Submucous fibrosis is characterized by devastating adverse effects and to date, it is a challenging clinical condition. The affected individual experiences both physical and mental trauma. Furthermore, restricted mouth opening leads to poor oral hygiene and also hinder social life. There is a number of treatment options available and reported in the literature to alleviate patient symptoms but to till date, there is no disease-specific treatment modality and not even a single treatment regime is satisfactory.
The affected patients complain mainly of the inability to open their mouth and the burning sensation on consuming spicy foods. So disease management focuses on alleviating the signs and symptoms of the disease. Preventive measures like counseling of patient to discontinue the habit of betel quid chewing, physiotherapy (adjuvant therapies), medical interventions like an intralesional injection of corticosteroids; immunomodulators; fibrinolytic agents; enzymes; anti-oxidants; vasodilators; placental extracts and other surgical interventions like flap surgeries; lasers have been tried for the management of submucous fibrosis.
Various studies from the literature have reported that there is upregulation or increased levels of TG2 in fibrotic disorders. Cystamine is a unique inhibitor of TG2 because it may have multiple inhibition mechanisms [Table 1]. Cystamine is a diamine capable of competitive amine inhibition. Indeed, the reduced form of cystamine, called cysteamine or 2-mercapto ethylamine (MEA), has been shown to be a competitive amine inhibitor of TG2.
Cysteamine (Cys) which is also known as 2-mercapto ethylamine (2-Aminoethanethiol) is synthesized endogenously during the metabolic cycle of co-enzyme A. In the year 1953, Cys was considered as most effective thiol derivative drug used against ionizing radiation. The inhibition of transglutaminase by cysteamine was well established by year 1984. Author Carla Atallah et al. has reported a narrative review with a compilation of all the data on the stability, quantification, and its various biological applications.
MATERIAL AND METHODS
Animal study design
A study was carried out in the Department of Oral Medicine and Radiology, Faculty of Dental Sciences, M S Ramaiah University of Applied Sciences with the objective of induction of oral submucous fibrosis in male Wistar rats. Institutional Animal Ethics Committee (IAEC) ethical clearance was obtained to conduct the animal experimental study. Registration number 220/PO/ABC/2000/CPCSEA and reference number MSRFPH/P/62/2015. Healthy Wistar rats (control-10; experimental animal-10) weighing approximately about 200 mg were obtained from private animal breeders. Wistar rats were maintained under standard laboratory guidelines in clean and hygienic cages. Regular 12 h light/dark cycle, controlled room temperature at 25°C ± 2°C, and humidity were maintained. Standard prescribed diet and drinking water ad libitum were provided to the animals.
Arecoline was procured from Sigma Aldrich Pharmaceuticals. A dosage of 10 mg/kg of freshly prepared arecoline was injected into the submucosa of the right buccal mucosa in experimental animals over a period of 3 months (90 days) on every alternate day.
Blood samples of 1 ml were collected from retro-orbital plexus in EDTA coated vials at the start of the experiment from healthy Wistar rats and also at the end of the experiment of induction of oral sub-mucous fibrosis.
5 experimental animals were sacrificed to check the induction of submucous fibrosis. The right buccal mucosa was dissected and subjected to histopathological examination. Tissue and blood samples of those 5 Wistar rats were subjected to ELISA for estimation of levels of transglutaminase.
Following the induction of oral submucous fibrosis in experimental animals, the Remaining 5 animals in the experimental group were treated with cysteamine hydrochloride. The drug was procured from Sigma Aldrich Pharmaceuticals, weighed for its therapeutic dose, and administered to experimental animals through drinking water. A dose of 48 mg/kg was administered through drinking water. On the 91st day, the drug treatment was started.
Whole blood samples were collected (post-treatment) at regular intervals i.e., on the 97th day (1st week), 104th day (2nd week), and 111st day (third week), and subjected to ELISA to assess the level of transglutaminase. On the 111st day all the experimental animals were sacrificed and the right buccal mucosa was subjected to the same enzyme estimation.
ELISA was performed based on the protocol mentioned in the Bioassay Technology Laboratory, Rat Transglutaminase ELISA kit (No. E1513Ra).
Reconstitution of the standard:
- From the Standard vial (24 ng/ml), 120 μl of the Original standard was taken to which 120 μl of standard diluent buffer was added making a concentration of 12 ng/ml. Further dilutions were prepared similarly making serial dilutions of 6 ng/ml, 3 ng/ml, 1.5 ng/ml, and 0.75 ng/ml.
Preparation of wash buffer
- 20 ml of 30X Wash buffer concentrate was diluted with distilled water to yield 500 ml of 1X wash buffer.
- In the standard wells, 50 μl of standards were added.
- In the control well (Zero well) 50 μl of the standard diluent buffer was added.
- 40 μl of received samples were added in sample wells following which 10 μl of anti-TG antibody was added.
- Streptavidin-HRP 50 μl was added to both standard and sample wells, other than blank wells.
- The sealed plate was incubated at a temperature of 37°C for a time period of 60 min.
- Following the incubation, the sealing cover was removed. The contents of the plate were discarded, and using a wash buffer the plate was washed 5 times.
- Substrate solution A followed by substrate solution B of 50 μl is added to each well in the plate.
- Later the plate was sealed and subjected to incubation for a time period of 10 minutes at a temperature of 37°C in the dark.
- The change in color was observed after adding a stop solution of 50 μl.
- Following the stop solution, optical density (O.D) was calculated in the microplate reader within 30 minutes of time at 450 nm.
- The method/formulae for calculation of relative O.D. 450 is as mentioned below:
(The relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zero well).
With the concentration of the standard solution (X) and relative O.D.450 of each standard solution (Y), the standard curve was plotted. The transglutaminase concentration of the samples was interpolated from the standard curve [Figure 1].
The histopathological section [Figure 2a] of the right buccal mucosa of a control group of Wistar rats showed Ortho-keratinized stratified squamous epithelium with blunt rete ridges and normal components of collagen fibers, few inflammatory cells, and normal vascularity in connective tissue. Whereas the histopathological section [Figure 2b] of the right buccal mucosa of an experimental group of Wistar rats showed areas of atrophy and hyperplasia seen with subepithelial hyalinization and increased density of collagen fibers, and inflammatory cells in connective tissue. After the successful induction of submucous fibrosis in the control group and experimental group of Wistar rats, it showed a significant increase in transglutaminase 2 both in tissues [Figure 3a] and serum [Figure 3b]. Following the induction of submucous fibrosis, the experimental group was treated with cysteamine hydrochloride, and they showed a significant decrease at the end of 1st week [Figure 4a] and 2nd week [Figure 4b]. By the end of 3rd week [Figure 4c] the tranglutaminase2 level has reached normal limits. Similarly, even in tissue samples [Figure 4d] significant reduction in transglutaminase2 levels at end of 3rd week was seen.
As the etiologic agents for submucous fibrosis (SMF) is a matter of conflict, the prime failure to obtain proper or definitive treatment for it may be the reason for its incomplete resolution of the disease. Stoppage of areca nut chewing is a foremost important measure to treat SMF. Various drugs alone or in combination are used to treat this crippling disease.
Induction of oral submucous fibrosis in various laboratory animal models was reported in the literature. Sirsat and Khanolkar, 1960 used capsaicin as a topical application in the palate in Wistar rat. Khrime et al., 1991 used instant pan masala as a topical application in albino rats. Huang et al., 1997 used aqueous areca nut injections as injections in Sprague Dawley rats. Majumdar et al., 2013 used a combination of areca nut; tobacco, and O-phenyl phenol in lagomorphs.
All the above-reported studies successfully induced oral submucous fibrosis but, the exact dosage and methodology were not discussed in detail. The time duration taken for induction was longer. Bleomycin-induced fibrosis is well documented in the literature.
The present study was attempted with a standardized methodology for the induction of oral submucous fibrosis. The histopathologic features of the affected Wistar rat tissues show similar histopathological characteristic features as SMF manifest in humans.
The enzyme, transglutaminase is essential in the biochemical reaction for the formation of isopeptide bonds i.e. cross-linkage of two amines namely glutamine and lysine. This enzyme is present in normal quantities under physiological conditions. The diverse functions of TG2 and its role in cell apoptosis, and fibrosis have been discussed in the literature. Increased levels of transglutaminase are various pathologies being reported in the literature. Examples include Huntington disease Lesort et al., 1999; alzheimers disease Appelt et al. 1996, Kim et al., 1999; Parkinson disease Andringa et al., 2004. In particular the inhibition of TG2 with cysteamine was studied in ocular cystinosis subjects. For different types of cancer this particular enzyme is upregulated or downregulated as studies conducted by various authors. It is also elevated in interstitial fibrosis, pulmonary fibrosis, and myocardial fibrosis.
So far studies have not been reported in the literature, on the involvement of this specific enzyme in oral submucous fibrosis. The present study evaluates the levels of transglutaminase in oral submucous fibrosis-affected Wistar rats. There is a significant increase in levels of this enzyme both in serum and tissue samples. This result is in agreement with G.S. Thangjam et al., 2009 who conducted an in vitro study and found a marked increase in transglutaminase levels.
Enzyme transglutaminase 2 is a multi-functional and multi-domain which involves post-translation modification of proteins. The biochemical reaction leads to the formation of an intermolecular isopeptide bond between the lysine and glutamine side chain.
The analysis of in vitro and in vivo studies correlating to the biological activities of proteins TG 2 cross-linking and formulation of specific TG 2 inhibitors is vital for the success of pharmacological therapy in several neurodegenerative diseases and also diseases involving TG 2 dysfunction.
In view of TG2 activity in the crosslink of proteins, thereby maturation of collagen fibers and stabilizing of extracellular matrix, the present study explores the TG2 inhibitor as a pharmacological therapy for the management of oral submucous fibrosis.
Cystamine, a common transglutaminase2 inhibitor undergoes reduction resulting in an aminothiol compound called cysteamine. Cystamine acts by promoting the oxidation of cysteine residues, whereas cysteamine acts as a competitive amine inhibitor in transamidation catalytic reactions.
In a similar concept of literature, this present study was formulated to finding the efficacy of cysteamine in the management of oral submucous fibrosis. The drug Cysteamine also known as 2-mercapto ethylamine, b-mercapto ethylamine, 2-amino ethanethiol, decarboxycysteine, thio-ethanol-amine and mercaptamine) was first considered as part of the coenzyme A pathway.
In the present study, the levels of transglutaminase were significantly increased in oral submucous fibrosis-affected serum and tissue samples. Oral submucous fibrosis was induced for 90 days, later for the treatment for which cysteamine hydrochloride was administered through drinking water for the Wistar rats. After successful induction of oral submucous fibrosis for 90 days of the time period, from the 91st day the drug was administered at regular intervals i.e. at end of 1st week, 2nd week, and 3rd week. The serum and tissue samples were evaluated for the transglutaminase enzyme.
In serum samples, at end of 1st week, the enzyme levels were 1.71 ng/dl, at 2nd week the enzyme levels were 1.52 ng/dl, and end of 3rd week the enzyme levels were 1.40 ng/dl. On all the intervals there is a significant reduction of the enzyme levels and on the 111st day the levels have reached near normal values. Tissue samples, at end of 3rd-week levels of enzyme, were 1.73 ng/dl and exhibited a significant reduction of enzyme levels.
The study was conducted by Alpaslan Dedeoglu et al. in 2002 with a similar methodology to assess the therapeutic effects of the drug cystamine in a mouse models affected by Huntington disease. The author concluded that transglutaminase activity was significantly reduced upon administration of the drug. A study conducted by Daryl M Okamura et al. in 2013 states that the fibrosis severity levels decreased significantly on the administration of cysteamine by drinking water in chronic kidney disease affected mice. Table 2 summarizes the application of cysteamine in various systemic disorders and its reference studies.
In contrast, Ju-Hong Jeon et al. in 2004 evaluated cystamine and cysteamine for inhibition of intracellular transglutaminase in various cell lines. The authors concluded that in situ TG2 activity was significantly inhibited by cystamine rather than cysteamine. In an in-vitro study, the inhibitory efficacy of cystamine largely depends on the availability of its reduced form. An vitro model reported by Lee S S et al. in 2016 used antioxidants [glutathione precursor N-acetyl-L-cysteine (NAC) and epigallocatechin-3 gallate (EGCG)] which could inhibit arecoline-upregulated TGM-2 expression.
From the results of the present study and literature survey, cysteamine hydrochloride used in the study strongly suggests the management of submucous fibrosis.
Despite decades of research, the pathogenesis and treatment regimen for submucous fibrosis is unclear and still evolving. The epidemiological and evidence-based studies have concluded as areca nut is the primary factor in causing submucous fibrosis. The directed education and national policy changes are desirable to reduce the usage of areca nut. Many treatment protocols have emerged through scientific studies, but there is no single universally accepted treatment regimen.
Among the transglutaminases enzyme family, TG2 is highly expressed and present in most mammalian cells which primarily involved the cross-linking of two proteins (glutamine and lysine). The cross-linking promotes extracellular matrix stabilization and deposition of highly dense fibrous collagen which is the characteristic feature of submucous fibrosis.
Transglutaminase 2 acts as a new therapeutic target for finding novel treatment modalities for fibrotic disorders. The drug cysteamine (competitive amine inhibitor), has been explored as a potential drug for nephropathic; liver, and pulmonary fibrosis, and clinical studies are reported in the literature. For the first time in literature, present study was carried out to exploring cysteamine as a potential drug in the management of submucous fibrosis.
The data from the present study shows that there is a significant inhibition of TG2 of cysteamine and thereby preventing stabilization of the extracellular matrix. This in turn helps in preventing the further progression of the disease.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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