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.     

γ-Secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway

Aim： Trans-4-[4-（3-adamantan-1-yl-ureido）-cyclohexyloxy]-benzoic acid （t-AUCB） is a soluble epoxide hydrolase inhibitor that suppresses glioblastoma cell growth in vitro. The aim of this study was to examine whether the γ-secretase inhibitor N-[N-（3,5-difluorophenacetyl）-l-alanyl]-S-phenylglycine t-butyl ester （DAPT） could sensitize glioma cells to t-AUCB-induced apoptosis.
Methods： Both U251 and U87 human glioblastoma cell lines were tested. Cell growth was assessed using the cell counting kit-8. Cell apoptosis was detected with caspase-3 activity assay kits and flow cytometry. The protein levels in the p38 MAPK/MAPKAPK2/Hsp27 pathway in the cells were analyzed using Western blots.
Results： Pretreatment with DAPT （2 μmol/L） substantially potentiated the growth inhibition caused by t-AUCB （200 μmol/L） in U251 and U87 cells. Furthermore, pretreatment with DAPT markedly increased t-AUCB-induced apoptosis of U251 and U87 cells. T-AUCB alone did not significant affect caspase-3 activity in the cells, but t-AUCB plus DAPT pretreatment caused significant increase of caspase-3 activity. Furthermore, pretreatment with DAPT completely blocked t-AUCB-induced phosphorylation of p38 MAPK, MAPKAPK2 and Hsp27 in the cells.
Conclusion： The γ-secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway, suggesting that the combination of t-AUCB and DAPT may be a potentially effective strategy for the treatment of glioblastoma.     

Synthesis of matrine derivative ZS10 and its mechanism of apoptosis induced by PI3K/AKT signaling pathway in human hepatocellular carcinoma cell line BEL-7402北大核心CSCD

Aim To explore the signaling pathway of matrine derivative ZS10 in inhibiting proliferation and inducing apoptosis of BEL-7402 cells. Methods ZS10 was synthesized by organic synthesis. The inhibitory effect of ZS10 on the proliferation of BEL-7402 cells was analyzed by MTT method at the time of 24 h, 48 h and 72 h, respectively, and IC50 was calculated. DAPI staining was used to observe the state of BEL-7402 cells. Clone formation method was used to observe the colony formation of BEL-7402 cells, flow cytometry was used to observe the cell cycle arrest and apoptosis of BEL7402 cells, and Western blot was used to detect the expression level of PI3K/AKT pathway and related proteins. Results MTT results showed that the IC50 was(6.62±1.11)μmol·L-1; DAPI staining showed that the cell state changed significantly with the increase of drug concentration, and the results of colony formation showed that ZS10 significantly inhibited the colony formation of BEL-7402 cells. The results of flow cytometry showed that ZS10 induced S phase arrest and cycle apoptosis of BEL-7402 cells. Western blot showed that ZS10 at the concentration of 0～8 μ mol·L-1 could regulate the PI3K/AKT pathway and its related proteins in a dose-dependent manner. Compared with the control group, the expression of PI3K, AKT, P-AKT and anti-apoptotic protein Bcl-2 significantly decreased, the expression of pro-apoptotic protein Bax significantly increased, the expression of Cyclin D1 and CDK2 significantly decreased, and the expression of EGFR and N-cadherin, Vimentin significantly decreased in the treatment group. The expression of E-cadherin increased. Conclusions Matrine derivative ZS10 can inhibit the growth and proliferation of hepatocellular carcinoma cell line BEL-7402. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Effect of total flavonoids of Rosa rugosa on PI3K/AKT pathway and endoplasmic reticulum stress apoptosis in rats with cerebral ischemia-reperfusion injury北大核心CSCD

Aim To investigate the effects of total flavonoids from Rosa rugosa (TFR) on cerebral ischemia reperfusion injury (CIRI) in rats, and to investigate whether TFR inhibited neuronal apoptosis by regulating phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway and endoplasmic reticulum stress (ERS) pathways. Methods SD rats were randomly divided into sham operation group, model group, low-dose group (50 mg · kg -1 · d -1), medium-does group (100 mg · kg -1 · d -1), and high-does group (200 mg· kg -1 · d -1). The injury model of middle cerebral artery occlusion/reperfusion (MCAO/R) was prepared following suture method. Neurobehavioral changes, cerebral infarct size and brain tissue water content were detected 24 h after surgery. HE and Nissl staining were performed to observe pathological indicators. TUNEL staining was used to detect the apoptosis of ischemic nerve cells in brain. Western blot was used to detect the protein levels of Bcl-2, Bax, and cleaved Caspase-3, PI3K, p-PI3K, AKT, p-AKT, GRP78, CHOP and Caspase-12. Results Compared with MCAO/R group, the rats in medium-dose group and high-dose group showed improvement in the neurobehavioral function, decrease in the cerebral infarction area and the degree of cerebral edema, and reduction of the pathological damage of cerebral cortex. Moreover, there was a significantly decrease in the apoptosis rate of nerve cells in medium-dose group and high-dose group. The expression of anti-apoptotic protein Bcl-2 increased, and the pro-apoptotic protein Bax and cleaved-Caspase-3 decreased, the expression of pPI3K/PI3K, p-Akt/AKT increased, and the expression of ERS-related protein GRP78, CHOP, Caspase-12 decreased. Conclusions TFR can inhibit neuronal apoptosis by regulating the PI3K/AKT signaling pathway and ERS pathway, thus playing a protective role in CIRI rats. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Preventive effect of dioscin against monosodium urate-mediated gouty arthritis through inhibiting inflammasome NLRP3 and TLR4/NF-κB signaling pathway activation: an in vivo and in vitro study

Journal of Natural Medicines - Monosodium urate (MSU)-mediated inflammation is closely related to gouty arthritis (GA). Dioscin, an active ingredient, has been reported to possess anti-inflammatory...     

Upregulation of Sirt1 by tyrosol suppresses apoptosis and inflammation and modulates extracellular matrix remodeling in interleukin-1β-stimulated human nucleus pulposus cells through activation of PI3K/Akt pathway

Intervertebral disc degeneration (IDD) is the major pathogenesis of lower back pain. Tyrosol is a polyphenolic compound that exhibits anti-oxidant, anti-apoptotic, and anti-inflammatory effects. Herein, we explored the effects and mechanisms of tyrosol on IDD progression in interleukin (IL)-1β-stimulated human nucleus pulposus cells (HNPCs). Cell viability and apoptosis were detected by CCK-8 and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α), IL-6, nitric oxide (NO), and prostaglandin E2 (PGE2) was examined to evaluate inflammation. The mRNA expression of matrix metalloproteinases (MMPs) (MMP-3/9/13), collagen type II, SRY-related high mobility group box 9 (SOX-9), and aggrecan was measured by qRT-PCR. Protein levels of silent information regulator 2 homolog 1 (Sirt1), phosphorylated protein kinase B (p-Akt), Akt, collagen type II, SOX-9, and aggrecan were determined by western blot. Results showed that tyrosol attenuated IL-1β-induced viability reduction, apoptosis, and caspase-3/7 activity in HNPCs. The increase in the production of TNF-α, IL-6, NO, and PGE2 in IL-1β-treated HNPCs was abolished by tyrosol treatment. Tyrosol treatment reversed IL-1β-induced upregulation of MMP-3, MMP-9, and MMP-13, and downregulation of collagen II, SOX-9, and aggrecan in HNPCs. Additionally, tyrosol treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in IL-1β-stimulated HNPCs. Sirt1 was upregulated by tyrosol, and Sirt1 silencing inhibited Akt phosphorylation in HNPCs. Sirt1 knockdown attenuated the effects of tyrosol on IL-1β-induced apoptosis, inflammation, and ECM remodeling in HNPCs. In summary, upregulation of Sirt1 by tyrosol suppressed apoptosis and inflammation and regulated ECM remodeling in IL-1β-stimulated HNPCs through activation of PI3K/Akt pathway.     

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.