Camptothecin suppresses expression of matrix metalloproteinase-9 and vascular endothelial growth factor in DU145 cells through PI3K/Akt-mediated inhibition of NF-κB activity and Nrf2-dependent induction of HO-1 expression

Though camptothecin (CPT) possesses potent anti-inflammatory, immunomodulatory, anticancerous, and antiproliferative effects, little is known about the mechanism by which CPT regulates the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). Therefore, the current study aimed to investigate the effects of CPT on the expression of MMP-9 and VEGF, which are important factors for the invasion of tumors. In vitro application of CPT resulted in a slight inhibition of cell proliferation and a significant reduction in the matrigel invasion of DU145 cells. Treatment with CPT also downregulated phorbol-12-myristate-13-acetate (PMA)- and tumor necrosis factor-α (TNF-α)-induced MMP-9 and VEGF expression by inhibiting nuclear factor-κB (NF-κB) activity. Downregulation of phosphoinositide 3-kinase (PI3K)/Akt phosphorylation in response to CPT was revealed as an upstream pathway regulating the expression of MMP-9 and VEGF accompanying the inhibition of NF-κB activity. We further confirmed that CPT inhibits PMA-induced MMP-9 and VEGF expression by upregulating nuclear factor-erythroid related factor-2 (Nrf2)-mediated heme oxygenase-1 (HO-1) induction. Taken together, these data indicate that CPT inhibits the invasion of cancer cells accompanied by suppression of MMP-9 and VEGF production by suppressing the PI3K/Akt-mediated NF-κB pathway and enhancing the Nrf2-dependent HO-1 pathway, suggesting that CPT may be a good candidate to inhibit MMP-9 and VEGF expression.     

Glycyrrhetinic acid inhibits ICAM-1 expression via blocking JNK and NF-KB pathways in TNF-α-activated endothelial cells

Aim:

To investigate the effects of glycyrrhetinic acid (GA), an active component extracted from the root of Glycyrrhizae glabra, on the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVEC).

Methods:

ICAM-1 mRNA and protein levels were detected using RT-PCR and cell enzyme-linked immunosorbent assays. The adherence of human monocytic THP-1 cells labeled with [³H]thymidine to HUVEC was determined by counting radioactivity with a scintillation counter. The activation of mitogen-activated protein kinases as well as the degradation of IκB and nuclear factor-κB (NF-κB) or phospho-c-Jun in the nucleus were detected by western blots. NF-κB binding activity was detected using electrophoretic mobility shift assay.

Results:

GA (50 and 100 μmol/L) significantly inhibits TNF-α-induced ICAM-1 mRNA and protein expressions, as well as THP-1 cell adhesiveness in HUVEC. GA selectively inhibited TNF-α-activated signal pathway of c-Jun N-terminal kinase (JNK), without affecting extracellular signal-regulated kinase 1/2 and p38. Furthermore, GA apparently inhibited IκB/NF-κB signaling system by preventing IκB degradation, NF-κB translocation, and NF-κB/DNA binding activity. Finally, pretreatment with GA or the inhibitors of NF-κB, JNK, and p38 reduced the ICAM-1 protein expression induced by TNF-α.

Conclusion:

GA inhibits TNF-α-stimulated ICAM-1 expression, leading to a decrease in adherent monocytes to HUVEC. This inhibition is attributed to GA interruption of both JNK/c-Jun and IκB/NF-κB signaling pathways, which decrease activator protein-1 (AP-1) and NF-κB mediated ICAM-1 expressions. The results suggest that GA may provide a beneficial effect in treating vascular diseases associated with inflammation, such as atherosclerosis.     

Coactivation of the PI3K/Akt and ERK signaling pathways in PCB153-induced NF-κB activation and caspase inhibition

Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxicity, endocrine disruption and reproductive abnormalities. In order to verify the hypothesis that the PI3K/Akt and MAPK pathways play important roles in hepatotoxicity induced by PCBs, Sprague–Dawley (SD) rats were dosed with PCB153 intraperitoneally at 0, 4, 16 and 32 mg/kg for five consecutive days; BRL cells (rat liver cell line) were treated with PCB153 (0, 1, 5, and 10 μM) for 24 h. Results indicated that the PI3K/Akt and ERK pathways were activated in vivo and in vitro after exposure to PCB153, and protein levels of phospho-Akt and phospho-ERK were significantly increased. Nuclear factor-κB (NF-κB) activation and caspase-3, -8 and -9 inhibition caused by PCB153 were also observed. Inhibiting the ERK pathway significantly attenuated PCB153-induced NF-κB activation, whereas inhibiting the PI3K/Akt pathway hardly influenced phospho-NF-κB level. However, inhibiting the PI3K/Akt pathway significantly elevated caspase-3, -8 and -9 activities, while the ERK pathway only synergistically regulated caspase-9. Proliferating cell nuclear antigen (PCNA), a reliable indicator of cell proliferation, was also induced. Moreover, PCB153 led to hepatocellular hypertrophy and elevated liver weight. Taken together, PCB153 leads to aberrant proliferation and apoptosis of hepatocytes through NF-κB activation and caspase inhibition, and coactivated PI3K/Akt and ERK pathways play critical roles in PCB153-induced hepatotoxicity.     

Wogonin inhibits H2O2-induced angiogenesis via suppressing PI3K/Akt/NF-κB signaling pathway

Wogonin, a natural monoflavonoid extracted from Scutellariae radix, has been reported for its ability of inhibiting tumor angiogenesis. In this study, we assessed the effect of wogonin on angiogenesis induced by low level of H₂O₂ (10 μM) in human umbilical vein endothelial cells (HUVECs). Wogonin suppressed H₂O₂-induced migration and tube formation of HUVECs as well as microvessel sprouting from rat aortic rings in vitro. Meanwhile, wogonin suppressed vessel growth in chicken chorioallantoic membrane (CAM) model in vivo. Mechanistic studies showed that wogonin suppressed H₂O₂-activated PI3K/Akt pathway and reduced the expression of vascular endothelial growth factor (VEGF) up-regulated by H₂O₂ in both protein and mRNA levels. In addition, wogonin also inhibited nuclear translocation of NF-κB, and decreased the binding ability of NF-κB with exogenous consensus DNA oligonucleotide. Then we further investigated the effect of wogonin on over-activated PI3K/Akt pathway by insulin-like growth factor-1 (IGF-1) and H₂O₂. We found that wogonin suppressed phosphorylation of Akt, up-regulation of VEGF and angiogenesis in vitro which was further induced by IGF-1 and H₂O₂. Moreover, in NF-κB overexpressed HUVECs, wogonin could also reduce the expression of VEGF and inhibited the migration and tube formation. Taken together, these results suggested that wogonin was potential in inhibiting H₂O₂-induced angiogenesis in vitro and in vivo via suppressing PI3K/Akt pathway and NF-κB signaling.     

Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1

Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE₂ without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE₂ production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE₂, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression.     

Proinflammatory activation of macrophages by bisphenol A-glycidyl-methacrylate involved NFκB activation via PI3K/Akt pathway

Aim: Bisphenol A-glycidyl-methacrylate (BisGMA), a dental composite resin and dentin bonding agent, might prompt inflammatory effects to adjacent tissues. Macrophages are a major cellular component of the inflammatory sites. Little is known about the mechanisms of BisGMA on macrophages activation. The aim of this study was to evaluate BisGMA on proinflammatory mediators generation of murine macrophage RAW264.7 cells.     

7,8-didehydrocimigenol from Cimicifugae rhizoma inhibits TNF-α-induced VCAM-1 but not ICAM-1expression through upregulation of PPAR-γ in human endothelial cells

Activators of PPAR have been demonstrated to inhibit the induction of VCAM-1 but not ICAM-1 in human endothelial cells (EC). During the screening of anti-inflammatory activity of traditional herbs, we found 7,8-didehydrocimigenol (7,8-DHC), one of active triterpenoids of Cimicifugae rhizoma (C. rhizoma) increases PPAR-γ expression in EC in a time- and dose-dependent manner. Therefore, we asked whether 7,8-DHC selectively inhibits the expression of VCAM-1 but not ICAM-1 in TNF-α-activated EC via upregulation of PPAR-γ. Treatment with 7,8-DHC or PPAR-γ agonists (GW1929, troglitazone) inhibited the expression of VCAM-1 but not ICAM-1. Furthermore, the selective inhibition of VCAM-1 expression was inhibited by PPAR-γ antagonist, GW9662, or siPPAR-γ-transfected cells. 7,8-DHC significantly inhibited NF-kB activity via inhibition of phosphorylation of IkB and it also inhibited phosphorylation of ERK1/2 and Akt but not PKC. Finally, attachment of monocytes (U937) to EC by TNF-α was significantly reduced by 7,8-DHC. These results indicate that upregualtion of PPAR-γ by 7,8-DHC in EC inhibits NF-kB activity of TNF-α-activated EC which leads to selective inhibition of VCAM-1 expression. In addition, ERK1/2 and Akt signal pathways are involved in differential regulation by 7,8-DHC. We concluded that 7,8-DHC can be used for the treatment of cardiovascular disorders such as atherosclerosis.     

Procyanidin B2 protects H2O2-induced oxidative damage in PC12 cells by regulating PI3K/Akt and Nrf2/HO-l signaling pathways北大核心CSCD

Aim To explore the protective effect of proanthocyanidin B2 (PC-B2) on oxidative damage of PC 12 cells induced by hydrogen peroxide (H2 O2 ) and the related mechanism. Methods PC12 cells were treated with H2 O2 (200 μmol • L -1) for 24 h to establish oxidative damage model in vitro, and were randomly divided into normal group, normal + PC-B2 group, H2O2-induced model group and H2 O2 +PC-B2 group. Cell proliferation and cytotoxicity in each group were detected by CCK-8 and LDH assays, respectively. The expressions of MDA, SOD, CAT and GSH-Px were detected by ELISA kit. Cell apoptosis was observed by TUNEL staining. The expressions of apoptosis-related factors Bax, Bcl-2, caspase-3, p-PI3 K, p-Akt, PI3 K/Akt and Nrf2/HO-l pathway were detected by RT-PCR and Western blot. Results Compared with the normal group, the cell proliferation ability of the H2 O2 model group decreased, the contents of LDH and MDA increased, the contents of SOD, CAT and GSH-Px decreased, and the apoptosis was aggravated. After PCB2 intervention, the proliferation ability of PC 12 cells increased, the contents of LDH and MDA decreased, and the contents of SOD, CAT and GSH-Px increased. PC-B2 effectively inhibited the apoptosis of PC12 cells and up-regulated the expression of PI3K/Akt and Nrf2/HO-l proteins. Conclusions PC-B2 can protect PC12 cells from oxidative damage induced by H2 O2 and improve the apoptosis of PC 12 cells. The mechanism may be related to the activation of PI3K/Akt and Nrf2/HO-l signaling pathways. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Schisandra chinensis α-iso-cubebenol induces heme oxygenase-1 expression through PI3K/Akt and Nrf2 signaling and has anti-inflammatory activity in Porphyromonas gingivalis lipopolysaccharide-stimulated macrophages

Heme oxygenase-1 (HO-1) is a potent anti-inflammatory molecule that regulates pro-inflammatory mediators. Several studies have indicated that HO-1 expression is induced by a variety of stimuli such as lipopolysaccharide (LPS), cytokines, oxidative stress, and antioxidant phytochemicals. In this study, we assessed the anti-inflammatory effects of a novel α-iso-cubebenol isolated from dried fruits of Schisandra chinensis in human macrophage THP-1 cells and investigated the involvement of HO-1 signaling. We first observed that α-iso-cubebenol induced HO-1 mRNA and protein expression in a dose- and time-dependent manner via activation of erythroid-specific nuclear factor-regulated factor 2 (Nrf2). We also found that α-iso-cubebenol induced phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt and extracellular-regulated kinase (ERK) in a time-dependent manner. Furthermore, treatment of THP-1 cells with inhibitors and siRNA specific for PI3K/Akt and ERK decreased the expression of HO-1. These results suggested that α-iso-cubebenol induced HO-1 expression through the activation of PI3K/Akt, ERK, and Nrf2 signaling. Next, α-iso-cubebenol strongly inhibited Porphyromonas gingivalis LPS-stimulated pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-12). Moreover, we observed that α-iso-cubebenol treatment inhibited nuclear levels and activity of NF-κB in a dose-dependent manner. Additionally, treatment with tin-protoporphyrin (SnPP), a selective inhibitor of HO-1, reversed the α-iso-cubebenol-mediated inhibition of P. gingivalis LPS-induced pro-inflammatory cytokines. Hence, α-iso-cubebenol might induce anti-inflammatory effects on P. gingivalis LPS-stimulated human THP-1 macrophages by mediating the activation of PI3k/Akt and ERK that leads to over-expression of HO-1 and Nrf-2. These findings suggest that α-iso-cubebenol may be considered as a novel therapeutic agent to ameliorate periodontitis.     

Ampelopsin reduces endotoxic inflammation via repressing ROS-mediated activation of PI3K/Akt/NF-κB signaling pathways

Ampelopsin (AMP), a plant flavonoid, has potent anti-inflammatory properties in vitro and in vivo. The molecular mechanisms of ampelopsin on pharmacological and biochemical actions of RAW264.7 macrophages in inflammation have not been clearly elucidated yet. In the present study, non-cytotoxic level of ampelopsin significantly inhibited the release of nitric oxide (NO) and pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in a dose-dependent manner. Consistent with NO inhibition, ampelopsin suppressed lipopolysaccharide (LPS)-induced expression of inducible NO synthase (iNOS) by inhibiting nuclear factor κB (NF-κB) activation, which highly correlated with its inhibitory effect on IκB kinase (IKK) phosphorylation, IκB phosphorylation and NF-κB nuclear translocation. Further study demonstrated that ampelopsin suppressed LPS-induced activation of Akt without effecting mitogen-activated protein kinases (MAPKs) phosphorylation. A pharmacological inhibitor of the phosphoinositide 3-kinase (PI3K)-Akt pathway, LY294002, abrogated IKK/IκB/NF-κB-mediated iNOS gene expression. Finally, we certificated that ampelopsin reduced reactive oxygen species (ROS) accumulation and an anti-oxidant N-acetyl-L-cysteine (NAC) significantly repressed LPS-induced PI3K/Akt phosphorylation and the downstream IKK/IκB activation. NAC thereby inhibited LPS-induced iNOS expression and NO production. The present results suggest that the anti-inflammatory effect of ampelopsin is due to inhibiting the interconnected ROS/Akt/IKK/NF-κB signaling pathways.     

Amlodipine inhibits TNF-α production and attenuates cardiac dysfunction induced by lipopolysaccharide involving PI3K/Akt pathway

Calcium channel blockers (CCBs) are widely used in the therapy of cardiovascular diseases. Recent studies have shown that several CCBs exerted distinct anti-inflammatory effect in myocardial dysfunction models. The purpose of the present study was to evaluate therapeutic effect and possible mechanism of action of amlodipine, one of the widely used CCBs, on rat cardiac dysfunction during sepsis induced by lipopolysaccharide (LPS). Pretreatment of the rats with amlodipine (10 or 30 mg/kg, i.v.) delayed the fall of mean arterial blood pressure caused by LPS. Amlodipine also significantly inhibited the elevation of plasma tumor necrosis factor α (TNF-α) and decreased levels of inducible nitric oxide synthase (iNOS) in response to LPS challenge. To investigate the mechanism of the action of amlodipine, neonatal rat cardiomyocytes were used as a model. Amlodipine concentration-dependently decreased the release of TNF-α and iNOS protein expression, and suppressed the degradation and phosphorylation of inhibitor of κB-α (IκB-α) in LPS-activated neonatal rat cardiomyocytes. Further studies revealed that amlodipine markedly activated phosphatidylinositiol 3-kinase (PI3K) and Akt, downstream of the PI3K signal cascade. Application of PI3K inhibitors, wortmannin and LY294002 attenuated the depression of TNF-α and iNOS expression by amlodipine in LPS-induced cardiomyocytes. These findings may explain some cardioprotective effects of amlodipine in LPS-mediated sepsis and suggest that the inhibition of TNF-α and iNOS expression by amlodipine is, at least in part, dependent on PI3K/Akt signaling pathway.     

The protective effect of kaempferol on heart via the regulation of Nrf2, NF-κβ, and PI3K/Akt/GSK-3β signaling pathways in isoproterenol-induced heart failure in diabetic rats

This study was designed to delineate the effect of kaempferol (KF) on heart failure (HF) in diabetic rats. Streptozotocin-induced male diabetic rats received KF orally at 10 and 20 mg/kg for 42 consecutive days. In last 2 days of the experimental period, isoproterenol was subcutaneously injected at 85 mg/kg to induce HF. The hearts were processed for hemodynamic, biochemical, molecular, and histological investigations. Systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure were elevated in KF-treated HF-induced diabetic rats. Moreover, KF treatment resulted in decreased fasting blood glucose and glycosylated hemoglobin levels with increased serum insulin levels. Besides, serum cardiac injury markers like troponin-I, creatine kinase-muscle/brain, lactate dehydrogenase, and brain natriuretic peptide levels were significantly reduced in KF treatment. KF treatment has shown decrease in cardiac heme oxygenase-1, nuclear factor erythroid 2–related factor 2 (Nrf-2), and γ-glutamylcysteine synthetase with increased Keap1 mRNA levels. The cardioprotection of KF was improved by inhibition of apoptosis via blocking phosphorylation of Akt/glycogen synthase kinase (GSK)-3β and p38 mitogen-activated protein-kinase/extracellular signal-regulated kinases signaling pathways in HF-induced diabetic rats. Moreover, reduced cardiac apoptosis in KF treatment was confirmed by decreased terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) positive cells, histopathological changes in HF-induced diabetic rats. Therefore, the cardioprotective effect of KF is attributed to the regulation of Nrf2, nuclear factor kappa-light-chain-enhancer of activated B cells, and Akt/GSK-3β signaling pathways in HF-induced diabetic rats.     

Piperine inhibits PMA-induced cyclooxygenase-2 expression through downregulating NF-κB,C/EBP and AP-1 signaling pathways in murine macrophages

Piperine is a major component of black (Piper nigrum Linn) and long (Piper longum Linn) peppers, and is widely used as a traditional food and medicine. It also exhibits a variety of biological activities, which include antioxidant, anti-tumor and anti-pyretic properties. In the present study, we investigated the inhibitory effects of piperine on phorbol 12-myristate 13-acetate (PMA)-induced cyclooxygenase-2 (COX-2) gene expression and analyzed the molecular mechanism of its activity in murine RAW 264.7 macrophages. Piperine dose-dependently decreased PMA-induced COX-2 expression and PGE₂ production, as well as COX-2 promoter-driven luciferase activity. Transient transfections utilizing COX-2 promoter deletion constructs and COX-2 promoter constructs, in which specific enhancer elements were mutagenized, revealed that the nuclear factor-κB (NF-κB), CCAAT/enhancer binding protein (C/EBP) and activator protein-1 (AP-1), were the predominant contributors to the effects of piperine. In addition, piperine inhibited PMA-induced NF-κB, C/EBP and c-Jun nuclear translocation. Furthermore, piperine significantly inhibited PMA-induced activation of the Akt and ERK. These findings demonstrate that piperine effectively attenuates COX-2 production, and provide further insight into the signal transduction pathways involved in the anti-inflammatory effects of piperine.     

FM19G11 reverses endothelial dysfunction in rat and human arteries through stimulation of the PI3K/Akt/eNOS pathway,independently of mTOR/HIF-1α activation

Background and Purpose

FM19G11 up-regulates mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α) and PI3K/Akt pathways, which are involved in endothelial function. We evaluated the effects of FM19G11 on defective endothelial vasodilatation in arteries from rats and humans and investigated the mechanisms involved.

Experimental Approach

Effects of chronic in vivo administration of FM19G11 on aortic endothelial vasodilatation were evaluated together with ex vivo treatment in aortic and mesenteric arteries from control and insulin-resistant rats (IRR). Its effects on vasodilator responses of penile arteries (HPRAs) and corpus cavernosum (HCC) from men with vasculogenic erectile dysfunction (ED) (model of human endothelial dysfunction) were also evaluated. Vascular expression of phosphorylated-endothelial NOS (p-eNOS), phosphorylated-Akt (p-Akt) and HIF-1α was determined by immunodetection and cGMP by elisa.

Key Results

Chronic administration of FM19G11 reversed the impaired endothelial vasodilatation in IRR. Ex vivo treatment with FM19G11 also significantly improved endothelium-dependent vasodilatation in aorta and mesenteric arteries from IRR. These effects were accompanied by the restoration of p-eNOS and cGMP levels in IRR aorta and were prevented by either NOS or PI3K inhibition. p-Akt and p-eNOS contents were increased by FM19G11 in aortic endothelium of IRR. FM19G11-induced restoration of endothelial vasodilatation was unaffected by mTOR/HIF-1α inhibitors. FM19G11 also restored endothelial vasodilatation in HPRA and HCC from ED patients.

Conclusions and Implications

Stimulation of the PI3K/Akt/eNOS pathway by FM19G11 alleviates impaired NO-mediated endothelial vasodilatation in rat and human arteries independently of mTOR/HIF-1α activation. This pharmacological strategy could be beneficial for managing pathological conditions associated with endothelial dysfunction, such as ED.