi et al., 2015;Kiliç et al., 2013).
The rats were then given treatment for the oral ulcer. The treatment was the topical application of distilled water (control), benzydamine hydrochloride (BHCL), and DLS-CS with a dose of 20 µl/20 g body weight once a day for 3, 5, and 7 days. The animals were terminated after the topical application of control, BHCL, and DLS-CS in each group, and a specimen of inferior labial fornix was taken to examine the macrophage, IL-6, IL-1β, and TNF-α expressions.

Oral ulcer tissue preparation
After all the groups had been treated for 3, 5, and 7 days, the oral ulcers in the inferior fornix labial region were biopsied. The oral ulcer tissue was fixed using a 10% formalin buffer solution and then processed in a paraffin block.

Macrophage count
A histological assessment was carried out by doing hematoxylin-eosin staining and counting macrophages using a microscope with 400x magnification assessed by a single operator in five fields of view.

Nrf2 expression
Histological assessment was carried out by doing immunohistochemistry staining for Nrf2 expression using a microscope with 400× magnification with a single operator in five fields of view (Papanicolaou et al., 2012). Nrf2 was used as the primary antibody (1:400; monoclonal; antibody online). The DAB system was used as the secondary antibody (Universal HRP Excell Stain; Biogear; Life Science). Hematoxylin 560 was used as the counterstain (Leica Biosystem).

Data analysis
Data obtained were analyzed with the one-sample Kolmogorov-Smirnov test for data distribution and the Levene test for data homogeneity. A one-way analysis of variance (ANOVA) with the least significant difference (LSD) was carried out for the post-hoc test to assess the difference between macrophage count and Nfr2, IL-6, IL-1β, and TNF-α expression between each group, with a significance number of p < 0.05.

Diabetic confirmation
The fasting glucose level and diabetic condition of all the animals were measured. Before injecting alloxan, the animals had a fasting blood glucose <200 mg/dl. The diabetic condition was confirmed by a fasting blood glucose >200 mg/dl. After treatment with sterile water, BHCL, and LS-CS, fasting blood glucose was >200 mg/dl, indicating that the condition still remained ( Table 1).

Macrophage count
The macrophages were counted in the area of the oral ulcer that was formed. The oral ulcer region is characterized by disintegration in the epithelial layer (blue box; Fig. 1A-C). The highest macrophage count was seen in the group treated with DLS-CS. The macrophage count of the DLS-CS group (9.50 ± 0.58), after 3 days of treatment, was significantly higher than that of the BHCL group (6.00 ± 1.42) and control group (5.50 ± 0.58) (p = 0.001; p = 0.000, resp.) (Fig. 2).
In the diabetes mellitus condition, there is a delay in macrophage recruitment, causing the accumulation of macrophages in inflamed tissue and an increase in proinflammatory cytokine secretion (Yan et al., 2018). Treatment with DLS-CS showed higher macrophage count and increased macrophage recruitment. The previous study confirmed that cellular response also increased after treatment, especially in the recruitment of neutrophils (Ernawati et al., 2020).

Nrf2 expression
Nrf2 expression was analyzed in the macrophages (Fig.  3). The Nrf2 profile increased after 5 days of treatment in the DLS-CS group, unlike the control group (p = 0.003). In the 7 days of treatment, the Nrf2 profile also increased more in the DLS-CS group (68.75 + 1.50) than the control groups (56.25 + 4.27) (p = 0.000). The increased Nrf2 profile in treatment with DLS-CS is the integral mechanism aimed at counterbalancing nuclear factor kappa B (NF-κB) activation (Ahmed et al., 2017). DLS-CS has a role in stimulating NF-κB activation in diabetes. NF-κB activation leads to the release of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β (Surboyo et al., 2019a). The increased Nrf2 profile leads to an increase in anti-inflammatory cytokine secretion and the growth factor. In other researches, treatment with DLS-CS was able to stimulate the fibroblast and secretion of the fibroblast and vascular endothelial growth factors (Ayuningtyas et al., 2020).

TNF-α, IL-6, and IL-1β expression
TNF-α, IL-6, and IL-1β expression was analyzed in the macrophages (Figs. 4-6, resp.). The highest cytokine profiles were found in the control group and the lowest in the DLS-CS group. The BHCL and DLS-CS groups showed a decrease in TNF-α, IL-6, and IL-1β expression, while the control group showed an increase in TNF-α, IL-6, and IL-1β expression.
The TNF-α profile showed no significant difference in the DLS-CS groups, compared to the BHCL and control group, after three (p = 0.09), five (p = 0.577), and seven (p = 0.256) days of treatment. TNF-α, IL-6, and IL-1β are proinflammatory cytokines with essential roles in the wound healing process in diabetes mellitus. Inhibition of excessive IL-1β in diabetes mellitus could promote oral ulcer healing by switching the phenotype of macrophages from proinflammatory to healing-associated and increasing wound growth factor production, while IL-6 is directly connected to the pathogenesis of diabetes mellitus (Mirza et al., 2013;Saiki et al., 2018). IL-1β is also associated with diabetes mellitus pathology. Accelerated healing of oral ulcers in diabetes mellitus patients will increase their quality of life by eliminating pain, thus improving nutrition intake and oral hygiene. Oral ulcer healing also decreases the risk of secondary infection caused by delayed healing (Lim et al., 2016).
The group treated with DLS-CS showed lower IL-6 and IL-1β profiles than the other groups. DLS-CS was able to decrease the IL-6 and IL-1β profiles consistently and progressively during treatment as well as increase macrophage migration to the inflammation sites. The mechanism that may have caused this process might be the phenolic compounds contained in DLS-CS. Phenolic compounds regulate the activity of transcription factors such as NF-κB (Surboyo et al., 2019a) and Nrf2, inhibiting the activity of the cyclooxygenase enzyme and the profiles of another inflammation mediator. DLS-CS proved to reduce NF-κB activity in the research conducted by Surboyo et al. (2019a). The research also states that the topical application of DLS-CS shows a lower TNF-α profile. Lower TNF-α profile synergies with lower IL-6 and IL-1β profiles are consequences of lower NF-κB activity due to treatment with DLS-CS (Surboyo et al., 2019a. The results of this research also support that DLS-CS is able to increase or stimulate Nfr2 as compensation for the suppression of NF-κB. Phenolic compounds, such as EMP and guaiacol (2-methoxyphenol), also have antioxidative properties, preventing oxidative stress and decreasing proinflammatory cytokine secretion, such as TNF-α, IL-6, and IL-1β. EMP is a guaiacol compound that possesses strong antioxidant properties, binding ONOO − and resulting in the inhibition of NF-κB activation and decrease of proinflammatory cytokine secretion. Guaiacol has two functional groups: a hydroxyl (−OH) and a methoxy group (− OCH 3 ), which able to bind free radicals due to their high ionization potential to form hydrogen bonds and inhibit superoxide radicals by donating hydrogen atoms to −OH, forming H 2 O. Prevention of oxidative stress could decrease NO synthase activity in     macrophages and reduce NF-κB activity and proinflammatory cytokine secretion. Lower profiles of TNF-α, IL-6, and IL-1β could increase the proliferation of fibroblasts and collagen synthesis and promote oral ulcer healing (Surboyo et al., 2019;Suryavanshi and Kulkarni, 2017;Tarawan et al., 2017;Surboyo et al., 2017;Surboyo et al., 2019b;Suryavanshi and Kulkarni, 2017).
Higher macrophage count was observed in groups treated with DLS-CS, but cytokine assessment showed a lower profile of proinflammatory cytokines. This indicates that DLS-CS promotes macrophage migration to inflammation sites but suppresses their activity to secrete proinflammatory cytokines, such as IL-6, IL-1β, and TNF-α. The TNF-α profile in this research did not show any difference between treatment with DLS-CS, BHCL, and control. This might happen because TNF-α is not the only proinflammatory cytokine secreted during the inflammation phase; there are a couple of proinflammatory cytokines that are secreted at the same time as TNF-α, such as IL-6 and IL-1β (Turner et al., 2014).
DLS-CS has proven effective in decreasing IL-6 and IL-1β profiles and increasing Nrf2 profiles and macrophage migration. IL-6 and IL-1β reduction might involve phenolic compounds contained by DLS-CS, which are able to regulate Nfr2 and NF--B activity and prevent oxidative stress and are proven to promote the clinical healing of oral ulcers.

FUNDING
This work was supported by Kementerian Riset Teknologi dan Pendidikan Tinggi Republik Indonesia in 2019 with grant number 6/E/KTP/2019.

AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.

CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.

ETHICAL APPROVALS
The protocol was approved by the Ethical Clearance of the Health Experiment Committee, Faculty of Dental Medicine, Airlangga University, Surabaya, with registered number 186/ HRECC.FODM/IV/2019, and all experiments were approved by the Animal Care and Use Committee of Universitas Airlangga.

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