Study on iNOS expression and levels of Malondialdehyde (MDA) in the White Rat (Rattus norvegicus) Exposed with Aggregatibacter actinomycetemcomitans
(Aa)
Reviana Setin1, Aulani’am1 and Chanif Mahdi1
1Department of Chemistry, Faculty of Mathematics and Natural Sciences, Brawijaya University, Jl. Veteran, Malang 65145, East Java Indonesia
ABSTRACT
Aggregatibacter actinomycetemcomitant (Aa) is gram-negative bacteria composed of lipopolysaccharides (LPS) in its outer membrane. This bacteria is normally found in dental plaque and periodontal pockets causing parotid gland damage when it is present in excess amounts. LPS is an endotoxin and a complex glycolipid composed of polysaccharides (hydrophilic) and lipid A (hydrophobic) which can induces Reactive Oxygen Species (ROS), and increase the levels of malonaldialdehyde (MDA) and tissue damage. The aims of this research are to find the expression of iNOS parotid gland cell and to determine levels of MDA using thiobarbituric acid (TBA) spectrophotometric test at 533 nm. The results showed a significantly difference (P<0.05) of MDA levels in control rats (3.135 ± 0.0436 ppm) and Aa rats (6.307 ± 0.0473 ppm). iNOS expression is confirmed by the brown spots formed on the parotid gland, affording the enhancements of iNOS expression in Aa rats and control rats of 2.292 ± 0.038 and 0.166 ± 0.003, respectively (P<0.05).
Keywords : Aggregatibacter actinomycetemcomitans, Reactive Oxygen Species, malondialdehyde, iNOS
Corresponding author : [email protected]
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Mobile : +6285246109179 INTRODUCTION
There are a variety of oral microorganisms that are beneficial and can be pathology in individual immunocompromised. There are about 1011 bacteria per milligram of dental plaque attached to the tooth surface, although not all that much harm as it takes the normal flora of the oral cavity6. In a state of decreased immunity, originally oral bacteria may well turn into pathogens and spread in the body causing systemic infections. Progress in the identification of oral bacteria increasingly convince the relationship between the incidence of infections of the mouth, gums and teeth with various systemic diseases such as heart disease, diabetes and stroke. It is increasingly clear that the oral cavity may be the origin of the spread of bacteria and other microorganisms to other organs in the human body.
Bacteria is one of the factors that cause periodontitis through its ability to induce tissue damage through stimulation of inflammatory cells. Inflammatory cells are capable of damaging tissue by releasing destructive enzymes, Reactive Oxygen Species (ROS) and pro-apoptotic factors2. One of various kinds of microorganisms grows in the oral cavity as a source of pathology in the mouth is an Aa bacteria.
These bacteria are classified as pathogenic gram-negative bacteria because their outer membrane is composed of lipopolysaccharide (LPS)1. LPS is an endotoxin and a glycolipid complex composed of polysaccharides (hydrophilic) and lipid A (hydrophobic)4.
Aa bacteria found in dental plaque and periodontal pockets and in excess amounts will cause various diseases. Previous studies have confirmed that Aa can impair many types of host cells. There is evidence for the ability of this organism to invade endothelial cells and epithelial3. So it can be said that Aa induced Reactive Oxygen Species (ROS) in the oral cavity producing free radicals that affect the increment levels of MDA, tissue damage and changes in protein profiles.
Production of ROS in the cells causes the oxidation of macromolecules essential protein oxidation, DNA oxidation and changes in the constituent bases of DNA, which then trigger cell and tissue damages10. ROS also causes peroxidation of membrane lipid which one of its final products is MDA. One of the tissues found in the oral cavity possibly affected directly because of excessive ROS production by bacteria Aa is the parotid gland tissue. Tissue parotid gland is the largest salivary gland located under the ear around the maxilla. Excess ROS especially in the oral cavity due to bacteria Aa exposure can directly stimulate the tissue damage by infecting and damaging the tissue. Aa bacteria in the body react with the blood, affecting the whole system of the body.
Based on some information aforementioned, this study is aimed to examine the effect of Aa bacteria on the expression of iNOS and MDA level of parotid gland using white rats (Rattus norvegicus) as experimental animals. This type of rat is chosen because of ideal species for toxicology testing and has similar physiological human5. EXPERIMENTAL
Treatment of Animals
10 males of white rats aged 3 months with weight of 150-180 grams and adapted for 12 days were used. Samples were divided into 2 groups, consisting of 5 rats for each group. Group 1 is the control and treated by injecting 200 L of physiological NaCl (0.9%) whereas group 2 is the Aa rats and treated by injecting 200 L of bacteria Aa 50
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with the dose of 108 CFU/mL. Injection is done twice on day 1 and day 7 in the upper gum located at the left gingiva. On the day 13, the white rat samples were surgered and subsequently their parotid gland and blood serum were harvested. All conditions and handling of animals in this study were conducted with protocols approved by the Brawijaya University Committee on Animal Use and Care (UB 106-UB-PEM).
Harvesting the parotid glands
After surgery on the day 13, parotid gland tissues of white rats are divided into two sections; ¼ part is treated with 10% paraformaldehyde (PFA) as a preparative sample while the remaining part is treated with a phosphate buffer saline-azide (PBS-azide) pH 7.4 for isolation of a protein tissue.
Protein isolation
Parotid gland is cut into small pieces and subsequently added with 1 mL PMSF solution, small amount of quartz sand, and crushed with a mortar placed on the ice block. Then, the homogenates are added with 2 mL PSMF solution and transferred into a sterilized polypropylene tube. The mixture is then homogenized with a vortex vibrator for 10 minutes, sonicated for 10 minutes, and centrifuged at 6000 rpm for 15 min. Furthermore, the supernatant is taken and added with cold absolute ethanol with a ratio of 1:1. The mixture is left overnight for the precipitate formation. After centrifugation for 15 minutes at 10,000 rpm, the precipitate is taken and dried to remove ethanol. Then the precipitate is homogenized with cold Tris-HCl (0.02 M, pH 6.5) with the ratio of 1:1 (w/v).
Preparation of Standard Curve of MDA
Each 100 L of Kit MDA standard (density 0.997 mg/mL) with a series concentration of 0,1,2,3,4,5,6,7 and 8 mg/mL is transferred into micro-tubes, and followed by adding 550 L of distilled water. Then, to each tube 100 L of 10% TCA, 250 L of 1N HCl, and 100 L of Na-thio 1% are added. These solutions are homogenized by vortex and incubated in a water bath at 100 ° C for 30 min, followed by cooling at room temperature. Measurements of each solution were conducted at the wavelength of 533 nm.
Measurement of MDA using Thio Barbituric Acid test (TBA)
Parotid gland is cut into small pieces, crushed using a mortar placed on an ice block and added with 1 mL of NaCl 0.9%. After homogenization, the mixture is transferred into micro tubes and centrifuged at 8000 rpm for 20 min. Then, 100 L of the parotid gland supernatant is taken, and subsequently added with 550 L of distilled water, 100 L of 10% TCA, 250 L of 1N HCl, and 100 L of Na-thio1%. Every addition of reagent solution, the mixture is homogenized by vortex, followed by centrifugation at 500 rpm for 10 min. The supernatant was taken and then incubated in a water bath at 100 ° C for 30 min. After cooling the sample solution to room temperature, the absorbance is measured at 533 nm. The absorbance is then plotted on a standard curve to calculate sample concentrations.
Expression of iNOS Immunohistochemistry method
Staining maxillary parotid tissue preparations involves the binding of antigen- antibody (avidin-biotin). The addition of primary antibody (antibody NOS) conducted for 1 hour at room temperature, and washed in PBS pH 7.4 for 3 x 5 min. Further, secondary antibodies Anti-Rabbit IgG Biotin Labeled were added for 1 hour at room 100
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temperature, and washed in PBS pH 7.4 for 3 x 5 min. SA-HRP (Strep Avidin- Horseradish Peroxidase) were added for 30-60 minutes at room temperature and washed in PBS pH 7.4 for 3 x 5 min. Furthermore, chromogen DAB (3,3- diaminobenzidine tetra hydrochloride) were added for 10-20 minutes at room temperature and washed in distilled water for 3 x 5 min. Then counterstain was conducted with Hematoxylen for 5 minutes at room temperature, washed in distilled water for 3 x 5 min, followed by the mounting with entellan. The observation was preformed using a microscope with 400x magnification. Expression of iNOS Aa is noticeably from a brown color on the tissue preparation.
RESULTS AND DISCUSSION
Levels of MDA white rats parotid gland (Rattus norvegicus) were exposed to Aa MDA is a major reactive aldehyde resulting from the lipid peroxidation reaction and usually used as a marker of oxidative stress. In a condition of high oxidative stress, MDA levels also increase significantly. Analysis of MDA level in rat parotid gland is be measured from each group of rats. MDA level in the parotid gland of rats exposed by Aa bacteria increases due to the LPS existing in the outer membrane of bacteria.
The incorporation of LPS with LBP is generated by dihydrogen phosphate (R- H2PO4) group of LPS and ammonium group (R-NH3+) derived from LBP. At physiological pH (6-7) of the parotid, hydrolysis of R-H2PO4 occurs by releasing hydrogen ion (H+) to form hydrogen phosphate (R-HPO4-). The loss of one hydrogen ion causes a negatively charged oxygen ion (O-). This anion tends to bind with ammonium group (R-NH3+) of LBP to form a new complex called LPS-LBP as displayed in Fig.1. This complex will interact with specific receptors of macrophages to stimulate the production as well as release inflammatory cells7.
The damage of the parotid gland tissue, which is caused by bacteria Aa exposure, would trigger ROS. ROS can deteriorate lipids and proteins as the major components of the tissue. Consequently, a lipid peroxidation reaction occurred and thus the levels of MDA in the parotid gland tissue increased. As shown in Table 1, the MDA level in Aa rats is significantly (P<0.05) rise in comparison to that of control rats.
Phagocytosis may stimulate the excessive production of ROS, which causes the oxidative stress, leading to inflammation. Oxidative stress is an imbalance situation between the amount of oxidants (free radicals) and antioxidants in the body.
Meanwhile, ROS are molecules or atoms that are formed due to the oxygen reduction reaction (O2) and it can be classified based on its radical activity, such as superoxide (O2•), hydrogen peroxide (H2O2) and hydroxyl radical (•OH). ROS serves to damage cells infected by a virus or bacteria. Highly reactive radicals can cause biochemical changes and damage to various components of living cells, such as proteins, lipids, carbohydrates and nucleic. The attachment of free radicals to cells membrane would lead to lipid peroxidation, which is indicated by the increase in MDA level. The latter compound is highly toxic to the cells since it is able to changes cell membrane fluidity and function. The mechanism of MDA formation is shown in Fig 2.
The CC bonds of polyunsaturated fatty acid (PUFA) as the component of lipid in cells membrane is attached by ROS. The double bonds weaken C-H bonds in lipid, causing the reduction of H due to the attachment of OH. As the result, the lipid radicals are formed. Moreover, the lipid radical is oxidized by O2 to form a peroxyl radical. Newly formed peroxyl radical can react with another PUFA by removing H, producing lipid hydro peroxides and other lipid radicals. This process is continuously 150
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propagated in a chain reaction. Lipid hydro peroxide is an unstable compound and it will produce MDA when fragmentation occurred. Additionally, peroxyl radical can also undergo cyclization reaction to produce cyclic peroxide. This radical can be reduced to form hydro peroxide or subsequent cyclization reaction to form the bicyclic peroxide which then produces a molecule analogous to endoperoxide. The latter compound generates MDA formation9. So, the excessive numbers of free radicals increase the lipid peroxidation production followed by the increasing number of MDA.
Expression of iNOS white rats parotid gland (Rattus norvegicus) were exposed to Aa
The expression of inducible Nitric Oxide Synthase (iNOS) causes inflammation in many types of cell as a response to the foreign substances (i.e. virus, bacteria, etc.).
The catalysis of NO by iNOS normally involves the reaction of L-arginine to form L- sitrulin in the presence of NADP as shown in Fig.3
Excess production of NO could damage the cells. iNOS showed NO production in large quantities for immune defense. The presence of iNOS in a tissue can be described by immunohistochemistry, which involves a combination of immunological and histological methods. In immunohistochemistry, method of staining substances or active ingredients in the tissue is based on the basic principles of immunology (binding of the antigen with antibodies). The results of antigen and antibody can be identified on the specimen since the antibody is bound by a marker for visualization, which can indicate the presence of the active ingredient in the tissue.
LPS could stimulate macrophage to produce and release inflammatory cells that causes pathophysiological effects such as infection up to tissue damage. Inflammatory processes in the cell will cause the migration of macrophages and produce cytokines8. Initially, LPS as an exogenous NO will activate macrophages. Then, the activated macrophages produce pro inflammatory cytokines and excessive inflammatory cells.
Consequently, the increase in the effectiveness of the inflammatory enzyme iNOS is obtained. Moreover, iNOS could trigger the formation of free radicals endogenous NO* through the reaction catalyzed by NOS Fig.4. Additionally, the expression formed between antigen and antibody indicates the condition of the infected tissue pathology in the expression of iNOS. As shown in Table 2, it was found that there is a significantly (P<0.05) increase of iNOS expression in Aa rats in comparison to the rats control.
Fig.5 showed expression of iNOS in cells of control rats (A) and Aa rats (B). It was found that there is a small number of at least the brown color shown on the tissue of control rats. This result reveals that there is always a process of regeneration of cells iNOS endogenous to produce NO in maintaining homeostasis in the body. In Aa rats (B), lots of brown spots were detected as the result of high expression of iNOS.
The high expression of iNOS in Aa rats indicates pathological conditions as a consequence of Aa bacteria exposure.
CONCLUSION
Based on the exposure to bacteria Aa known by relation between MDA and iNOS expression in the parotid gland. Levels of MDA for control and Aa rats by T-test respectively, 3.135 ± 0.0436 ppm and 6.307 ± 0.0473 ppm. Meanwhile the numbers of cells that express iNOS is 0.166 ± 0.003% for control rats and increase 2.292 ± 0.038 % for Aa rats.
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ACKNOWLEDGEMENTS
The author would like to thanks for Dr. drg. Rini Devijanti Ridwan, M.Kes which provides all facilities of research and Dr. Sc Akhmad Sabarudin who helped the forming of this manuscript.
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Mathison, P. S. Tobias and R.J. Ulevitch. 1990. Structure and function of lipopolysaccharide binding protein. Science 249, 1429–1431.
8. Tomlinson, J. L. and T.B. Anthony. 2004. Interaction between Lipopolysaccharide and The Intestinal Epithelium. J Am Vet Med Assoc 224(9):1446-1452
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Figure Caption
Figure 1. Reaction Mechanism of formation of LPS-LBP complex Figure 2. MDA formation through lipid peroxidation
Figure 3. Catalysis reaction of NO formation by iNOS
Figure 4. The mechanism of endogenous NO* formation catalyzed by iNOS as a result of inflammation
Figure 5. Parotid gland (Rattus norvegicus) by immunohistochemistry method 200x magnification
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Figures
Figure 1. Reaction Mechanism of formation of LPS-LBP complex
Figure 2. MDA formation through lipid peroxidation 350
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NH NH NH2
COOH NH2
+ O2 iNOS NADPH NADP+
NH
COOH NH2
O NH2
+ NO.
L-arginin L-Sitrulin
Figure 3. Catalysis reaction of NO formation by iNOS
Figure 4. The mechanism of endogenous NO* formation catalyzed by iNOS as a result of inflammation
LPSLPS
Pro inflammatory cytokines Pro inflammatory cytokines
iNOS protein iNOS protein
iNOSiNOS
NO* endogenous NO* endogenous
NO exogenous NO exogenous
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A B
Figure 5. Parotid gland (Rattus norvegicus) by immunohistochemistry method 200x magnification
description:
A. Control rats B. Aa rats 412411
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Tables
Table 1. MDA levels in parotid gland of white rats (n = 5) Treatment
group
Level of MDA (ppm) Control
Aa
3.135±0.0436 6.307±0.0473
Table 2. Profile of the number of cells expression of iNOS in Aa rats and control rats Treatment group
The average number of cells that express iNOS (%)
Control
Aa 0.166 0.003
2.292 0.038 452
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465
