Neurodegeneration resulting from pathogen invasion or tissue damage has been associated with
activation of microglia, and exacerbated by the release of neurotoxic mediators such as pro-inflammatory cytokines,
chemokines and reactive oxygen species. Activation of microglia stimulated by lipopolysaccharide is mediated in
part by GSK-3 signaling molecule. Induced IL-10 expression via GSK-3 inhibition is noteworthy since IL-10 has been
remarkably shown to suppress inflammation. Objectives: We aimed to inactivate microglia through inhibition of
GSK-3 signaling and to determine its effects on the production of pro- and anti-inflammatory mediators. Methods:
LPS-stimulated BV-2 cells were treated with a GSK-3 inhibitor (LiCl, NP12, SB216763 or CHIR99021). Inhibition
of GSK-3 was determined by the phosphorylation status of GSK-3β. The effects of GSK-3 inhibition on microglial
inflammatory response were investigated by examining various mediators and CD200R marker. Production of nitric
oxide (NO), glutamate and pro- and anti-inflammatory cytokines were measured using flow cytometry, Griess assay,
glutamate assay and Cytometric Bead Array (CBA) respectively. Results: GSK-3β signaling in LPS-stimulated microglia
was blocked by GSK-3 inhibitor through increased phosphorylation at Serine 9 residue. GSK-3 inhibitors had also
led to reducing in microglia activity via increased expression of CD200R. Inhibition of GSK-3 also diminished
inflammatory mediators such as nitric oxide (NO), glutamate, pro-inflammatory cytokines (TNF-α and IL-6) and
chemokine, MCP-1. Reduction of pro-inflammatory mediators by GSK-3 inhibitor was coincided with increased
IL-10 production. Conclusions: Suppression of microglia-mediated inflammatory response was facilitated by GSK-3
inhibition with associated increased in IL-10 production.
Introduction: Systemic lupus erythematosus (SLE) has a broad spectrum of clinical presentations. The diagnosis of SLE remains a challenge and largely depends on the presence of several serum autoantibodies including anti-nuclear antibody (ANA), anti-double-stranded DNA antibody (anti-dsDNA) and anti-Smith antibody (anti-Sm). ANA, a highly sensitive but not specific marker is used for SLE screening Anti-dsDNA and anti-Sm are SLE-specific biomarkers but has lower sensitivity of 80% and 30% for SLE, respectively. However, it is noted that there are SLE patients who are persistently negative for SLE-specific autoantibodies. Anti-dsDNA and anti-Sm were reported to be negative in up to 51.2% and 62.4% of SLE, respectively. This limitation can lead to misdiagnosis and halter proper treatment to SLE patients. Previous studies have suggested that cell membrane DNA (cmDNA) can act as a specific target for the autoantibodies in SLE patients. Autoantibodies towards cmDNA (anti-cmDNA) were reported to have promis-ing value as a reliable biomarker for SLE. In this study, we would like to determine the usefulness of anti-cmDNA in diagnosing SLE as compared to the standard SLE-specific autoantibodies. Methods: Serum samples from 83 SLE patients, 86 other connective tissue diseases and 61 healthy subjects were included in this study. The other connec-tive tissue diseases include samples from 10 Sjogren’s syndrome, 56 rheumatoid arthritis, 12 scleroderma and eight mixed connected tissues disease (MCTD) patients. All samples were analysed by indirect immunofluorescence (IIF) technique using Raji cells as substrate to detect the presence of anti-cmDNA. Anti-cmDNA was reported as positive if there was presence of a fluorescent ring, either continuous or punctate. Sera from SLE patients were also tested for anti-dsDNA and anti-Sm antibodies by using enzyme-immunoassays. Results: Anti-cmDNA positivity was highest in SLE (55.4%) than in other connective tissue diseases (9.3%) and healthy subjects (0%). Anti-cmDNA was 100% spe-cific at differentiating SLE from healthy subjects and 90.7% specific at differentiating SLE from other connective tissue diseases. There was no difference in the sensitivity (55.4%) of anti-cmDNA at differentiating SLE from both groups. Anti-cmDNA were present in 46 SLE samples negative for standard SLE-specific autoantibodies. It was detected in 11 (42.3%) of anti-dsDNA, 23 (63.9%) of anti-Sm and 8 (12.9%) of both anti-Sm and anti-dsDNA negative samples. Conclusion: The high specificity of anti-cmDNA detection using IIF method makes it an excellent diagnostic tool for SLE. Anti-cmDNA is potentially a very useful biomarker for SLE with negative anti-dsDNA or/and anti-Sm antibodies.
Introduction:Alternative treatment for cancer from herbal medicine has gained interest due to its benefits on im-mune modulation, improving the survival and quality of life. Mitragyna speciosa (M. speciosa) or Kratom is an indig-enous plant that can be found in Thailand and northern part of Peninsular Malaysia has becomes popular in recent years due to its ability to exhibit the opioid-like effects of analgesia. Mitragynine is the main alkaloid in M. speciosa which is found to reduce gastrointestinal motility and has been used by local communities as traditional treatment for diarrhoea and many other diseases. However, there is lack of scientific evidence to show that M. speciosa has anti-oxidative and anti-cancer properties especially in colorectal cancer. Therefore, our study aims to evaluate the anti-oxidative properties of M. Speciosa methanolic extract (MSME) and its effects on colorectal cancer cell line, SW480. Methods: The anti-oxidant content and scavenging activity of MSME were determined by total phenolic content (TPC) assay and total flavonoid content (TFC) assay as well as 2,2’-azino-bis (3-ethylbenzothiazoline-6-sul-phonic acid) (ABTS) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay respectively. Cytotoxicity and cytokine inhibitory effects of MSME on SW480 cells were determined by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) and cytokine beads array (CBA), respectively. Results: The TPC of MSME (0.1mg/ml = 85.85 ± 8.25 mg GAE/g extract; 1mg/ml = 167.43 ± 13.50 mg GAE/g extract; 10mg/ml = 408.94 ±7.17 mg GAE/g extract) was lower than pterostilbene, the positive control drug (76.37 ± 2.75; 230.52 ± 10.92; 835.44 ± 6.84 mg GAE/g extract). Conversely, the TFC of MSME (0.1mg/ml = 32.17 ± 27.92 mg QE/g extract; 1mg/ml = 347.72 ± 15.97 mg QE /g extract; 10mg/ml = 739.81 ± 5.56 mg QE /g extract) was slightly higher than pteros-tilbene (ND; 212.73 ± 17.92; 700.50 ± 3.47 mg QE/g extract). In DPPH assay, MSME showed comparatively similar antioxidant scavenging activity (IC50=4.34μg/ml) with pterostilbene (IC50=4.393μg/ml). However, MSME showed lower anti-oxidant scavenging activity (IC50=4.26μg/ml) than pterostilbene (IC50=1.556μg/ml) as measured by ABTS assay. In cytotoxicity assay, IC50 of MSME on SW480 cells was determined to be at 1.486 mg/ml. Overexpression of cytokines such as IL-6, IL-8 (CXCR8) and IL-10 could potentially promote tumour cell proliferation, growth and metastasis. Increased production of these cytokines through LPS stimulation in SW480 was slightly reduced by treat-ment with MSME. Conclusion: MSME could have a potential bioactive compound that possesses anti-oxidative and anti-cancer properties that would be beneficial as an alternative treatment of colorectal cancer.
Introduction: Glycogen synthase kinase-3 (GSK-3) is an important immune regulator that controls inflammation via inhibition of its protein kinase activities. Persistent inflammatory responses through the activation of
immune cells and excessive production of immune mediators may cause tissue destruction and implicated in the
development of chronic inflammatory diseases. The objective of this study was to examine the role of Tideglusib,
a GSK-3 inhibitor, in inflammatory responses elicited through macrophage activation by investigating the
expression of cell surface biomarkers and inflammatory molecule levels. Method: The effects of GSK-3 inhibition by Tideglusib on the expression of CD11b and CD40 and secretion of pro-inflammatory cytokines in the
lipopolysaccharide (LPS)-activated macrophage-derived RAW 264.7 cells were determined by flow cytometry, while
the presence of nitric oxide (NO) was determined by Griess assay. Results: Stimulation of RAW 264.7 cells with LPS
increased substantial levels of CD11b and CD40 expressions, and secretion of NO, TNF-α, and MCP-1. However,
the expression of these molecules was suppressed through inhibition of GSK-3. Conclusion: These findings suggest
the significant role of Tideglusib to limit the upregulation of immune responses in activated macrophages, and as a
potential anti-inflammatory drug for the intervention and treatment of inflammatory diseases.