Renin activates PI3K-Akt-eNOS signalling through the angiotensin AT₁ and Mas receptors to modulate central blood pressure control in the nucleus tractus solitarii

BACKGROUND AND PURPOSE

The renin-angiotensin system (RAS) is critical for the control of blood pressure by the CNS. Recently, direct renin inhibitors were approved as antihypertensive agents. However, the signalling mechanism of renin, which regulates blood pressure in the nucleus tractus solitarii (NTS) remains unclear. Here we have investigated the signalling pathways involved in renin-mediated blood pressure regulation, at the NTS.

EXPERIMENTAL APPROACH

Depressor responses to renin microinjected into the NTS of Wistar-Kyoto rats were elicited in the absence and presence of the endothelial nitric oxide synthase (eNOS)-specific inhibitor, N(5)-(-iminoethyl)-L-ornithine, Akt inhibitor IV and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor and GP antagonist-2A [G_q inhibitor]. Lisinopril (angiotensin converting enzyme inhibitor), losartan, valsartan (angiotensin AT₁ receptor antagonists), D-Ala7-Ang-(1-7) (angiotensin-(1-7) receptor antagonist) were used to study the involvement of RAS on renin-induced depressor effects.

KEY RESULTS

Microinjection of renin into the NTS produced a prominent depressor effect and increased NO production. Pretreatment with G_q-PI3K-Akt-eNOS pathway-specific inhibitors significantly attenuated the depressor response evoked by renin. Immunoblotting and immunohistochemical studies further showed that inhibition of PI3K significantly blocked renin-induced eNOS-Ser¹¹⁷ and Akt-Ser⁴⁷³ phosphorylation in situ. In addition, pre-treatment of the NTS with RAS inhibitors attenuated the vasodepressor effects evoked by renin. Microinjection of renin also increased Ras activation in the NTS.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results suggest renin modulated blood pressure at the NTS by AT₁ and Mas receptor-mediated activation of G_q and Ras to evoke PI3K-Akt-eNOS signalling.     

Ursolic acid induces apoptosis in human leukaemia cells and exhibits anti-leukaemic activity in nude mice through the PKB pathway

BACKGROUND AND PURPOSE

Ursolic acid (UA) has been extensively used as an anti-leukaemic agent in traditional Chinese medicine. In the present study, we investigated the ability of UA to induce apoptosis in human leukaemia cells in relation to its effects on caspase activation, Mcl-1 down-regulation and perturbations in stress-induced signalling pathways such as PKB and JNK.

EXPERIMENTAL APPROACH

Leukaemia cells were treated with UA after which apoptosis, caspase activation, PKB and JNK signalling pathways were evaluated. The anti-tumour activity of UA was evaluated using xenograft mouse model.

KEY RESULTS

UA induced apoptosis in human leukaemia cells in a dose- and time-dependent manner; this was associated with caspase activation, down-regulation of Mcl-1 and inactivation of PKB accompanied by activation of JNK. Enforced activation of PKB by a constitutively active PKB construct prevented UA-mediated JNK activation, Mcl-1 down-regulation, caspase activation and apoptosis. Conversely, UA lethality was potentiated by the PI3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Interruption of the JNK pathway by pharmacological or genetic (e.g. siRNA) attenuated UA-induced apoptosis. Furthermore, UA-mediated inhibition of tumour growth in vivo was associated with induction of apoptosis, inactivation of PKB as well as activation of JNK.

CONCLUSIONS AND IMPLICATIONS

Collectively, these findings suggest a hierarchical model of UA-induced apoptosis in human leukaemia cells in which UA induces PKB inactivation, leading to JNK activation and culminating in Mcl-1 down-regulation, caspase activation and apoptosis. These findings indicate that interruption of PKB/JNK pathways may represent a novel therapeutic strategy in haematological malignancies.     

Inhibitory actions of the phosphatidylinositol 3-kinase inhibitor LY294002 on the human Kv1.5 channel

Background and purpose:

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I_Kur), and in humans, Kv1.5 channels are highly expressed in cardiac atria but are scarce in ventricles. Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as effective for prevention and treatment of re-entry-based atrial tachyarrhythmias. Here we examined blockade of hKv1.5 channels by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a well-known inhibitor of phosphatidylinositol 3-kinase (PI3K).

Experimental approach:

hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. Effects of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on wild-type and mutant (T462C, H463C, T480A, R487V, A501V, I502A, I508A, L510A and V516A) hKv1.5 channels were examined by using the whole-cell patch-clamp method.

Key results:

{"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner (IC₅₀ of 7.9 µmol·L⁻¹). In contrast, wortmannin, a structurally distinct inhibitor of PI3K, had little inhibitory effect on hKv1.5 current. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 block of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. The apparent binding (k₊₁) and unbinding (k₋₁) rate constants were calculated to be 1.6 µmol·L⁻¹⁻¹·s⁻¹ and 5.7 s⁻¹ respectively. Inhibition by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was significantly reduced in several hKv1.5 mutant channels: T480A, R487V, I502A, I508A, L510A and V516A.

Conclusions and implications:

{"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 acts directly on hKv1.5 currents as an open channel blocker, independently of its effects on PI3K activity. Amino acid residues located in the pore region (Thr480, Arg487) and the S6 segment (Ile502, Ile508, Leu510, Val516) appear to constitute potential binding sites for {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.     

Resveratrol reduces endothelial progenitor cells senescence through augmentation of telomerase activity by Akt-dependent mechanisms

Background and purpose:

Recent studies have shown that resveratrol increased endothelial progenitor cells (EPCs) numbers and functional activity. However, the mechanisms remain to be determined. Previous studies have demonstrated that increased EPC numbers and activity were associated with the inhibition of EPC senescence, which involves activation of telomerase. Therefore, we investigated whether resveratrol inhibits the onset of EPC senescence through telomerase activation, leading to potentiation of cellular activity.

Experimental approach:

After prolonged in vitro cultivation, EPCs were incubated with or without resveratrol. The senescence of EPCs were determined by acidic β-galactosidase staining. The bromo-deoxyuridine incorporation assay or a modified Boyden chamber assay were employed to assess proliferative or migratory capacity, respectively. To further examine the underlying mechanisms of these effects, we measured telomerase activity and the phosphorylation of Akt by western blotting.

Key results:

Resveratrol dose dependently prevented the onset of EPCs senescence and increased the proliferation and migration of EPCs. The effect of resveratrol on senescence could not be abolished by eNOS inhibitor or by an oestrogenic receptor antagonist. Resveratrol significantly increased telomerase activity and Akt phosphorylation. Pre-treatment with the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, significantly attenuated resveratrol-induced telomerase activity.

Conclusions and implications:

Resveratrol delayed the onset of EPC senescence and this effect was accompanied by activation of telomerase through the PI3K-Akt signalling pathway. The inhibition of EPCs senescence by resveratrol might protect EPCs against dysfunction induced by pathological factors in vivo and improve EPC functional activities in a way that may be important for cell therapy.     

Rosiglitazone via upregulation of Akt/eNOS pathways attenuates dysfunction of endothelial progenitor cells,induced by advanced glycation end products

Background and purpose:

Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play key roles in pathogenesis of diabetes-related vascular complications. AGEs can induce dysfunction in EPCs. The peroxisome proliferator-activated receptor-gamma (PPARγ) agonists are widely used in the treatment of type 2 diabetes, and it remains unknown if they could attenuate EPC dysfunction induced by AGEs.

Experimental approach:

EPCs isolated from healthy adults were cultured with various concentrations of AGEs (0, 50, 100 and 200 mg·L⁻¹) with or without rosiglitazone (10 nM), antibody for the receptors for AGE-human serum albumin (anti-receptor for advanced glycation end products (RAGE); 50 µg·mL⁻¹), phosphatidylinositol-3-kinase (PI3K) inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, 5 µM), nitric oxide (NO) synthase inhibitor (L-N^G-nitro-arginine methyl ester (L-NAME), 100 µM) or sodium nitroprusside (SNP, 25 µM). Proliferation, apoptosis, cell adhesion, migration and NO production in EPCs were assessed, and expressions of endothelial NO synthase (eNOS) and Akt were determined.

Key results:

Number, proliferation/migration capacities, eNOS and Akt phosphorylation as well as NO synthesized by EPCs were increased by rosiglitazone and reduced by AGEs. AGEs promoted while rosiglitazone reduced EPC apoptosis. The AGE-induced effects were significantly ameliorated by pre-incubation with rosiglitazone, RAGE antibody and SNP. The beneficial effects of rosiglitazone could be blocked by pretreatment with L-NAME and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions and implications:

The PPARγ agonist rosiglitazone increased EPC function and attenuated EPC dysfunction induced by AGEs via upregulating the Akt-eNOS signal pathways of EPCs.     

Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose

Aim:

To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway.

Methods:

Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H₂O₂ (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-β1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay.

Results:

Treatment of the cells with HG, H₂O₂, or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Both HG and H₂O₂ significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H₂O₂ significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, the treatment with HG or H₂O₂ significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusion:

The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.     

Cannabinoid receptor agonists modulate oligodendrocyte differentiation by activating PI3K/Akt and the mammalian target of rapamycin (mTOR) pathways

BACKGROUND AND PURPOSE

The endogenous cannabinoid system participates in oligodendrocyte progenitor differentiation in vitro. To determine the effect of synthetic cannabinoids on oligodendrocyte differentiation, we exposed differentiating cultures of oligodendrocytes with cannabinoid CB₁, CB₂ and CB₁/CB₂ receptor agonists and antagonists. The response of the PI3K/Akt and the mammalian target of rapamycin (mTOR) signalling pathways were studied as effectors of cannabinoid activity.

EXPERIMENTAL APPROACH

Purified oligodendrocyte progenitor cells (OPC) obtained from primary mixed glial cell cultures were treated for 48 h with CB₁, CB₂ and CB₁/CB₂ receptor agonists (ACEA, JWH133 and HU210, respectively) in the presence or absence of the antagonists AM281 (CB₁ receptor) and AM630 (CB₂ receptor). Moreover, inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR pathways ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and rapamycin, respectively) were used to study the involvement of these pathways on cannabinoid-induced OPC maturation.

KEY RESULTS

ACEA, JWH133 and HU-210 enhanced OPC differentiation as assessed by the expression of stage specific antigens and myelin basic protein (MBP). Moreover, this effect was blocked by the CB receptor antagonists. ACEA, JWH133 and HU210 induced a time-dependent phosphorylation of Akt and mTOR, whereas the inhibitors of PI3K/Akt ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or of mTOR (rapamycin) reversed the effects of HU-210 on oligodendrocyte differentiation and kinase activation.

CONCLUSIONS AND IMPLICATIONS

Activation of cannabinoid CB₁ or CB₂ receptors with selective agonists accelerated oligodendrocyte differentiation through the mTOR and Akt signalling pathways.

LINKED ARTICLES

This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

The PI3K/Akt pathway mediates the protection of SO2 preconditioning against myocardial ischemia/reperfusion injury in rats

Aim:

To explore the mechanisms underlying the protection by SO₂ preconditioning against rat myocardial ischemia/reperfusion (I/R) injury.

Methods:

Male Wistar rats underwent 30-min left coronary artery ligation followed by 120-min reperfusion. An SO₂ donor (1 μmol/kg) was intravenously injected 10 min before the ischemia, while {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (0.3 mg/kg) was intravenously injected 30 min before the ischemia. Plasma activities of LDH and CK were measured with an automatic enzyme analyzer. Myocardial infarct size was detected using Evans-TTC method. The activities of caspase-3 and -9 in myocardium were assayed using a commercial kit, and the levels of p-Akt, Akt, PI3K and p-PI3K were examined with Western blotting.

Results:

Pretreatment with SO₂ significantly reduced the myocardial infarct size and plasma LDH and CK activities, as well as myocardial caspase-3 and -9 activities in the rats. Furthermore, the pretreatment significantly increased the expression levels of myocardial p-Akt and p-PI3K p85. Administration of the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked all the effects induced by SO₂ pretreatment.

Conclusion:

The results suggest that the PI3K/Akt pathway mediates the protective effects of SO₂ preconditioning against myocardial I/R injury in rats.     

Raloxifene protects endothelial cell function against oxidative stress

Background and purpose:

Maintaining a delicate balance between the generation of nitric oxide (NO) and removal of reactive oxygen species (ROS) within the vascular wall is crucial to the physiological regulation of vascular tone. Increased production of ROS reduces the effect and/or bioavailability of NO, leading to an impaired endothelial function. This study tested the hypothesis that raloxifene, a selective oestrogen receptor modulator, can prevent endothelial dysfunction under oxidative stress.

Experimental approach:

Changes in isometric tension were measured in rat aortic rings. The content of cyclic GMP in aortic tissue was determined by radioimmunoassay. Phosphorylation of endothelial NOS (eNOS) and Akt was assayed by Western blot analysis.

Key results:

In rings with endothelium, ACh-induced relaxations were attenuated by a ROS-generating reaction (hypoxanthine plus xanthine oxidase, HXXO). The impaired relaxations were ameliorated by acute treatment with raloxifene. HXXO suppressed the ACh-stimulated increase in cyclic GMP levels; this effect was antagonized by raloxifene. The improved endothelial function by raloxifene was abolished by ICI 182,780, and by wortmannin or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Raloxifene also protected endothelial cell function against H₂O₂. Raloxifene increased the phosphorylation of eNOS at Ser-1177 and Akt at Ser-473; this effect was blocked by ICI 182,780. Finally, raloxifene was not directly involved in scavenging ROS, and neither inhibited the activity of xanthine oxidase nor stimulated that of superoxide dismutase.

Conclusion and implications:

Raloxifene is effective against oxidative stress-induced endothelial dysfunction in vitro through an ICI 182,780-sensitive mechanism that involves the increased phosphorylation and activity of Akt and eNOS in rat aortae.     

PI3K/AKT/mTOR Signaling-Mediated Neuropeptide VGF in the Hippocampus of Mice Is Involved in the Rapid Onset Antidepressant-Like Effects of GLYX-13

Background:

VGF (nonacryonimic) and phosphatidylinositol 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB)/mammalian target of rapamycin (mTOR) signaling play pivotal roles in depression. However, whether phosphatidylinositol 3-kinase/AKT/mTOR signaling-mediated VGF participates in rapid-acting antidepressant-like actions of GLYX-13 is unclear.

Methods:

Herein, we evaluated the effects of acute treatment of GLYX-13 (0.5, 5, and 10mg/kg, i.p.) in the forced swim test. In addition, we assessed whether the acute treatment with GLYX-13 reverses the depressive-like behaviors induced by chronic unpredictable mild stress. Furthermore, we determined whether the Vgf knockdown in hippocampus of mice blocks the effects of GLYX-13. Moreover, we also demonstrated the effects of intra-hippocampus infusion of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (10 nmol/side), a specific phosphatidylinositol 3-kinase inhibitor prior to the treatment of GLYX-13 in the forced swim test. Lastly, whether alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mTOR activation involves in the antidepressant-like effects of GLYX-13 was examined.

Results:

Our results shown that GLYX-13 dose-dependently reversed the depressive-like behaviors in forced swim test. Additionally, GLYX-13 significantly reversed the downregulation of phosphorylation of AKT, mTOR, and eukaryotic elongation factor 2 as well as VGF induced by chronic unpredictable mild stress in hippocampus. Further, Vgf knockdown in hippocampus of mice significantly blocked the rapid-acting antidepressant-like effects and upregulation on phosphatidylinositol 3-kinase/AKT/mTOR/VGF signaling of GLYX-13. Moreover, intra-hippocampus infusion of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 significantly abolished the antidepressant-like effects and upregulation on phosphatidylinositol 3-kinase/AKT/mTOR/VGF signaling of GLYX-13. Finally, antidepressant-like effects of GLYX-13 required AMPA receptor and mTOR activation, as evidenced by the ability of NBQX and rapamycin to block the effects of GLYX-13, respectively.

Conclusions:

Our results suggest that phosphatidylinositol 3-kinase/AKT/mTOR signaling-mediated VGF in hippocampus may be involved in the antidepressant-like effects of GLYX-13.     

Atorvastatin enhances neurite outgrowth in cortical neurons in vitro via up-regulating the Akt/mTOR and Akt/GSK-3β signaling pathways

Aim:

To investigate whether atorvastatin can promote formation of neurites in cultured cortical neurons and the signaling mechanisms responsible for this effect.

Methods:

Cultured rat cerebral cortical neurons were incubated with atorvastatin (0.05–10 μmol/L) for various lengths of time. For pharmacological experiments, inhibitors were added 30 min prior to addition of atorvastatin. Control cultures received a similar amount of DMSO. Following the treatment period, phase-contrast digital images were taken. Digital images of neurons were analyzed for total neurite branch length (TNBL), neurite number, terminal branch number, and soma area by SPOT Advanced Imaging software. After incubation with atorvastatin for 48 h, the levels of phosphorylated 3-phosphoinoside-dependent protein kinase-1 (PDK1), phospho-Akt, phosphorylated mammalian target of rapamycin (mTOR), phosphorylated 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K), and glycogen synthase kinase-3β (GSK-3β) in the cortical neurons were evaluated using Western blotting analyses.

Results:

Atorvastatin (0.05–10 μmol/L) resulted in dose-dependent increase in neurite number and length in these neurons. Pretreatment of the cortical neurons with phosphatidylinositol 3-kinase (PI3K) inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (30 μmol/L) and wortmannin (5 μmol/L), Akt inhibitor tricribine (1 μmol/L) or mTOR inhibitor rapamycin (100 nmol/L) blocked the atorvastatin-induced increase in neurite outgrowth, suggesting that atorvastatin promoted neurite outgrowth via activating the PI3K/Akt/mTOR signaling pathway. Atorvastatin (10 μmol/L) significantly increased the levels of phosphorylated PDK1, Akt and mTOR in the cortical neurons, which were prevented by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (30 μmol/L). Moreover, atorvastatin (10 μmol/L) stimulated the phosphorylation of 4E-BP1 and p70S6K, the substrates of mTOR, in the cortical neurons. In addition, atorvastatin (10 μmol/L) significantly increased the phosphorylated GSK-3β level in the cortical neurons, which was prevented by both {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and tricribine.

Conclusion:

These results suggest that activation of both the PI3K/Akt/mTOR and Akt/GSK-3β signaling pathways is responsible for the atorvastatin-induced neurite outgrowth in cultured cortical neurons.     

Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

Aim： To investigate the mechanisms underlying the protective effects of sodium tanshinone IIA sulfonate （STS） in an ischemia- reperfusion （I/R）-induced rat myocardial injury model. Methods： Male SD rats were iv injected with STS, STS＋LY294002, or saline （NS） for 15 d. Then the hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. Cardiac function, infarction size and area at risk were assessed. Cell apoptosis was evaluated with TUNEL staining, DNA laddering and measuring caspase-3 activity. In addition, isolated cardiomyocytes of neonatal rats were pretreated with the above drugs, then exposed to H202 （200 μmol/L） for I h. Cell apoptosis was detected using flow cytometric assay. The levels of p-Akt, p-FOXO3A and Bim were examined with immunoblotting. Results： Compared to NS group, administration of STS （20 mg/kg） significantly reduced myocardial infarct size （40.28%＋5.36% in STS group vs 59.52%±7.28% in NS group）, and improved the myocardial function as demonstrated by the increased values of dp/dt LVDP and coronary flow at different reperfusion time stages. Furthermore, STS significantly decreased the rate of apoptotic cells （15.11%±3.71% in STS group vs 38.21%±7.83% in NS group）, and reduced caspase-3 activity to nearly a quarter of that in NS group. Moreover, STS significantly increased the phosphorylation of Akt and its downstream target FOXO3A, and decreased the expression of pro-apoptotic gene Bim. Co-treatment with the PI3K inhibitor LY294002 （40 mg/kg） partially countered the protective effects induced by STS treatment. In isolated cardiomyocytes, STS exerted similar protective effects as shown in the ex vivo I/R model. Conclusion： STS pretreatment reduces infarct size and improves cardiac function in an I/R-induced rat myocardial injury model via activation of Akt/FOXO3A/Bim-mediated signal pathway.     

LKB1 gene inactivation does not sensitize non-small cell lung cancer cells to mTOR inhibitors in vitro

Aim:

Previous study has shown that endometrial cancers with LKB1 inactivation are highly responsive to mTOR inhibitors. In this study we examined the effect of LKB1 gene status on mTOR inhibitor responses in non-small cell lung cancer (NSCLC) cells.

Methods:

Lung cancer cell lines Calu-1, H460, H1299, H1792, and A549 were treated with the mTOR inhibitors rapamycin or everolimus (RAD001). The mTOR activity was evaluated by measuring the phosphorylation of 4EBP1 and S6K, the two primary mTOR substrates. Cells proliferation was measured by MTS or sulforhodamine B assays.

Results:

The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells. However, the LKB1 mutant A549 and H460 cells were not more sensitive to the mTOR inhibitors than the LKB1 wild-type Calu-1 and H1792 cells. Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors. Treatment with rapamycin or RAD001 significantly increased the phosphorylation of AKT in both LKB1 wild-type and LKB1 mutant NSCLC cells, which was attenuated by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Furthermore, RAD001 combined with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells.

Conclusion:

LKB1 gene inactivation in NSCLC cells does not increase the sensitivity to the mTOR inhibitors. The negative feedback activation of AKT by mTOR inhibition may contribute to the resistance of NSCLC cells to mTOR inhibitors.     

The integrin α2β1 agonist,aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production

Background and purpose:

During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin α2β1 agonist.

Experimental approach:

Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques.

Key results:

Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin α2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-β. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by α2β1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-β mAb, or a phosphoinositide-3 kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Aggretin stimulated the phosphorylation of PDGFR-β, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and PD98059.

Conclusions and implications:

Aggretin induces VSMC proliferation and migration mainly through binding to integrin α2β1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.     

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.