Lixia Guo

Neuroprotection of geniposide against hydrogen peroxide induced PC12 cells injury : involvement of PI3 kinase signal pathway

AbstractAim:Oxidative stress plays a critical role in the pathogenic cascade leading to neuronal degeneration in AD. Consequently, the induction of endogenous antioxidative proteins by antioxidants seems to be a very reasonable strategy for delaying the diseases progression. In previous work, we identified the neurotrophic and neuroprotective effects of geniposide, which result from the activation of glucagon-like peptide 1 receptor (GLP-1R). In this study, we explore the role of PI3 kinase signaling pathway in the neuroprotection of geniposide in PC12 cells.Methods:Cell viability was determined by MTT assay. Apoptosis was detected by Hoechst and PI double staining. The protein expression of Bcl-2 and phosphorylation of Akt308, Akt473, GSK-3β, and PDK1 was measured by Western blot.Results:Geniposide induced the expression of the antiapoptotic protein Bcl-2, which inhibited apoptosis in PC12 cells induced by H2O2, and this effect could be inhibited by preincubation with LY294002, a selective inhibitor of PI3K. Furthermore, geniposide enhanced the phosphorylation of Akt308, Akt473, GSK-3β and PDK1 under conditions of oxidative stress.Conclusion:These results demonstrate that the PI3K signaling pathway is involved in the neuroprotection of geniposide in PC12 cells against the oxidative damage induced by H2O2 in PC12 cells.

Neurotrophic property of geniposide for inducing the neuronal differentiation of PC12 cells.

The emerging data show that the insulinotrophic hormone glucagon‐like peptide‐1(GLP‐1) and its agonist extendin‐4 have neurotrophic function to inducing neuronal differentiation of PC12 cells and prevent neurons damage challenged by oxidative stress. Here, with the model of high throughput screen for GLP‐1 receptor agonists, we screen and identify that geniposide is a novel agonist for GLP‐1 receptor. Furthermore, geniposide induces the neuronal differentiation of PC12 cells with resulting neurites outgrowth; we also observe an increase in expression of growth‐associated protein‐43. U0126, a selective MEK inhibitor, prevents neurites out growth and phosphorylation of mitogen‐activated kinase proteins in PC12 cells induced by geniposide. All these results show that activation of GLP‐1 receptor by geniposide to induce the neuronal differentiation of PC12 cells involves in MAPK signaling cascade.

GLP-1 receptor plays a critical role in geniposide-induced expression of heme oxygenase-1 in PC12 cells

AbstractAim:To explore the role of activation of glucagon-like peptide 1 receptor (GLP-1R) and its relative cell signaling pathway in the cytoprotection of geniposide.Methods:Cell viability was determined by MTT assay. Knockdown of the Glp-1r gene was carried out with shRNA. The levels of HO-1 protein and cAMP response element binding protein (CREB) phosphorylation were measured by Western blotting.Results:Geniposide protected PC12 cells from oxidative damage induced by 3-morpholinosydnonimine hydrochloride (SIN-1) by enhancing the expression of heme oxygenase 1 (HO-1) via the cAMP-PKA-CREB signal pathway. After transfecting PC12 cells with the AB1 enhancer from the HO-1 gene, luciferase activity induced by geniposide increased in a dose-dependent manner, but not in the PC12 cells whose Glp-1r gene was disrupted. Additionally, inhibition of HO-1 activity by Sn-protoporphyrin IX (SnPP) or shRNA-mediated knockdown of Glp-1r decreased the neuroprotection of geniposide in PC12 cells.Conclusion:GLP-1R plays a critical role in geniposide-induced HO-1 expression to attenuate oxidative insults in PC12 cells.

Geniposide Regulates Glucose-Stimulated Insulin Secretion Possibly through Controlling Glucose Metabolism in INS-1 Cells

Glucose-stimulated insulin secretion (GSIS) is essential to the control of metabolic fuel homeostasis. The impairment of GSIS is a key element of β-cell failure and one of causes of type 2 diabetes mellitus (T2DM). Although the KATP channel-dependent mechanism of GSIS has been broadly accepted for several decades, it does not fully describe the effects of glucose on insulin secretion. Emerging evidence has suggested that other mechanisms are involved. The present study demonstrated that geniposide enhanced GSIS in response to the stimulation of low or moderately high concentrations of glucose, and promoted glucose uptake and intracellular ATP levels in INS-1 cells. However, in the presence of a high concentration of glucose, geniposide exerted a contrary role on both GSIS and glucose uptake and metabolism. Furthermore, geniposide improved the impairment of GSIS in INS-1 cells challenged with a high concentration of glucose. Further experiments showed that geniposide modulated pyruvate carboxylase expression and the production of intermediates of glucose metabolism. The data collectively suggest that geniposide has potential to prevent or improve the impairment of insulin secretion in β-cells challenged with high concentrations of glucose, likely through pyruvate carboxylase mediated glucose metabolism in β-cells.

Geniposide Regulates Insulin-Degrading Enzyme Expression to Inhibit the Cytotoxicity of Aβ1-42 in Cortical Neurons

We reported previously that geniposide showed neurotrophic and neuroprotective activities with the activation of glucagons-like peptide 1 receptor (GLP-1R) in neurons. The current study was designed to further investigate the protective effect of geniposide on β-amyloid (Aβ)-induced cytotoxicity. Our results showed that pre-incubation with geniposide prevented Aβ₁₋₄₂-induced cell injury in primary cultured cortical neurons. Geniposide also induced the expression of insulin-degrading enzyme (IDE), a major degrading protease of Aβ, in a dose-dependent manner. Moreover, bacitracin, an inhibitor of IDE, and RNAi on Glp-1r gene decreased the neuroprotection of geniposide in Aβ₁₋₄₂-treated cortical neurons. Our findings indicated that geniposide activating GLP-1R to against Aβ-induced neurotoxicity involved in its regulation on the expression of IDE in cortical neurons, which provided an additional mechanistic insight into the role of GLP-1R in neuroprotection.

Glucagon-like peptide 1 receptor plays an essential role in geniposide attenuating lipotoxicity-induced β-cell apoptosis

β-Cell apoptosis is considered to be a major cause of loss of β cells in diabetes. Geniposide could prevent oxidative stress-induced neuron apoptosis, and improved glucose stimulated insulin secretion by activating glucagon-like peptide 1 receptor (GLP-1R) in INS-1 cells. Here we have investigated whether geniposide can exert a direct effect against pancreatic β-cell lipoapoptosis. The results indicated that pretreatment pancreatic INS-1 cells with geniposide for 7h attenuated palmitate-induced β-cell apoptosis and active caspase-3 expression, but this effect was disappeared at 18 h. Long-term incubation with palmitate decreased GLP-1R expression in INS-1 cells, and exendin (9-39), an antagonist for GLP-1R, inhibited the effect of geniposide on palmitate-induced apoptosis in INS-1 cells. Moreover, geniposide also improved the impairment of GLP-1R signaling through enhancing the phosphorylation of Akt and Foxo1, and increased the expression of PDX-1 in palmitate-treated INS-1 cells. These results suggest that geniposide inhibits early stage of lipotoxicity-induced β-cell apoptosis, and GLP-1R plays a critical role in geniposide counteracting the action of lipotoxicity in INS-1 pancreatic β cells.

Geniposide Suppresses Hepatic Glucose Production via AMPK in HepG2 Cells.

Geniposide is one of the main compounds in Gardenia jasminoides ELLIS and has many pharmacological activities, but its anti-hyperglycemic activity has not yet been fully explored. This study was designed to determine, for the first time, how geniposide from G. jasminoides regulates hepatic glucose production, and the underlying mechanisms. During in vitro study, we found the inhibitory effect of geniposide on the hepatic glucose production is partly through AMP-activated protein kinase (AMPK) activation in HepG2 cells. Geniposide significantly inhibited hepatic glucose production in a dose-dependent manner. AMPK, acetyl coenzyme A synthetase (ACC) and forkhead box class O1 (FoxO1) phosphorylation were stimulated by different concentrations of geniposide. In addition, the enzyme activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were all significantly suppressed. What is important is that these effects were partly reversed by (1) inhibition of AMPK activity by compound C, a selective AMPK inhibitor, and by (2) suppression of AMPKα expression by small interfering RNA (siRNA). In summary, geniposide potentially ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis by modulation of the AMPK-FoxO1 signaling pathway. Geniposide or geniposide-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes on gluconeogenesis.

Development of a cell-based high-throughput peroxisome proliferator-activated receptors (PPARs) screening model and its application for evaluation of the extracts from Rhizoma Coptis

To date, peroxisome proliferator-activated receptors (PPARs) are becoming the new therapeutic targets for the treatment of metabolic diseases, such as Type 2 diabetes, obesity, and cardiovascular disease. In this study, a cell-based high-throughput PPARs (PPARα/β/γ) model was developed for the screening of PPARs agonists. The screening conditions were evaluated through analyzing the expression value of luciferase. Finally, 24 h of drug acting time, 5 times of the dilution factor of luciferase zymolyte, and about 2 × 104 cells/ well on HeLa cells in 96-well plates were used, respectively. Furthermore, the quality of high-throughput screening (HTS) in stability and reliability was evaluated by the Z′-factor. Additionally, different extracts of Rhizoma Coptis and berberine were tested by the developed method. The results suggested that both the EtOAc extract and berberine were able to activate PPARα/β/γ, and Rhizoma Coptis contains potential natural agonists of PPARs besides berberine. In conclusion, the developed HTS assay is a simple, rapid, stable, and specific method for the screening of PPARs natural agonists.

Antitumor effects and mechanisms of total saponin and total flavonoid extracts from Patrinia villosa (Thunb.) Juss

Patrinia villosa (Thunb.) Juss is a Chinese edible herbal widely used in China for treatment of carbuncles, acute appendicitis, hepatitis and stasis for hundreds of years. In this study, the antitumor effects and the possible mechanisms of total saponin extract from P. villosa (SPV) and total flavonoid extract from P. villosa (FPV) were investigated in four cancer cell lines including mouse melanoma cell line B16, MCF-7 human breast cancer cells, Hela human epithelial cervical cancer cells and L1210 mouse lymphocytic leukemia cells. The antiproliferative effects of SPV and FPV on these cells were observed by 3-(4,5-Dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The cell cycle was detected by flow cytometry. The expression of CDK4 and cyclin D1 were measured by western blot. The results of MTT assay suggested that FPV showed much stronger antiproliferative effects on L1210 cells in a dose-dependent manner. On the other hand, SPV showed better antiproliferative effect than FPV on the other three cell lines in a dose-dependent manner. The mechanism of antitumor effect of SPV and FPV might be the inhibition of expression of CDK4 and cyclin D1, and accordingly arrested four cancer cell lines in G0/G1 phase, decreased the number of cells in S phase, and finally induced antiproliferative effect. In summary, pharmacological data obtained from this study suggested that SPV and FPV possessed cancer chemopreventive potential on different types of cancer cells. These results were much more favorable on bioactivity-guided isolations of SPV and FPV.

Possible Role of AMPK/SIRT1 Signaling on Energy Balance in Geniposide- Treated INS-1 Cells

Our previous work showed that in pancreatic INS-1 cells, geniposide exerted a contrary role on both glucosestimulated insulin secretion (GSIS) and glucose uptake and metabolism in the presence of low and high glucose. But the molecular mechanisms are presently not well understood. In the present study, we design to probe the role of AMP-activated protein kinase (AMPK) and NAD+- dependent deacetylase sirtuin-1 (SIRT1) on geniposide regulating GSIS, and analyze the interaction between AMPK and SIRT1 in pancreatic β cells. Our results indicate that geniposide induce the phosphorylation of AMPK and enhance the expression of SIRT1 in the presence of low concentration of glucose, but in the presence of high concentrations of glucose, geniposide played a contrary role on those. Furthermore, Compound C (an AMPK inhibitor) and Ex527 (a potent and selective inhibitor of SIRT1) prevent the effects of geniposide on glucose uptake, ATP production and GSIS in INS-1 cells. Taken together, our findings suggest that AMPK/SIRT1 are associated with the role of geniposide on energy balance in INS-1 cells.