Nicotine stimulates human lung cancer cell growth by inducing fibronectin expression

The mechanisms by which tobacco promotes lung cancer remain incompletely understood. Herein, we report that nicotine, a major component of tobacco, promotes the proliferation of cultured non-small cell lung carcinoma (NSCLC) cells; this effect was most noticeable at 5 days. However, nicotine had no effect on apoptosis of NSCLC cells. In experiments designed to unveil the mechanisms for this effect, we found that nicotine also stimulated mRNA and protein expression of fibronectin. Fibronectin is a matrix glycoprotein that regulates important cellular processes (e.g., adhesion, proliferation, and differentiation) and is highly expressed in tobacco-related lung disorders. Of note, reagents against the integrin alpha5beta1 (antibodies, RGD peptides, alpha5 shRNA) blocked the mitogenic effects of nicotine. Thus, nicotine stimulated NSCLC cell proliferation indirectly via fibronectin induction. We then focused on the mechanisms responsible for nicotine-induced fibronectin expression in NSCLC cells and found that nicotine stimulated the surface expression of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), and that alpha-bungarotoxin, an inhibitor of alpha7 nAChR, abolished the nicotine-induced fibronectin response. The fibronectin-inducing effects of nicotine were associated with activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/mammalian target of rapamycin (mTOR) signaling pathways, and were abrogated by inhibitors of ERK (PD98059), PI3-K (LY294002), and mTOR (rapamycin), but not by inhibitors of protein kinase (PK)C (calphostin C) and PKA (H89). These observations suggest that nicotine stimulates NSCLC proliferation through induction of fibronectin, and that these events are mediated through nAChR-mediated signals that include ERK and PI3-K/mTOR pathways. This work highlights the role of fibronectin and alpha5beta1 integrins as potential targets for anti-lung cancer therapies.     

NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells

Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC12 cells. We also found that LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI 3-K), reduced the survival of cells treated with NGF for 24h in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY 294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI 3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal.     

原花青素对Aβ_(25-35)作用下PC12细胞的保护作用

目的:探讨原花青素对Aβ_(25-35)作用下PC12细胞的保护作用及机制。方法:25μmol/L的Aβ_(25-35)作用于PC12细胞48 h,预先加入25、50和100 mg/L的原花青素干预24 h。采用MTT法检测细胞存活率,DCFH-DA单染法检测细胞活性氧簇(ROS)的含量,JC-10单染法检测细胞线粒体膜电位,AnnexinⅤ/PI双染法检测细胞凋亡,Western blot检测凋亡蛋白caspase-3的蛋白水平。结果:原花青素可提高Aβ_(25-35)作用下PC12细胞的存活率,降低胞内ROS含量,阻止线粒体膜电位下降,抑制caspase-3的活化(P0.05或P0.01),从而抑制Aβ_(25-35)诱导的PC12细胞凋亡。结论:原花青素对Aβ_(25-35)作用下的PC12细胞具有明显的保护作用,其作用机制可能是通过清除Aβ_(25-35)诱导产生的ROS而减轻对线粒体膜的损伤作用,从而抑制细胞凋亡的发生。     

PI3K/Akt signaling pathway-induced heme oxygenase-1 upregulation mediates the adaptive cytoprotection of hydrogen peroxide preconditioning against oxidative injury in PC12 cells

Both the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and heme oxygenase-1 (HO-1) create a survival signal against oxidative stress-induced injuries. Although we have demonstrated that hydrogen peroxide (H2O2) preconditioning confers adaptive cytoprotection against oxidative stress-induced injury in PC12 cells, it remains unknown whether these defense systems are involved in the protective effect of H2O2 preconditioning. In the current study, PC12 cells were preconditioned with 100 μM H2O2 for 90 min, followed by 24 h recovery and subsequent exposure to 300 μM H2O2 for further 12 h. The findings showed that preconditioning with 100 μM H2O2 upregulated HO-1 expression. Zinc protoporphyrin IX (ZnPP), a selective inhibitor of HO-1, at a concentration of 15 μM, significantly attenuated H2O2 preconditioning-elicited cytotoxicity, apoptosis, oxidative stress and mitochondrial membrane potential (ΔΨm) loss in PC12 cells. In addition, H2O2 preconditioning enhanced phosphorylation of Akt. Treatment with 25 μM LY294002, a selective inhibitor of PI3K, for 20 min before H2O2 preconditioning blocked not only H2O2 preconditioning-induced HO-1 induction, but also the protective effect of H2O2 preconditioning against cytotoxicity. The present study provides novel evidence for the effect of preconditioning with H2O2 on the induction of HO-1, which contributes to the adaptive cytoprotection of H2O2 preconditioning against oxidative stress-induced cellular injury via a PI3K/Akt-dependent mechanism in PC12 cells.     

Protective effect of 7-difluoromethoxy-5,4'-Di-hydroxyl isoflavone against the damage induced by glutamate in PC12 cells

7-difluoromethoxy-5,4'-Di-hydroxyl isoflavone (dFGEN), prepared by the difluoromethylation of genistein, is an active chemical entity. In this study, our main purpose was to investigate whether dFGEN had an effect on glutamate-induced apoptosis in cultured PC12 cells. The PC12 cells were treated with different glutamate concentrations for 24 h in vitro. The PC12 cells impaired by glutamate were used as the cell model of excitability. Cells were incubated for 30 min with genistein, dFGEN, vitamin E, and exposed to 10 mM glutamate for 24 h. Cell morphology was observed by light microscopy. The growth and proliferation of PC12 cells were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was determined by flow cytome-try (FCM) with propidium iodide (PI) staining. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and the content of malondialdelyde (MDA) were measured by kits, respectively. Acridine orange (AO) staining was used to detect characteristics of cell apoptosis. When PC12 cells were incubated with glutamate for 24 h, cells appeared to have significant changes in shape. The cellular viability was reduced and the apoptotic rate was increased. The levels of LDH and the content of MDA were increased. The activity of SOD was decreased. After PC12 cells were pretreated with dFGEN, dFGEN significantly improved cell morphology, cell growth and proliferation, suppressed apoptosis of cells, reduced the release of LDH, improving SOD activity and decreased MDA content in a concentration-dependent manner. AO staining displayed that apoptosis was decreased. These results suggested that dFGEN has a protective effect against glutamate-induced damage in PC12 cells. dFGEN seemed to have a better protective effect than the lead compound genistein in a concentration-dependent manner. The mechanism of protective effect of dFGEN may be mainly related to its antioxidative activity.     

Subtypes of neuronal nicotinic acetylcholine receptors involved in nicotine-induced phosphorylation of extracellular signal-regulated protein kinase in PC12h cells

Although many kinds of nicotinic acetylcholine receptor (nAChR) subtypes have been reported in the neuronal tissues, subtype differences in the nAChR-mediated intracellular signaling remains obscure. Using nAChR agonists and antagonists, the involvement of nAChRs in extracellular signal-regulated protein kinase (ERK) phosphorylation in PC12h cells was investigated. Cytisine and nicotine induced the phosphorylation of ERKs in a dose-dependent manner, whereas RJR-2403 had no effect. Cytisine, but not RJR-2403, also induced phosphorylation of CREB. Mecamylamine, dextromethorphan and 18-methoxycoronaridine inhibited nicotine-induced ERK phosphorylation with much higher affinity than dihydro-beta-erythroidine and alpha-conotoxin MII. These results suggest the involvement of alpha3beta4 nAChRs in ERK phosphorylation in PC12h cells.     

Epigallocatechin gallate inhibits nitric oxide-induced apoptosis in rat PC12 cells

Nitric oxide (NO) is associated with many pathophysiology of the central nervous system including brain ischemia, neurodegeneration and inflammation. Epigallocatechin gallate (EGCG) is a major compound of green tea polyphenol that has shown the protective activity against neuronal diseases. This study examined the effect of EGCG on NO-induced cell death in PC12 cells. The administration of sodium nitroprusside (SNP), a NO donor, decreased the cell viability and induced apoptosis showing characterization such as cell shrinkage and chromatin condensation as well as subG1 fraction of cell cycles. EGCG inhibited the cytotoxicity and apoptotic morphogenic changes induced by SNP. EGCG attenuated the production of reactive oxygen species (ROS) by SNP, and ameliorated the SNP-induced Bax to Bcl-2 expression ratio leading to apoptosis. In addition, EGCG prevented the release of cytochrome c from the mitochondria into the cytosol as well as the upregulation of the voltage-dependent anion channel (VDAC), a cytochrome c releasing channel, in the mitochondria of SNP-treated cells. EGCG abrogated the activation of caspase-9, caspase-8 and caspase-3 induced by SNP. These results demonstrate that EGCG has a protective effect against SNP-induced apoptosis in PC12 cells by scavenging ROS and modulating the signal molecules associated with cytochrome c, caspases, VDAC and the Bcl-2 family. These findings suggest that EGCG might be a natural neuroprotective substance.     

Up-regulation of epidermal growth factor-receptors (EGF-R) by nicotine in cervical cancer cell lines: this effect may be mediated by EGF

PROBLEM: Over-expression of epidermal growth factor-receptors (EGF-R) has been described in a variety of cancers, including cervical cancer. Nicotine may increase cellular proliferation rates through a mechanism involving EGF or EGF-R. In this study, we ascertain the effect of EGF antibodies on nicotine-enhanced proliferation rates in two cervical cancer cell lines. METHOD OF STUDY: We studied (a) nicotine-induced increase in the cellular expression of EGF-R in human papillomavirus (HPV)-positive ME-180 and HPV-negative HT-3 cervical cancer cell line cultures, using a semi-quantitative immunofluorescent antibody assay; (b) alterations in cellular proliferation in association with changes in EGF-R levels; and (c) the EGF-R mediation by EGF. RESULTS: Nicotine exposure at physiologically attainable plasma concentrations caused increased expression of EGF-R in both cervical cancer cell lines. Up-regulation of EGF-R was associated with increased cellular proliferation. Decreased expression of EGF-R was associated with decreased cellular proliferation. These data were consistent with EGF-R expression as a mechanism for the control of proliferation of the cervical cancer cells. The action of nicotine was abrogated when antibodies to EGF were added, implying that nicotine up-regulation of EGF-R may be mediated by EGF. CONCLUSIONS: Our data show that nicotine-induced proliferation of cervical cancer cells is mediated through EGF-R over-expression and that this action of nicotine utilizes EGF.     

Nicotine upregulates nerve growth factor expression and prevents apoptosis of cultured spinal cord neurons

Modulation of neurotrophic factor expression may constitute an important part of neuroprotective effects of nicotine. Therefore, the effects of nicotine on expression of nerve growth factor (NGF) and its receptor, tyrosine receptor kinase A (trkA), were studied in cultured spinal cord neurons treated with arachidonic acid. Because injury to spinal cord is associated with elevated levels of arachidonic acid, this cell culture system has been developed in our laboratory as an in vitro model of neuronal injury in spinal cord trauma. Treatment with nicotine markedly upregulated NGF mRNA and protein expression in spinal cord neurons. In addition, a 12h treatment with nicotine increased mRNA levels of trkA. Both nicotine and exogenous NGF inhibited arachidonic acid induced apoptosis of spinal cord neurons. However, the blockage of the trkA receptor prevented nicotine-mediated anti-apoptotic effects. The present results indicate that increased expression of NGF may be an important element of the neuroprotective effects of nicotine in injured spinal cord neurons.     

ErbB-4 activation inhibits apoptosis in PC12 cells.

Neuregulins, a large family of polypeptide growth factors, exert various distinctive effects in the nervous system. neuregulins and their receptors are widely expressed in neurons implying important roles in neuronal cell functions. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate neuregulin-induced differentiation. In the present study we demonstrate that in the PC12-ErbB-4 cells, neuregulin rescues cells from apoptosis induced by serum deprivation or tumor necrosis factor (TNF)alpha treatment. The neuregulin-induced survival is comparable to the effect mediated by the neurotrophic factor nerve growth factor (NGF). Both neuregulin and NGF protect cells from apoptosis induced by serum deprivation and TNF alpha treatment. Moreover, neuregulin like NGF induces the survival of neuronal differentiated PC12-ErbB-4 cells. The survival effect of neuregulin is probably mediated by the phosphoinositide 3-kinase (PI3K) and protein kinase B/Akt signaling pathways. Neuregulin induces the activation of PI3K and prolonged activation of protein kinase B/Akt. In addition, inhibition of the PI3K activity prevented the neuregulin-induced survival effect.Taken together, these results indicate that survival induced by neuregulin in PC12-ErbB-4 cells requires PI3K signaling networks.     

S-allyl-L-cysteine selectively protects cultured rat hippocampal neurons from amyloid beta-protein- and tunicamycin-induced neuronal death

S-allyl-L-cysteine (SAC), one of the organosulfur compounds found in aged garlic extract, has been shown to possess various biological effects including neurotrophic activity. In our previous experiments, we found that SAC could protect against amyloid beta-protein (Abeta)- and tunicamycin-induced cell death in differentiated PC12 cells. In the study described here, we characterized the neuronal death induced by Abeta, 4-hydroxynonenal (HNE), tunicamycin, and trophic factor deprivation, and investigated whether and how SAC could prevent this in cultured rat hippocampal neurons. Treatment with SAC protected these cells against Abeta- and tunicamycin-induced neuronal death, which is mediated predominantly through caspase-12-dependent pathway in a concentration-dependent manner. In contrast, it afforded no protection against HNE- and trophic factor-deprivation-induced cell death, which has been shown to be mediated by caspase-3-dependent pathway. SAC also attenuated the Abeta-induced increase of intracellular reactive oxygen species in hippocampal neurons. SAC had no effect on Abeta-induced cell death in cultured cerebellar granule neurons, which was prevented by a caspase-3 inhibitor. These results suggest that SAC could protect against the neuronal cell death that is triggered by ER dysfunction in the hippocampus, and that it has no effect on neuronal cell death that is dependent upon the caspase-3 mediated pathway.     

Glucagon-like peptide-1 (GLP-1) protects against methylglyoxal-induced PC12 cell apoptosis through the PI3K/Akt/mTOR/GCLc/redox signaling pathway

Patients with long-standing diabetes commonly develop diabetic encephalopathy, which is characterized by cognitive impairment and dementia. Oxidative stress–induced neuronal cell apoptosis is a contributing factor. Glucagon-like peptide (GLP)-1 has recently become an attractive treatment modality for patients with diabetes. It also readily enters the brain, prevents neuronal cell apoptosis, and improves the cognitive impairment characteristic of Alzheimer's disease. Therefore, we investigated whether GLP-1 could protect against oxidative stress–induced neuronal cell apoptosis in pheochromocytoma (PC12) cells. PC12 cells were exposed to 1 mM methylglyoxal (MG) or MG plus 3.30 μg/ml GLP-1. Cell apoptosis, expression and phosphorylation of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin/γ-glutamylcysteine ligase catalytic subunit (GCLc), and redox balance were then determined. The data showed that MG induced PC12 apoptosis in accordance with the redox (glutathione (GSH) and GSH/glutathione disulfide [GSSG]) imbalance. GLP-1 protected against this MG-induced apoptosis, which corresponded to the phosphorylation of PI3K, Akt, and mTOR, as well as the upregulation of GCLc and the restoration of the redox imbalance. Inhibitors of PI3K (LY294002), Akt (Akt-I), and mTOR (rapamycin) reduced the GLP-1-induced GCLc upregulation and its protection against MG-induced PC12 apoptosis. The GLP-1-induced redox restoration was also attenuated by rapamycin. In conclusion, the neuroprotective effect of GLP-1 is due to an enhancement of PI3K/Akt/mTOR/GCLc/redox signaling.     

Neuroglobin protects PC12 cells against beta-amyloid-induced cell injury

Excessive accumulation of amyloid beta (Aβ) has been proposed as a pivotal event in the pathogenesis of Alzheimer's disease. Possible mechanisms underlying Aβ-induced neuronal cytotoxicity include excess production of reactive oxidative species (ROS) and apoptosis. Neuroglobin (Ngb), a newly discovered globin in vertebrates that exhibits neuroprotective functions, may have a potential role in scavenging ROS. To examine the potential protective role of Ngb in Aβ-induced cytotoxicity, PC12 cells were treated with Aβ (1-42 fragment) for 24 h. Aβ treatments increased ROS production in PC12 cells. Overexpression of Ngb but not Ngb mutant in the PC12 cells significantly attenuated Aβ-induced ROS production and lipids peroxidation. Furthermore, overexpression of Ngb also attenuated Aβ-induced mitochondrial dysfunction and apoptosis, and promoted cell survival in PC12 cells. Therefore, Ngb may act as an intracellular ROS scavenger, and such antioxidant properties may play a protective role against Aβ-induced cell injury.     

The expression pattern of Nischarin after lipopolysaccharides (LPS)-induced neuroinflammation in rats brain cortex

Objective

To investigate whether Nischarin participated in neuronal apoptosis induced by neuroinflammation and via the phosphatidylinositol 3-kinase (PI3K) and PKB-dependent pathway.

Material

Use of male Sprague–Dawley rats, rat pheochromocytoma (PC12), and murine microglial cells (BV-2). Treatment lipopolysaccharides (LPS) were injected into the brain lateral ventricle of the rat. The BV-2 cells were treated by LPS. The PC12 cells were pretreated by or not pretreated by conditioned media and siRNA.

Methods

Western blotting was used for analyzing the expression level of Nischarin, pAKT, BAD and Bcl-2. Immunohistochemistry and immunofluorescence were used to perform the morphology and localization of Nischarin. The siRNA could down-regulate the protein level of endogenous Nischarin.

Results

The expression level of Nischarin was elevated after LPS injection; meanwhile, Nischarin was located in the neuron. Nischarin was involved in regulating the PI3K/PKB patway.

Conclusion

Nischarin might be involved in mediating the process of PI3K/PKB pathway-dependent neuronal apoptosis. After the silencing of Nischarin in cultured PC12 (pheochromocytoma) by siRNA, these results showed that it would induce a reduction of pAKT and Bcl-2 proteins expression; meanwhile, it induces an increase of BAD and active caspase-3.     

Testosterone up-regulates seladin-1 expression by iAR and PI3-K/Akt signaling pathway in C6 cells

The previous study indicated that DHCR24/seladin-1 was an important neuroprotective effector. However, the molecular mechanisms that androgen modulates the expression of seladin-1 remain incompletely defined. In this paper, we showed that the expression of seladin-1 was significantly increased by testosterone at all concentrations tested at the protein and mRNA levels in C6 cells, the selective AR antagonist flutamide obviously inhibited the effect in a concentration-dependent manner. Furthermore, we found that testosterone significantly increased the phosphorylation level of V-akt murine thymoma viral oncogene (Akt), a key effector of the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway, while a specific PI3-K inhibitor LY294002 obviously prevented the activation of Akt phosphorylation. In addition, the PI3-K inhibitor LY294002 also markedly blocked the up-regulation expression of seladin-1 gene induced by testosterone at the protein and mRNA levels. Collectively, the above results suggested that testosterone regulated the expression of seladin-1 by the intracellular androgen receptor (iAR)-mediated genomic signaling pathway and the non-genomic PI3-K/Akt signaling pathway in C6 glial cells.     

The effect of formalin pretreatment on nicotine-induced antinociceptive effect: the role of mu-opioid receptor in the hippocampus

Nicotine is attractive as an analgesic component despite that its antinociceptive mechanism is not well known until now. In the present study, we examined the antinociceptive effect of nicotine administered supra-spinally on acetic acid-induced visceral pain induction (writhing test), and found that the antinociceptive effect of nicotine was abolished by mu-, delta-, and kappa-opioid receptor antagonist administered i.c.v. In addition, s.c. 5% formalin pretreatment at 5 h, 20 h, 40 h, and 1 week prior to i.c.v. nicotine injection abolished the antinociceptive effect of nicotine in the writhing test, suggesting that s.c. formalin pretreatment induced tolerance to the antinociceptive effect of nicotine in the supra-spinal region. Furthermore, neuronal loss of the hippocampal cornus ammonis (CA) 3 region reduced nicotine-induced an antinociceptive effect in the writhing test. In Western blot assay, we examined s.c. formalin injection down-regulated mu-opioid receptor in the hippocampus after 40 h, and its effect was maintained for 1 week. However, various acetylcholine receptor subunits and delta-, and kappa-opioid receptors were not altered. These results suggest that s.c. formalin pretreatment can contribute to induce tolerance on nicotine-induced antinociception as down-regulating mu-opioid receptor in the hippocampus, especially 40 h after s.c. formalin injection.     

Mesenchymal stem cells inhibition of chronic ethanol-induced oxidative damage via upregulation of phosphatidylinositol-3-kinase/Akt and modulation of extracellular signal-regulated kinase 1/2 activation in PC12 cells and neurons

It is well known that chronic ethanol consumption damages CNS through oxidative stress which results in many dysfunctions. Recently, it has been demonstrated that as a promising strategy to treat several neurological diseases, transplanted bone marrow-derived mesenchymal stem cells (MSCs) can secrete lots of protective factors that in turn promote function recovery. In the present study, we assessed the potential effects of MSCs conditioned medium (MSC-CM) against chronic ethanol-associated damage on PC12 cells and primary cortical neurons. We found that pretreatment with MSC-CM notably improved cell survival, prevented chronic ethanol-associated apoptosis and abolished the robust deterioration in oxidative status. In addition, we also discovered that chronic ethanol exposure induced an inactivation of phosphatidylinositol-3-kinase (PI3K)/Akt and a lasting activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in both PC12 cells and primary cortical neurons which were able to be reversed by MSC-CM. The PI3K inhibitor (LY294002) was able to reduce the antioxidative and cytoprotective effects conferred by MSC-CM, in part, and the ERK1/2 inhibitor (PD98059) was able to elicit significant protection from chronic ethanol cytotoxicity but not rescue the deterioration in oxidative status induced by chronic ethanol. Taken together, these findings provide the first evidence that MSCs might have potent antioxidant action to shield the apoptotic impairment from chronic ethanol exposure in PC12 cells and neurons, which is involved in upregulation of PI3K/Akt and modulation of ERK1/2 activation, at least partially.     

骨髓间充质干细胞分泌基质细胞衍生因子1保护心肌细胞

背景：有研究显示表达CXCR4的干细胞能够沿着基质细胞衍生因子1的浓度梯度迁移到心肌梗死部位再生心肌和血管而改善心脏的功能。 目的：探索间充质干细胞通过其分泌的基质细胞衍生因子1对心肌细胞的保护作用。 方法：收集培养2 d的间充质干细胞条件培养基。在缺氧条件,利用基质细胞衍生因子1受体CXCR4阻断剂AMD3100或PI3-K/Akt途径阻断剂LY294002预处理H9C2细胞后,利用AnnexinV/PI双标法流式细胞术分析间充质干细胞条件培养基作用下H9C2细胞凋亡的变化;Western blotting分析H9C2细胞磷酸化Akt蛋白的表达;RT-PCR分析间充质干细胞基质细胞衍生因子1的表达。 结果与结论：RT-PCR结果显示间充质干细胞表达基质细胞衍生因子1,Western blotting结果显示间充质干细胞条件培养基增加了H9C2细胞磷酸化Akt蛋白的水平。AnnexinV/PI分析发现间充质干细胞条件培养基明显降低了H9C2细胞缺氧复氧后的凋亡,且这种抗凋亡作用能被CXCR4阻断剂AMD3100或PI3-K/Akt途径阻断剂LY294002所阻断。说明间充质干细胞通过其分泌的基质细胞衍生因子1通过激活PI3-K/Akt途径保护H9C2细胞,增加H9C2细胞的幸存能力。     

米诺环素通过调控PI3K/Akt通路抑制硝普钠诱导的PC12细胞凋亡

 目的： 探讨PI3K/Akt信号通路在米诺环素（minocycline,MC）抑制硝普钠(sodium nitoprusside,SNP)诱导的PC12细胞凋亡中的作用。方法：将体外培养的PC12细胞分为4组：空白对照组、SNP组、MC+SNP组和PI3K抑制剂LY294002+ MC+SNP组。用四甲基偶氮唑盐（MTT）法检测细胞活力,流式细胞术检测细胞凋亡;Western blotting检测不同时点（0.5、1、2、3 h）各处理组PI3K/Akt通路蛋白p-Akt和Akt的表达。结果：SNP处理PC12细胞24 h能抑制细胞生长,加入10 μmol/L MC预处理30 min可明显提高细胞活力,降低细胞凋亡率（P<0.05）,抑制SNP诱导的PC12细胞凋亡。MC组的p-Akt表达高于其它组,而加入LY294002后可阻断MC的上述效应。结论：MC可通过调控PI3K/Akt通路抑制SNP诱导的PC12细胞凋亡。