ANTITUBERCULOSIS ACTION OF URSOLIC ACID AND ITS REGULATION IN C57BL/6 MOUSE BONE MARROW MACROPHAGES INFECTED WITH MYCOBACTERIUM AVIUM

• Main Author: Ayu Eka Pitaloka, Dian
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48732
• Institution: Institut Teknologi Bandung

Tuberculosis (TB) is one of the top ten causes of death worldwide induced by Mycobacterium tuberculosis (MTB). These mycobacteria are known to have immune escape such as macrophages regulation to prevent apoptosis, the formation of granuloma structure, and the ability to dormancy or inactive. Based on these facts, regulation of cytokines or endogenous compounds which are responsible for those process is critically important. The lack of regulatory effects on the macrophage by antituberculosis (anti-TB) drugs also needs attention. Therefore, the discovery of new chemical compounds that are effective in TB treatment urgently needed to kill MTB especially intracellular MTB. Ursolic acid (UA) is a pentacyclic triterpenoid compound which is known to have pharmacological activities. UA isolated from medicinal plants was found to have anti- TB activity, both sensitive and resistant strains of MTB. UA decreased mycolic acid concentration extracted from avirulent of MTB H37Ra. UA is also reported to regulate the Mitogen-Activated Protein Kinase (MAPK) signaling pathway in leukemia cells which is involved in cytokines production. Thus, UA was chosen for further investigation as anti-TB and its regulation on infected macrophages. The purpose of this study was to examine the mechanism of UA as anti-TB and its effect on the macrophages during infection. The investigation started with in vitro anti-TB activity of UA on MTB H37Rv, clinically isolated of isoniazid-ethambutol (HE) resistant of MTB, clinically isolated rifampicin-streptomycin (RS) resistant of MTB, and Mycobacterium avium 724. Colony Forming Unit (CFU) was used to estimate the number of viable bacteria. The study continued with combination study of UA and anti- TB drugs (isoniazid, ethambutol, rifampicin, and streptomycin) to observe the effect of UA on their activity. The result of UA on the morphology of MTB was analyzed using Scanning Electron Microscopy (SEM). Furthermore, the molecular mechanism of UA in MTB was carried out by using molecular docking. Regulation of UA on the MAPK signaling pathway in infected macrophages was determined by analyzing the UA's ability to phosphorylate three MAPK signaling pathways such as Extracellular signalregulated Kinase (ERK) 1/2, Stress-Activated Protein Kinase (SAPK) / c-Jun NH2- terminal Kinase (JNK), and MAPK p38. Mycobacterium avium was used as an infectious agent in this study because of its parallels characteristics of intracellular activity and similarity of modulation macrophages function caused by MTB. The ability of UA and MTB to activate the MAPK pathway in Mycobacterium avium-infected macrophages was also investigated by analyzing the concentration of TNF-?, IL-1?, IL-6, and NO2 -. Homology of the amino acid sequence between InhA protein in MTB and Mycobacterium avium was analyzed using web-based software such as NCBI blast and MultAlin. The acute toxicity test of UA was performed using the zebrafish model. The results showed UA was able to kill all test mycobacteria used at concentration of 50 ?g/ml. Isoniazid, rifampicin, ethambutol, and streptomycin were used as drug control and only appeared to kill H37Rv strain of MTB with concentration 0.2, 40, 2, and 4 ?g/ml respectively. In vitro combination of UA with anti-TB drugs was found to have a synergistic effect of killing H37Rv strain of MTB with the Fractional Inhibitory Concentration Inhibition (FICI) of 0.24-0.5. UA also showed a synergistic effect when combined with rifampicin in killing clinically isolated of RS resistant strain of MTB with FICI value of 0.5. This compound found to cause cell wall damage and holes formation in H37Rv strain of MTB observed with SEM. Isoniazid as drug control also showed loss of cell integrity and rigidity.UA activity on the cell wall is strengthened by the results of UA molecular docking which showed the affinity in InhA enzyme (an enzyme that involve in the synthesis of mycolic acid in the Fatty Acid Synthase II pathway) with the predicted binding energy of -9,30 kcal/mol. Moreover, amino acid sequences of InhA enzyme in MTB and Mycobacterium avium had a similarity percentage of 85.50%. Mycobacterium avium was found to increase the production of TNF-?(9 fold), IL-6 (195 fold), IL-1?(4 fold), and nitrite (4 fold) compare to control, while the treatment of UA was able to inhibit the production of these parameters significantly. The CFU analysis also showed no Mycobacterium avium colonies were found both in cell lysates and supernatant with the lowest concentration of UA 50 ?g / ml. UA has found to increase the concentration of IL-1?(2.5 fold) and nitrite (1.7 fold) in Mycobacterium avium-pretreated macrophages compare to control, but to decrease the concentration of TNF-?(6.6 fold) and IL-6 (4 fold) compare to control. UA was able to inhibit the activation of the ERK 1/2 pathways and MEK (3 fold) pathway, but could activate the SAPK / JNK (8 fold) signaling pathway compare to control in Mycobacterium avium-infected macrophages. The LC50 value of UA in the zebrafish model was 152.5 ± 3.5 mg/l and categorized as non-toxic. In conclusion, this study revealed that UA has a bactericidal effect on MTB by causing cell wall damage which is supported by the result of molecular docking that showed affinity to InhA enzyme. UA was found to regulate MAPK signaling pathways, cytokine production, and nitrite and is expected to increase the ability of anti-TB drugs to eliminate intracellular MTB.