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The Ability of Earthworm Lumbricus Rubellus Extract in Slowing Down the Activation of Nfkb and TNF-Α in Lipopolysaccharide-Induced Rattus Norvegicus

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The Ability of Earthworm Lumbricus Rubellus Extract in Slowing Down the Activation of Nfkb and TNF-Α in Lipopolysaccharide-Induced Rattus Norvegicus ORIGINAL ARTICLE Bali Medical Journal (Bali Med J) 2019, Volume 8, Number 2: 347-352 P-ISSN.2089-1180, E-ISSN.2302-2914 The ability of earthworm Lumbricus rubellus extract ORIGINAL ARTICLE in slowing down the activation of NFkB and TNF-α in lipopolysaccharide-induced Rattus norvegicus Published by DiscoverSys CrossMark Doi: http://dx.doi.org/10.15562/bmj.v8i2.1405 Neiny Prisy Foekh,1 I Dewa Made Sukrama,2 Anak Agung Wiradewi Lestari3* Volume No.: 8 ABSTRACT Background: Earthworm (L. rubellus) contains many compounds such Treatment was performed for 14 days. The containment of NFkB and Issue: 2 as Lumbricin I, G-90 glycoprotein, and polyphenols as antimicrobial, TNF-α was measured on the 16th day using quantitative technique antioxidant, and hepatoprotective toward bacterial infections. Tumor sandwich enzyme immunoassay (ELISA). necrosis factor (TNF-α) has been identified as a mediator for necrosis Results: The results of the Shapiro-Wilk test and Levene’s test showed tumor inside animal serum treated by lipopolysaccharide (LPS). High that the data were normally distributed and homogeneous, with First page No.: 347 concentrated LPS can cause tissue damage and death. The increasing p > 0.05. As the data were normally distributed, the one way ANOVA number of Nuclear Factor Kappa Beta (NFkB) activation has been was used to analyze the data, followed by the Least Significant responded by macrophage to produce cytokines pro-inflammation Difference (LSD) test. There was a significant difference between the TNF-α as an indicator of inflammation. average amount of TNFα found in each treatment group p = 0.001 P-ISSN.2089-1180 Methods: The research was conducted in the Animal Laboratory (< 0.05). There were also significant differences between the average Pharmacology Unit, Biomolecular Laboratorium, Faculty of Medicine amount of NFkB found in each treatment group p = 0.001 (< 0.05). Udayana University and the Pathology Clinic Sanglah General Hospital Advanced test using LSD shows more significant differences by p < 0.05. E-ISSN.2302-2914 with a post-test only control group design. Thirty-two experimental Conclusion: It can be concluded that earthworm extract (L.rubellus) rats were made as samples and divided into four groups, which are raises the amount of NFkB and TNF-α in male experimental rats negative control, positive control, treatment 1, and treatment 2. injected with LPS. Keywords: Earthworm extract, Lumbricus rubellus, lipopolysaccharide, NFkB, TNF-α Cite this Article: Foekh, N.P., Sukrama, I.D.M., Lestari, A.A.W. 2019. The ability of earthworm Lumbricus rubellus extract in slowing down the activation of NFkB and TNF-α in lipopolysaccharide-induced Rattus norvegicus. Bali Medical Journal 8(2): 347-352. DOI:10.15562/bmj.v8i2.1405 1Master Program of Biomedicine, INTRODUCTION Faculty of Medicine, Udayana University, Bali, Indonesia. Infectious disease is a major problem in different contributes to the systemic inflammatory response.6 2Department of Microbiology, tropical countries around the world, including LPS is a membrane component of gram-negative Faculty of Medicine, Udayana Indonesia. A clinical syndrome that occurs due bacteria, which is a pathogenic cause of sepsis.7 University, Sanglah General Hospital, Bali, Indonesia. to the body's excessive systemic inflammatory Several types of antibiotics such as chloram- 3Department of Clinical Pathology, response to infection is called sepsis,1 which is also phenicol, ampicillin, and cotrimoxazole have been Faculty of Medicine, Udayana referred to as Systemic Inflammatory Response utilized for quite a long time for the treatment of University, Sanglah General Syndrome (SIRS), particularly in the intestines, typhoid fever until the resistance issue emerged, Hospital, Bali, Indonesia. lungs, kidneys, and other organs.2 In the digestive known as the multidrug-resistant (MDR) S. thypi. tract, sepsis prompts the intestinal hypoperfusion The causes of resistance include irrational adminis- as inflammatory disorders. tration, consumption of antibiotics that are not in Sepsis is caused by gram-negative bacteria (60 – accordance with guidelines, and intrinsic changes in 70% of cases) and gram-positive bacteria including the microbes themselves. The issue of antibiotic resis- Staphylococci and Pneumococci (20 – 40% of cases). tance is what underlies the researchers to attempt to *Correspondence to: On the other hand, fungi, virus, or protozoa were find additional medicinal ingredients, specifically Anak Agung Wiradewi Lestari, also reported to cause sepsis.3 Sepsis is the leading an ingredient from the Indonesian nature. One of Department of Clinical Pathology, 4 Faculty of Medicine, Udayana cause of mortality in the world. The occurrence of the natural ingredients obtained from animals that University, Sanglah General Hospital, sepsis continues to rise in the extent to the human can be used as an additional medicine is earth- Bali, Indonesia population.5 worm (Lumbricus rubellus). Extracts of earthworm [email protected] Over the last 30 years, numerous investigations (Lumbricus sp) contain antibacterial properties that on sepsis have been carried out at no little cost. can inhibit the growth of pathogenic bacteria from Received: 2018-12-01 Endotoxin utilizing Lipopolysaccharide (LPS) is both gram-positive and gram-negative bacteria. Accepted: 2019-01-23 usually used in sepsis models. Endotoxin originates Cytokines are mediators (in the form of proteins Published: 2019-08-01 from one component of another cell wall, which also or glycoproteins) produced by cells in inflammatory Open access: www.balimedicaljournal.org and ojs.unud.ac.id/index.php/bmj 347 ORIGINAL ARTICLE or immunologic responses that serve as signals The sample size in this study was estimated between cells to form communication networks in based on standard procedures in determining the the immune response. Included in cytokines are number of samples using mouse animal models. interleukin (IL-1, IL-2), interferon (IFN α, β, and Based on the calculation, the minimum number ᵧ), Tumor Necrotic Factor (TNF), growth factors of replications (r) required was six mice per group. and chemokines (chemotactic cytokines). Almost The study comprised of 4 groups: (1) no treatment all inflammatory processes result in the activation group, (2) the positive control group, (3) treatment of tissue macrophages and the infiltration of blood group, and (4) treatment group; subsequently the monocytes. total number of rats required was 24. To anticipate Cytokines serve to maintain the inflammatory dropouts, the sample size was increased to 32 rats to processes to be always interconnected. For exam- allow adjustment for an attrition rate of 10%. ple, in sepsis syndrome, endotoxins produced by bacteria and other toxic products will stimulate the Study variable production of TNF-α and IL-1. The above condi- The measured variables in this study were inde- tions occur in not only infection but also other pendent variables and dependent variables. The pathobiology processes. The cytokine stimulation independent variable was earthworm extract of depends on the characteristics of the cell and the Lumbricus rubellus. The dependent variables were type of tissue. NFkB and TNF-α levels in the blood of LPS-induced Nuclear Factor Kappa Beta (NFkB) can be acti- rats (Rattus norvegicus). The controlled variables vated by various stimuli, cytokines, and LPS, which were gender, age, rat weight, temperature, humid- results in the release of inhibitory kB binding so that ity, nutrition, and cage. the NFkB is within the cell nucleus and regulates various inflammatory mediators such as TNF-α. Research procedure TNF-α acts as a mediator of endogenous inflam- This was an experimental study with a posttest- mation and directs numerous cellular responses, only control group design (Federer, 2008). The including the activation of genes involved in mice were kept in a well-ventilated room with an immune inflammation and regulation, cell prolif- adequate light source and were singly-confined. eration, antiviral response, growth inhibition, and Wistar strain male mice were adapted for one week cell death. in a room with a temperature of around 20°C and Complications that occur are usually due to an a cage measuring 23 cm x 17 cm x 9.5 cm. Food imbalance of pro- and anti-inflammatory mediator and drinks were given ad libitum in the form of production. Prolonged production of anti-inflam- pellets and mice food. The lighting was regulated matory mediators could result in harm to some with a 12-hour bright-dark cycle (the bright period tissues and organs. The cytokine is an inflammatory started at 6:00 a.m. to 6:00 p.m.). mediator. Inflammatory reactions that occur in the The animal models were assigned to four groups. digestive tract can instigate endothelial cell death The negative control group (P0) consisted of six (necrosis). mice that were given standard food and drink, Regarding the above prelude, this study aimed while the positive control group (P1) consisted of to investigate the ability of earthworm extracts in six mice that were injected with LPS on the first day inhibiting the activation of NFkB and TNFα in and given standard food and drink until day 14. In lipopolysaccharide-induced Rattus norvegicus. This treatment group 1 (P2), subjects were injected with study used animal models of white rats (Rattus LPS on the first day and given earthworm extract norvegicus) because it is a standard model that is until day 14, whereas in treatment group 2 (P3), often utilized to examine the mechanism of infec- subjects were given earthworm extracts on days 1-6, tion caused by LPS exposure.8,9 and on day-7, they were injected with LPS followed with earthworm extract until day 14. MATERIALS AND METHODS Blood sampling was done using anesthesia with 0.3 ml of ketamil per mouse. Blood was taken using Animal models 3 – 4 cc syringe and collected in a vacuum tube with The study subject was male rats (Ratus norvegicus; a gel separator. Blood sampling was carried out at Wistar strain), which were 3-4 months old with the medical canthus of the orbital sinus because a body weight of 200-250 grams.
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