阿魏酸钠对抗谷氨酸诱导的鼠皮层神经元凋亡作用的影响

目的探讨MEK/ERK信号转导通路是否参与了阿魏酸钠(SF)对抗谷氨酸诱导的鼠皮层神经元凋亡作用。方法以谷氨酸诱导大鼠皮层神经元凋亡为模型。采用Westernblot观察Bcl-2、caspase-3、磷酸化ERK1/2表达的改变。结果阿魏酸钠能够显著降低谷氨酸诱导的神经细胞caspase-3的表达,提高Bcl-2、磷酸化ERK1/2的表达。PD98059可以减弱SF的保护作用。结论 MEK/ERK1/2通路参与阿魏酸钠对抗谷氨酸诱导的鼠皮层神经元凋亡。     

阿托伐他汀对谷氨酸引起大鼠神经元损伤保护作用机制的研究

目的探讨阿托伐他汀对谷氨酸所致培养大鼠皮层神经元损伤保护作用的机制。方法 MTT法测定细胞存活率;Hoechst33258核染色观察细胞凋亡的形态学改变;Western blot检测活性的半胱氨酸天冬氨酸蛋白酶-3(caspase-3)和钙蛋白酶Ⅰ蛋白表达水平。结果谷氨酸(100μmol.L-1)可使神经元细胞存活率下降,细胞凋亡百分比明显增加,活性的caspase-3和钙蛋白酶Ⅰ蛋白表达增加。阿托伐他汀明显对抗谷氨酸诱导的神经元存活率下降及细胞凋亡百分比增加,同时明显抑制活性的caspase-3和钙蛋白酶Ⅰ蛋白表达增加。磷酯酰肌醇-3激酶(phos-phoinositide3-kinase,PI3K)/磷酸化蛋白激酶B(protein ki-nase B,Akt)通路特异性阻断剂LY294002(10μmol.L-1)能抑制阿托伐他汀对抗谷氨酸引起神经元细胞存活率下降,细胞凋亡百分比增加及活性caspase-3和钙蛋白酶Ⅰ蛋白表达增加的作用。结论阿托伐他汀能够明显对抗谷氨酸引起的皮层神经元损伤作用,这种作用可能与激活PI3K/Akt信号转导通路有关。     

Carboxymethylated chitosan stimulates proliferation of Schwann cells in vitro via the activation of the ERK and Akt signaling pathways

Proliferation of Schwann cell in the injured peripheral nerve supports axonal regeneration and also is critical for the regeneration of injured nerves. In this publication, carboxymethylated chitosan (CMCS) was studied to determine its capacity (i) to induce proliferation and synthesis of proliferating cell nuclear antigen (PCNA) and (ii) to activate mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) and phosphatidylinositil-3 kinase (PI3K)/Akt signaling pathways in rat Schwann cells. CMCS was found to induce proliferation and PCNA synthesis in Schwann cells in a dose and time dependent manner. CMCS was shown to phosphorylate ERK1/2 and Akt in Schwann cell proliferation. The phosphorylation of ERK1/2 and Akt in Schwann cells was blocked by the MEK inhibitor PD98059 and the PI3K inhibitor wortmannin. In addition, inhibition of the MEK/ERK or the PI3K/Akt signaling pathways significantly decreased the proliferative effects of CMCS in Schwann cells. Overall, the above results indicate that CMCS stimulates proliferation of Schwann cells by activating the intracellular signaling cascades of ERK1/2 and PI3K/Akt.     

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.     

Honokiol-induced neurite outgrowth promotion depends on activation of extracellular signal-regulated kinases (ERK1/2)

We have found that honokiol [4-allyl-2-(3-allyl-4-hydroxy-phenyl)-phenol] can promote neurite outgrowth and mobilize intracellular Ca2+ store in primary cultured rat cortical neurons. In this study, we examined the effects of honokiol on extracellular signal-regulated kinases (ERK1/2) and Akt, and their possible relationship to neurite outgrowth and Ca2+ mobilization. Honokiol-induced neurite outgrowth in the cultured rat cortical neurons was significantly reduced by PD98059, a mitogen-activated protein kinase kinase (MAPKK, MAPK/ERK kinase MEK, direct upstream of ERK1/2) inhibitor, but not by LY294002, a phosphoinositide 3-kinase (PI3K, upstream of Akt) inhibitor. Honokiol also significantly enhanced the phosphorylation of ERK1/2 in a concentration-dependent manner, whereas the effect of honokiol on Akt phosphorylation was characterized by transient enhancement in 10 min and lasting inhibition after 30 min. The phosphorylation of ERK1/2 enhanced by honokiol was inhibited by PD98059 as well as by KN93, a Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor. Moreover, the products of the phosphoinositide specific phospholipase C (PLC)-derived inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG) were measured after honokiol treatment. Together with our previous findings, these results suggest that the signal transduction from PLC, IP3, Ca2+, and CaMK II to ERK1/2 is involved in honokiol-induced neurite outgrowth.     

Targeted molecular strategies for cancer therapy based on the blockage of oncogenic pathways in human tumor cells

Constitutive activation of the extracellular signal-regulated kinase (ERK) and/or phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathways is associated with neoplastic transformation of a variety of human tumor cells. Treatment of such tumor cells with agents that specifically inhibit the activity of mitogen-activated protein kinase/ERK kinase (MEK) or PI3K completely suppresses their proliferation. However, treatment of cells with these inhibitors leads to only a modest increase in apoptotic cell death. Efficient induction of apoptosis is essential for the development of effective cancer chemotherapy. Therefore, we have examined whether specific blockade of these two signaling pathways affects the efficacy of the induction of apoptosis by various anti-cancer agents in tumor cells. We found that MEK inhibitors markedly enhance the efficacy of histone deacetylase inhibitors to induce the generation of reactive oxygen species and apoptosis. This effect was only observed in tumor cells in which the ERK pathway was constitutively activated. Furthermore, we found that PI3K inhibitors selectively and markedly enhanced the efficacy of the induction of apoptosis by doxorubicin. This latter effect was only detected in tumor cells in which the PI3K/Akt pathway was constitutively activated and in which the p53 pathway was functional. These combination treatments provide an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway or PI3K/Akt pathway is constitutively activated. This review gives an overview of the development of new targeted molecular strategies for cancer therapy based on the suppression of the activity of oncogenic pathways involved in the proliferation or survival of tumor cells.     

Andrographolide acts through inhibition of ERK1/2 and Akt phosphorylation to suppress chemotactic migration

We now evaluated the anti-inflammatory mechanisms of andrographolide on complement 5a (C5a)-induced macrophage recruitment in vitro. Andrographolide concentration dependently inhibited cell migration toward C5a with an IC50 of 5.6+/-0.7 microM. With relatively specific kinase inhibitors (PD98059, SB203580, SP600125, wortmannin and LY294002, respectively) the results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol-3-kinase (PI3K) were necessary for C5a-induced migration, whereas c-Jun N-terminal kinase (JNK) was nonessential. Andrographolide significantly attenuated C5a-stimulated phosphorylation of ERK1/2, and of its upstream activator, MAP kinase-ERK kinase (MEK1/2). C5a-activated ERK1/2 phosphorylation was 86+/-9% inhibited by 30 microM andrographolide. Under the same conditions, however, andrographolide failed to affect C5a-stimulated p38 MAPK and JNK phosphorylation. Andrographolide also strongly abolished C5a-stimulated Akt phosphorylation, a downstream target protein for PI3K. These results indicate that inhibition of cell migration by interfering with ERK1/2 and PI3K/Akt signal pathways may contribute to the anti-inflammatory activity of andrographolide.     

Elastin peptides activate extracellular signal-regulated kinase 1/2 via a Ras-independent mechanism requiring both p110gamma/Raf-1 and protein kinase A/B-Raf signaling in human skin fibroblasts

Elastin peptides (EPs) produced during cancer progression bind to the elastin binding protein (EBP) found at the surface of dermal fibroblasts, leading to the expression of collagenase-1 gene. The production of this enzyme involved in stromal reaction is caused by the sustained activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway via cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K). However, the mechanism of these signaling events remains unknown. We show that kappa-elastin (kappaE), a commonly used EP, induces maximum phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)1/2 and ERK1/2 after 30 min. The simultaneous inhibition of PKA and PI3K, by N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H89) and 2-(4-morpholynil)-8-phenyl-4H-1-bemzopyran-4-one (LY294002), respectively, blocked MEK1/2 and ERK1/2 phosphorylation, as did lactose, an EBP antagonist. kappaE induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110gamma is expressed and activated by betagamma-derived subunits from a pertussis toxin-sensitive G protein after fibroblast stimulation. Pertussis toxin also blocks the Raf-1/MEK1/2/ERK1/2 phosphorylation cascade. In addition, we found that B-Raf is expressed in dermal fibroblasts and activated in a PKA-dependent manner after kappaE treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by kappaE was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Ras-independent ERK1/2 activation system in which p110gamma/Raf-1/MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110gamma and B-Raf seem to be important modulators of dermal fibroblasts physiology and should now qualify as therapeutic targets in strategies aiming at limiting elastin degradation contribution to cancer progression.     

Platelet-activating factor increases pH(i) in bovine neutrophils through the PI3K-ERK1/2 pathway

1. Platelet-activating factor (PAF) is known to stimulate a variety of neutrophil activities, including chemotaxis, phagocytosis, degranulation, reactive oxygen species production and intracellular pH increase. The purpose of this study was to investigate the effect of PAF on pH((i)), specifically if these changes in pH are the result of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway activation in bovine neutrophils. 2. PAF caused intracellular alkalinization in 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester-loaded bovine neutrophils. This phenomenon seems to be mediated by amiloride-sensitive Na(+)/H(+) exchange, and is inhibited by WEB2086 (a selective PAF receptor antagonist), genistein (a tyrosine kinase inhibitor), wortmannin and LY294002 (PI3K inhibitors), and PD98059 and UO126 (MEK inhibitors). 3. PAF 100 nm induced an increase in tyrosine phosphorylation of proteins 62, 44 and 21 kDa with a maximum response at 2 min of incubation. 4. Unlike human neutrophils, bovine neutrophils are strongly stimulated by PAF via phosphorylation of ERK1/2 (extracellular-signal-regulated protein kinase) with an EC(50) of 30 and 13 nm, respectively. 5. PAF MAPK activation was also inhibited by WEB2086, pertussis toxin (PTX), genistein, wortmannin, LY294002, PD98059 and UO126 in bovine neutrophils. The ERK1/2 activation is dependent on PI3K pathway, because protein kinase B was phosphorylated by PAF and inhibited by wortmannin and LY294002, but not by U0126. 6. Our results suggest that PAF induces intracellular alkalinization via PI3K-MAPK activation. This effect is upstream regulated by PAF receptor, PTX-sensitive G protein, tyrosine kinase, PI3K and MEK1/2 in bovine neutrophils.     

Stimulation of 90- and 70-kDa ribosomal protein S6 kinases by arginine vasopressin and lysophosphatidic acid in rat cardiomyocytes

Arginine vasopressin (AVP) and lysophosphatidic acid (LPA) have been shown to stimulate protein kinase C (PKC) and mitogen-activated protein (MAP) kinases and the proliferation of vascular smooth muscle cells. However, the actions of these two agents in cardiomyocytes are less well understood. To investigate the signal transduction pathways of AVP and LPA, freshly isolated adult rat cardiomyocytes were examined. Both AVP and LPA induced concentration- and time-dependent stimulation of the phosphotransferase activities of p90 ribosomal S6 kinases (RSK) and their upstream activators, extracellularly regulated kinases (ERK) 1 and 2. The activation of ERK1 and ERK2 by LPA was PKC- and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent. However, AVP-induced activation of RSK2, a downstream substrate of ERK1 and ERK2, was PKC-dependent and PI 3-kinase-independent. AVP and LPA were also observed to increase the phosphotransferase activity of p70 ribosomal protein S6 kinase (p70 S6K) in a time- and concentration-dependent manner. The activation of p70 S6K by LPA and AVP was PI 3-kinase-dependent. PKC was necessary in AVP- but not in LPA-induced activation of p70 S6K. Since RSK and p70 S6K have been implicated in the regulation of translational control of protein synthesis, we concluded that AVP and LPA may stimulate the growth of cardiomyocytes through these two protein kinase cascades.     

Stearic acid protects primary cultured cortical neurons against oxidative stress

AIM: To observe the effects of stearic acid against oxidative stress in primary cultured cortical neurons. METHODS: Cortical neurons were exposed to glutamate, hydrogen peroxide (H2O2), or NaN3 insult in the presence or absence of stearic acid. Cell viability of cortical neurons was determined by MTT assay and LDH release. Endogenous antioxidant enzymes activity[superoxide dismutases (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT)] and lipid peroxidation in cultured cortical neurons were evaluated using commercial kits. {3-[1(p-chlorobenzyl)- 5-(isopropyl)-3-t-butylthiondol-2-yl]-2,2-dimethylpropanoic acid, Na} [MK886; 5 micromol/L; a noncompetitive inhibitor of proliferator-activated receptor (PPAR) alpha], bisphenol A diglycidyl ether (BADGE; 100 micromol/L; an antagonist of PPAR gamma), and cycloheximide (CHX; 30 micromol/L, an inhibitor of protein synthesis) were tested for their effects on the neuroprotection afforded by stearic acid. Western blotting was used to determine the PPAR gamma protein level in cortical neurons. RESULTS: Stearic acid dose-dependently protected cortical neurons against glutamate or H2O2 injury and increased glutamate uptake in cultured neurons. This protection was concomitant to the inhibition of lipid peroxidation and to the promotion activity of Cu/Zn SOD and CAT in cultured cortical neurons. Its neuroprotective effects were completely blocked by BADGE and CHX. After incubation with H2O2 for 24 h, the expression of the PPAR gamma protein decreased significantly (P<0.05), and the inhibitory effect of H2O2 on the expression of PPAR gamma can be attenuated by stearic acid. CONCLUSION: Stearic acid can protect cortical neurons against oxidative stress by boosting the internal antioxidant enzymes. Its neuroprotective effect may be mainly mediated by the activation of PPAR gamma and new protein synthesis in cortical neurons.     

Molecular signalling mediating the protective effect of A1 adenosine and mGlu3 metabotropic glutamate receptor activation against apoptosis by oxygen/glucose deprivation in cultured astrocytes

Astrocyte death may occur in neurodegenerative disorders and complicates the outcome of brain ischemia, a condition associated with high extracellular levels of adenosine and glutamate. We show that pharmacological activation of A(1) adenosine and mGlu3 metabotropic glutamate receptors with N(6)-chlorocyclopentyladenosine (CCPA) and (-)2-oxa-4-aminocyclo-[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), respectively, protects cultured astrocytes against apoptosis induced by a 3-h exposure to oxygen/glucose deprivation (OGD). Protection by CCPA and LY379268 was less than additive and was abrogated by receptor blockade with selective competitive antagonists or pertussis toxin. Both in control astrocytes and in astrocytes exposed to OGD, CCPA and LY379268 induced a rapid activation of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2)/mitogen-activated protein kinase (MAPK) pathways, which are known to support cell survival. In cultures exposed to OGD, CCPA and LY379268 reduced the activation of c-Jun N-terminal kinase and p38/MAPK, reduced the levels of the proapoptotic protein Bad, increased the levels of the antiapoptotic protein Bcl-X(L), and were highly protective against apoptotic death, as shown by nuclear 4'-6-diamidino-2-phenylindole staining and measurements of caspase-3 activity. All of these effects were attenuated by treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which inhibit the MAPK and the PI3K pathways, respectively. These data suggest that pharmacological activation of A(1) and mGlu3 receptors protects astrocytes against hypoxic/ischemic damage by stimulating the PI3K and ERK1/2 MAPK pathways.     

Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells

Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of G(i). AKT activation could be detected 5min after relaxin stimulation, and was still detected after 24h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was G(i)- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to G(i) and activation of both MEK/ERK1/2 and PI3K/AKT pathways.     

Molecular mechanisms of ZD1839 （Iressa）-induced apoptosis in human leukemic U937 cells

Aim： To investigate the molecular mechanisms of ZD 1839-induced apoptosis in human leukemic U937 cells. Methods： The inhibition of human leukemic U937 cell growth was assessed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphnyl-2H-tetrazolim bromide （MTT） assays, lactate dehydrogenase （LDH） release, and cell cycle distribution. The expression of anti- and pro-apoptotic proteins was detected by Western blot analysis. Results： This study demonstrated that ZD1839 induced apoptosis in leukemic U937 cells by the downregulation of Bcl-2, caspase activation and subsequent apoptotic features. Cotreatment with ZD 1839 and the caspase- 3 inhibitor z-DEVD-fmk blocked apoptosis, indicating that caspase-3 activation is at least partially responsible for ZD 1839-induced apoptosis. The ectopic expression of Bcl-2 attenuated caspase-3 activation, PARP cleavage, and subsequent indicators of apoptosis, including sub-G1 DNA content and LDH release. These results indicate that the downregulation of Bcl-2 plays a major role in the initiation of ZD1839-induced apoptosis, and that the activation of a caspase cascade is involved in the execution of apoptosis. Furthermore, ZD1839 treatment triggered the activation of p38 mitogen-activated protein kinase （MAPK） and the downregulation of c-Jun-N-terminal kinase （JNK）, extracellular signal-regulated kinase （ERK） and phosphatidyl inositol 3-kinase （PI3K）/Akt. The inhibition of the ERK and PI3K/Akt pathways also significantly increased cellular death. Conclusion： ZD 1839 activated caspase-3 and the inhibited Bcl-2 in human leukemic U937 cells through the downregulation of the ERK and PI3K/Akt pathways.     

Extracellular taurine induces angiogenesis by activating ERK-, Akt-, and FAK-dependent signal pathways

Taurine, a non essential sulfur-containing amino acid, plays a critical role in cardiovascular functions. We here examined the effect of taurine on angiogenesis and its underlying signal pathway. Taurine treatment increased angiogenesis in vitro and in vivo, which was followed by activation of the phosphatidylinositol 3-kinase (PI3K)/Akt, MEK/ERK, and Src/FAK signaling pathways. Further, taurine promoted endothelial cell cycle progression to the S and G2/M phases by up-regulating the positive cell cycle proteins, particularly cyclins D1 and B, as well as down-regulating the negative cell cycle proteins, p53 and p21(WAF1/CIP1), resulting in Rb phosphorylation. This angiogenic event was inhibited by inhibitors of PI3K and MEK. In addition, a PI3K inhibitor blocked the activation of Akt and ERK, while Akt knockdown did not affect taurine-induced ERK activation, indicating that PI3K is an upstream mediator of both MEK and Akt. Taurine-induced endothelial cell migration was suppressed by Src inhibitor, but not by other inhibitors, suggesting that the increase in cell migration is regulated by Src-dependent pathway. Moreover, inhibition of cellular taurine uptake by β-alanine and taurine transporter knockdown promoted taurine-induced cell proliferation, ERK and Akt activation, and in vivo angiogenesis, suggesting that extracellular taurine induces angiogenesis. However, taurine did not induce vascular inflammation and permeability in vitro and in vivo. These data demonstrate that extracellular taurine promotes angiogenesis by Akt- and ERK-dependent cell cycle progression and Src/FAK-mediated cell migration without inducing vascular inflammation, indicating that it is potential use for the treatment of vascular dysfunction-associated human diseases.     

Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways

Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H(2)O(2)), superoxide dismutase (SOD) (scavenger of O(2)(?-)), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H(2)O(2) or O(2)(?-) showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.     

阿托伐他汀通过激活PI3K/Akt/mTOR信号转导而促进神经元突起生长

目的探讨阿托伐他汀(Ato)对体外培养大鼠皮质神经元突起生长促进作用的信号转导机制。方法 取培养7 d大脑皮质神经元,分为Ato 10μmo.lL-1作用48 h组和阻断剂+Ato组,先分别加入阻断剂PD98059 50μmo.l L-1、LY294002 30μmol.L-1、曲西立滨(TCBN)2.5μmol.L-1和西罗莫司(雷帕霉素,Rapa)100 nmo.l L-1作用1 h,再加入Ato共同作用48 h。应用倒置相差显微镜观察神经元突起生长状况;Western印迹法检测磷酸化的磷酸肌醇依赖激酶1(PDK1)、磷酸化蛋白激酶B(Akt)、磷酸化西罗莫司靶蛋白(mTOR)、磷酸化的核糖体S6激酶(p70S6K)和磷酸化的真核翻译起始因子4E结合蛋白1(p-4E-BP1)的表达。结果 形态学观察结果显示,Ato 10μmo.lL-1组可明显促进突起生长,表现为突起总长度增加、一级突起数目增多、末端分支数增多及胞体面积增大。PD98059,LY294002,TCBN和Rapa均可阻断Ato对神经元突起生长的促进作用。Western印迹结果显示,Ato 10μmo.lL-1可显著上调p-PDK1,p-Akt(Ser473),p-mTOR,p-p70S6K和p-4E-BP1蛋白表达水平(P<0.01)。LY294002可显著阻断Ato引起的p-PDK1,p-Akt(Ser473)蛋白表达水平增加(P<0.01)。TCBN可显著阻断Ato引起的p-mTOR蛋白表达水平增加(P<0.01)。Rapa可明显阻断Ato引起的p-p70S6K和p-4E-BP1蛋白表达水平增加(P<0.01)。结论 Ato对体外培养皮质神经元突起发育的促进作用可能与激动MEK/ERK信号转导通路有一定的关系,主要可能与通过激活PI3K/Akt/mTOR信号转导通路有关。     

Major role of the PI3K/Akt pathway in ischemic tolerance induced by sublethal oxygen-glucose deprivation in cortical neurons in vitro

Ischemic preconditioning can provide protection to neurons from subsequent lethal ischemia. The molecular mechanisms of neuronal ischemic tolerance, however, are still not well-known. The present study, therefore, examined the role of MAPK and PI3K/Akt pathways in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. Ischemic tolerance was simulated by preconditioning of the neurons with sublethal 1-h OGD imposed 12 h before lethal 3-h OGD. The time-course studies of relative phosphorylation and expression levels of ERK1/2, JNK and p38 MAPK showed lack of their involvement in ischemic tolerance. However, there were significant increases in Akt phosphorylation levels during the reperfusion period following preconditioned lethal OGD. In addition, Bcl-2 associated death promoter (Bad) and GSK-3β were also found to be inactivated during that reperfusion period. Finally, treatment with an inhibitor of PI3K, wortmannin, applied from 15 min before and during lethal OGD abolished not only the preconditioning-induced neuroprotection but also the Akt activation. Concomitant with blockade of the Akt activation, PI3K inhibition also resulted in activation of Bad and GSK-3β. The results suggest that ischemic tolerance induced by sublethal OGD preconditioning is primarily mediated through activation of the PI3K/Akt pathway, but not the MAPK pathway, in rat cortical neurons.     

Dexamethasone inhibits basic fibroblast growth factor-stimulated gastric epithelial cell proliferation

Basic fibroblast growth factor (bFGF) is essential for gastric ulcer healing, whereas glucocorticoids delay gastric ulcer healing. We found that dexamethasone inhibited bFGF-stimulated rat gastric epithelial RGM-1 cells proliferation and attempted to elucidate the possible mechanistic pathway. Flowcytometry was used to determine cell proliferation. Western blot and RT-PCR were performed to evaluate changes in signaling pathways. Results showed that bFGF significantly increased mRNA expression of FGF receptor (FGFR)1 and FGFR2 at 10 min and increased expression of phosphorylated extracellular signal-regulated kinase (pERK1/pERK2) but not phosphorylated p38 mitogen-activated protein kinase (MAPK) or phosphorylated phosphatidylinositol 3-kinase (PI3K) within 30 min. This was followed by an increase of cyclooxygenase (COX)-2 mRNA and protein expression at 30 and 240 min, respectively. Mitogen-activated protein kinase kinase (MEK) inhibitor-PD98059 (10(-5) M) markedly suppressed bFGF-stimulated COX-2 expression and cell proliferation, but neither p38 MAPK inhibitor-SB203580 nor PI3K inhibitor-Wortmannin had any effect. Dexamethasone (10(-6)M) substantially reduced bFGF-stimulated ERK activation at 10 min, COX-2 mRNA and protein expression at 30 and 240 min, respectively, and prostaglandin E(2) synthesis at 8 h. Dexamethasone (10(-6) M) also significantly decreased mRNA expression of FGFR1 and FGFR2 at basal and bFGF-stimulated conditions at 10 min. This study indicated that bFGF-stimulated gastric epithelial RGM-1 cells proliferation via up-regulating FGFR1 and FGFR2, activating ERK1/ERK2 signal transduction pathway and COX-2 pathway. Dexamethasone significantly suppresses bFGF-stimulated RGM-1 cells proliferation in part via down-regulation of FGFR1/FGFR2, then decreasing bFGF-stimulated activation of ERK1/ERK2, followed by inhibition of COX-2 activation, and finally DNA synthesis.