A NAD(P)H oxidase mimic for catalytic tumor therapy via a deacetylase SIRT7-mediated AKT/GSK3ß pathway.

Nicotinamide adenine dinucleotide (NADH) and its phosphorylated form, NADPH, are essential cofactors that play critical roles in cell functions, influencing antioxidation, reductive biosynthesis, and cellular pathways involved in tumor cell apoptosis and tumorigenesis. However, the use of nanomaterials to consume NAD(P)H and thus bring an impact on signaling pathways in cancer treatment remains understudied. In this study, we employed a salt template method to synthesize a carbon-coated-cobalt composite (C@Co) nanozyme, which exhibited excellent NAD(P)H oxidase (NOX)-like activity and mimicked the reaction mechanism of natural NOX. The C@Co nanozyme efficiently consumed NAD(P)H within cancer cells, leading to increased production of reactive oxygen species (ROS) and a reduction in mitochondrial membrane potential. Meanwhile, the generation of the biologically active cofactor NAD(P)+ promoted the expression of the deacetylase SIRT7, which in turn inhibited the serine/threonine kinase AKT signaling pathway, ultimately promoting apoptosis. This work sheds light on the influence of nanozymes with NOX-like activity on cellular signaling pathways in tumor therapy and demonstrates their promising antitumor effects in a tumor xenograft mouse model. These findings contribute to a better understanding of NAD(P)H manipulation in cancer treatment and suggest the potential of nanozymes as a therapeutic strategy for cancer therapy.

A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42-mediated GSK-3ß/ß-catenin Signaling.

Kidney fibrosis is a common fate of chronic kidney diseases (CKDs), eventually leading to renal dysfunction. Yet, no effective treatment for this pathological process has been achieved. During the bioassay-guided chemical investigation of the medicinal plant Wikstroemia chamaedaphne, a daphne diterpenoid, daphnepedunin A (DA), is characterized as a promising anti-renal fibrotic lead. DA shows significant anti-kidney fibrosis effects in cultured renal fibroblasts and unilateral ureteral obstructed mice, being more potent than the clinical trial drug pirfenidone. Leveraging the thermal proteome profiling strategy, cell division cycle 42 (Cdc42) is identified as the direct target of DA. Mechanistically, DA targets to reduce Cdc42 activity and down-regulates its downstream phospho-protein kinase Cζ(p-PKCζ)/phospho-glycogen synthase kinase-3ß (p-GSK-3ß), thereby promoting ß-catenin Ser33/37/Thr41 phosphorylation and ubiquitin-dependent proteolysis to block classical pro-fibrotic ß-catenin signaling. These findings suggest that Cdc42 is a promising therapeutic target for kidney fibrosis, and highlight DA as a potent Cdc42 inhibitor for combating CKDs.

An LGR4 agonist activates the GSK­3ß pathway to inhibit RANK­RANKL signaling during osteoclastogenesis in bone marrow­derived macrophages.

The binding between receptor­activated nuclear factor­κB (RANK) and the RANK ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. The leucine­rich repeat­containing G­protein­coupled receptor 4 (LGR4) acts as a negative regulator of RANK­RANKL that suppresses canonical RANK signaling during osteoclast differentiation. Therefore, LGR4 agonists may be useful in inhibiting osteoclastogenesis and effectively treating osteoporosis. In the present study, bone marrow­derived macrophages and a mouse model of RANKL­induced bone loss were used to investigate the effect of mutant RANKL (MT RANKL), which was previously developed based on the crystal structure of the RANKL complex. In the present study, the binding affinity of wild­type (WT) RANKL and MT RANKL to RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT­glycogen synthase kinase­3ß (GSK­3ß)­nuclear factor of activated T cells, cytoplasmic, calcineurin­dependent 1 (NFATc1) signaling cascade was investigated using western blotting and confocal microscopy. In addition, the expression of LGR4 and the colocalization of LGR4 with MT RANKL were analyzed in a mouse model of RANKL­induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4 and increased GSK­3ß phosphorylation independently of AKT, resulting in the inhibition of NFATc1 nuclear translocation. In the mouse model, MT RANKL colocalized with LGR4 and inhibited bone resorption. These results indicated that MT RANKL may inhibit RANKL­induced osteoclastogenesis through an LGR4­dependent pathway and this could be exploited to develop new therapies for osteoporosis.

Neuroprotection and Mechanism of Gas-miR36-5p from Gastrodia elata in an Alzheimer's Disease Model by Regulating Glycogen Synthase Kinase-3ß.

Alzheimer's disease (AD) is currently the most common neurodegenerative disease. Glycogen synthase kinase 3ß (GSK-3ß) is a pivotal factor in AD pathogenesis. Recent research has demonstrated that plant miRNAs exert cross-kingdom regulation on the target genes in animals. Gastrodia elata (G. elata) is a valuable traditional Chinese medicine that has significant pharmacological activity against diseases of the central nervous system (CNS). Our previous studies have indicated that G. elata-specific miRNA plays a cross-kingdom regulatory role for the NF-κB signaling pathway in mice. In this study, further bioinformatics analysis suggested that Gas-miR36-5p targets GSK-3ß. Through western blot, RT-qPCR, and assessments of T-AOC, SOD, and MDA levels, Gas-miR36-5p demonstrated its neuroprotective effects in an AD cell model. Furthermore, Gas-miR36-5p was detected in the murine brain tissues. The results of the Morris water maze test and western blot analysis provided positive evidence for reversing the learning deficits and hyperphosphorylation of Tau in AD mice, elucidating significant neuroprotective effects in an AD model following G. elata RNA administration. Our research emphasizes Gas-miR36-5p as a novel G. elata-specific miRNA with neuroprotective properties in Alzheimer's disease by targeting GSK-3ß. Consequently, our findings provide valuable insights into the cross-kingdom regulatory mechanisms underlying G. elata-specific miRNA, presenting a novel perspective for the treatment of Alzheimer's disease.

LQB-118 Suppresses Migration and Invasion of Prostate Cancer Cells by Modulating the Akt/GSK3ß Pathway and MMP-9/Reck Gene Expression.

BACKGROUND/AIM: Prostate cancer (PCa) is one of the most common malignancies in adult men. LQB-118 is a pterocarpanquinone with antitumor activity toward prostate cancer cells. It inhibits cell proliferation by down-regulating cyclins D1 and B1 and up-regulating p21. However, the effects of LQB-118 on PCa cell migration are still unclear. Herein, the LQB-118 effects on PCa metastatic cell migration/invasion and its mechanism of action were evaluated. MATERIALS AND METHODS: PC3 cells were treated with LQB-118 or Paclitaxel (PTX), and cell migration (wound healing and Boyden chamber assays) and invasion (matrigel assay) were determined. The LQB-118 mechanisms were evaluated by αVßIII protein expression (flow cytometry), protein phosphorylation (Western blot), and mRNA expression (qPCR). RESULTS: LQB-118 impaired PCa cell migration and invasion, down-regulated Akt phosphorylation, and also reduced GSK3ß phosphorylation, through a FAK-independent pathway. Also, it was observed that LQB-118 controlled the invasiveness behavior by reducing matrix metalloproteinase-9 (MMP-9) and up-regulating reversion-inducing cysteine rich protein with Kazal motifs (Reck) mRNA levels. Interestingly, LQB-118 increased integrin αvßIII expression, but this effect was not related to its activation, since the cell adhesion ability was reduced after LQB-118 treatment. CONCLUSION: These data highlight novel LQB-118 mechanisms in prostate cancer cells. LQB-118 acts as a negative regulator of the Akt/GSK3 signaling pathway and can modulate PCa cell proliferation, death, and migration/invasion. The results also support the use of LQB-118 for the treatment of metastatic PCa, alone or combined with another chemotherapeutic agent, due to its demonstrated pleiotropic activities.

The α7 nAChR allosteric modulator PNU-120596 amends neuroinflammatory and motor consequences of parkinsonism in rats: Role of JAK2/NF-κB/GSk3ß/ TNF-α pathway.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and a leading cause of disability. The current gold standard for PD treatment, L-Dopa, has limited clinical efficacy and multiple side effects. Evidence suggests that activation of α7 nicotinic acetylcholine receptors (α7nAChRs) abrogates neuronal and inflammatory insults. Here we tested whether PNU-120596 (PNU), a type II positive allosteric modulator of α7 nAChR, has a critical role in regulating motor dysfunction and neuroinflammation correlated with the associated PD dysfunction. Neuroprotective mechanisms were investigated through neurobehavioral, molecular, histopathological, and immunohistochemical studies. PNU reversed motor incoordination and hypokinesia induced via the intrastriatal injection of 6-hydroxydopamine and manifested by lower falling latency in the rotarod test, short ambulation time and low rearing incidence in open field test. Tyrosine hydroxylase immunostaining showed a significant restoration of dopaminergic neurons following PNU treatment, in addition to histopathological restoration in nigrostriatal tissues. PNU halted striatal neuroinflammation manifested as a suppressed expression of JAK2/NF-κB/GSk3ß accompanied by a parallel decline in the protein expression of TNF-α in nigrostriatal tissue denoting the modulator anti-inflammatory capacity. Moreover, the protective effects of PNU were partially reversed by the α7 nAChR antagonist, methyllycaconitine, indicating the role of α7 nAChR modulation in the mechanism of action of PNU. This is the first study to reveal the positive effects of PNU-120596 on motor derangements of PD via JAK2/NF-κB/GSk3ß/ TNF-α neuroinflammatory pathways, which could offer a potential therapeutic strategy for PD.

Protein phosphatase 1 regulatory inhibitor subunit 14C promotes triple-negative breast cancer progression via sustaining inactive glycogen synthase kinase 3 beta.

Triple-negative breast cancer (TNBC) is fast-growing and highly metastatic with the poorest prognosis among the breast cancer subtypes. Inactivation of glycogen synthase kinase 3 beta (GSK3ß) plays a vital role in the aggressiveness of TNBC; however, the underlying mechanism for sustained GSK3ß inhibition remains largely unknown. Here, we find that protein phosphatase 1 regulatory inhibitor subunit 14C (PPP1R14C) is upregulated in TNBC and relevant to poor prognosis in patients. Overexpression of PPP1R14C facilitates cell proliferation and the aggressive phenotype of TNBC cells, whereas the depletion of PPP1R14C elicits opposite effects. Moreover, PPP1R14C is phosphorylated and activated by protein kinase C iota (PRKCI) at Thr73. p-PPP1R14C then represses Ser/Thr protein phosphatase type 1 (PP1) to retain GSK3ß phosphorylation at high levels. Furthermore, p-PPP1R14C recruits E3 ligase, TRIM25, toward the ubiquitylation and degradation of non-phosphorylated GSK3ß. Importantly, the blockade of PPP1R14C phosphorylation inhibits xenograft tumorigenesis and lung metastasis of TNBC cells. These findings provide a novel mechanism for sustained GSK3ß inactivation in TNBC and suggest that PPP1R14C might be a potential therapeutic target.

Dapagliflozin diminishes memory and cognition impairment in Streptozotocin induced diabetes through its effect on Wnt/ß-Catenin and CREB pathway.

Diabetic neuropathy is a chronic condition that affects a significant number of individuals with diabetes. Streptozotocin injection intraperitoneally to rodents produces pancreatic islet ß-cell destruction causing hyperglycemia, which affect the brain leading to memory and cognition impairment. Dapagliflozin may be able to reverse beta-cell injury and alleviate this impairment. This effect may be via neuroprotective effect or possible involvement of the antioxidant, and anti-apoptotic properties. Forty rats were divided into four groups as follows: The normal control group, STZ-induced diabetes group, STZ-induced diabetic rats followed by treatment with oral dapagliflozin group and normal rats treated with oral dapagliflozin. Behavioral tests (Object location memory task and Morris water maze) were performed. Serum biomarkers (blood glucose and insulin) were measured and then the homeostatic model assessment for insulin resistance (HOMA-IR) was calculated. In the hippocampus the followings were determined; calmodulin, ca-calmodulin kinase Ⅳ (CaMKIV), protein kinase A (PKA) and cAMP-responsive element-binding protein to determine the transcription factor CREB and its signaling pathway also Wnt signaling pathway and related parameters (WnT, B-catenin, lymphoid enhancer binding factor LEF, glycogen synthase kinase 3ß). Moreover, nuclear receptor-related protein-1, acetylcholine and its hydrolyzing enzyme acetylcholine esterase, oxidative stress parameter malondialdehyde (MDA) and apoptotic parameter caspase-3 were determined. STZ was able to cause destruction to pancreatic ß-cells which was reflected on glucose levels causing diabetes. Diabetic neuropathy was clear in the rats performing the behavioral tests. Memory and cognition parameters in the hippocampus were negatively affected. Oxidative stress and apoptotic parameter were elevated while the electrical activity was declined. Dapagliflozin was able to reverse the previously mentioned parameters and behavior. Thus, to say dapagliflozin significantly showed neuroprotective action along with antioxidant, and anti-apoptotic properties.

Decursin prevents melanogenesis by suppressing MITF expression through the regulation of PKA/CREB, MAPKs, and PI3K/Akt/GSK-3ß cascades.

Abnormal melanin synthesis upon UV exposure causes excessive oxidative stress, which leads to skin hyperpigmentation disorders such as freckles, melisma, and age spots. The present study investigated the anti-melanogenic effects of decursin and the underlying mechanism using multiple approaches. Decursin exhibited no cytotoxicity and significantly reduced intracellular tyrosinase activity and melanin content in B16F10 melanoma cells. Decursin also inhibited the expression of melanogenic enzymes such as tyrosinase and tyrosinase-related protein (TRP)- 1, but not TRP-2. Mechanistically, decursin suppressed melanin synthesis through cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein (CREB)-dependent downregulation of microphthalmia-associated transcription factor (MITF), a master transcription factor in melanogenesis. Further, decursin exerted anti-melanogenic effects by downregulating the p38 signaling pathway and upregulating extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthesis kinase-3ß (GSK-3ß) cascades. in silico analysis showed that decursin formed specific interactions with residues of upstream regulators of MITF and exhibited optimal pharmacokinetic profiles, including permeability and skin sensitization. Finally, the anti-melanogenic effects of decursin were confirmed ex vivo in 3D human skin models, suggesting its applicability as a protective agent against hyperpigmentation.

Cannabidiol reverses memory impairments and activates components of the Akt/GSK3ß pathway in an experimental model of estrogen depletion.

Clinical and preclinical evidence has indicated that estrogen depletion leads to memory impairments and increases the susceptibility to neural damage. Here, we have sought to investigate the effects of Cannabidiol (CBD) a non-psychotomimetic compound from Cannabis sativa, on memory deficits induced by estrogen depletion in rats, and its underlying mechanisms. Adult rats were subjected to bilateral ovariectomy, an established estrogen depletion model in rodents, or sham surgery and allowed to recover for three weeks. After that, they received daily injections of CBD (10 mg/kg) for fourteen days. Rats were tested in the inhibitory avoidance task, a type of emotionally-motivated memory. After behavioral testing they were euthanized, and their hippocampi were isolated for analysis of components of the Akt/GSK3ß survival pathway and the antiapoptotic protein Bcl2. Results revealed that ovariectomy impaired avoidance memory, and CBD was able to completely reverse estrogen depletion-induced memory impairment. Ovariectomy also reduced Akt/GSK3ß pathway's activation by decreasing the phosphorylation levels of Akt and GSK3ß and Bcl2 levels, which were ameliorated by CBD. The present results indicate that CBD leads to a functional recovery accompanied by the Akt/GSK3ß survival pathway's activation, supporting its potential as a treatment for estrogen decline-induced deterioration of neural functioning and maintenance.

GSK-3ß as a target for apigenin-induced neuroprotection against Aß 25-35 in a rat model of Alzheimer's disease.

Glycogen synthase kinase-3 (GSK-3) is a critical molecule in Alzheimer's disease (AD) that modulates two histopathological hallmarks of AD: Amyloid beta (Aß) plaques and neurofibrillary tangles composed of aberrant hyper-phosphorylation of tau protein. This study was performed to investigate the protective effect of flavone apigenin through inhibition of GSK-3 and the involvement of this kinase in the inhibition of BACE1 expression and hyperphosphorylation of tau protein in an AD rat model. 15 nM of aggregated amyloid-beta 25-35 was microinjected into the left lateral ventricle of an AD rat. Apigenin (50 mg/kg) was administered orally 45 min before the Aß injection and continued daily for three weeks. Immunohistochemistry and western blot analysis showed that apigenin significantly reduced the hyperphosphorylation of tau levels in the hippocampus. Real-time PCR analysis revealed significant inhibition of the mRNA level of ß secretase (BACE1) and GSK-3ß, but Apigenin had no effect on the level of GSK-3α. The results demonstrate that apigenin has a protective effect against amyloid-beta 25-35 by decreasing the expression of GSK-3ß with the consequence of lowering the hyperphosphorylation of tau protein and suppressing BACE1 expression.

Sevoflurane preconditioning protects experimental ischemic stroke by enhancing anti-inflammatory microglia/macrophages phenotype polarization through GSK-3ß/Nrf2 pathway.

AIMS: Sevoflurane preconditioning (SPC) results in cerebral ischemic tolerance; however, the mechanism remains unclear. Promoting microglia/macrophages polarization from pro-inflammatory state to anti-inflammatory phenotype has been indicated as a potential treatment target against ischemic stroke. In this study, we aimed to assess the effect of SPC on microglia polarization after stroke and which signaling pathway was involved in this transition. METHODS: Mouse primary microglia with SPC were challenged by oxygen-glucose deprivation (OGD) or lipopolysaccharide (LPS), and mice with SPC were subjected to middle cerebral artery occlusion (MCAO). Then, the mRNA and protein levels of pro-inflammatory/anti-inflammatory factors were analyzed. GSK-3ß phosphorylation and Nrf2 nuclear translocation were measured. The mRNA and protein expression of pro-inflammatory/anti-inflammatory factors, neurological scores, infarct volume, cellular apoptosis, the proportion of pro-inflammatory/anti-inflammatory microglia/macrophages, and the generation of super-oxidants were examined after SPC or GSK-3ß inhibitor TDZD treatment with or without Nrf2 deficiency. RESULTS: Sevoflurane preconditioning promoted anti-inflammatory and inhibited pro-inflammatory microglia/macrophages phenotype both in vitro and in vivo. GSK-3ß phosphorylation at Ser9 was increased after SPC. Both SPC and TDZD administration enhanced Nrf2 nuclear translocation, reduced pro-inflammatory microglia/macrophages markers expression, promoted anti-inflammatory markers level, and elicited a neuroprotective effect. Nrf2 deficiency abolished the promoted anti-inflammatory microglia/macrophages polarization and ischemic tolerance induced by TDZD treatment. The reduced percentage of pro-inflammatory positive cells and super-oxidants generation induced by SFC or TDZD was also reversed by Nrf2 knockdown. CONCLUSIONS: Our results indicated that SPC exerts brain ischemic tolerance and promotes anti-inflammatory microglia/macrophages polarization by GSK-3ß-dependent Nrf2 activation, which provides a novel mechanism for SPC-induced neuroprotection.

GSK-3ß inhibitor TWS119 alleviates hypoxic-ischemic brain damage via a crosstalk with Wnt and Notch signaling pathways in neonatal rats.

Preterm infant brain injury is a leading cause of morbidity and disability in survivors of preterm infants. Unfortunately, the effective treatment remains absent. Recent evidence suggests that GSK-3ß inhibitor TWS119 has a neuroprotectiverole in adult brain injury by activation of Wnt/ß-catenin signaling pathway. However, the role on neonatal brain injury is not yet explored. The study aims to evaluate the effect of TWS119 at 7 d after hypoxic-ischemic brain damage and investigate the mechanism that it regulates Wnt and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats. Three-day-old rats were randomly divided into 3 groups: sham group, HI group and TWS119 group. The neonatal rats were subjected to left carotid artery ligation followed by 2 h of hypoxia (8.0% O2). A single dose of TWS119 (30 mg/kg) was intraperitoneally injected 20 min prior to hypoxia-ischemia (HI). At 7 d after HI, TWS119 improved the tissue structure, reduced cell apoptosis, up-regulated bcl-2 expression, up-regulated the expression of PSD-95 and Synapsin-1. At 24 h after HI, it activated Wnt/ß-catenin signaling pathway by up-regulation of ß-catenin protein expression and wnt3a/wnt5a/wnt7a mRNA expression. Simultaneously, it suppressed Notch signaling pathway by down-regulation of Notch1 and HES-1 proteins expression. Our study suggested that TWS119 performed a neuroprotective function at 7 d after hypoxic-ischemic brain damage via a crosstalk with Wnt/ß-catenin and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats.

Dimethyl fumarate abridged tauo-/amyloidopathy in a D-Galactose/ovariectomy-induced Alzheimer's-like disease: Modulation of AMPK/SIRT-1, AKT/CREB/BDNF, AKT/GSK-3ß, adiponectin/Adipo1R, and NF-κB/IL-1ß/ROS trajectories.

Since the role of estrogen in postmenauposal-associated dementia is still debatable, this issue urges the search for other medications. Dimethyl fumarate (DMF) is a drug used for the treatment of multiple sclerosis and has shown a neuroprotective effect against other neurodegenerative diseases. Accordingly, the present study aimed to evaluate the effect of DMF on an experimental model of Alzheimer disease (AD) using D-galactose (D-Gal) administered to ovariectomized (OVX) rats, resembling a postmenopausal dementia paradigm. Adult 18-month old female Wistar rats were allocated into sham-operated and OVX/D-Gal groups that were either left untreated or treated with DMF for 56 days starting three weeks after sham-operation or ovariectomy. DMF succeeded to ameliorate cognitive (learning/short- and long-term memory) deficits and to enhance the dampened overall activity (NOR, Barnes-/Y-maze tests). These behavioral upturns were associated with increased intact neurons (Nissl stain) and a reduction in OVX/D-Gal-mediated hippocampal CA1 neurodegeneration and astrocyte activation assessed as GFAP immunoreactivity. Mechanistically, DMF suppressed the hippocampal contents of AD-surrogate markers; viz., apolipoprotein (APO)-E1, BACE1, Aß42, and hyperphosphorylated Tau. Additionally, DMF has augmented the neuroprotective parameters p-AKT, its downstream target CREB and BDNF. Besides, it activated AMPK, and enhanced SIRT-1, as well as antioxidant defenses (SOD, GSH). On the other hand, DMF inhibited the transcription factor NF-κB, IL-1ß, adiponectin/adiponectin receptor type (AdipoR)1, GSK-3ß, and MDA. Accordingly, in this postmenopausal AD model, DMF treatment by pursuing the adiponectin/AdipoR1, AMPK/SIRT-1, AKT/CREB/BDNF, AKT/GSK-3ß, and APO-E1 quartet hampered the associated tauo-/amyloidopathy and NF-κB-mediated oxidative/inflammatory responses to advance insights into its anti-amnesic effect.

Enhanced anticancer activity by the combination of vinpocetine and sorafenib via PI3K/AKT/GSK-3ß signaling axis in hepatocellular carcinoma cells.

Vinpocetine is widely used to treat cerebrovascular diseases. However, the effect of vinpocetine to treat hepatocellular carcinoma (HCC) has not been investigated. In this study, we revealed that vinpocetine was associated with antiproliferative activity in HCC cells, but induced cytoprotective autophagy, which restricted its antitumor activity. Autophagy inhibitors improved the antiproliferative activity of vinpocetine in HCC cells. Sorafenib is effective to treat advanced HCC, but the effect of autophagy induced by sorafenib is indistinct. We demonstrated vinpocetine plus sorafenib suppressed the cytoprotective autophagy activated by vinpocetine in HCC cells and significantly induced apoptosis and suppressed cell proliferation in HCC cells. In addition, vinpocetine plus sorafenib activates glycogen synthase kinase 3ß (GSK-3ß) and subsequently inhibits cytoprotective autophagy induced by vinpocetine in HCC cells. Meanwhile, overexpression of GSK-3ß was efficient to increase the apoptosis induced by vinpocetine plus sorafenib in HCC cells. Our study revealed that vinpocetine plus sorafenib could suppress the cytoprotective autophagy induced by vinpocetine and subsequently show synergistically anti-HCC activity via activating GSK-3ß and the combination of vinpocetine and sorafenib might reverse sorafenib resistance via the PI3K/protein kinase B/GSK-3ß signaling axis. Thus, vinpocetine may be a potential candidate for sorafenib sensitization and HCC treatment, and our results may help to elucidate more effective therapeutic options for HCC patients with sorafenib resistance.

IL-37-induced activation of glycogen synthase kinase 3ß promotes IL-1R8/Sigirr phosphorylation, internalization, and degradation in lung epithelial cells.

Interleukin (IL)-37 diminishes a variety of inflammatory responses through ligation to its receptor IL-1R8/Sigirr. Sigirr is a Toll like receptor/IL-1R family member. We have shown that Sigirr is not stable in response to IL-37 treatment. IL-37-induced Sigirr degradation is mediated by the ubiquitin-proteasome system, and the process is reversed by a deubiquitinase, USP13. However, the molecular mechanisms by which USP13 regulates Sigirr stability have not been revealed. In this study, we investigate the roles of glycogen synthesis kinase 3ß (GSK3ß) in Sigirr phosphorylation and stability. IL-37 stimulation induced Sigirr phosphorylation and degradation, as well as activation of GSK3ß. Inhibition of GSK3ß attenuated IL-37-induced Sigirr phosphorylation, while exogenous expressed GSK3ß promoted Sigirr phosphorylation at threonine (T)372 residue. Sigirr association with GSK3ß was detected. Amino acid residues 51-101 in GSK3ß were identified as the Sigirr binding domain. These data indicate that GSK3ß mediates IL-37-induced threonine phosphorylation of Sigirr. Further, we investigated the role of GSK3ß-mediated phosphorylation of Sigirr in Sigirr degradation. Inhibition of GSK3ß attenuated IL-37-induced Sigirr degradation, while T372 mutant of Sigirr was resistant to IL-37-mediated degradation. Furthermore, inhibition of Sigirr phosphorylation prevented Sigirr internalization and association with USP13, suggesting GSK3ß promotes Sigirr degradation through disrupting Sigirr association with USP13.

Intranasal insulin ameliorates cognitive impairment in a rat model of Parkinson's disease through Akt/GSK3ß signaling pathway.

AIMS: Parkinson's disease dementia (PDD) is one of the most common non-motor symptoms of advanced Parkinson's disease (PD). This study aimed to determine whether intranasal insulin has protective effects on cognition in the rat PD model induced by 6-hydroxylase dopamine (6-OHDA) through the insulin signaling pathway. MATERIALS AND METHODS: The rats were given intranasal insulin administration for six weeks after unilateral medial forebrain bundle (MFB) injection of 6-OHDA. Then a series of cognitive-behavioral tests, immunofluorescence, and immunoblotting was performed on the rats. KEY FINDINGS: The results demonstrated that the injection of 6-OHDA in the unilateral MFB damaged working memory and long-term habituation of rats in the T-maze rewarded alternation test and hole-board test. Besides, rats with unilateral 6-OHDA injury performed poorly in terms of escape latency and average speed during the hidden platform training phase rather than in the probe trial of the Morris Water Maze (MWM) test. Immunofluorescence results showed that unilateral 6-OHDA injury in MFB led to the massive death of ipsilateral-substantia nigra (SN) tyrosine hydroxylase (TH)-positive neurons. Western blot results further indicated that 6-OHDA-induced necrosis of ipsilateral-SN dopaminergic neurons reduced the levels of p-Akt (Ser473) and p-GSK3ß (Ser9) in the ipsilateral-hippocampus. SIGNIFICANCE: These findings provide a solid evidence base for the relationship between PD cognitive impairment and insulin signaling pathways.

Arctigenin attenuates platelet activation and clot retraction by regulation of thromboxane A2 synthesis and cAMP pathway.

Pathophysiological reaction of platelets in the blood vessel is an indispensable part of thrombosis and cardiovascular disease, which is the most common cause of death in the world. In this study, we performed in vitro assays to evaluate antiplatelet activity of arctigenin in human platelets and attempted to identify the mechanism responsible for thromboxane A2 (TXA2) generation, integrin αIIbß3 activation and cAMP pathway. Arctigenin exhibited obvious inhibitory effects on collagen-, thrombin-, and ADP-induced human platelet aggregation, granule secretion, TXA2 generation, integrin αIIbß3 activation, and clot retraction. Additionally, we found that arctigenin attenuated PI3K/Akt/mTOR/GSK-3ß and MAPK signaling pathways, and increased cAMP level. Accordingly, the findings support that arctigenin attenuates thrombotic events through the inhibition of platelet activation and platelet plug formation. Therefore, we suggest that arctigenin may have therapeutic potential as an antiplatelet and antithrombotic agent.

Di-n-hexyl phthalate causes Leydig cell hyperplasia in rats during puberty.

Di-n-hexyl phthalate (DNHP) is commonly used as a plasticizer. However, whether DNHP influences Leydig cell development during puberty remains unexplored. In this study, DNHP (0, 10, 100, and 1000 mg/kg) was administered via gavage to 35-day-old male Sprague-Dawley rats for 21 days. Serum levels of testosterone, luteinizing hormone, follicle-stimulating hormone, Leydig cell number, the expression of Leydig and Sertoli cell genes and proteins were investigated. DNHP significantly increased serum testosterone levels at 10 mg/kg but lowered its level at 1000 mg/kg. DNHP significantly increased luteinizing hormone levels at 1000 mg/kg without affecting follicle-stimulating hormone levels. DNHP increased Leydig cell number at all doses but down-regulated the expression of Lhcgr, Hsd3b1, Hsd17b3, and Hsd11b1 in Leydig cell per se at 1000 mg/kg. DNHP elevated phosphorylation of ERK1/2 and GSK-3ß at 10 mg/kg but decreased SIRT1 and PGC-1α levels at 1000 mg/kg. In conclusion, DNHP exposure causes Leydig cell hyperplasia possibly via stimulating phosphorylation of ERK1/2 and GSK-3ß signaling pathways.

Pyrroloquinoline Quinine and LY294002 Changed Cell Cycle and Apoptosis by Regulating PI3K-AKT-GSK3ß Pathway in SH-SY5Y Cells.

To verify the role of PI3K-AKT-GSK3ß pathway during manganese (Mn)-induced cell death, apoptosis, related indicators were investigated. SH-SY5Y cells were directly exposed to different concentrations of MnCl2. Then, cell viability, apoptosis, necrosis rate, and cell cycle were detected by MTT, FITC Annexin V Apoptosis Detection Kit with PI and PI staining. Then, in two intervention groups, cells were preconditioned with agonist (PQQ) and suppressant (LY294002). The cell viability decreased with a dose-response relationship (p < 0.05), while apoptosis and necrosis increased (p < 0.05). The ratio of G0/G1 and G2/M also decreased, but the percentage of S phase increased (p < 0.05). During above process, PI3K-AKT-GSK3ß pathway was involved by regulating the expression of PI3K, AKT, p-AKT, and GSK3ß (p < 0.05). For further research, cell cycle and apoptosis were detected pretreatment with PQQ and LY294002 before Mn exposure. The result showed cell ability, apoptosis, and necrosis rate changed obviously compared with non-pretreated group (p < 0.05). The variance of G0/G1 and G2/M ratio and percentage of S phase were also different, especially in 2.0 mM (p < 0.05). Mn can cause apoptosis and necrosis, varying cell cycle of SH-SY5Y cells, which could be changed by PQQ and LY294002 by regulating PI3K-AKT-GSK3ß pathway.