Salidroside protects PC12 cells from MPP-induced apoptosis via activation of the PI3K/Akt pathway

Oxidative stress plays an important role in the pathogenesis of Parkinson’s disease (PD). Salidroside (SAL), a phenylpropanoid glycoside isolated from Rhodiola rosea L., can exert potent antioxidant properties. In this study, we investigated the protective effects, and the possible mechanism of action, of SAL against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell damage in rat adrenal pheochromocytoma PC12 cells. Pretreatment of PC12 cells with SAL significantly reduced the ability of MPP⁺ to induce apoptosis in a dose and time-dependent manner. SAL significantly and dose-dependently inhibited MPP⁺-induced chromatin condensation and MPP⁺-induced release of lactate dehydrogenase by PC12 cells. SAL enhanced Akt phosphorylation in PC12 cells, and the protective effects of SAL against MPP⁺-induced apoptosis were abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation. These findings suggest that SAL prevents MPP⁺-induced apoptosis in PC12 cells, at least in part through activation of the PI3K/Akt pathway.     

Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells

Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gβ1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation, which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gβ1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.     

转录因子Cup2对白念珠菌铜离子代谢、氧化应激调控作用的初步研究

目的 利用白念珠菌（Candida albicans）不同转录因子基因缺失菌,考察参与调控铜离子（Cu²⁺）代谢和氧化应激的重要转录因子。方法 点板实验（spot assay）、生长曲线法。结果 spot assay筛选发现,转录因子Cup2缺失菌Cup2Δ/Δ对Cu²⁺的敏感性增加,进一步研究表明Cup2Δ/Δ在含5 mmol/L的Cu²⁺培养液中生长缓慢;Cup2Δ/Δ对H₂O₂的敏感性也有所增加,而且Cu²⁺协同H₂O₂发挥抑菌作用,在BCS螯合Cu²⁺后,增加了Cup2Δ/Δ和亲本菌SN250对H₂O₂诱导的氧化应激的耐受性。在氟康唑、咪康唑和酮康唑敏感性实验中,Cup2Δ/Δ并未表现出对唑类药物敏感。结论 敲除转录因子Cup2,可增加白念珠菌对Cu²⁺和H₂O₂的敏感性。转录因子Cup2可能参与调控白念珠菌对Cu²⁺的代谢和H₂O₂诱导的氧化应激反应,但并未参与对唑类药物耐药性的调控。     

抑制PI3K/Akt信号通路对HER-2/neu诱导的Ishikawa细胞增殖的影响

目的研究抑制磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路对表皮生长因子受体2(HER-2/neu)诱导的雌激素依赖性子宫内膜癌细胞增殖的拮抗作用。方法①表皮生长因子(EGF)处理Ishika-wa细胞株及转染HER-2/neu的Ishikawa细胞株,蛋白印迹法检测转染细胞前后总Akt(t-Akt)及磷酸化Akt(p-Akt)蛋白、环氧化酶(COX)-2蛋白的表达。酶联免疫吸附试验(ELISA)方法检测细胞培养上清液中雌二醇(E2)的含量。②用PI3K/Akt的抑制剂LY294002抑制信号通路,以不同时间(浓度为20μmol/L时,分别作用10,20,40和60min)和不同浓度(5,10,20和40μmol/L)作用30min后,再次检测COX-2的表达水平,ELISA检测E2水平。结果 HER-2/neu可引起子宫内膜癌细胞Akt活化,经EGF刺激后p-Akt/t-Akt比值、COX-2及细胞上清液中E2的表达量在转染组显著高于未转染组(P<0.05)。应用抑制剂抑制PI3K/Akt通路后,转染HER-2/neu的Ishikawa细胞株中COX-2的表达水平低于正常的Ishikawa细胞株,同时细胞上清液中肿瘤E2的含量于转染HER-2/neu的Ishikawa细胞株明显低于正常Ishikawa细胞株(P<0.05);随药物浓度的增加及作用时间的延长,2组细胞COX-2及E2的表达均逐渐减少,且抑制作用与浓度及作用时间呈依赖关系。结论 HER-2/neu可能通过PI3K/Akt通路来诱导COX-2的转录,进而导致雌激素的分泌增多,使子宫内膜癌细胞无限生长。     

The modulation of PI3K/Akt pathway by 3β hydroxylup-12-en-28-oic acid isolated from Thymus linearis induces cell death in HCT-116 cells

The presence of intricate carbon skeletons in natural compounds enhances their bioactivity spectrum with unique modes of action at several targets in various dreadful diseases like cancer. The present study was designed to purify the molecules from Thymus linearis and elucidate their antiproliferative activity. The compounds were isolated from the active methanolic extract of Thymus linearis through column chromatography and characterized by various spectroscopic techniques. Antiproliferative activity of isolated compounds was evaluated using MTT assay on cancer and normal cell lines. Mechanism of cell death was elucidated using flow cytometric, microscopic, and Western blot analysis. Four compounds, Sitosterol, Chrysin, 3β-hydroxylup-12-en-28-oic acid (3BH), and β-Sitosterol glycoside, were isolated. Among these, 3BH was most potent antiproliferative agent across all cell lines under study, HCT-116 being the most affected one. 3BH was demonstrated to downregulate PI3Ksubunits (p110α and p85α), downstream pAktSer⁴⁷³ and prompted G₁ phase cell cycle arrest. The cell cycle CDK inhibitor p27 and p21 were upregulated with simultaneous downregulation of cyclin D1 and cyclin E in HCT-116 cells. This was accompanied by apoptosis, as depicted by decrease in Bcl-2/Bax ratio, with increase in active caspases-3 and caspase-9, cleavage of PARP-1, the generation of reactive oxygen species (ROS), and the loss of mitochondrial membrane potential. The findings established that 3BH induced cell death in HCT-116 cells by modulating PI3K/Akt signaling axis, impeding cell cycle, and instigating apoptosis.     

bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways

Extensive research has focused on finding effective strategies to prevent or improve recovery from brain ischemia and reperfusion (I/R) injury. The basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some central nervous system (CNS) disorders, including ischemic injury. In this study, we demonstrate that bFGF administration can improve locomotor activity and inhibit the ER stress induced in the CA1 region of the hippocampus in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response proteins CHOP, XBP-1, ATF-6 and caspase-12 that are induced by H(2)O(2) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signaling pathways, PI3K/Akt and ERK1/2. Inhibition of the PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and U0126, respectively, partially reduce the protective effect of bFGF. Taken together, our results indicate that the neuroprotective role of bFGF involves the suppression of ER stress in the ischemic oxidative damage models and oxidative stress-induced PC12 cell injury, and these effects is underlying the activation of the PI3K/Akt and ERK1/2 signal pathway.     

Andrographolide-induced pi class of glutathione S-transferase gene expression via PI3K/Akt pathway in rat primary hepatocytes

Andrographis paniculata is an herb widely used in China, Korea, and India for its anti-hepatotoxic, anti-viral, and anti-inflammatory effects. Andrographolide is the major bioactive diterpene lactone in A. paniculata. The pi class of glutathione S-transferase (GSTP) is one of the phase II biotransformation enzymes. Our previous study indicated that andrographolide upregulates the expression of GSTP. The aim of this study was to investigate the mechanism by which andrographolide induces GSTP gene expression in rat primary hepatocytes. In hepatocytes treated with 40 μM andrographolide, immunoblots showed maximal Akt phosphorylation at 0.5 h and maximal c-jun phosphorylation at 3 h. However, pretreatment with PI3K inhibitors, wortmannin and LY294002, or siPI3K inhibited the andrographolide-induced phosphorylation of c-jun and GSTP protein expression. EMSA showed that pretreatment with wortmannin, LY294002, or siPI3K attenuated the AP-1-DNA-binding activity caused by andrographolide. Results of immunoprecipitation indicated that nuclear c-fos/c-jun heterodimer increases with andrographolide treatment. Addition of antibodies against c-jun and c-fos decreased nuclear protein bound to the AP-1 consensus DNA sequence. In summary, andrographolide induces GSTP gene expression in rat primary hepatocytes through activation of the PI3K/Akt, phosphorylation of c-jun, nuclear accumulation of AP-1, and subsequent binding to the response element in the gene promoter region.     

Kurarinone attenuates hydrogen peroxide-induced oxidative stress and apoptosis through activating the PI3K/Akt signaling by upregulating IGF1 expression in human ovarian granulosa cells

Dysregulated follicular development may lead to follicular atresia, and this is associated with oxidative stress in granulosa cells. Kurarinone is a natural compound possessing multiple activities, including antioxidative ability. However, the role of kurarinone in granulosa cell damage during follicular atresia remains unknown. Human ovarian granulosa KGN cells were treated with hydrogen peroxide (H₂O₂) to induce cellular damage. Cytotoxicity was investigated by lactate dehydrogenase (LDH) release assay. Oxidative stress was evaluated by detection of reactive oxygen species (ROS) generation and oxidative biomarker levels. Cell apoptosis was evaluated by flow cytometry, a Cell Death Detection ELISA Kit, and a Caspase-3 Assay Kit. The downstream target and related signaling pathway were analyzed by western blotting. Kurarinone attenuated H₂O₂-induced LDH release in KGN cells. Kurarinone relieved H₂O₂-induced increase in ROS generation and malondialdehyde level as well as decrease in superoxide dismutase-1 activity and heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 mRNA levels. Kurarinone inhibited H₂O₂-induced apoptosis in KGN cells. Kurarinone targeted insulin-like growth factor 1 (IGF1) and upregulated IGF1 expression to activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling. IGF1 silencing attenuated the suppressive effects of kurarinone on H₂O₂-induced oxidative stress and apoptosis in KGN cells. In conclusion, kurarinone attenuates H₂O₂-induced oxidative stress and apoptosis in KGN cells through activating the PI3K/Akt signaling by upregulating IGF1 expression, indicating the therapeutic potential of kurarinone in follicular atresia.     

The neuroprotective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K/Akt pathway

Pyrroloquinoline quinone (PQQ), a cofactor in several enzyme-catalyzed redox reactions, possesses a potential capability of scavenging reactive oxygen species (ROS) and inhibiting cell apoptosis. In this study, we investigated the effects of PQQ on glutamate-induced cell death in primary cultured hippocampal neurons and the possible underlying mechanisms. We found that glutamate-induced apoptosis in cultured hippocampal neurons was significantly attenuated by the ensuing PQQ treatment, which also inhibited the glutamate-induced increase in Ca2+ influx, caspase-3 activity, and ROS production, and reversed the glutamate-induced decrease in Bcl-2/Bax ratio. The examination of signaling pathways revealed that PQQ treatment activated the phosphorylation of Akt and suppressed the glutamate-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). And inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt cascade by LY294002 and wortmannin significantly blocked the protective effects of PQQ, and alleviated the increase in Bcl-2/Bax ratio. Taken together, our results indicated that PQQ could protect primary cultured hippocampal neurons against glutamate-induced cell damage by scavenging ROS, reducing Ca2+ influx, and caspase-3 activity, and suggested that PQQ-activated PI3K/Akt signaling might be responsible for its neuroprotective action through modulation of glutamate-induced imbalance between Bcl-2 and Bax.     

Salvianolic acid B protects human endothelial progenitor cells against oxidative stress-mediated dysfunction by modulating Akt/mTOR/4EBP1, p38 MAPK/ATF2, and ERK1/2 signaling pathways

The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Protection against reactive oxygen species (ROS)-mediated oxidative damage via antioxidant mechanisms is essential for tissue maintenance and shows therapeutic potential for patients suffering from cardiovascular and metabolic disorders. Salvianolic acid B (SalB), a natural bioactive component known from Traditional Chinese Medicine, has been reported to exert cellular protection in various types of cells. However, the underlying mechanisms involved are not fully understood. Here, we showed that SalB significantly promoted the migratory and tube formation abilities of human bone marrow derived-endothelial progenitor cells (BM-EPCs) in vitro, and substantially abrogated hydrogen peroxide (H₂O₂)-induced cell damage. SalB down-regulated Nox4 and eNOS, as well as nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase expression upon H₂O₂ induction that in turn prevents oxidative-induced endothelial dysfunction. Moreover, SalB suppressed the Bax/Bcl-xL ratio and caspase-3 activation after H₂O₂ induction. Furthermore, our results provide mechanistic evidence that activation of the mTOR/p70S6K/4EBP1 pathways is required for both SalB-mediated angiogenic and protective effects against oxidative stress-induced cell injury in BM-EPCs. Suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways by SalB significantly protected BM-EPCs against cell injury caused by oxidative stress via reduction of intracellular ROS levels and apoptosis. Taken together, by providing a mechanistic insight into the modulation of redox states in BM-EPCs by SalB, we suggest that SalB has a strong potential of being a new proangiogenic and cytoprotective therapeutic agent with applications in the field of endothelial injury-mediated vascular diseases.     

Edible wild vegetable, Gymnaster koraiensis protects retinal ganglion cells against oxidative stress

This study was conducted to determine whether Gymnaster koraiensis is effective at blunting the negative influence of N-methyl-d-aspartate (NMDA) on the retinas of rats and on oxidative stress induced cell death in transformed retinal ganglion cells (RGC-5).The ethyl acetate fraction of G. koraiensis (EAGK) and the isolated compound, 3,5-di-O-caffeoylquinic acid (3,5-DCQA), were shown to significantly attenuate the negative effect of H₂O₂ on the RGC-5 cells tested by various procedures.The inclusion of EAGK or 3,5-DCQA in the culture reduced the reactive oxygen species (ROS) and replenished the reduced glutathione levels caused by various radical species such as H₂O_2, O₂⁻ or OH. Moreover, EAGK or 3,5-DCQA inhibited lipid peroxidation caused by sodium nitroprusside (SNP) in rat brain homogenates.From in vivo experiments, the presence of NMDA in the retina affected the thickness of the inner plexiform layer (IPL) and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) in positive ganglion cells. EAGK or 3,5-DCQA protected the thinning of the IPL and increased TUNEL positive cells in the ganglion cell layer (GCL).Our results clearly demonstrate the neuroprotective effect of EAGK both in vitro and in vivo. Moreover, 3,5-DCQA is suggested to be the active compound of EAGK.     

Daphnetin inhibits TNF‐α and VEGF‐induced angiogenesis through inhibition of the IKKs/IκBα/NF‐κB,Src/FAK/ERK1/2 and Akt signalling pathways

Coumarins, identified as plant secondary metabolites possess diverse biological activities including anti‐angiogenic properties. Daphnetin (DAP), a plant derived dihydroxylated derivative of coumarin has shown significant pharmacological properties such as anticancer, anti‐arthritic and anti‐inflammatory. The present study was performed to investigate the anti‐angiogenic potential of DAP, focusing on the mechanism of action. The in vivo anti‐angiogenic potential of DAP was evaluated by vascular endothelial growth factor (VEGF)‐induced rat aortic ring (RAR) assay and chick chorioallantoic membrane (CAM) assay. For in vitro evaluation, wounding migration, transwell invasion, tube formation and apoptosis assays were performed on VEGF (8 ng/mL)‐induced human umbilical vein endothelial cells (HUVECs). The cellular mechanism of DAP was examined on TNFα (10 ng/mL) and VEGF‐induced HUVECs by extracting the mRNA and protein levels using RT‐qPCR and western blotting. Our data demonstrated that DAP inhibited the in vivo angiogenesis in the RAR and CAM assay. DAP also inhibited the different steps of angiogenesis, such as migration, invasion, and tube formation in HUVECs. DAP inhibited nuclear factor‐κB signalling together including TNF‐α induced IκBα degradation; phosphorylation of IκB kinase (IKKα/β) and translocation of the NF‐κB‐p65 protein. Furthermore, western blotting revealed that DAP significantly down‐regulated the VEGF‐induced signalling such as c‐Src, FAK, ERK1/2 and the related phosphorylation of protein kinase B (Akt) and VEGFR2 expressions. DAP reduced the elevated mRNA expression of iNOS, MMP2 and also, induced apoptosis in VEGF‐stimulated HUVECs by the caspase‐3 dependent pathway. Taken together, this study reveals that DAP may have novel prospective as a new multi‐targeted medication for the anti‐angiogenesis and cancer therapy.     

The compound isolated from the leaves of Phyllostachys nigra protects oxidative stress-induced retinal ganglion cells death

This study was performed to determine whether the compound isolated from Phyllostachys nigra could attenuate oxidative stress in transformed retinal ganglion cells (RGC-5 cells) death.     

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb2+ -induced neuronal death in cultured hippocampal neurons

Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb(2+) causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb(2+). Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb(2+)-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb(2+) treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 microM) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb(2+). And that Pb(2+)-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb(2+) and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.     

The facilitatory actions of snake venom phospholipase A2 neurotoxins at the neuromuscular junction are not mediated through voltage-gated K channels

Electrophysiological investigations have previously suggested that phospholipase A₂ (PLA₂) neurotoxins from snake venoms increase the release of acetylcholine (Ach) at the neuromuscular junction by blocking voltage-gated K⁺ channels in motor nerve terminals.We have tested some of the most potent presynaptically-acting neurotoxins from snake venoms, namely β-bungarotoxin (BuTx), taipoxin, notexin, crotoxin, ammodytoxin C and A (Amotx C & A), for effects on several types of cloned voltage-gated K⁺ channels (mKv1.1, rKv1.2, mKv1.3, hKv1.5 and mKv3.1) stably expressed in mammalian cell lines. By use of the whole-cell configuration of the patch clamp recording technique and concentrations of toxins greater than those required to affect acetylcholine release, these neurotoxins have been shown not to block any of these voltage-gated K⁺ channels. In addition, internal perfusion of the neurotoxins (100 μg/ml) into mouse B82 fibroblast cells that expressed rKv1.2 channels also did not substantially depress K⁺ currents. The results of this study suggest that the mechanism by which these neurotoxins increase the release of acetylcholine at the neuromuscular junction is not related to the direct blockage of voltage-activated Kv1.1, Kv1.2, Kv1.3, Kv1.5 and Kv3.1 K⁺ channels.     

Oxidative stress and alpha1-adrenoceptor-mediated stimulation of the Cl-/HCO3- exchanger in immortalized SHR proximal tubular epithelial cells

BACKGROUND AND PURPOSE: This study evaluated the signalling coupled to the alpha1-adrenoceptor-induced stimulation of the Cl-/HCO3- exchanger in hypertension. EXPERIMENTAL APPROACH: The Na+ -independent HCO3- transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rat (SHR) and their normotensive control (Wistar Kyoto rat; WKY). KEY RESULTS: Noradrenaline increased Cl-/HCO3- exchanger activity with EC50 values of 0.6 and 5.3 microM in SHR and WKY cells, respectively. These effects were abolished by prazosin, but not by yohimbine. Phenylephrine increased Cl-/HCO3- exchanger activity in SHR and WKY cells (EC50 of 2.6 and 4.9 microM, respectively). Phenylephrine-mediated increase in Cl-/HCO3- exchanger activity in WKY and SHR cells was inhibited by protein kinase C (PKC), MAPK/ERK kinase (MEK) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. The expression of alpha1A- and alpha1B-adrenoceptors was identical in WKY and SHR cells. SHR cells generated more H2O2 than WKY cells. In SHR cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H2O2 and abolished their hypersensitivity to phenylephrine, but failed to affect basal Cl-/HCO3- exchanger activity. H2O2-dependent stimulation of Cl-/HCO3- exchange activity was significantly higher in SHR than in WKY cells. CONCLUSIONS AND IMPLICATIONS: Differences between WKY and SHR cells on their sensitivity to alpha1-adrenoceptor stimulation did not correlate with the abundance of alpha1A- and alpha1B-adrenoceptors and may be related to the increased generation of H2O2, which may amplify the response downstream of alpha1-adrenoceptor activation.     

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway

Aim:

(−)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro.

Methods:

BDL rats were treated with EGCG (25 mg·kg⁻¹·d⁻¹, po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-β1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR.

Results:

In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1β, TGF-β1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 μmol/L) dose-dependently suppressed TGF-β1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-β1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-β1-stimulated LX-2 cells. Application of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-β1-stimulated LX-2 cells, but co-application of EGCG and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not produce additive effects.

Conclusion:

EGCG exerts anti-fibrotic effects in BDL rats and TGF-β1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.     

Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As(+3)- and MMA(+3)-induced apoptosis through inhibition of telomerase activity via JNK activation

The effects of six arsenic compounds including As(+3), MMA(+3), DMA(+3), As(+5), MMA(+5), and DMA(+5) on the viability of NIH3T3 cells were examined. As(+3) and MMA(+3), but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As(+3) and MMA(+3) were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As(+3) and MMA(+3) treatments. An increase in the intracellular peroxide level was examined in As(+3)- and MMA(+3)-treated NIH3T3 cells, and As(+3)- and MMA(+3)-induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As(+3)- and MMA(+3)-induced cytotoxicity. Suppression of JNKs significantly inhibited As(+3)- and MMA(+3)-induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As(+3)- and MMA(+3)-induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As(+3) or MMA(+3). These data provide the first evidence to indicate that apoptosis induced by As(+3) and MMA(+3) is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved.     

Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: Insight into the receptor activation switch

A₃ Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A₃ adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A₃ adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N⁶-position. In contrast, the adenosine-5′-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A₃ agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5′-substituents on ligand–target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation.