Macrophages provide the first line of defense against Shigella flexneri infection in the gastrointestinal tract by inducing a variety of inflammatory and antimicrobial responses. Secondary metabolites of plants are used as drugs against infections that are resistant to common antibiotics. In this study, the innate effects of asiaticoside on the proinflammatory activity of mouse macrophages infected with S. flexneri were investigated. The viability of the infected mouse macrophages were examined using viability assay, while the pro-inflammatory cytokines productions were determined using the enzyme-linked immunosorbent assay (ELISA) for determination of IL-1β, IL-12 p40 and TNF-α levels. The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) protein were determined using the Griess assay and western blot, respectively. Statistical analyses were performed using the Statistical Package of Social Sciences (SPSS) software, version 20. The data obtained from independent experiments (n = 3) were presented as the mean ± standard error of mean (SEM). The results showed that, asiaticoside stimulated the infected macrophages by stimulating increased production of TNF-α, IL-12 p40 and NO as well as increased expression of iNOS in a dose-dependent manner. In contrast the viability of the cells and the production of IL-1β and were reduced also in a dose-dependent manner when compared to untreated cells. These results indicate that asiaticoside has immunomodulatory effects on the innate immune function of infected macrophages, showing the potential use of this compound to reduce the clinical symptoms of the infections.
Induction of apoptosis is one of the targeted approaches in cancer therapies. As previously reported, natural products can induce apoptosis in in vitro cancer treatments. However, the underlying mechanisms of cancer cell death are poorly understood. The present study aimed to elucidate cell death mechanisms of gallic acid (GA) and methyl gallate (MG) from Quercus infectoria toward human cervical cancer cell lines (HeLa). The antiproliferative activity of GA and MG was characterised by an inhibitory concentration using 50% cell populations (IC50) by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Cervical cancer cells, HeLa, were treated with GA and MG for 72 h and calculated for IC50 values. The IC50 concentration of both compounds was used to elucidate the apoptotic mechanism using acridine orange/propidium iodide (AO/PI) staining, cell cycle analysis, the Annexin-V FITC dual staining assay, apoptotic proteins expressions (p53, Bax and Bcl-2) and caspase activation analysis. GA and MG inhibited the growth of HeLa cells with an IC50 value of 10.00 ± 0.67 µg/mL and 11.00 ± 0.58 µg/mL, respectively. AO/PI staining revealed incremental apoptotic cells. Cell cycle analysis revealed an accumulation of cells at the sub-G1 phase. The Annexin-V FITC assay showed that cell populations shifted from the viable to apoptotic quadrant. Moreover, p53 and Bax were upregulated, whereas Bcl-2 was markedly downregulated. Activation of caspase 8 and 9 showed an ultimate apoptotic event in HeLa cells treated with GA and MG. In conclusion, GA and MG significantly inhibited HeLa cell growth through apoptosis induction by the activation of the cell death mechanism via extrinsic and extrinsic pathways.
Enhanced resonance search (ERS) is a nondestructive testing method that has been created to evaluate the quality of a pavement by means of a special instrument called the pavement integrity scanner (PiScanner). This technique can be used to assess the thickness of the road pavement structure and the profile of shear wave velocity by using the principle of surface wave and body wave propagation. In this study, the ERS technique was used to determine the actual thickness of the asphaltic pavement surface layer, while the shear wave velocities obtained were used to determine its dynamic elastic modulus. A total of fifteen locations were identified and the results were then compared with the specifications of the Malaysian PWD, MDD UKM, and IKRAM. It was found that the value of the elastic modulus of materials is between 3929 MPa and 17726 MPa. A comparison of the average thickness of the samples with the design thickness of MDD UKM showed a difference of 20 to 60%. Thickness of the asphalt surface layer followed the specifications of Malaysian PWD and MDD UKM, while some of the values of stiffness obtained are higher than the standard.
Macrophage apoptosis exerts an efficient mechanism in controlling intracellular infection during innate immune response against various pathogens including malaria parasites. This study was carried out to determine the apoptosis activity in mouse macrophage cell line J774A.1 infected with a Mycobacterium bovis bacille Calmette-Guerin (BCG) clone and a recombinant BCG clone expressing the C-terminus of merozoite surface protein-1 (BCG-MSP1C) of Plasmodium falciparum for 48 h. In this study, a parent BCG cells was used as a control. The nuclear staining with Hoechst 33342 showed that the BCG-MSP1C cells was capable of increasing the nuclear condensation and morphological stages of apoptosis in the infected cells compared to the BCG-infected cells and the lipopolysaccharide (LPS)-stimulated cells. The flow cytometric analysis using Annexin-V and Propidium iodide (PI) staining confirmed that the BCG-MSP1C cells significantly increased the percentage of early apoptotic activity in the infected macrophage higher than the one stimulated by the parent BCG cells and LPS. This apoptotic response corresponded with the reduction of the anti-apoptotic Bcl-2 protein expression and higher p53 expression. The colorimetric assay demonstrated that the BCG cells capable of stimulating higher production of caspase-1, -3, -8 and -9 while the BCG-MSP1C cells stimulated the expression of caspase-1 and -9 in the infected macrophages, suggesting the involvement of mitochondrial-mediated (intrinsic) pathway of apoptosis. In conclusion, both the BCG and BCG-MSP1C cells are capable of inducing macrophage apoptosis activity in the mouse macrophage cell line J774A.1. This mechanism is important for the elimination of pathogens such as malaria parasite during the phagocytosis activity of macrophage. However, the BCG-MSP1C cells showed higher apoptosis activity than those produced by the parent BCG cells.