Phosphorylation Of Glycogen Synthase Kinase-3beta Research Articles

The GLP-1 Agonist Semaglutide Ameliorates Cognitive Regression in P301S Tauopathy Mice Model via Autophagy/ACE2/SIRT1/FOXO1-Mediated Microglia Polarization.

Tau hyper-phosphorylation has been recognized as an essential contributor to neurodegeneration in Alzheimer's disease (AD) and related tauopathies. In the last decade, tau hyper-phosphorylation has gained considerable concern in AD therapeutic development. Tauopathies are manifested with a broad spectrum of symptoms, from dementia to cognitive decline and motor impairments. Tau undergoes conformational changes and abnormal phosphorylation that mediate its detaching from microtubules, forming neurofibrillary tangles (NFTs). In the current study, a widely used P301S transgenic mice model of tauopathy was employed to evaluate the possible neuroprotective effects of semaglutide as an autophagy regulator through modifications of the brain renin-angiotensin system (RAS). Mice were divided into two groups according to their genotypes (wild type (Wt) and P301S), which were further subdivided to receive either vehicle (saline) or semaglutide (25 nmol/kg, i.p.), once every 2 days for 28 days. Current data suggest that semaglutide ameliorated the hyperactive pattern and alleviated the cognitive decline of P301S mice. It also hastened the autophagic flux through augmenting angiotensin-converting enzyme 2/sirtuin 1/forkhead box protein O1 signaling. Semaglutide also hindered the expression of phosphorylated adenosine monophosphate-activated protein kinase and phosphorylated glycogen synthase kinase-3 beta at serine 9, reducing the propagation of neuroinflammatory cytokines and oxidative reactions. Finally, semaglutide protected against hippocampal degeneration and reduced the immunoreactivity for total tau and ionized calcium-binding adapter molecule. Semaglutide showed promising neuroprotective implications in alleviating tauopathy-related AD's molecular and behavioral deficits through controlling autophagy and brain RAS.

LT-102, an AMPA receptor potentiator, alleviates depression-like behavior and synaptic plasticity impairments in prefrontal cortex induced by sleep deprivation

BackgroundSleep loss is closely related to the onset and development of depression, and the mechanisms involved may include impaired synaptic plasticity. Considering the important role of glutamate α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) in synaptic plasticity as well as depression, we introduce LT-102, a novel AMPARs potentiator, to evaluate the potential of LT-102 in treating sleep deprivation-induced depression-like behaviors. MethodsWe conducted a comprehensive behavioral assessment to evaluate the effects of LT-102 on depression-like symptoms in male C57BL/6J mice. This assessment included the open field test to measure general locomotor activity and anxiety-like behavior, the forced swimming test and tail suspension test to assess despair behaviors indicative of depressive states, and the sucrose preference test to quantify anhedonia, a core symptom of depression. Furthermore, to explore the impact of LT-102 on synaptic plasticity, we utilized a combination of Western blot analysis to detect protein expression levels, Golgi-Cox staining to visualize neuronal morphology, and immunofluorescence to examine the localization of synaptic proteins. Additionally, we utilized primary cortical neurons to delineate the signaling pathway modulated by LT-102. ResultsTreatment with LT-102 significantly reduced depression-like behaviors associated with sleep deprivation. Quantitative Western blot (WB) analysis revealed a significant increase in GluA1 phosphorylation in the prefrontal cortex (PFC), triggering the Ca2+/calmodulin-dependent protein kinase II/cAMP response element-binding protein/brain-derived neurotrophic factor (CaMKII/CREB/BDNF) and forkhead box protein P2/postsynaptic density protein 95 (FoxP2/PSD95) signaling pathways. Immunofluorescence imaging confirmed that LT-102 treatment increased spine density and co-labeling of PSD95 and vesicular glutamate transporter 1 (VGLUT1) in the PFC, reversing the reductions typically observed following sleep deprivation. Golgi staining further validated these results, showing a substantial increase in neuronal dendritic spine density in sleep-deprived mice treated with LT-102. Mechanistically, application of LT-102 to primary cortical neurons, resulted in elevated levels of phosphorylated AKT (p-AKT) and phosphorylated glycogen synthase kinase-3 beta (p-GSK3β), key downstream molecules in the BDNF signaling pathway, which in turn upregulated FoxP2 and PSD95 expression. LimitationsIn our study, we chose to exclusively use male mice to eliminate potential influences of the estrous cycle on behavior and physiology. As there is no widely accepted positive drug control for sleep deprivation studies, we did not include one in our research. ConclusionOur results suggest that LT-102 is a promising therapeutic agent for counteracting depression-like behaviors and synaptic plasticity deficits induced by sleep deprivation, primarily through the activation of CaMKII/CREB/BDNF and AKT/GSK3β/FoxP2/PSD95 signaling pathways.

Cilostazol Combats Lipopolysaccharide-Induced Hippocampal Injury in Rats: Role of AKT/GSK3β/CREB Curbing Neuroinflammation.

Neuroinflammation is important in the pathophysiology of several degenerative brain disorders. This study looked at the potential neuroprotective benefits of cilostazol, a phosphodiesterase inhibitor, against LPS-induced hippocampus damage in rodents and the principal molecular involvement of AKT/GSK3β/CREB signaling pathways. Behavioral tests revealed that cilostazol successfully corrected LPS-induced neurobehavioral impairments. Furthermore, cilostazol therapy lowered hippocampal levels of amyloid beta 1-42 (Aβ1-42) and p-tau protein, both of which are critical pathological indicators of neurodegenerative disorders. Furthermore, cilostazol administration suppressed LPS-induced rises in hippocampus caspase-3 and NF-κB levels while elevating rat B-cell/lymphoma 2 (BCL2) and brain-derived neurotrophic factor (BDNF) levels, which are implicated in neuronal survival and synaptic plasticity. Cilostazol treatment also restored the decreased phosphorylation of protein kinase B (p-AKT) and reduced the elevated levels of phosphorylated glycogen synthase kinase-3 beta (p-GSK3β) and cAMP response element-binding protein (CREB) in the hippocampus of LPS-treated rats. Histopathological examination revealed that cilostazol ameliorated LPS-induced brain damage with reduced neuronal loss and gliosis. Immunohistochemistry analysis showed a decrease in Iba-1 expression, indicating a reduction in microglial activation in the cilostazol-treated group compared to the LPS group. The findings advocate that cilostazol exerts neuroprotective effects against LPS-induced hippocampal injury by modulating the AKT/GSK3β/CREB pathway and curbing neuroinflammation. Cilostazol may hold promise as a therapeutic agent for neuroinflammatory conditions associated with neurodegenerative diseases.

The Wnt/β-catenin signaling pathway inhibits osteoporosis by regulating the expression of TERT: an in vivo and in vitro study.

Our study was performed to investigate whether the Wingless and int-1 (Wnt) signaling pathway promotes osteogenic differentiation and inhibits apoptosis in bone marrow mesenchymal stem cells (BMSCs) by regulating telomerase reverse transcriptase (TERT) expression. An in vivo model of osteoporosis (OP) in C57BL/6J mice by bilateral ovariectomy (OVX) and an in vitro model of H2O2-induced BMSCs were established separately. Western blotting was used to detect the expression of the pathway-related proteins TERT, β-catenin, and phosphorylated-glycogen synthase kinase-3beta (p-GSK3β)/GSK3β, the osteogenic-related markers osteopontin (OPN), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (Runx2), and the apoptosis-related indicators B-cell lymphoma-2 (Bcl-2) and BAX. Osteoblastic phenotypes were also evaluated by alkaline phosphatase (ALP) staining and serum ALP activity assays. Osteogenic differentiation phenotypes in mice were verified by H&E staining, micro-CT, and parameter analysis of the femur. Western blotting results showed that the expression of the pathway-related proteins TERT, β-catenin, p-GSK3β/GSK3β was reduced in OVX mice and H2O2-induced BMSCs, accompanied by downregulated protein expression of osteogenic-related markers and antiapoptotic indicators and upregulated protein expression of apoptotic proteins compared to those in the control group. Mechanistic studies showed that the activation of Wnt signaling pathway in BMSCs promoted β-catenin translocation to the nucleus, as verified by immunofluorescence and facilitated colocalization between β-catenin and TERT, as verified by double-labeling immunofluorescence, thereby promoting osteogenic differentiation and reducing apoptosis. In summary, our experiments confirmed that the GSK3β/β-catenin/TERT pathway could regulate the osteogenic differentiation and apoptosis of BMSCs and that TERT might be a promising target for the future treatment of osteoporosis.

Stratifin Promotes Hepatocellular Carcinoma Progression by Modulating the Wnt/β-Catenin Pathway.

Abnormal stratifin (SFN) expression is closely related to the progression of several human cancers, but the potential roles of SFN in hepatocellular carcinoma (HCC) remain largely unknown. In this study, we found that SFN was upregulated in HCC cell lines and tissues and was positively associated with tumor size, poor differentiation, Tumor Node Metastasis (TNM) stage, and vascular invasion. In addition, high expression levels of SFN were associated with poor overall survival and disease-free survival. Biologically, downregulation of SFN suppressed tumor cell proliferation, epithelial-mesenchymal transition (EMT), invasion, and migration in vitro and tumor growth in vivo. However, overexpression of SFN promoted cell proliferation, EMT, invasion, and migration in vitro and tumor growth in vivo. Mechanistically, overexpression of SFN activated the Wnt/β-catenin pathway by promoting Glycogen synthase kinase-3 beta (GSK-3β) phosphorylation, decreasing β-catenin phosphorylation, promoting β-catenin transport into the nucleus, and enhancing the expression of c-Myc, whereas depletion of SFN inhibited the Wnt/β-catenin pathway. In addition, TOPFlash/FOPFlash reporter assays showed that overexpression or downregulation of SFN obviously increased or decreased, respectively, the activity of the Wnt/β-catenin pathway. Our results indicated that SFN plays an important role in HCC, possibly providing a prognostic factor and therapeutic target for HCC.

Astragalin alleviates cognitive deficits and neuronal damage in SAMP8 mice through upregulating estrogen receptor expression.

Senile plaques composed of β-amyloid protein (Aβ) and neurofibrillary tangles (NFTs) composed of intracellular hyper-phosphorylated tau are major causes of cognitive impairment and neuronal damage in Alzheimer disease (AD). Astragalin (AST), a naturally-occurring flavonoid compound, was reported to have neuroprotective effects in the brain, but its effects in AD remain unknown. Herein, the learning and memory deficits were alleviated and neuronal damage in the hippocampus were inhibited after the senescence-accelerated mouse prone 8 (SAMP8) mouse were given AST (5mg/kg or 10mg/kg) daily by gavage for 2 months. Furthermore, AST reduced Aβ1-40 and Aβ1-42 deposition, decreased β-carboxyl-terminal fragment (β-CTF) protein level and tau hyper-phosphorylation, but increased α-CTF protein level and glycogen synthase kinase-3beta (GSK-3β) phosphorylation in hippocampus of SAMP8 mice. Meanwhile, the effects of AST on AD were also explored in vitro by treating primary neurons with amyloid-β1-42 oligomers (Aβ1-42O). Consistently, AST also alleviated amyloid-β1-42 oligomers (Aβ1-42O)-induced neuronal damage, amyloid plaques, and tau phosphorylation in vitro model. Of note, estrogen receptor (ER)α and ERβ expression in the hippocampus of SAMP8 mice and Aβ1-42O-treated neurons was significantly decreased but their levels were increased by AST. Moreover, in vivo and in vitro experiments revealed that ER antagonist, Fulvestrant, reversed the effects caused by AST. Altogether, our investigation indicates that AST may ameliorate cognitive deficits and AD-type pathologies in SAMP8 mice and Aβ1-42O-treated neurons through upregulating ERα and ERβ expression. Our findings indicate the value of AST as a potential reagent for AD treatment.

Sulforaphane Inhibits Proliferation and Apoptosis of Colorectal Cancer Cells by Down-Regulating the Cyclooxygenase-2/Protein Kinase B/Glycogen Synthase Kinase-3 Beta Signaling Pathway

To determine the effects of sulforaphane on the proliferation and apoptosis of colorectal cancer cell line HCT116 and the levels of proteins related to the cyclooxygenase-2/protein kinase B/glycogen synthase kinase-3 beta signal transduction. Colorectal cancer HCT116 cell lines were cultured in vitro and assigned into sulforaphane (30, 60 and 120 μM) groups, simple cisplatin group and cisplatin+sulforaphane group. The relative survival rate and apoptosis rate of HCT116 cells were determined by cell counting kit-8 assay and flow cytometry, respectively. Besides, the expression levels of proteins survivin, caspase-3, cyclooxygenase-2, protein kinase B, phosphorylated-protein kinase B, glycogen synthase kinase-3 beta and phosphorylated-glycogen synthase kinase-3 beta in HCT116 cells were measured through Western blotting. Compared with that in control group, the relative survival rate of cells in 60 and 120 μM sulforaphane groups, simple cisplatin group and cisplatin+sulforaphane group declined significantly (p<0.05). Sulforaphane can exert its anti-tumor effect by inducing apoptosis and suppressing proliferation of HCT116 cells and its mechanism may be associated with inhibition on the activation of the cyclooxygenase-2/protein kinase B/ glycogen synthase kinase-3 beta signaling pathway.

Effect of l-carnitine supplementation on lead acetate-induced liver cell apoptosis and inflammation: role of caspase-3 and glycogen synthase kinase-3β enzymes

AimThe study aimed at studying the hepatoprotective effect of l-carnitine against lead (Pb) acetate-induced hepatocellular injury, emphasizing the role of caspase-3 and glycogen synthase kinase-3β in hepatocellular apoptosis and inflammation. Materials and methodsMale Wistar rats were used. The experimental approach involved estimation of the liver enzymes' serum levels. Oxidative and inflammatory biomarkers were measured in hepatic tissue homogenates. Paraffin-embedded hepatic sections were prepared for histopathology and immunohistochemistry. Quantitative determination of the phosphorylated glycogen synthase kinase-3 beta was performed. Key findingsThe serum showed a significant elevation in ALT, AST, and LDH; tissue homogenates showed significant elevation in lipid peroxide and inflammatory biomarkers with significant reduction in reduced glutathione in the Pb acetate-treated group. Co-administration of l-carnitine with Pb acetate produced significant reduction in liver enzymes with significant improvement in oxidant, antioxidant and inflammatory markers. Lead acetate treatment significantly reduced the phosphorylated glycogen synthase kinase-3 beta, while l-carnitine enhanced its phosphorylation. Histopathological examination showed inflammatory reaction around blood vessels with fatty degeneration in hepatocytes of the Pb acetate intoxicated group. l-Carnitine caused a decrease in hepatic damage with minimal vascular alterations in central vein. Caspase-3 expression in hepatocytes was decreased in Pb-treated group supplemented with l-carnitine. SignificanceOur study reveals that oxidative stress and inflammation participate in Pb acetate-induced hepatocellular injury. Glycogen synthase kinase-3β and caspase-3 play role in Pb acetate-induced hepatic damage. l-Carnitine shows significant protective effects against hepatocellular apoptosis and inflammation induced by Pb acetate through antioxidant, anti-inflammatory and anti-apoptotic pathways in part mediated by GSK-3β inhibition.

Moringa oleifera Lam. seed extract protects kidney function in rats with diabetic nephropathy by increasing GSK-3β activity and activating the Nrf2/HO-1 pathway

BackgroundMoringa oleifera Lam. (M. oleifera) seeds are widely used in traditional folk medicine and as nutritional supplements in the Middle East, Africa, and other regions. Published research showed that M. oleifera seeds (MOS) have pharmacological activities such as blood glucose-lowering, anti-inflammatory, and antitumor effects. However, experimental evidence on the use of MOS to treat diabetic nephropathy and its underlying mechanisms were rarely reported. PurposeTo evaluate the therapeutic effects of MOS extract on the kidneys of high-fat diet (HFD)-fed rats with streptozotocin-induced diabetic nephropathy and reveal its underlying mechanisms. Study designHFD-fed rats with streptozotocin-induced diabetic nephropathy and high-glucose induced Human Renal Mesangial Cells (HRMC) were used to explore the protective effect of MOS on diabetic nephropathy in vivo and in vitro. Methods: HRMC were used to preliminarily evaluate the effect of MOS extract under high glucose conditions. For the in vivo study, rats were divided into the following 6 groups (n = 5): normal control group (NC), diabetic nephropathy model group (DN), high dose of MOS-treatment group (DN + MOS-H, 200 mg/kg/d); medium dose of MOS-treatment group (DN + MOS-M, 100 mg/kg/d); low dose of MOS-treatment group (DN + MOS-l, 50 mg/kg/d), and metformin-treatment group (DN + MET, 200 mg/kg/d). After 4 weeks of treatment, the damage caused by DN was assessed based on the related parameters of urine and blood. Periodic acid-Schiff (PAS) staining and hematoxylin and eosin (HE) staining were used to assess pathological tissue damage. Immunohistochemistry was used to detect nuclear factor erythroid-derived 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and phosphorylated-glycogen synthase kinase-3beta (P-GSK-3β) levels, whereas western blotting was used to detect Nrf2, HO-1, nephrin, GSK-3β, and p-GSK-3β levels. ResultsMOS extract could inhibit the proliferation of HRMCs induced by high glucose levels. Compared with the rats in the DN group, MOS not only significantly reduced blood glucose levels and oxidative stress in the experimental rats but also improved their kidney function and reduced kidney tissue damage. Additionally, MOS extract increased GSK-3β activity and the expression of Nrf2 and HO-1. ConclusionsThis study showed that MOS could activate GSK-3β and Nrf2/HO-1 pathways to exert antioxidant and anti-renal fibrosis activities, and delay the progression of diabetic nephropathy.

Deletion or inhibition of prolyl oligopeptidase blocks lithium-induced phosphorylation of GSK3b and Akt by activation of protein phosphatase 2A.

Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5-triphosphate (IP3 ) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP-related lithium insensitivity is dependent on PP2A. To assess this, HEK-293 and SH-SY5Y cells with PREP deletion or PREP inhibition (KYP-2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium-induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP-2047 did not have effect on lithium-induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation.

Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3β/NRF2 Pathway in Diabetic Hindlimb Ischemia.

Background Peripheral arterial disease (PAD) is a typical disease of atherosclerosis, most commonly influencing the lower extremities. In patients with PAD, revascularization remains a preferred treatment strategy. Buyang Huanwu decoction (BHD) is a popular Chinese herbal prescription which has showed effects of cardiovascular protection through conducting antioxidant, antiapoptotic, and anti-inflammatory effects. Here, we intend to study the effect of BHD on promoting revascularization via the Akt/GSK3β/NRF2 pathway in diabetic hindlimb ischemia (HLI) model of mice. Materials and Methods All db/db mice (n = 60) were randomly divided into 6 groups by table of random number. (1) Sham group (N = 10): 7-0 suture thread passed through the underneath of the femoral artery and vein without occlusion. The remaining 5 groups were treated differently on the basis of the HLI (the femoral artery and vein from the inguinal ligament to the knee joint were transected and the vascular stump was ligated with 7-0 silk sutures) model: (2) HLI+NS group (N = 15): 0.2 ml NS was gavaged daily for 3 days before modeling and 14 days after occlusion; (3) HLI+BHD group (N = 15): 0.2 ml BHD (20 g/kg/day) was gavaged daily for 3 days before modeling and 14 days after occlusion; (4) HLI+BHD+sh-NC group (N = 8): local injection of adenovirus vector carrying the nonsense shRNA (Ad-GFP) in the hindlimbs of mice before treatment; (5) HLI+BHD+sh-NRF2 group (N = 8): knockdown of NRF2 in the hindlimbs of mice by local intramuscular injection of adenovirus vector carrying NRF2 shRNA (Ad-NRF2-shRNA) before treatment; and (6) HLI+BHD+LY294002 group (N = 4): intravenous injection of LY294002 (1.5 mg/kg) once a day for 14 days on the basis of the HLI+BHD group. Laser Doppler examination, vascular cast, and immunofluorescence staining were applied to detect the revascularization of lower limbs in mice. Western blot analysis was used to detect the expression of vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor- (TNF-) α, heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone-1 (NQO-1), catalase (CAT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphorylated protein kinase B (p-AKT), and phosphorylated glycogen synthase kinase-3 beta (p-GSK3β). HE staining was used to assess the level of muscle tissue damage and inflammation in the lower extremities. Local multipoint injection of Ad-NRF2-shRNA was used to knock down NRF2, and qPCR was applied to detect the mRNA level of NRF2. The blood glucose, triglyceride, cholesterol, MDA, and SOD levels of mice were tested using corresponding kits. The SPSS 20.0 software and GraphPad Prism 6.05 were used to do all statistics. Values of P < 0.05 were considered as statistically significant. Results and Conclusions. BHD could enhance the revascularization of lower limbs in HLI mice, while BHD has no effect on blood glucose and lipid level in db/db mice (P > 0.05). BHD could elevate the protein expression of VEGF, HO-1, NQO-1, and CAT (P < 0.05) and decrease the expression of IL-1β, IL-6, and TNF-α (P < 0.05) in HLI mice. Meanwhile, BHD could activate NRF2 and promote the phosphorylation of AKT/GSK3β during revascularization (P < 0.05). In contrast, knockdown of NRF2 impaired the protective effects of BHD on HLI (P < 0.05). LY294002 inhibited the upregulation of NRF2 activated by BHD through inhibiting the phosphorylation of the AKT/GSK3β pathway (P < 0.05). The present study demonstrated that BHD could promote revascularization on db/db mice with HLI through targeting antioxidation, anti-inflammation, and angiogenesis via the AKT/GSK3β/NRF2 pathway.

Effects of Aerobic Exercise on Tau and Related Proteins in Rats with the Middle Cerebral Artery Occlusion.

Although Alzheimer’s disease (AD)-like pathology is frequently found in patients with post-stroke dementia, little is known about the effects of aerobic exercise on the modifications of tau and related proteins. Therefore, we evaluated the effects of aerobic exercise on the phosphorylation and acetylation of tau and the expressions of tau-related proteins, after middle cerebral artery occlusion (MCAO) stroke. Twenty-four Sprague–Dawley rats with MCAO infarction were used in this study. The rehabilitation group (RG) received treadmill training 40 min/day for 12 weeks, whereas the sedentary group (SG) did not receive any type of training. Functional tests, such as the single pellet reaching task, rotarod, and radial arm maze tests, were performed monthly for 3 months. In ipsilateral cortices in the RG and SG groups, level of Ac-tau was lower in the RG, whereas levels of p-tauS396, p-tauS262, and p-tauS202/T205 were not significantly lower in the RG. Level of phosphorylated glycogen synthase kinase 3-beta Tyr 216 (p-GSK3βY216) was lower in the RG, but levels of p-AMPK and phosphorylated glycogen synthase kinase 3-beta Ser 9 (p-GSK3βS9) were not significantly lower. Levels of COX-2 and BDNF were not significantly different between the two groups, while SIRT1 significantly decreased in ipsilateral cortices in RG. In addition, aerobic training also improved motor, balance, and memory functions. Rehabilitation with aerobic exercise inhibited tau modification, especially tau acetylation, following infarction in the rat MCAO model, which was accompanied with the improvement of motor and cognitive functions.

Aliskiren and captopril improve cognitive deficits in poorly controlled STZ-induced diabetic rats via amelioration of the hippocampal P-ERK, GSK3β, P-GSK3β pathway

Learning and memory deficits are obvious symptoms that develop over time in patients with poorly controlled diabetes. Hyperactivity of the renin-angiotensin system (RAS) is directly associated with β-cell dysfunction and diabetic complications, including cognitive impairment. Here, we investigated the protective and molecular effects of two RAS modifiers, aliskiren; renin inhibitor and captopril; angiotensin converting enzyme inhibitor, on cognitive deficits in the rat hippocampus. Injection of low dose streptozotocin for 4 days resulted in type 1 diabetes. Then, poorly controlled diabetes was mimicked with ineffective daily doses of insulin for 4 weeks. The hyperglycaemia and pancreatic atrophy caused memory disturbance that were identifiable in behavioural tests, hippocampal neurodegeneration, and the following significant changes in the hippocampus, increases in the inflammatory marker interleukin 1β, cholinesterase, the oxidative stress marker malondialdehyde and protein expression of phosphorylated extracellular-signal-regulated kinase and glycogen synthase kinase-3 beta versus decrease in the antioxidant reduced glutathione and protein expression of phosphorylated glycogen synthase kinase-3 beta. Blocking RAS with either drugs along with insulin amended all previously mentioned parameters. Aliskiren stabilized the blood glucose level and restored normal pancreatic integrity and hippocampal malondialdehyde level. Aliskiren showed superior protection against the hippocampal degeneration displayed in the earlier behavioural modification in the passive avoidance test, and the aliskiren group outperformed the control group in the novel object recognition test. We therefore conclude that aliskiren and captopril reversed the diabetic state and cognitive deficits in rats with poorly controlled STZ-induced diabetes through reducing oxidative stress and inflammation and modulating protein expression.

Isoflurane aggravates peripheral and central insulin resistance in high-fat diet/streptozocin-induced type 2 diabetic mice

Isoflurane anesthesia is reported to induce insulin resistance (IR) in the peripheral tissues. However, researches on the impact of isoflurane on insulin-related metabolism in the central nervous system, especially in type 2 diabetes mellitus (T2DM), are scarce. This study sought to explore whether isoflurane anesthesia had a negative effect on insulin sensitivity both in peripheral and central tissues. Moreover, the possible role of isoflurane anesthesia in T2DM mice with pre-existing IR was analyzed. T2DM model in C57BL/6J mice was established by high fat diet (HFD) and single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). Both HFD/STZ-induced T2DM mice and normal mice received 6 h isoflurane exposure. Blood glucose level and serum insulin concentration were detected and the homeostasis model assessment of IR (HOMA-IR) index was calculated to estimate peripheral IR. Relative levels of genes and proteins in the insulin-dependent signaling pathway in mouse prefrontal cortex and hippocampus were determined to measure central IR. Results indicated that 6 h isoflurane exposure induced hyperglycemia, hyperinsulinemia and raised HOMA-IR index. Meanwhile, phosphorylated insulin receptor substrate-1 (pIRS1) (Ser639) and phosphorylated insulin receptor substrate-2 (pIRS2) (Ser731) were upregulated, while phosphorylated protein kinase B (pAKT) (Ser473) and phosphorylated glycogen synthase kinase-3 beta (pGSK3β) (Ser9) were downregulated in the prefrontal cortex and hippocampus of anesthetized mice. Notably, isoflurane anesthesia significantly aggravated the degree of central IR in the aspects of gene transcriptions and protein expressions in HFD/STZ-induced T2DM mice with pre-existing IR. This study suggested that isoflurane anesthesia induced peripheral and central IR and aggravated pre-existing insulin resistance in T2DM mice.

Baicalin Promotes Osteogenic Differentiation of Human Cementoblast Lineage Cells Via the Wnt/β Catenin Signaling Pathway.

Baicalin constitutes a natural bioactive flavonoid extracted from Scutellaria baicalensis Georgi that mediates bone formation. However, the biological functions of baicalin in cementoblasts remain unclear. The purpose of this study was to examine the effects of baicalin on osteogenic differentiation of human cementoblast (HCEM) cells. HCEM cells were cultured and treated with 0, 0.01, 0.1 or 1 µM baicalin. Alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) mRNA and protein levels were examined by real-time polymerase chain reaction and western blot analysis, respectively. Cell mineralization was assessed using Alizarin red staining. Glycogen synthase kinase-3 beta (GSK3β) phosphorylation was measured in 1 µM baicalin-treated HCEM cells with or without the Wnt signaling pathway inhibitor, DKK-1 using ELISA and western blotting. The protein levels of ALP and Runx2 and the intensity of Alizarin red staining were enhanced by baicalin in a dose-dependent manner compared to that of the non-treated control. The ratio of phosphorylated to total GSK3β increased in the presence of baicalin but was reduced by the addition of DKK-1. Treatment of HCEMs with baicalin up-regulated mRNA levels of ALP and Runx2, which were reduced by DKK-1. In addition, the protein levels of ALP and Runx2, ALP activity, and calcium deposition were also enhanced by baicalin, and these parameters were inhibited by DKK-1. Baicalin enhanced osteogenic differentiation of HCEM cells through the Wnt/beta catenin signaling pathway which may be useful for periodontal tissue regeneration.

Role of Wnt/β-catenin signaling pathway in lithium chloride preconditioning-induced improvement in postoperative cognitive function of aged rats

Objective To evaluate the role of Wnt/β-catenin signaling pathway in lithium chloride preconditioning-induced improvement in postoperative cognitive function of aged rats. Methods A total of 100 pathogen-free healthy male Sprague-Dawley rats, aged 18 months, weighing 540-650 g, were divided into 5 groups(n=20 each)using a random number table: control group(group C), surgery group(group S), lithium chloride preconditioning group(group L), secreted frizzled-related protein 1(sFRP-1) group(group F)and lithium chloride preconditioning plus sFRP-1 group(group L+ F). Lithium chloride 2 mmol/kg was intraperitoneally injected once a day for 5 consecutive days before operation in L and L+ F groups.In F and L + F groups, sFRP-1 10 μl(concentration 10 μg/ml)was injected into the ventricle at 1 day before operation.Ten rats in each group were randomly sacrificed at 1 day after operation, and the hippocampi were removed for determination of the expression of phosphorylated glycogen synthase kinase-3 beta(p-GSK-3β), β-catenin and Wnt in hippocampal tissues(by Western blot). The rest rats underwent Morris water maze test at day 3-7 after operation, and the concentrations of amyloid beta 42(Aβ-42)and phosphorylated tau-181 protein(p-tau-181)in cerebrospinal fluid(CSF)were detected by enzyme-linked immunosorbent assay at day 7 after operation. Results Compared with group C, the escape latency was significantly prolonged at 3-7 days after operation, and the concentration of Aβ-42 in CSF was increased in the other four groups, and the expression of Wnt, p-GSK-3β and β-catenin in hippocampal tissues was down-regulated in group S, the expression of Wnt, p-GSK-3β and β-catenin in hippocampal tissues was significantly up-regulated in L and L+ F groups, and the concentration of p-tau-181 in CSF was significantly increased in S and L+ F groups(P<0.05). Compared with group S, the escape latency was significantly shortened at 3-7 days after operation in group L and at 5-7 days after operation in group L+ F, and the expression of Wnt, p-GSK-3β and β-catenin was significantly up-regulated, and the concentrations of Aβ-42 and p-tau-181 in CSF were decreased in L and L+ F groups(P<0.05). Compared with group L, the escape latency was significantly prolonged at 3-6 days after operation, the expression of Wnt, p-GSK-3β and β-catenin was down-regulated, and the concentration of p-tau-181 in CSF was increased in group L+ F(P<0.05). Compared with group F, the escape latency was significantly shortened at 3-7 days after operation, the expression of Wnt, p-GSK-3β and β-catenin was up-regulated, and the concentrations of Aβ-42 and p-tau-181 in CSF were decreased in group L+ F(P<0.05). Conclusion The mechanism by which lithium chloride preconditioning improves in postoperative cognitive function is partially related to activating Wnt/β-catenin signaling pathway in aged rats. Key words: Lithium chloride; Wnt proteins; Aged; Cognition disorders; Preconditioning

The role of glycogen synthase kinase-3β phosphorylation in the protective effects of preconditioning with endoplasmic reticulum stress induced by low dose tunicamycin in myocardial ischemia-reperfusion injury

Objective To evaluate the role of glycogen synthase kinase-3beta phosphorylation (p-GKS-3β) in the protective effects of preconditioning with endoplasmic reticulum stress (ERS) induced by tunicamycin (TM) against myocardial ischemia reperfusion (I/R) injury. Methods Forty Sprague-Dawley rats were randomly divided into four groups: Sham, TM [Rats were treated by TM (0.6 mg/kg) at 30 min before model established], I/R, and TM+ LY [Rats were treated by TM (0.6 mg/kg) and protein kinase B (Akt) inhibitor LY294002 (10 mg/kg) before model established]. The models of I/R were established by occlusion of the left anterior descending coronary artery for 0.5 h followed by reperfusion for 2 h. Blood samples were withdrawn from carotid artery after thoracotomy and 2 h after reperfusion. The levels of creatine kinase isozyme (CK-MB) and lactic dehydrogenase (LDH) were detected. The expression of phosphorylated Akt (p-Akt) and p-GSK-3β in the heart at the end of the I/R was examined by Western blotting in all groups. The myocardial apoptosis index (AI) was measured using terminal deoxynucleotidyl nick-end labeling in all groups. Results The levels of CK-MB, LDH and AI in TM+ LY group were higher than TM group[(987±103) U/L vs. (746±99) U/L, t=2.945, P=0.006; (4 761±636) U/L vs. (3 681±518) U/L, t=2.789, P=0.008; (18.7±2.9)% vs. (14.4±2.8)%, t=2.864, P=0.007; respectively] at 2 h after reperfusion. The expressions of p-Akt, p-GSK-3β protein in TM group (0.613±0.068, 0.717±0.086 respectively) and TM+ I/R group (0.364±0.046, 0.452±0.053) were higher than I/R group (0.251±0.032, 0.232±0.031), but expressions in TM group were higher than TM+ LY group at 2 h after reperfusion (t=2.925, P=0.006; t=2.858, P=0.007; respectively). Conclusion Preconditioning with ERS induced by low dose TM attenuates I/R injury, which may be associated with GSK-3β phosphorylation level by Akt-GSK-3β pathway in rats. Key words: Myocardial ischemia reperfusion injury; Endoplasmic reticulum stress; Preconditioning; Glycogen synthase kinase-3β

Dihydromyricetin exerts a rapid antidepressant-like effect in association with enhancement of BDNF expression and inhibition of neuroinflammation.

Major depressive disorder (MDD) is a highly prevalent illness that affects large populations across the world, and increasing evidence suggests that neuroinflammation and levels of brain-derived neurotrophic factor (BDNF) are closely related to depression. Dihydromyricetin (DHM) is a kind of flavonoid natural product that has been reported to display multiple pharmacological effects, including anti-inflammatory and anti-oxidative properties, and these may contribute to ameliorate MDD. This study investigated the effect of DHM on depression-related phenotypes in various experimental animal models. The antidepressant-like effect of DHM was validated via depression-related behavioral tests in naïve male C57BL/6 mice, as well as in the acute lipopolysaccharide-induced mouse model of depression. The chronic unpredicted mild stress (CUMS) mouse model of depression was also used to assess the rapid antidepressant-like effect of DHM by tail suspension test (TST), forced swimming test (FST), locomotor activity, and sucrose preference test (SPT). The expression of BDNF and inflammatory factors were determined through Western blotting and enzyme-linked immunosorbent assay, respectively. DHM reduced immobility time in the TST and FST both in mice and the acute LPS-induced mouse model of depression. Seven days of DHM treatment ameliorated depression-related behaviors induced by CUMS, whereas similar treatment with the typical antidepressant venlafaxine did not. DHM activated the ERK1/2-CREB pathway and increased glycogen synthase kinase-3 beta (GSK-3β) phosphorylation at ser-9, with upregulation of BDNF expression, in both hippocampal tissues and cultured hippocampal cells. The present data indicate that DHM exerts a more rapid antidepressant-like effect than does a typical antidepressant, in association with enhancement of BDNF expression and inhibition of neuroinflammation.

Zeaxanthin ameliorates high glucose-induced mesangial cell apoptosis through inhibiting oxidative stress via activating AKT signalling-pathway

Oxidative stress is a critical factor in the pathophysiology of diabetic kidney disease. Previous study shows that hyperglycaemia aggravates renal injury through oxidative stress in diabetic model, and antioxidants have beneficial effect on diabetic kidney disease. However, the role of antioxidants in the progression of diabetic kidney disease is poorly understood. The aim of this study was to clarify whether zeaxanthin, an antioxidant, could ameliorate mesangial cell injury and if so, identify the related mechanism underlying this protective effect. To that end, superoxide dismutase (SOD) activity and methane dicarboxylic aldehyde (MDA) levels were measured by an assay kit, and mesangial cell apoptosis and ROS levels were assessed using flow cytometry analysis. Furthermore, The levels of a phosphorylated ser/thr protein kinase (p-AKT), phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β), Bcl-2 associated X protein (Bax) and cleaved cysteinyl aspartate-specific proteinase-3 (caspase-3) were detected by western blot. We found that zeaxanthin decreases MDA levels and increased SOD activity, as well as inhibits apoptosis and decreases ROS levels in mesangial cells in a high sugar environment. Furthermore, zeaxanthin increased p-AKT levels while decreased the levels of p-GSK-3β, Bax and cleaved-caspase-3. In addition, LY294002 reversed the protective effect of zeaxanthin on mesangial cells. In conclusion, zeaxanthin ameliorated mesangial cell apoptosis may be involved in inhibiting oxidative stress through activating of the AKT signalling pathway.

Bamboo leaf extract ameliorates diabetic nephropathy through activating the AKT signaling pathway in rats

Diabetic nephropathy (DN) is one of the most severe diabetic complication and it is becoming become a worldwide epidemic, accounting for approximately one-third of all case of end-stage renal disease. However, the underlying mechanism and strategy to alleviate renal injury remain unclear. In the present study, we assessed the protective effect of bamboo leaf extract on the DN, and investigated the underlying mechanism by which bamboo leaf extract ameliorating DN. Diabetic rats were induced by 4 weeks high sugar and high fat diet, and then injected a single dose of STZ (35mg/kg) into abdominal cavity. Different dose of bamboo extract (50mg/kg, 100mg/kg and 200mg/kg) were orally administered every day for a period of 12 weeks. Body weight, blood glucose, glycosylated hemoglobin A1c (HbAlc), blood urea nitrogen (BUN), serum creatinine (Scr), and 24-hour urinary protein (24 h-UP) were assessed. Total superoxide dismutase (T-SOD) activity and MDA (methane dicarboxylic aldehyde, MDA) level were tested by assay kit. Microstructural changes were observed by hematoxylin–eosin (HE) staining and electron microscopy. Expression of phosphorylated ser/thr protein kinase (P-AKT), phosphorylated glycogen synthase kinase-3 beta (P-GSK-3β), B cell lymphoma/leukemia 2-associated X protein (BAX) and cleaved-cysteinyl aspartate-specific proteinase-3 (Cleaved Caspase-3) were measured by Western-Blotting (WB). Results showed that diabetic rats had weight loss, high blood glucose, HbAlc, BUN, Scr and 24-UP and T-SOD activity were increased and MDA level was decreased in diabetic rats. Moreover, hyperglycemia could injury renal tissue ultrastructure, inhibit P-AKT level and increase P-GSK-3β, BAX and Cleaved Caspase-3 levels in rats. However, bamboo leaf extract treatment could reduce body weight loss, BUN, Scr, 24 h-UP and MDA level, improve T-SOD activity and alleviate renal injury in diabetic rats. Furthermore, bamboo leaf extract increased P-AKT level, decreased P-GSK-3β, BAX and Cleaved Caspase-3 levels in STZ-diabetic rats. In conclusion, our study suggested that bamboo leaf extract ameliorated DN in diabetic rats, and this protective effect is possibly related to suppressing oxidative stress through activating AKT signaling pathway. Bamboo leaf extract treatment may be a potential promising therapy for DN.