AIM OF THE STUDY: To determine the inhibitory properties of FD aqueous extract on pro-inflammatory mediators involved in lipopolysaccharide (LPS)-induced microglial cells.

METHODS: Vitexin and isovitexin in the extract were quantified via high performance liquid chromatography (HPLC). The extract was evaluated for its cytotoxicity activity via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Pre-treatment with the extract on LPS-induced microglial cells was done to determine its antioxidant and anti-neuroinflammatory properties by measuring the level of reactive oxygen species (ROS), nitric oxide (NO), tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) via 2'-7'-dichlorofluorescin diacetate (DCFDA) assay, Griess assay and Western blot respectively.

RESULTS: The extract at all tested concentrations (0.1 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL) were not cytotoxic as the percentage viability of microglial cells were all above ~80%. At the highest concentration (100 μg/mL), the extract significantly reduced the formation of ROS, NO, TNF-α, IL-1β and IL-6 in microglial cells induced by LPS.

MATERIALS AND METHODS: The influx of immune cells, release of pro-inflammatory cytokines and subsequent accumulation of synovial fluid (swelling) and pain manifest into the disease. The present study used CFA induced Balb/c mice model and treated them intraperitoneally with andrographolide and dexamethasone (used as a positive control) on alternate days for six days. After 6 days, blood and peritoneal macrophages were collected to evaluate the expression of various arthritic markers and paw edema was measured on all days.

RESULTS: The in vitro and ex vivo experiments showed that andrographolide treated animal group had reduced paw edema, cell cytotoxicity and nitric oxide production than dexamethasone treated animal group. Further, the study revealed the mechanistic role of andrographolide in treatment of arthritis by suppressing battery of molecules like COX-2, NF-κB, p-p38, CD40, TNF-α, IL-1β and IL-6 involved in arthritis.

METHODS: The effects of LPS-induced NLRP3 activation in the presence or absence of MCC950, NLRP3-specific inhibitor, was tested on a panel of three pancreatic cancer cell lines (SW1990, PANC1 and Panc10.05). Western blotting, cell viability kits and ELISA kits were used to examine the effects of LPS-induced NLRP3 activation and inhibition by MCC950 on NLRP3 expression, cell viability, caspase-1 activity and cytokine IL-1β, respectively.

RESULTS: LPS-induced inflammation in the presence of ATP activates NLRP3 that subsequently increases pancreatic cancer cell proliferation by increasing caspase-1 activity leading to overall production of IL-1β. The inhibition of the NLRP3 inflammasome activation via the specific NLRP3 antagonist MCC950 was able to reduce the cell viability of pancreatic cancer cells. However, the efficacy of MCC950 varies between cell types which is most probably due to the difference in ASC expressions which have a different role in inflammasome activation.

METHODS: Release of interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and production of prostaglandin E2 (PGE2) were determined by using enzyme-linked immunosorbent assay (ELISA). Immunoblot technique was executed to determine the activation of MAPKs molecules, NF-κB, PI3K-Akt and cyclooxygenase-2 (COX-2) protein. Determination of pro-inflammatory cytokines and COX-2 relative gene expression levels was by performing the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). A reversed-phase HPLC method was developed and validated to standardize the T. crispa extract and chemical profiling of its secondary metabolites was performed by LC-MS/MS.

RESULTS: Qualitative and quantitative analyses of chromatographic data indicated that syringin and magnoflorine were found as the major components of the extract. T. crispa-treatment prompted activation of NF-κB by enhancing IKKα/β and NF-κB (p65) phosphorylation, and degradation of IκBα. The extract upregulated COX-2 protein expression, release of pro-inflammatory mediators and MAPKs (ERK, p38 and JNK) phosphorylation as well as Akt dose-dependently. T. crispa extract also upregulated the upstream signaling adaptor molecules, toll-like receptor 4 (TLR4) and MyD88. T. crispa-treatment also upregulated the pro-inflammatory markers mRNA expression.