|Year : 2016 | Volume
| Issue : 1 | Page : 379-385
Evaluation of transforming growth factor beta1 gene in oral submucous fibrosis induced in Sprague-Dawley rats by injections of areca nut and pan masala (commercial areca nut product) extracts
Shilpa Maria, Venkatesh V Kamath, Krishnanand Satelur, Komali Rajkumar
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Bangalore, Karnataka, India
|Date of Web Publication||13-Apr-2016|
Venkatesh V Kamath
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Bangalore - 560 037, Karnataka
Source of Support: None, Conflict of Interest: None
Context: Oral submucous fibrosis (OSF) is a potentially malignant oral disorder causally linked to the habit of chewing arecanut. The pathogenesis of the disorder is multifactorial and transforming growth factor beta (TGF-beta) is a prominent player in the induction of fibrosis. The alkaloids of the arecanut seem to target the TGF-beta and the deposition of collagen is predominantly mediated through this cytokine.
Aims and Objectives: The present study attempts to induce OSF in Sprague-Dawley rats by injections with solutions of arecanut and pan masala extracts. The tissues were then analysed for the TGF-beta1 gene by real time polymerase chain reaction. (rtPCR).
Materials and Methods: Buccal mucosa of Sprague-Dawley rats were injectedwith arecanut and pan masala solutions on alternate days over a period of 48. weeks. Quantitative real time PCR was done to assess the expression of TGF-beta1 in the tissues.
Results: OSF-like lesions were seen in both the arecanut and pan masala.treated groups. The histological changes included atrophic epithelium, partial or complete loss of rete ridges, juxta-epithelial hyalinization, inflammation and accumulation of dense bundles of collagen fibers subepithelially. Quantitative real-time PCR showed a significant upregulation of TGF beta1. A. peak fold change of 4.74 in the 18th. week was observed for the arecanut group while the pan masala group recorded a peak change of 4.9 in the 24th. week.
Conclusion: The study provides further evidence that arecanut and pan masala induce oral submucous fibrosisvia the TGF beta1 pathway.
Keywords: Areca nut, oral submucous fibrosis, pan masala, Sprague-Dawley rats, transforming growth factor beta
|How to cite this article:|
Maria S, Kamath VV, Satelur K, Rajkumar K. Evaluation of transforming growth factor beta1 gene in oral submucous fibrosis induced in Sprague-Dawley rats by injections of areca nut and pan masala (commercial areca nut product) extracts. J Can Res Ther 2016;12:379-85
|How to cite this URL:|
Maria S, Kamath VV, Satelur K, Rajkumar K. Evaluation of transforming growth factor beta1 gene in oral submucous fibrosis induced in Sprague-Dawley rats by injections of areca nut and pan masala (commercial areca nut product) extracts. J Can Res Ther [serial online] 2016 [cited 2019 Nov 15];12:379-85. Available from: http://www.cancerjournal.net/text.asp?2016/12/1/379/148729
| > Introduction|| |
Documental evidence on the habit of chewing arecanut in India can be traced back to the Sanskrit manuscripts from the 1st century BC, thereby testifying to the existence of this practice since several centuries. Arecanutranks 4th among the most commonly used drugs globally after tobacco, alcohol and caffeine. The habit is prevalent throughout the Indian subcontinent, in most parts of Asia, Africa, and North America andamong the migrated communities in Europe. The widespread prevalence of this habit is in itself an indication of its indisputable antiquity.
Arecanut, an important agricultural product globally, is the fruit of the oriental palm Areca catechu. It has been incorporated into a variety of widely used chewing products. Two of these principal commodities which have gained immense popularity in India, are pan masala and gutka. Pan masalais a dry, relatively non-perishable commercial preparation containing arecanut, catechu, lime, unspecified spices and flavoring agents. The same mixture with tobacco is called gutka. These products are inexpensive and affordable owing to lower taxes imposed on them, thus easily accessible even to minors. These commercially available products are said to contain high concentrates of arecanut per chew and appear to cause OSF. The extensive promotion of these products as an aid to smoking cessation has added to the already existing menace.,,
Oral submucous fibrosis (OSF) is a chronic insidious disease affecting the oral cavity and sometimes the pharynx, associated with juxta-epithelial inflammatory reaction followed by fibroelastic change of the lamina propria, with epithelial atrophy leading to stiffness of the oral mucosa, trismus, reduced mouth opening and inability to eat. Although there have been a few animal models developed in the past for OSF, they were usually focused on assessing a single etiological agent. Perera et al. studied the effects of aqueous arecanut extracts in the buccal mucosa of a group of 20 female BALB/c strain miceand reported development of OSF-like lesions. Khrime et al. evaluated the effects of pan masala on the oral mucosa of albino ratsand found a 88% increase in collagen deposition at the end of 6 months. In addition the epithelium also exhibited cellular changes indicative of leukoplakia. In the present study we have attempted to develop an animal model using Sprague-Dawley rats to evaluate the effects of both pan masala and arecanut extracts at regular time intervals.
Transforming growth factor beta 1 or TGF-β1 is a polypeptide member of the transforming growth factor beta super family of cytokines. It was first identified in human platelets as a protein with a molecular mass of 25 kilo Daltons with a potential role in wound healing. It was later characterized as a large protein precursor (containing 390 amino acids) that was proteolytically processed to produce a mature peptide of 112 amino acids. TGF-β1 plays an important role in controlling the immune system, and shows different activities on different types of cell, or cells at different developmental stages. Most immune cells (or leukocytes) secrete TGF-β1.
Pro-fibrogenic cytokines such as transforming growth factor-beta (TGF-beta) become key mediators of fibrogenesis by differentiating fibroblasts into myofibroblast phenotype in connective tissue disorders. TGF-betahas been shown to be up regulated in OSF tissues and probably serves as the major pathway in the fibrogenesis. TGF-b1 is the best characterized between three TGF-b isoforms (TGF-b1, TGF-b2, TGF-b3) encountered in mammalian species and plays a major role in the fibro-genetic pathways.
The present study aims to develop oral submucous fibrosis-like lesions in Sprague-Dawley rats by injections of arecanut and pan masala solutions. The buccal mucosa tissues were then analysed histologically for changes and for the expression and regulation of TGF-beta1 gene by real-time quantitative PCR.
| > Materials and Methods|| |
Development of an animal model for OSF
Thirty Sprague-Dawley rats weighing 120-150gwere chosen for the study. The experimental animals were divided into threegroups: Arecanut group-10, pan masala group-10 and control-10. The animals were housed in a clean, hygienic and well-ventilated environment atthe animal facility. They received regular diet and water.
The arecanut extract was prepared by dissolving 0.2 g of powdered arecanut in 6 ml of distilled water. It was then centrifuged at 15000 rpm for 30 minutes. The supernatant was collected and used for injection into the rat buccal mucosa. Similar protocol was followed for the preparation of pan masala solution. Rats in the control group were injected with 0.2 ml of sterile saline on every alternate day for 48 weeks. Experimental rats in the arecanut and panmasala groups were injected with 0.2 ml (33 mg/ml) of the prepared arecanut and panmasala extracts respectively on every alternate day for 48 weeks. The site of injection was the right buccal mucosa.
Animals from each group were randomly sacrificed at an interval of every 6 weeks. The right buccal mucosae were dissected out. Part of the tissue was preserved in RNAlater, to be used for gene expression studies and the rest was fixed in 10% formalin followed by conventional processing, sectioning and H and E staining for histopathological assessment.
RNA extraction, semi-quantitative and real-time RT-PCR
To isolate RNA, rat tissues were weighed and crushed to powder in liquid nitrogen in a mortar and pestle followed by transfer to homogenization tube. TRI-reagent was then added as recommended; according to the tissue weight (1 ml for 100 mg of tissue) and homogenized. Homogenized tissue was processed for RNA isolation as per the manufacturer's protocol.
Two microgram of the total RNA extracted was reverse transcribed using a High Capacity cDNA synthesis kit (Applied Biosystems, Foster City, USA). 1/100th of the cDNA was used per 20 μl of PCR reaction. PCR reactions were performed with 1x DyNAZYME Master Mix (Finnzymes, Finland) along with gene-specific primer pair. RPL19 was used as the normalizing gene. The primer sequences used in this study, respective amplicon sizes and PCR conditions are enlisted in [Table 1]. All PCR reactions were performed at sub-saturating cycle numbers for each gene. The PCR products were resolved on 2% Agarose gel containing Ethidium bromide and the band intensities were determined using Gel documentation system (UviPro platinum, Uvitec, UK).
Real time PCR quantifications were performed in ABI Prism 7000 sequence detection system and analyzed with SDS 2.1 software (Applied Biosystems, USA). The reactions were same as described above; except that DynamoTM SYBERgreen 2X mix (Finnzymes, Finland) was used in place of DyNAZYME mix. The reactions were performed in triplicate for each sample. The differential expression was determined by the formula:
δCT = CT gene-CT RPL19
δδCT= δCT untreated-δCT untreated
Fold Change (FC) =2-δδCT
Log2 FC=- δδCT
In the above formula CT (threshold cycle) reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold and is inversely correlated to the logarithm of the initial copy number. All the primers used in the study gave a single peak in the dissociation curve suggesting that there is a single amplicon.
| > Results|| |
Monitoring clinical changes by visual examination was hindered owing to the small size of the rat oral cavity.
Assessment of the H and E-stained tissue sections under the light microscope showed OSF-like lesions in different degrees in both the arecanut and pan masala treated groups. The histologic changes observed were atrophic epithelium, partial or complete loss of rete ridges, presence of inflammatory cells, juxta-epithelial hyalinization and accumulation of dense bundles of collagen fibers in the lamina propria. Although the changes were not uniformly progressive our findings bear a close semblance to the histological traits of OSMF as seen in humans and documented in literature. [Table 2] gives a detailed description of histopathological changes observed at every 6 weeks, beginning from the 6th week and extending up to the 48th week.
Results obtained from real time PCR
In the 6th week the arecanut treated group showed an upregulation ofTGF beta1 as compared to the control which was injected with saline. The levels were upregulated by 1.53 folds. However, in the pan masala group the amplification plot was not seen on the RT-PCR due to contamination of the sample. In the 12th week the levels of upregulation were almost the same in both the treatment groups. Evaluation of samples obtained after 18 weeks of treatment showed an upregulation of 4.74 fold in the arecanut group whereas the fold change plotted for the pan masala group stood at 1.42. Surprisingly at 24 weeks the arecanut-treated group showed a two-fold lower expression (2.7) as compared to the 18th week sample. Whereas the pan masala treated group showed a 4.9-fold upregulation.
In the sample obtained following 30 weeks of arecanut treatment the expression of TGF-beta1 was almost close to its expression in the 24th week. The value obtained on RT-PCR was2.29. In the pan masala treated group the fold change plotted was 1.25. At 36 weeks the expression of TGF Beta1 was similar in both the treatment groups. After slumping down to expression levels slightly below that of the control group in the 36th week, there was a slight upregulation in the expression of the gene after 42 weeks of treatment. The values recorded for the arecanut treated group was 1.47 and 1.09 for the pan masala-treated group. At the end 48 weeks the gene expression in both the treatment groups showed a downswing and the expression levels were comparable to that of the control group.
A comparative evaluation of the expression ofTGF-beta1 following treatment with arecanut and panmasala extracts at various time intervals against their respective controls has been given in [Table 3].
|Table 3: Comparison of fold changes in the expression of transforming growth factor beta1 gene following treatment with arecanut and panmasala extracts at various time intervals as compared to corresponding controls|
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| > Discussion|| |
The present study aimed to develop an animal model for OSFto assess histological changes following treatment with arecanut and pan masala solutions over a period of 48 weeks and also to check the expression of TGF-beta1 in the fibrotic rats as compared to the controls.
Unlike earlier animal models where only one etiological agent was assessed, in the present study we have evaluated changes induced by both arecanut and pan masala solutions at regular time intervals. The results emerging from our study showed OSF-like lesions in different degrees in both the arecanut and pan masala-treated groups. The histologic changes seen included: Atrophic epithelium, partial or complete loss of rate ridges, inflammation, juxta-epithelial hyalinization and accumulation of dense bundles of collagen fibers.
Interestingly the pan masala-treated group showed atrophic changes in the epithelium beginning from the 6th week itself, with the lamina propria showing no evidence of fibrosis. Subepithelial fibrosis was noticeable only from the 12th week onwards.
On the other hand in the arecanut treated group, thinning of epithelium was evident only after 12 weeks, with atrophic changes in the epithelium manifested predominantly following 18 weeks of treatment. However, fibrosis in the subepithelial region was noticeable from the 6th week onwards.
Arecanut extracts and arecoline have been shown to be cytotoxic to epithelial cells. The cell death induced by arecoline was shown to be due to production of ROS resulting from a suppression of catalase activity in the epithelium. Thus, arecanut-induced cytotoxicity on epithelial cells could be mediating epithelial atrophy, a hallmark of OSMF.
The early manifestation of atrophic epithelium in the pan masala-treated group could be due to the combined effects of arecanut and other deleterious agents in pan masala such as polycyclic aromatic hydrocarbons, nitrosamines, toxic metals and pesticide residues.,
Canniff and Harvey in 1981 have reported that treatment of human fibroblasts with arecanut extract increases collagen production. Besides more evidence has been provided to support that arecanut extracts decrease the collagen phagocytosis by fibroblasts and increase the collagen stability rendering them resistant to collagenase activity., Arecanut is an essential ingredient of pan masala. Hence, the induction of fibrosis in both the arecanut and panmasala-treated groups could be due to the proliferative action of arecanut on the fibroblasts.
The subepithelial fibrosis that developed may also be stemming from an attempt to repair the damage caused by irritation of tissue following injection of the arecanut and pan masala solutions.
In our study the histological changes that were manifested in the SD rats at different time intervals following treatment with arecanut and panmasala solutions were not uniformly progressive. This is probably because each individual experimental animal responded differently to the treatment solutions depending on its immune status and genetic predisposition. Also the animals used were crossbred, which could also be one of the contributing factors for differing responses.
[Figure 1],[Figure 2],[Figure 3] show the histological evidence of the changes induced in the rat buccal mucosa by the arecanut and pan masala extracts.
|Figure 1: A composite set of photomicrographs showing effects of extracts of arecanut and pan masala on the buccal mucosa of SD rats at 6 weeks. (a) Control – Note the epithelial hyperplasia, inflammation in subepithelial connective tissue secondary to injection of saline.|
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|Figure 2: A composite set of photomicrographs showing effects of extracts of arecanut and pan masala on the buccal mucosa of SD rats at 18 weeks. (a) Control – Note the epithelial hyperplasia has regressed, inflammation is minimal. (b) Arecanutgoup – Note the epithelial atrophy and well-established fibrosis in the connective tissue (c) Pan masala group – Note the epithelial atrophy and fibrosis. (H and E stain, original magnification ×10)|
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|Figure 3: A composite set of photomicrographs showing effects of extracts of arecanut and pan masala on the buccal mucosa of SD rats at 36 weeks. (a) Control – Changes are minimal (b) Arecanutgoup – Note the severe epithelial atrophy and fibrosis extending and involving muscle layer in the connective tissue (c) Pan masala group – Note the epithelial atrophy and fibrosis, but much less in intensity than the arecanut group. (H and E stain, original magnification ×10)|
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Gene expression analysis
Histopathological findings suggest that an imbalance between production and degradation of extracellular matrix (ECM), essentially collagen in the oral sub-mucosal region leads to OSF. Collagen production is driven and sustainedby a means of a delicate balance between pro- and anti-fibrogenic cytokines. The three most importantpro-fibrogenic cytokines include transforming growth factor-b (TGF-b), endothelin-1 and connective tissue growth factor (CTGF). Bone morphogenetic proteins 4 and 7 (BMP4, 7) function as anti-fibrogenic cytokines.
TGF-beta1 is a ubiquitous, multifunctional growth factor, prototype of the TGF-beta superfamily. It was first identified in human platelets as a protein with a molecular mass of 25 kilodaltons, later characterized as a large protein precursor (containing 390 amino acids) that was proteolytically processed to produce a mature peptide of 112 amino acids.
TGF-beta1 has been reckoned to play a major role in synthesis and deposition of extracellular matrix components such as collagen type I, III, and IV, fibronectin, tenascin, elastin, osteonectin, biglycan, and decorin. In addition to its fibrogenic effect, TGF-b1 decreases the degradation of extracellular matrix by inhibition of metalloproteinasesand activation of tissue inhibitors of metalloproteinases.
Since TGF-b1 is the prototype and best characterized of three TGF-beta isoforms (TGF-b1, TGFb2, TGF-b3) encountered in mammalian species, in the present study we have assessed the gene expression levels of TGF-b1 at regular time intervals as per the study design.
In the gene expression analysis performed using the quantitative real time PCR, arecanut group showed maximum upregulation of TGF-β1 gene after 18 weeks of treatment, whereas in the pan masala group, maximum peak of gene expression was seen after 24 weeks of treatment. This corresponds with the histological findings in the respective treatment groups. However, the lag between the twotreatment groups in attaining the maximum peak of gene expression could be attributed to the difference in composition of arecanut and pan masala solutions.
The major constituents of arecanut are carbohydrates, fats, proteins, crude fiber, polyphenols (flavonols and tannins), alkaloids and mineral matter. Water extract of arecanut mainly contains alkaloids and polyphenols. Arecoline is approximately 0.2% in arecanut compared to other compounds such as polyphenols, which are approximately 11–17.8% in arecanut.
Khan et al. (2012) in their study have shown that both arecanut alkaloids and polyphenols can induce the pro fibrotic cytokine TGF-beta in the epithelium. They have postulated that when arecanut comes in contact with epithelial cells both the alkaloids and polyphenols act on the epithelial cells and induces TGF-beta signaling. In the connective tissue, arecanut acts on fibroblast cells along with TGF-beta produced from the epithelium and potentiates its action in activating fibroblast cells responsible for inducing fibrosis. This lends support to the observation that total arecanut extract can indeed result in OSF progression.
The constituents listed on the packets of various pan masala include arecanut, catechu, lime, sandal oil, menthol, cardamom, flavors, spices, aniseed, sugar, waxes, oil seeds, colors, etc. This implies that pan masala is a blend of several components and “arecanut” constitutes only a small portion of the prepared pan masala solution which was used to treat the experimental rats in our study design. The presence of arecanut in smaller proportion could have caused a delayed upregulation of TGF-β1 in the pan masala-treated group.
Towards the end of our study the expression levels of TGFβ1 gene showed a downswing in both the treatment groups. This could be attributed to two different factors, one of which is appearance of fibrotic lesions in the control group. Hence when the gene expression levels in the treated tissues were compared against their respective controls, there was no significant fold change. Also the histological assessment of samples from the 36th, 42nd and 48th week did not show extensive fibrosis. However, epithelial atrophy, the hallmark of OSF was not manifested by any of the controls.
Secondly some in vitro tissue culture experiments have demonstrated that arecanut alkaloids may have cytotoxic properties. The suggested mechanism for arecoline-induced cytotoxicity was through depletion of Thiol in the fibroblasts. Hence it is tenable to postulatethat arecanut alkaloids may be cytotoxic to fibroblasts, eventually causing a reduction in cellularity. Thus atrophic epithelium and reduced cellularity in the lamina propria could have also led to a diminished expression on TGF-beta1 gene in the terminal weeks.
| > Conclusions|| |
The present study has examined in detail the development of an animal model for OSF based on histopathological evaluation and gene expression analysis of TGF-beta1. We demonstrated in this in vivo rat model the effects of arecanut extracts and pan masala solutions on epithelial thickness leading to atrophy and connective tissue fibrosis. Moreover, fibrotic rats overexpress the pro-fibrotic cytokine TGF-beta1 which is observed in OSF patients as well. The results from our study, therefore, provide further support to the data that arecanut and pan masala can have deleterious effects on the oral mucosa contributing to the development of oral submucous fibrosis. The TGF-beta cytokine seems to play an important role in the development of the fibrosis.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]