Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells.

AIM: To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells. METHODS: Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca(2+) was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit. RESULTS: The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC(50)) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular [Ca(2+)](i), loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3. CONCLUSION: Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway, and produces a slight cytotoxicity to L02 cells.

Anti-inflammatory flavonoids from Cryptocarya chingii.

Six flavonoids named cryptogiones A-F, and nine known compounds were isolated from an ethanol extract of stems of Cryptocaryachingii. The structures of the compounds were elucidated by interpretation of comprehensive spectroscopic data and X-ray analysis. A majority of these flavonoids contained an acetic acid/lactone moiety, a possible taxonomic marker. Anti-inflammatory effects of the compounds were evaluated using in vitro assays. At 20 µM concentration, three compounds significantly inhibited TNFα-induced NF-кB activation and LPS-induced IL-1ß expression.

Fumigaclavine C inhibits tumor necrosis factor α production via suppression of toll-like receptor 4 and nuclear factor κB activation in macrophages.

AIMS: Atherosclerosis is the main cause of cardiovascular disease and is widely treated with statins. However, there are a few cases of intolerable adverse reactions by statins; thus, there is still a need for new drugs to prevent atherosclerosis. The inflammation associated with the activation of Toll-like receptor 4 (TLR4) and nuclear factor κB (NFκB) has been shown to be an important factor in the development of atherosclerosis. In the current study, we investigated the anti-inflammatory action of the fungal alkaloid fumigaclavine C (FC), its effects on the TLR4 and NFκB signaling pathway, and its potential relevance as an anti-atherosclerotic agent. MAIN METHODS: The inhibitory effects of FC on tumor necrosis factor α (TNFα) production were determined by enzyme-linked immunosorbent assays (ELISA). The mRNA and protein expression of TLR4 and p65NFκB were detected by quantitative real-time polymerase chain reaction and western blot analysis, respectively. The effect of FC on NFκB was determined using the Dual-Luciferase reporter assay. KEY FINDINGS: FC reduced TNFα production in LPS-stimulated human whole blood and RAW 264.7 macrophages via reduced IκBα phosphorylation associated with the decreased expression of p65NFκB. FC also suppressed LPS-induced TLR4 overexpression at the mRNA and protein level. SIGNIFICANCE: FC attenuated TNFα via the TLR4-NFκB signaling transduction pathway, suggesting that this alkaloid might serve as a promising molecule for anti-inflammatory treatment of atherosclerosis.

Icariin protects against brain injury by enhancing SIRT1-dependent PGC-1alpha expression in experimental stroke.

Icariin (ICA) has neuroprotection in oxygen-glucose deprivation (OGD) neurons by increasing Sirtuin1 (SIRT1). However, little is known about the role of ICA on stroke. SIRT1 is a class III histone deacetylase and activates peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) which stimulates mitochondrial activity. This study aims to investigate the expression of SIRT1 and PGC-1alpha during ICA's neuroprotection against ischemia. In vivo, behavioral test, infarct size and brain water content were evaluated on middle cerebral artery occlusion (MCAO) mouse models treated by ICA/saline. In vitro, primary cortical neurons were tortured by OGD in the presence of ICA or SIRT1 inhibitor III or PGC-1alpha siRNA. Cell viability and mortality were measured by MTT and flow cytometer assay. Knockdown efficiency of PGC-1alpha siRNA was measured by real time PCR. Expressions of SIRT1 and PGC-1alpha were also investigated. In result, neurological scores, infarct size and brain edema were all significantly improved, the cortical expressions of SIRT1 and PGC-1alpha were higher with ICA compared to the control (P < 0.05), and reversed by SIRT1 inhibitor III/PGC-1alpha siRNA. In conclusion, ICA protects against brain ischemic injury by increasing the SIRT1 and PGC-1alpha expression, potentially to be a neuroprotectant for ischemic brain injury.

Human urinary kallidinogenase suppresses cerebral inflammation in experimental stroke and downregulates nuclear factor-kappaB.

The purpose of this study is to investigate the possible mechanism and the neuroprotective effect of human urinary kallidinogenase (HUK) in cerebral ischemia. The mouse middle cerebral artery occlusion (MCAO) model was used. Mice were treated with HUK (20 PNAU/g per day, intravenous) or saline as control, from the beginning of reperfusion to 72 h. Neurological deficits, infarct size, and BWC were measured at 6, 24, 48, and 72 h after MCAO, respectively. Pathological changes of brain were observed by TUNEL assay. Inflammatory factors were measured by real-time PCR and western blotting. Activation of MAPKs, Akt, and nuclear factor-kappaB (NF-kappaB) was detected by western blotting. Our results indicated that HUK significantly improved neurofunction, decreased infarct size, and suppressed edema, as well as inflammatory mediators as compared with the vehicle group. Furthermore, HUK inhibited the NF-kappaB pathway and activated the MAPK/ERK pathway in this neuroprotection.

Alteration of Aß metabolism-related molecules in predementia induced by AlCl3 and D-galactose.

The purpose of this study was to look for alterations in ß-amyloid peptide (Aß) metabolism-related molecules in predementia, the early stage of Alzheimer's disease (AD). AlCl3 (Al) and d-galactose (D-gal) were used to induce the mouse model for predementia and AD. Protein expression of ß-amyloid (Aß), ß-secretase (BACE1), neprilysin (NEP), insulin degrading enzyme (IDE) and receptor for advanced glycation end products (RAGE) in the brain was measured. The results indicated that Al + D-gal induced an AD-like behavioral deficit at 90 days. The period from 45 to 75 days showed no significant behavioral deficit, and we tentatively define this as predementia in this model. A significant increase in BACE1 and decreasing NEP characterized days 45­90 in the cortex and hippocampus. However, high Aß occurred at day 60. IDE increased from day 60 to day 75. There was no change in RAGE. The results suggest that the observed changes in BACE1, NEP and Aß in predementia might relate to a different stage of the AD-like pathology, which may be developed into useful biomarkers for the diagnosis of very early AD.