Neuroprotective effects of astaxanthin against oxygen and glucose deprivation damage via the PI3K/Akt/GSK3β/Nrf2 signalling pathway in vitro

Astaxanthin (ATX), which is the most abundant flavonoid in propolis, has previously shown neuroprotective properties against cerebral ischaemia‐induced apoptosis. However, the mechanisms by which ATX mediates its therapeutic effects are unclear. At present, we explored the underlying mechanisms involved in the protective effects of ATX via the phosphoinositide 3‐kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling pathway in SH‐SY5Y cells. The PI3K/Akt inhibitor LY294002 and GSK3β inhibitor LiCl were employed in this study. Pre‐treatment with ATX for 24 hours significantly decreased the oxygen and glucose deprivation (OGD)‐induced viability loss, reduced the proportion of apoptosis and regulated OGD‐mediated reactive oxygen species (ROS) production. Furthermore, ATX suppressed OGD‐caused mitochondrial membrane potential and decomposition of caspase‐3 to cleaved caspase‐3, and heightened the B‐cell lymphoma 2 (Bcl‐2)/Bax ratio. PI3K/Akt/GSK3β/Nrf2 signalling pathway activation in SH‐SY5Y cells was verified by Western blot. ATX and LiCl treatment raised the protein levels of p‐Akt, p‐GSK3β, nucleus Nrf2 and haeme oxygenase 1 (HO‐1). However, these protein expression levels decreased by treatment of LY294002. The above in vitro data indicate that ATX can confer neuroprotection against OGD‐induced apoptosis via the PI3K/Akt/GSK3β/Nrf2 signalling pathway.     

Pravastatin-induced proangiogenic effects depend upon extracellular FGF-2

The HMG-CoA reductase inhibitors (statins) have been shown to exert several protective effects on the vasculature that are unrelated to changes in the cholesterol profile, and to induce angiogenesis. The proangiogenic effect exerted by statins has been attributed to the activation of the PI3K/Akt pathway in endothelial cells; however, it is unclear how statins activate this pathway. Pravastatin-mediated activation of Akt and MAPK occurs rapidly (within 10 min.) and at low doses (10 nM). Here, we hypothesized that FGF-2 contributes to the proangiogenic effect of statins. We found that pravastatin, a hydrophilic statin, induced phosphorylation of the FGF receptor (FGFR) in human umbilical vein endothelial cells. SU5402, an inhibitor of FGFR, abolished pravastatin-induced PI3K/Akt and MAPK activity. Likewise, anti-FGF-2 function-blocking antibodies inhibited Akt and MAPK activity. Moreover, depletion of extracellular FGF-2 by heparin prevented pravastatin-induced phosphorylation of Akt and MAPK. Treatment with FGF-2 antibody inhibited pravastatin-enhanced endothelial cell proliferation, migration and tube formation. These observations indicate that pravastatin exerts proangiogenic effects in endothelial cells depending upon the extracellular FGF-2.     

Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

Dynorphins act as endogenous anticonvulsants via activation of kappa opioid receptor (KOR). However, the mechanism underlying the anticonvulsant role remains elusive. This study aims to investigate whether the potential protection of KOR activation by dynorphin against epilepsy was associated with the regulation of PI3K/Akt/Nrf2/HO-1 pathway. Here, a pilocarpine-induced rat model of epilepsy and Mg²⁺-free-induced epileptiform hippocampal neurons were established. Decreased prodynorphin (PDYN) expression, suppressed PI3K/Akt pathway, and activated Nrf2/HO-1 pathway were observed in rat epileptiform hippocampal tissues and in vitro neurons. Furthermore, dynorphin activation of KOR alleviated in vitro seizure-like neuron injury via activation of PI3K/Akt/Nrf2/HO-1 pathway. Further in vivo investigation revealed that PDYN overexpression by intra-hippocampus injection of PDYN-overexpressing lentiviruses decreased hippocampal neuronal apoptosis and serum levels of inflammatory cytokines and malondialdehyde (MDA) content, and increased serum superoxide dismutase (SOD) level, in pilocarpine-induced epileptic rats. The protection of PDYN in vivo was associated with the activation of PI3K/Akt/Nrf2/HO-1 pathway. In conclusion, dynorphin activation of KOR protects against epilepsy and seizure-induced brain injury, which is associated with activation of the PI3K/Akt/Nrf2/HO-1 pathway.     

α-Lipoic acid alleviates ferroptosis in the MPP+-induced PC12 cells via activating the PI3K/Akt/Nrf2 pathway

Parkinson's disease (PD) is a typical neurodegenerative disease. α-Lipoic acid (α-LA) can reduce the incidence of neuropathy. The present study explored the role and mechanism of α-LA in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell model of PD. The PD model was induced via treating PC12 cells with MPP⁺ at different concentrations. MPP⁺ and α-LA effects on PC12 cells were assessed from cell viability and ferroptosis. Cell viability was detected using the cell counting kit-8 assay. Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, reactive xygen species (ROS), and glutathione (GSH) concentrations, and ferroptosis-related protein SLC7A11 and GPx4 expressions were used for ferroptosis evaluation. p-PI3K, p-Akt, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels were detected. The PI3K/Akt/Nrf2 pathway inhibitors were applied to verify the role of the PI3K/Akt/Nrf2 pathway in α-LA protection against MPP⁺-induced decreased cell viability and ferroptosis. MPP⁺-reduced cell viability and induced ferroptosis as presented by increased MDA, 4-HNE, iron, and ROS concentrations, and reduced levels of GSH and ferroptosis marker proteins (SLC7A11 and GPx4). α-LA attenuated MPP⁺-induced cell viability decline and ferroptosis. The PI3K/Akt/Nrf2 pathway was activated after α-LA treatment. Inhibiting the PI3K/Akt/Nrf2 pathway weakened the protection of α-LA against MPP⁺ treatment. We highlighted that α-LA alleviated MPP⁺-induced cell viability decrease and ferroptosis in PC12 cells via activating the PI3K/Akt/Nrf2 pathway.     

Gastrodin prevents homocysteine-induced human umbilical vein endothelial cells injury via PI3K/Akt/eNOS and Nrf2/ARE pathway

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine-induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3-kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200-800 μg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl-2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L-NAME but enhanced by Cav-1 knockdown, and MK-2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p-Akt, and eNOS and decreased Cav-1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine-induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.     

Nrf2 Negatively Regulates Melanogenesis by Modulating PI3K/Akt Signaling

Nrf2 plays a role in protection of cells against oxidative stress and xenobiotic damage by regulating cytoprotective genes. In this study, we investigated the effect of Nrf2 on melanogenesis in normal human melanocytes (NHMCs). When NHMCs were transduced with a recombinant adenovirus expressing Nrf2, melanin synthesis was significantly decreased. Consistent with this result, overexpression of Nrf2 decreased the expression of tyrosinase and tyrosinase-related protein 1. The inhibitory effect of Nrf2 was reversed by overexpression of Keap1, an intracellular regulator of Nrf2. Interestingly, Nrf2 overexpression resulted in marked activation of PI3K/Akt signaling. Conversely, inhibition of PI3K activity by treatment with wortmannin reversed the depigmentary effects of Nrf2. Taken together, these results strongly suggest that Nrf2 negatively regulates melanogenesis by modulating the PI3K/Akt signaling pathway.     

Pyrroloquinoline quinone rescues hippocampal neurons from glutamate-induced cell death through activation of Nrf2 and up-regulation of antioxidant genes

Pyrroloquinoline quinone (PQQ) has been shown to protect primary cultured hippocampal neurons from glutamate-induced cell apoptosis by scavenging reactive oxygen species (ROS) and activating phosphatidylinositol-3-kinase (PI3K)/Akt signaling. We investigated the downstream pathways of PI3K/Akt involved in PQQ protection of glutamate-injured hippocampal neurons. Western blot analysis indicated that PQQ treatment following glutamate stimulation triggers phosphorylation of glycogen synthase kinase 3β, accompanied by maintenance of Akt activation. Immunostaining and quantitative RT-PCR revealed that PQQ treatment promotes nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), and up-regulates mRNA expression of Nrf2 and the antioxidant enzyme genes, heme oxygenase-1 and glutamate cysteine ligase catalytic in glutamate-injured hippocampal neurons; this is a process dependent on the PI3K/Akt pathway, as evidenced by blocking experiments with PI3K inhibitors. In addition, increased ROS production and decreased glutathione levels in glutamate-injured hippocampal neurons were found to be reduced by PQQ treatment. Collectively, our findings suggest that PQQ exerts neuroprotective activity, possibly through PI3K/Akt-dependent activation of Nrf2 and up-regulation of antioxidant genes. However, the ability of PQQ to scavenge ROS was not totally regulated by PI3K/Akt signaling; possibly it is governed by other mechanisms.     

Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

Electroacupuncture at select acupoints have been verified to protect against organ dysfunctions during endotoxic shock. And, heme oxygenase (HO)-1 as a phase II enzyme and antioxidant contributed to the protection of kidney in septic shock rats. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway mediated the activation of NF-E2 related factor-2 (Nrf2), which was involved in HO-1 induction. To understand the efficacy of electroacupuncture stimulation in ameliorating acute kidney injury (AKI) through the PI3K/Akt/Nrf2 pathway and subsequent HO-1 upregulation, a dose of LPS 5mg/kg was administered intravenously to replicate the rabbit model of AKI induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Neiguan acupoints for five consecutive days while sham electroacupuncture at non-acupoints as control. Results displayed that electroacupuncture stimulation significantly alleviated the morphologic renal damage, attenuated renal tubular apoptosis, suppressed the elevated biochemical indicators of AKI caused by LPS, enhanced the expressions of phospho-Akt, HO-1protein, Nrf2 total and nucleoprotein, and highlighted the proportions of Nrf2 nucleoprotein as a parallel. Furthermore, partial protective effects of elecroacupuncture were counteracted by preconditioning with wortmannin (the selective PI3K inhibitor), indicating a direct involvement of PI3K/Akt pathway. Inconsistently, wortmannin pretreatment made little difference to the expressions of HO-1, Nrf2 nucleoprotein and total protein, which indicated that PI3K/Akt may be not the only pathway responsible for electroacupuncture-afforded protection against LPS-induced AKI. These findings provide new insights into the potential future clinical applications of electroacupuncture for AKI induced by endotoxic shock instead of traditional remedies.     

Knockdown of LASP2 inhibits the proliferation,migration, and invasion of cervical cancer cells

LIM and SH3 protein 2 (LASP2) belongs to nebulin family. It has been proven that LASP2 is involved in several cancers; however, its role in cervical cancer is unclear. Herein, we showed that LASP2 was highly expressed in cervical cancer tissues and cell lines. To knockdown LASP2 in cervical cancer cells, small interfering RNAs (siRNAs) targeting LASP2 (si-LASP2) were used. We found that cell proliferation, migration/invasion were markedly reduced after si-LASP2 transfection. A significant increase in E-cadherin expression, and decrease in N-cadherin and vimentin expressions were observed in si-LASP2 transfected cervical cancer cells. Knockdown of LASP2 caused significant inhibitory effect on the PI3K/Akt pathway. Treatment with the activator of the PI3K/Akt pathway, 740Y-P, abolished the effects of si-LASP2 transfection on cervical cancer cells. These findings suggested that LASP2 may be an oncogene through regulating the PI3K/Akt pathway in cervical cancer.