The Indonesian Biomedical Journal

139-47

Authors: Nor Raihan Mohammad Shabani, Che Muhammad Khairul Hisyam Ismail, Chiuan Herng Leow, Munirah Mokhtar, Kirnpal Kaur Banga Singh, Chiuan Yee Leow

BACKGROUND: Shigella is a Gram-negative rod-shaped intracellular bacterial pathogen that causes bacterial dysentery or shigellosis among children under five years old. Antibiotics have been less effective in treating shigellosis due to the multi-drug resistance of Shigella. Therefore, an effective vaccine is urgently needed to prevent this disease. The present study aims to determine the peptides presented by major histocompatibility complex (MHC) class II molecules of Shigella-infected macrophages using mass spectrometry-based immunopeptidomics approaches. The MHC class II-associated peptides derived from Shigella-infected macrophages are candidates for developing subunit-based Shigella vaccine. METHODS: THP-1-derived macrophages were infected with Shigella flexneri 2a at the multiplicity of infection equal to 10. The lysate was immunoprecipitated and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The sequences retrieved were analyzed using bioinformatics tools. RESULTS: The Shigella-infected THP-1-derived macrophages contained sample peptides from source proteins of almost all subcellular localizations. Eight peptides from S. flexneri 2a-infected macrophages were predicted to be localized at the outer membrane proteins (OMPs) of S. flexneri 2a by the PSORTb server. Two of the OMP-associated peptides were predicted as antigenic, non-allergenic, and non-toxic by respective bioinformatics tools. CONCLUSION: The findings in this study showed two selected OMPs have great potential for vaccine development against shigellosis.

156-63

Authors: Hiqmah Yusi Yana, Lisna Hidayati, Nastiti Wijayanti, Tri Rini Nuringtyas

BACKGROUND: Aquilaria malaccensis has been consumed as herbal medicine, and in vitro study showed that the leaf extract possesses high antioxidant activities. A brief preliminary study indicated that A. malaccensis showed a promising immunomodulatory activity when evaluated using latex beads. This current study aimed to evaluate the immunomodulatory activity of A. malaccensis leaf extract on the macrophage, which was challenged with pathogenic bacteria Staphylococcus aureus. METHODS: Bioactivity was determined by evaluating the phagocytic capacity of macrophages isolated from Mus musculus against S. aureus. First, the cytotoxicity of extracts on macrophages was evaluated using MTT assays, and the IC50 value was used to determine the dose of immunomodulatory assays. The highest toxicity was observed on chloroform extract with an IC50 value of 111.4 µg/mL. Therefore, the treatment was 100 and 50 µg/mL. Two parameters, including the phagocytic activity and phagocytic capacity of macrophages infected with S. aureus, were used to evaluate immunomodulatory activity. The analysis of variance was done at p<0.05 to determine the significant difference among treatments. RESULTS: Chloroform and ethanol extracts at a 50 µg/mL concentration showed the best results with the phagocytic activity of 82.33%±9.61% and 80.33±1.53%. The ethyl acetate showed lower phagocytic activities of 70.67±1.53. All extracts significantly increased phagocytic activity and phagocytic capacity, and the results differed significantly between negative and positive controls. Thin-layer chromatography indicated that the extract contained terpenoid, flavonoid, phenolic, and tannin. CONCLUSION: A. malaccensis leaf extracts showed immunomodulatory activity. Both chloroform and ethanol extracts showed comparable activity, while the ethyl acetate extract was lower. The extracts contained diverse bioactive compounds that may support activating macrophage cells for immunomodulatory activity.

172-9

Authors: Ferdian Ramadhan, Jamaluddin Jamaluddin, Ismi Zuhria, Luki Indriaswati, Evelyn Komaratih

BACKGROUND: Pterygium is an ocular surface disease that often occurs in tropical countries with a high recurrence rate. Matrix metalloproteinase-3 (MMP-3) play a key role in the inflammatory process of pterygium. This study aims to investigate the ability of curcumin and fibrin glue (FG) in suppressing the expression of MMP-3, and whether can be expected as adjuvant therapy to reduce pterygium recurrence. METHODS: Human pterygium fibroblasts (HPF) obtained from primary cultured of pterygium were treated with no treatment, curcumin, mitomycin-C (MMC), and FG. MMP-3 expression was analyzed using immunocytochemistry and the intensity measurement was done using ImageJ software. Cell migration was measured by scratching and stratification of fibroblast culture after cell confluence, and assessed for 48 hours. RESULTS: The expression of MMP-3 were lower in the HPF treated with 100 mol/mL curcumin, 200 mol/mL, and FG (2205.84±86.1 pg/mL, 1002.51±25.22 pg/mL, 1131.55±17.71 pg/mL, respectively) in comparison with untreated HPF (4703.49±108.9 pg/mL). The expression of MMP-3 were significantly different between groups (p<0.001). Cell migration of HPF after scratching with curcumin intervention at 200 mol/mL decrease from 178.67±2.85 (24 hours) to 88.83±1.48 (48 hours). Meanwhile the migration in FG group also decrease from 180.4±2.56 (24 hours) to 72.45±1.25 (48 hours). CONCLUSION: Curcumin and FG able to reduce the expression of MMP-3 and inhibit the migration of HPF cells.

186-92

Authors: Erlina Sih Mahanani, Ikhsan Nur Arifin, Arya Nur Ihsan, Yusrina Lukitasari, Ferry Sandra

BACKGROUND: Parkia speciosa seeds have been reported to have an anticancer property due to the presence of various antioxidant compounds. Since the potential uses of P. speciosa for the tongue cancer has not been clearly disclosed, we conducted a study to investigate anticancer properties of P. speciosa seed ethanol extract (PSSEE) as well as its effect on cardiac cells. METHODS: Tongue cancer rat model were treated with/without Doxorubicin and various concentrations of PSSEE. After treatment, tongue and heart samples were collected and processed further for histological examinations. Tongue epithelium thickness and damaged heart tissues was observed by HE staining, while tongue cancer cell proliferation was assessed by Ki-67 immunohistochemistry. Analyses were performed under an upright light microscope to measure tongue epithelium thickness, state of cancer cell proliferation, and degree of heart tissue damage. RESULTS: Addition of 400 mg/kg body weight (BW) PSSEE to 4.6 mg/kg BW Doxorubicin reduced the average tongue epithelial thickness and Ki-67+ cells number. Upon addition of PSSEE to Doxorubicin, the damage of heart tissue was reduced in a concentration dependent manner. Among all groups, the group of tongue cancer treated with 4.6 mg/kg BW Doxorubicin and 400 mg/kg BW PSSEE had the lowest percentage as well as the lowest degree of heart tissue damage. CONCLUSION: Since addition of PSSEE to Doxorubicin reduced epithelial thickness, number of Ki-67+ cells and heart tissue damage, PSSEE could be a potential co-chemotherapeutic agent for Doxorubicin toward tongue cancer.

199-205

Authors: Bertoka Fajar Surya Perwira Negara, Jae Suk Choi

BACKGROUND: Bifidobacterium is a genus of lactic acid bacteria that lives in the large intestine of humans and animals. The health benefits of this genus are well established; however, the anti-inflammatory activity of this genus, specifically Bifidobacterium lactis, has not been well defined. Therefore, in this study, we evaluated anti-inflammatory activity of B. lactis hydrolysates using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. METHODS: RAW 264.7 cells were cultured using Dulbecco’s Modified Eagle’s Medium in 5 % CO2 incubator at 37 ℃. One µg/mL of LPS was used to stimulate RAW 264.7 cells. Nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were measured to evaluate anti-inflammatory activity of B. lactis hydrolysates. The cytotoxicity of the inhibitor was also measured in present study through 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. RESULTS: The results showed that B. lactis hydrolysates at 25–200 μg/mL inhibited NO production. In concentration-dependent manner, B. lactis hydrolysate showed inhibition of iNOS expression. However, no inhibition on COX-2 expression was observed. The MTS assay of the B. lactis hydrolysates showed no side effects on the cell viability at all concentrations. CONCLUSION: The current study revealed that B. lactis hydrolysates possess specific anti-inflammatory effects by inhibiting iNOS expression without cytotoxicity and therefore could potentially be developed as a new iNOS inhibitor.

211-7

Authors: Ni Ketut Susilawati, I Wayan Putu Sutirta Yasa, Wayan Suardana, Sri Maliawan, I Made Jawi, Achmad Chusnu Romdhoni, Hamsu Kadriyan, I Wayan Gede Artawan Eka Putra, I Gusti Kamasan Nyoman Arijana

BACKGROUND:Purple sweet potato tuber ethanol extract (PSPTEE) has been known to have benefits in various disease, including to prevent nasopharyngeal carcinoma (NPC). However, there has been no research on PSPTEE in preventing the proliferation of nasopharyngeal mucosal epithelial cells induced by formaldehyde through the oxidative stress mechanisms and inflammatory process. Studies have proven the effect of PSP on various cancer cells, but the carcinogenesis process of the nasopharyngeal mucosal epithelium is still limited. Hence, this study aimed to demonstrate the mechanism for preventing histopathology changes with PSPTEE due to formaldehyde exposure. METHODS: Thirty-two formaldehyde-induced Wistar rats were treated with or without 1g/kgBW/day PSPTEE for 16 weeks. Malondialdehyde (MDA) level were examined with spectrophotometry method, while tumor necrosis factor (TNF)-α and p53 with enzyme linked immunosorbent assay (ELISA) method. The histopathology appearance of the nasopharyngeal epithelium was observed with hematoxylin-eosin (HE) staining. RESULTS: MDA and TNF-α levels in control and treatment group were 22.89±2.84 μM; 9.83±0.89 μM and 84.18±11.58 ng/L; 73.92±10.59 ng/L, respectively, and they showed significant difference. Meanwhile, the level of p53 showed no significant difference. Histopathology appearance showed a significant difference. Path analysis for MDA, TNF-α and p53 levels contributed 28.7% to histopathology appearance. MDA have an effect on significant direct effect on TNF-α. TNF-α has a significant direct effect to histopathological appearance. Indirect influence MDA on histopatological appearance were smaller than the direct effect. TNF-α has the greatest influence on histopatological appearance. CONCLUSION: Formaldehyde induces the histopathology appearance in nasopharyngeal epithelium. This study also demonstrates the mechanism to prevent the nasopharynx epithelial histopathology appearance by administration of PSPTEE.