[Protective effect of ligustilide against low potassium induced apoptosis in cultured rat cerebellar granule neurons].

OBJECTIVE: To investigate the effect of ligustilide (LIG) on low potassium-induced apoptosis in primary cultured cerebellar granule neurons (CGN). METHODS: Apoptosis was induced by low potassium in cultured neonatal rat CGN in vitro. The CGN was divided into control/model/CGP54626 + LIG and LIG group. The neuronal viability of each group was measured by MTT assay. The protein expression levels of the key insulin-like growth factor 1 (IGF)-1 signaling effectors,including the phosphorylated IGF-1 receptor (IGF-1R), Akt, ERK1/2, CREB and activated caspase 3 were examined by Western blot analysis. RESULTS: LIG ranging from 2.5 to 20 micromol/L could protect against low potassium-induced apoptosis of CGN ini a concentration-dependent manner. 20 micromol/L LIG significantly induced upregulation of the phosphorylated levels of IGF-1, Akt, ERK1/2 and CREB, and downregulation of cleaved-caspase 3 expression, which could be blocked by a selective gamma-aminobutyric acid B (GABAs) receptor antagonist CGP54626. CONCLUSION: LIG concentration-dependently protects against low potassium-induced apoptosis in CGN at least partly through GABAa receptor activation and its downstream IGF-1 signaling pathway.

Pennogenyl saponins induce cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Pennogenyl saponins, the characterized components of Rhizoma Paridis, have been reported to have anticancer activity through induction of apoptosis or anti-metastasis in cultured cells or animal models. The aim of the study was to evaluate the anticancer properties of four pennogenyl saponins (PS1-PS4) on a panel of human cancer and normal cell lines, and explore the potential mechanisms underlying the selective anticancer effects of the steroidal saponins in cancer cells. MATERIALS AND METHODS: Differences in the anticancer activity of pennogenyl saponins were examined by MTT assay in human cancer cell lines (HepG2 hepatocellular carcinoma cells, UACC-257 melanoma cells, MCF-7 breast and PC-3 prostate cancer cells) and normal human cell lines (L-02 liver cells and HEK293 kidney cells). Flow cytometry analysis, JC-1 staining and western blot analysis were applied to detect the effects of anticancer pennogenyl saponins on apoptosis, cell cycle, and expression and/or activation of main effectors involved in the potential signaling pathways. RESULTS: Among the tested four saponins, only PS1 and PS2 selectively inhibited cell growth in HepG2, MCF-7 and PC-3 cells. Moreover, PS1 and PS2 could significantly induce apoptosis and cell cycle G2/M arrest in HepG2 cells, which were at least associated with activation of mitochondrial caspase-dependent and -independent apoptotic cascades, inhibition of cyclin-dependent kinase 1 and PI3K/Akt signaling pathway, and modulation of mitogen-activated protein kinases. CONCLUSIONS: PS1 and PS2 had potent and selective anticancer activity to breast, liver and prostate cancer cells. Furthermore, the anticancer effects of PS1 and PS2 were associated with induction of apoptosis and blockage of cell cycle progression through multiple targets in HepG2 cells. These findings suggest that PS1 and PS2 can be considered as potential agents for the treatment of some cancers such as hepatoma.

Therapeutic efficacy of a novel non-peptide αvß3 integrin antagonist for pathological retinal angiogenesis in mice.

αvß3 integrin has been reported as a promising therapeutic target for angiogenesis. In the present study, we tested the antiangiogenic activity of 3-[3-(6-guanidino-1-oxoisoindolin-2-yl) propanamido]-3-(pyridin-3-yl) propanoic acid dihydrochloride (GOPPP), a novel non-peptide αvß3 antagonist. Both human umbilical vein endothelial cells (HUVECs) and a mouse model of oxygen-induced retinopathy (OIR) were investigated separately. HUVEC adhesion, proliferation, migration, ERK1/2 and Akt phosphorylation were assessed. C57BL/6 mice were used for the studies in the OIR model. After exposure to 75% oxygen from postnatal day (PD) 7 to PD12, the mice were returned to room air, and GOPPP was intravitreally administered on PD12. Retinal neovascularization was evaluated on PD17. Hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein levels and ERK1/2 phosphorylation were determined by Western blot analysis of retina proteins. GOPPP significantly inhibited the pro-angiogenic effects of vitronectin on HUVECs, including adhesion, proliferation, and migration, and inhibited ERK1/2 and Akt phosphorylation. Retinal neovascularization in the OIR model was significantly suppressed by intravitreal administration of 50 ng GOPPP. The pro-angiogenic factors HIF-1α and VEGF induced by hypoxia were significantly inhibited by GOPPP in OIR mice. GOPPP administration also inhibited ERK1/2 phosphorylation in the OIR model. These results indicate that GOPPP, a novel αvß3 integrin antagonist, may have potential for the treatment of pathological retinal angiogenesis.

Ligustilide ameliorates neuroinflammation and brain injury in focal cerebral ischemia/reperfusion rats: involvement of inhibition of TLR4/peroxiredoxin 6 signaling.

Blocking TLR4/peroxiredoxin (Prx6) signaling is proposed to be a novel therapeutic strategy for ischemic stroke because extracellular Prx6 released from ischemic cells may act as an endogenous ligand for TLR4 and initiate destructive immune responses in ischemic brain. Our previous studies showed that ligustilide (LIG) exerted antineuroinflammatory and neuroprotective effects against ischemic insult, but the underlying mechanisms remain unclear. This study investigated whether the TLR4/Prx6 pathway is involved in the protective effect of LIG against postischemic neuroinflammation and brain injury induced by transient middle cerebral artery occlusion (MCAO) in rats. Intraperitoneal LIG administration (20 and 40 mg/kg/day) at reperfusion onset after MCAO resulted in a reduction of brain infarct size and improved neurological outcome over 72 h. LIG-induced neuroprotection was accompanied by improvement of neuropathological alterations, including neuron loss, astrocyte and microglia/macrophage activation, neutrophil and T-lymphocyte invasion, and regulation of inflammatory mediators expression. Moreover, LIG significantly inhibited the expression and extracellular release of Prx6 and activation of TLR4 signaling, reflected by decreased TLR4 expression, extracellular signal-regulated kinase 1/2 phosphorylation, and transcriptional activity of NF-κB and signal transducer and activator of transcription 3 in the ischemic brain. Our results demonstrate that LIG may provide an early and direct neuroprotection by inhibiting TLR4/Prx6 signaling and subsequent immunity and neuroinflammation after cerebral ischemia. These findings support the translational potential of blocking TLR4/Prx6 signaling for the treatment of ischemic stroke.

Klotho upregulation contributes to the neuroprotection of ligustilide in an Alzheimer's disease mouse model.

Klotho, an aging-suppressor gene, encodes a protein that potentially acts as a neuroprotective factor by modulating insulin-like growth factor 1 signaling and oxidative stress. In the present study, we investigated the potential role of Klotho in the therapeutic effect of ligustilide against Alzheimer's disease (AD)-like neuropathologies and memory impairment in aged senescence-accelerated mouse prone-8 (SAMP8) mice. Ligustilide treatment (10 and 40 mg/kg for 8 weeks, intragastrically) in 10-month-old SAMP8 mice reduced memory deficits, amyloid-ß(1)-42 accumulation, tau phosphorylation, and neuron loss, increased mitochondrial manganese-superoxide dismutase and catalase expression and activity, and decreased malondialdehyde, protein carbonyl, and 8-hydroxydesoxyguanosine levels in the brain. Ligustilide upregulated Klotho expression in the cerebral choroid plexus and serum, decreased Akt and Forkhead box class O1 phosphorylation. Moreover, ligustilide inhibited the insulin-like growth factor 1 pathway and induced Forkhead box class O1 activation in 293T cells along with Klotho upregulation. An inverse correlation was found between Klotho expression and the AD phenotype, suggesting that Klotho might be a novel therapeutic target for age-related AD, and Klotho upregulation might contribute to the neuroprotective effect of ligustilide against AD.

Neuroprotective effect of kaempferol glycosides against brain injury and neuroinflammation by inhibiting the activation of NF-κB and STAT3 in transient focal stroke.

BACKGROUND: Ischemic brain injury is associated with neuroinflammatory response, which essentially involves glial activation and neutrophil infiltration. Transcription factors nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) contribute to ischemic neuroinflammatory processes and secondary brain injury by releasing proinflammatory mediators. Kaempferol-3-O-rutinoside (KRS) and kaempferol-3-O- glucoside (KGS) are primary flavonoids found in Carthamus tinctorius L. Recent studies demonstrated that KRS protected against ischemic brain injury. However, little is known about the underlying mechanisms. Flavonoids have been reported to have antiinflammatory properties. Herein, we explored the effects of KRS and KGS in a transient focal stroke model. METHODOLOGY/PRINCIPAL FINDINGS: Rats were subjected to middle cerebral artery occlusion for 2 hours followed by 22 h reperfusion. An equimolar dose of KRS or KGS was administered i.v. at the beginning of reperfusion. The results showed that KRS or KGS significantly attenuated the neurological deficits, brain infarct volume, and neuron and axon injury, reflected by the upregulation of neuronal nuclear antigen-positive neurons and downregulation of amyloid precursor protein immunoreactivity in the ipsilateral ischemic hemisphere. Moreover, KRS and KGS inhibited the expression of OX-42, glial fibrillary acidic protein, phosphorylated STAT3 and NF-κB p65, and the nuclear content of NF-κB p65. Subsequently, these flavonoids inhibited the expression of tumor necrosis factor α, interleukin 1ß, intercellular adhesion molecule 1, matrix metallopeptidase 9, inducible nitric oxide synthase, and myeloperoxidase. CONCLUSION/SIGNIFICANCE: Our findings suggest that postischemic treatment with KRS or KGS prevents ischemic brain injury and neuroinflammation by inhibition of STAT3 and NF-κB activation and has the therapeutic potential for the neuroinflammation-related diseases, such as ischemic stroke.