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Current treatments for Parkinson's Disease (PD) only provide symptomatic relief; however, they don't delay the disease progression, hence new treatment options should be considered. Carvedilol is a nonselective βα1 blocker with additional effects as an antioxidant, anti-inflammatory and neuro protective properties. In this research, an insilico study was conducted to primarily evaluate carvedilol as an anti-parkinsonian and anti-tau protein target. PASS prediction was performed followed by a docking study of carvedilol. Carvedilol yielded promising results and forward guided this study onto its in vivo evaluation. The in vivo study aimed to assess the neuro-protective effects of carvedilol in rotenone-induced rat model of PD and investigate the potential underlying mechanisms. The effects of carvedilol (2.5, 5, and 10 mg/kg) on the measured parameters of open field, catalepsy, Y-maze tests as well as brain histology, and tyrosine hydroxylase (TH) were evaluated. The effective doses (5 and 10 mg/kg) were further tested for their potential anti-tau protein effects. Carvedilol (5 and 10 mg/kg) prevented rotenone-induced motor deficits, spatial memory dysfunction, and histological damage. Additionally, carvedilol significantly inhibited rotenone-induced decrease in TH expression in the striata of the rats. These effects were associated with reduction of rotenone-induced neuro-inflammation, microglial activation and release of glial fibrillary acidic protein (GFAP), along with reduction in N-methyl-D-aspartate receptors activation, alpha-synculein and phospho-Tau (P-Tau) protein expression. Carvedilol also reduced tau protein hyper-phosphosrylation by Glycogen synthase 3β (GSK 3β) inhibition and Phosphoinositide 3-kinase (PI3K) stimulation. Collectively, these results suggest that carvedilol might be a possible candidate for management of PD.

Metastasis is the leading cause of death in melanoma patients. Aerobic glycolysis is a common metabolic feature in tumor and is closely related to cell growth and metastasis. Kaempferol (KAM) is one of the active ingredients in the total flavonoids of Chinese traditional medicine Sparganii Rhizoma. Studies have shown that it interferes with the cell cycle, apoptosis, angiogenesis and metastasis of tumor cells, but whether it can affect the aerobic glycolysis of melanoma is still unclear. Here, we explored the effects and mechanisms of KAM on melanoma metastasis and aerobic glycolysis. KAM inhibited the migration and invasion of A375 and B16F10 cells, and reduced the lung metastasis of melanoma cells. Extracellular acidification rates (ECAR) and glucose consumption were obviously suppressed by KAM, as well as the production of ATP, pyruvate and lactate. Mechanistically, the activity of hexokinase (HK), the first key kinase of aerobic glycolysis, was significantly inhibited by KAM. Although the total protein expression of HK2 was not significantly changed, the binding of HK2 and voltage-dependent anion channel 1 (VDAC1) on mitochondria was inhibited by KAM through AKT/GSK-3β signal pathway. In conclusion, KAM inhibits melanoma metastasis via blocking aerobic glycolysis of melanoma cells, in which the binding of HK2 and VDAC1 on mitochondria was broken.

Cells are fundamental units of life. To ensure the maintenance of homeostasis, integrity of structural and functional counterparts is needed to be essentially balanced. The cytoskeleton plays a vital role in regulating the cellular morphology, signalling and other factors involved in pathological conditions. Microtubules, actin (microfilaments), intermediate filaments (IF) and their interactions are required for these activities. Various proteins associated with these components are primary requirements for directing their functions. Disruption of this organization due to faulty genetics, oxidative stress or impaired transport mechanisms are the major causes of dysregulated signalling cascades leading to various pathological conditions like Alzheimer's (AD), Parkinson's (PD), Huntington's disease (HD) or amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia (HSP) or any traumatic injury like spinal cord injury (SCI). Novel or conventional therapeutic approaches may be specific or non-specific, targeting either three basic components of the cytoskeleton or various cascades that serve as a cue to numerous pathways like ROCK signalling or the GSK-3β pathway. An enormous number of drugs have been redirected for modulating the cytoskeletal dynamics and thereby may pave the way for inhibiting the progression of these diseases and their complications.

A series of novel scopariusicide derivatives were designed and synthesized starting from the main diterpenoid from the aerial parts of Isodon scoparius. Sis-25 was the most effective compound among them. The potential mechanism(s) of its immunosuppressive activity in vitro, as well as its effects on delayed type hypersensitivity (DTH) reaction and imiquimod-induced dermatitis in vivo were investigated in this study. Sis-25 inhibited anti-CD3/anti-CD28 mAbs, PHA or alloantigen-induced T cell proliferation without obvious cytotoxicity. Sis-25 was a highly selective inhibitor of GSK3-β and inhibited the mTOR/p70S6K pathway but not the PI3K/Akt, p38 MAPK/ERK 1/2 and JAK3/STAT5 pathways. Furthermore, Sis-25 significantly inhibited IFN-γ, IL-6 and IL-17 expression but not IL-10 expression in activated T cells. Finally, Sis-25 treatment mitigated the DNFB-induced DTH reaction and ameliorated imiquimod-induced dermatitis. In summary, Sis-25 exerted significant immunosuppressive activity by targeting GSK3β in vitro and in vivo. Sis-25 may guide the design of new drugs for more effective and safer treatments of autoimmune diseases and provide new insight into developing utilizations of Isodon scoparius.

We previously demonstrated that GSK-3β mediates NLRP3 inflammasome activation and IL-1β production in cardiac fibroblasts (CFs) after myocardial infarction (MI). In this study, we show how GSK-3β-mediated activation of the NLRP3 inflammasome/caspase-1/IL-1β pathway leads to apoptosis and pyroptosis of cardiomyocytes (CMs) and CFs. Administration of lipopolysaccharide (LPS)/ATP to primary newborn rat cardiac fibroblasts (RCFs) led to increase in proteins of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, IL-1β, and IL-18. Additionally, the expression of caspase-3 and N-terminal fragments of gasdermin D (N-GSDMD) and the Bax/Bcl-2 ratio increased. Administration of the GSK-3β inhibitor SB216763 reduced the levels of apoptosis- and pyroptosis-related proteins regulated by NLRP3 inflammasome activation in RCFs. Next, we transferred the culture supernatant of LPS/ATP-treated RCFs to in vitro primary newborn rat cardiomyocytes (RCMs). The results showed that SB216763 attenuate the upregulation of the ratios of Bax/Bcl-2 and the expression of caspase-3 and N-GSDMD in RCMs. Direct stimulation of RCMs and H9c2 cells with recombinant rat IL-1β increased the p-GSK-3β/GSK-3β and Bax/Bcl-2 ratios and the expression of caspase-3 and N-GSDMD, while both SB216763 and TLR1 (an IL-1β receptor inhibitor) markedly reduced these effects, as assessed using propidium iodide positive staining and the lactate dehydrogenase release assay. The caspase-11 inhibitor wedelolactone decreased the expression level of N-GSDMD but did not alter the p-GSK-3β/GSK-3β ratio. Lastly, we established a Sprague-Dawley rat MI model to confirm that SB216763 diminished the increase in caspase-3 and N-GSDMD expression and the Bax/Bcl-2 ratio in the ischemic area. These data demonstrate that GSK-3β regulates apoptosis and pyroptosis of RCMs and RCFs due to NLRP3 inflammasome activation in RCFs.

Our previous findings indicated that tanshinone IIA (tan IIA), a natural component extracted from the root and rhizome of danshen, significantly attenuated β-amyloid accumulation, neuroinflammation, and endoplasmic reticulum stress, as well as improved learning and memory deficits in APP/PS1 transgenic mouse model of Alzheimer's disease (AD). However, whether tan IIA can ameliorate tau pathology and the underlying mechanism in APP/PS1 mice remains unclear. In the current study, tan IIA (15 mg/kg and 30 mg/kg) or saline was intraperitoneally administered to the 5-month-old APP/PS1 mice once daily for 4 weeks. The open-field test, novel object recognition test, Y-maze test, and Morris water maze test were performed to assess the cognitive function. Nissl staining, immunohistochemistry, TUNEL, and western blotting were conducted to explore tau hyperphosphorylation, neuronal injury, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt)/glycogen synthase kinase-3β (GSK-3β) signaling pathway. The activity of GSK-3β, acetylcholinesterase (AChE), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), and the level of malondialdehyde (MDA) were measured using commercial kits. Our results revealed that tan IIA treatment significantly ameliorated behavioral deficits and improved spatial learning and memory ability of APP/PS1 mice. Additionally, tan IIA markedly attenuated tau hyperphosphorylation and prevented neuronal loss and apoptosis in the parietal cortex and hippocampus. Simultaneously, tan IIA reversed cholinergic dysfunction and reduced oxidative stress. Furthermore, tan IIA activated the PI3K/Akt signaling pathway and suppressed GSK-3β. Taken together, the above findings suggested that tan IIA improves cognitive decline and tau pathology may through modulation of PI3K/Akt/GSK-3β signaling pathway.

Targeting cancer stem cells (CSCs) may be an efficacious strategy against cancer. We were devoted to exploring the role of neogambogic acid in characteristics and growth of colorectal CSCs.SW480 and HCT116 cells were treated with neogambogic acid at different concentrations and transfected with siDLK1 and pcDNA3.1-DLK1 plasmids. The effect of neogambogic acid on the viability of SW480 and HCT116 cells was assessed by MTT assay. Spheroid formation assay was adopted to enrich colorectal CSCs from SW480 and HCT116 cells. The effect of neogambogic acid on colony number, aldehyde dehydrogenase (ALDH) level, apoptosis and cell cycle of SW480 and HCT116 CSCs was detected by colony formation and flow cytometry assays. The expressions of CSC markers, proliferation marker (proliferation nuclear antigen (PCNA)), apoptosis markers (cleaved caspase-3, cleaved caspase-9), Wnt/β-catenin pathway markers (P-GSK3β, GSK3β, β-catenin and Wnt) and DLK1 were determined by qRT-PCR or Western blot.Neogambogic acid suppressed viability, the spheroid formation ability and the levels of CSC markers in colorectal cancer (CRC) cells, accompanied with inhibition of colony-formation and ALDH level, apoptosis induction and G0/G1 phase arrest. Furthermore, neogambogic acid inhibited expressions of PCNA, P-GSK3β, P-GSK3β/GSK3β, β-catenin and Wnt, but promoted those of cleaved caspase-3, cleaved caspase-9 and GSK3β in colorectal CSCs. DLK1 silencing caused opposite results. DLK1 overexpression abrogated the effects of neogambogic acid on colorectal CSCs.Neogambogic acid could be an efficacious natural compound targeting colorectal CSCs via inhibition of DLK1 and Wnt/β-catenin pathway. Thus, neogambogic acid may be an attractive agent against CRC.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease and one of the most severe diabetic complications. However, there is lack of effective treatments for DN and the underlying mechanisms of the renal injury remain unclear. In current study, we evaluated the effects of silibinin on DN and further explored the underlying mechanisms. We administrated silibinin to db/db mice for 10 weeks. Then we monitored the diabetic metabolic parameters, kidney function, oxidative stress and AKT signaling pathway in db/db mice. Administration of silibinin to db/db mice improved diabetic condition, as evidenced by the decrease of body weight, HbAc1level and serum insulin level in db/db mice. Silibinin prevented kidney injury and attenuated oxidative stress in db/db mice. Silibinin activated AKT signaling pathway and decreased the levels of p-GSK-3β, Bax and cleaved caspase-3. Silibinin ameliorates diabetic nephropathy by activating the AKT signaling pathway.

Withaferin A (WFA), a withanolide, is isolated from plants of Withania somnifera (L.) Dual (Solanaceae), known as Indian ginseng, Indian winter cherry or Ashwagandha. It has been reported to exert multifaceted anti-neoplastic effects. Here, we analyzed the impact of WFA on apoptosis and autophagy activation in different human colorectal cancer cell lines. We observed that WFA exposure caused an increased aggregation of cells in the subG1 arrest in cell cycle, and increased the number of late apoptotic cells. WFA also induced the apoptosis via PARP and caspase-3 cleavage accompanied with suppression of levels of anti-apoptotic proteins like Bcl-2 and Bcl-xl. The influence of WFA on autophagy was validated by acridine orange, MDC staining, and immunocytochemistry of LC3. It was found that 24 h treatment of WFA increased the acridine and MDC stained autophagosome with induced the LC3 and other autophagy markers Atg7 and beclin-1 activation. We used Z-DEVD-FMK, a caspase-3 blocker, and 3-MA, an autophagy inhibitor, to confirm whether these effects were specific to apoptosis and autophagy, and observed the recovery of both these processes upon exposure to WFA. Moreover, the activation of β-catenin protein was attenuated by WFA. Interestingly, small interfering RNA (siRNA)-promoted β-catenin knockdown augmented the WFA-induced active form of p-GSK-3β, and stimulated autophagy and apoptosis through PARP and LC3 activation. These findings suggested that WFA could stimulate activation of both apoptosis and autophagy process via modulating β-catenin pathway.

Intracerebral hemorrhagic (ICH) stroke is a major cause of death and disability globally, with no proper treatment available so far. Rutin, a dietary flavonoid, has shown protection against cerebral ischemic stroke due to its antioxidant and anti-inflammatory attributes. However, the efficacy of rutin against ICH stroke remained unexplored. Therefore, in the current study, we investigated the effect of rutin in an ICH stroke zebrafish larva model. The larvae were exposed to atorvastatin (1.25 μM) in system water for induction of experimental ICH. Rutin treatment reduced the hematoma size, ROS production and decreased apoptosis in the zebrafish larvae brains. Reduction in the malondialdehyde and protein carbonyl level in the rutin-treated larvae also indicated quenching of the free radicals. The treatment increased the expression of tight junction claud5a gene and decreased the mRNA level of matrix metalloproteases (mmp2 and mmp9). Furthermore, rutin treatment also attenuated the genomic expression of oxidative markers (nrf2, hmox1a, sod1, and gpx) and inflammatory genes (il6, tnfa, il10, and irf2a) related to ICH. The Gsk-3β activity was also downregulated, and a normal pool of β-catenin and Nrf2 was maintained in the larvae treated with rutin. The current study suggested that rutin protects ICH stroke via suppressing oxidative stress and inflammatory events in a zebrafish model.

The loss of cholinergic neurons has been a major issue in researches on Alzheimer's disease (AD) for about 40 years. Therefore, the scopolamine model of amnesia has been widely used in AD researches. Recently, it was reported that the early stage amnesia of AD is related to memory retrieval deficit. Curcumin, as the main ingredient of turmeric, has been suggested to decrease the prevalence of AD in human population. This study was conducted to assess if curcumin prevents retrieval deficit induced by scopolamine in passive avoidance task. Moreover, according to the proposed link between cholinergic system and Akt/GSK-3β (Glycogen synthase kinase 3 beta) signaling, the hippocampal contents of these proteins were determined. Male NMRI mice (20–25 g body weight) were treated with 50 or 100 mg/kg/po curcumin or its vehicle for 10 days. On day 10, the animals were trained in passive avoidance apparatus. The retention trial was performed 24 h later. Scopolamine (1 mg/kg/i.p.) or its vehicle was administered 30 min before retention test. At the completion of behavioral studies, the hippocampi were removed and western blot analysis was performed to determine hippocampal phosphorylated and total Akt and GSK-3β and beta actin contents. The results showed that curcumin treatment at 50 and 100 mg/kg doses prevented scopolamine-induced memory retrieval deficit and restored Akt and GSK dephosphorylation caused by scopolamine. Overall, these findings showed that pre-test scopolamine administration disrupts memory retrieval along with the diminished Akt and GSK-3β phosphorylation in hippocampus while curcumin administration prevented those changes.

The cellular and tissue damage induced by oxidative stress (OS) contribute to a variety of human diseases, which include gastrointestinal diseases. Salvianolic acid B (Sal B), which is a natural polyphenolic acid in Salvia miltiorrhiza, exhibits prominent antioxidant properties. However, its precise function and molecular mechanisms in protecting normal intestine epithelium from OS-induced damage are still poorly defined. In this study, we tried to clarify this relationship. Here, we found Sal B addiction in the rat intestinal epithelial cell, IEC-6, prevented H2O2-induced cell viability decrease and apoptosis induction, ameliorated H2O2-induced intestinal epithelial barrier dysfunction and mitochondrial dysfunction, and suppressed H2O2-induced production of ROS to varying degrees, ranging from 10% to 30%. Moreover, by employing an ischemia reperfusion model of rats, we also discovered that Sal B treatment reversed ischemia and a reperfusion-caused decrease in villus height and crypt depth, decreased proliferation of enterocytes, and increased the apoptotic index in the jejunum and ileum. Mechanistically, Sal B treatment up-regulated the phosphorylated level of Akt and GSK3β in enterocytes in vitro and in vivo, and PI3K inhibitor LY294002 treatment abrogated the protective effects of Sal B. Meanwhile, the inactivation of GSK3β reversed the oxidative stress-induced apoptosis and mitochondrial dysfunction in IEC-6 cells. Together, our results demonstrated that the damage of intestinal epithelial cells in in vitro and in vivo models were both attenuated by Sal B treatment, and such antioxidant activity might very possibly be attributed to the activation of Akt/GSK3β signaling.

Intermittent fasting as an adjuvant therapy in clinical practice is an emerging treatment modality to target tumor growth by reducing glucose utilization. Berberine, an alkaloid extracted from the traditional Chinese medicine Coptidis Rhizoma, has been shown to be a safe and effective antitumor agent in several cancers. Hence, the purpose of the present study was to investigate the effects of the combination of berberine and low glucose on gastric cancer. Our results showed that the combination of berberine and low glucose effectively inhibited cell viability, promoted apoptosis, and reduced the migration ability of MGC803 cells. In addition, the combination was shown to activate the PP2A/GSK3β signaling axis, leading to the downregulation of the downstream pro-survival protein MCL-1, which leads to the death of gastric cancer cells. In addition, the inhibitor of GSK3β partially reversed the effect of this combination on MGC803 cells. In vivo experiments demonstrated that berberine effectively impaired the growth of xenograft tumors, when administered during intermittent fasting (hypoglycemic conditions), and was well tolerated by nude mice without the occurrence of any adverse effects. Based on these results, we conclude that the berberine/low-glucose combination can inhibit the growth of gastric cancer through the PP2A/GSK3β/MCL-1 signaling pathway. Accordingly, this combination of drugs and lifestyle may become a new type of safe and effective anti-cancer therapy.

Several studies have suggested that phosphodiesterase (PDE) inhibitors may be a disease-modifying for Alzheimer's disease (AD). Cilostazol (CSZ) has been shown to be a new treatment for cognitive impairment with limited efficacy. Our aim was to investigate the effect of caffeine on the efficacy of CSZ against STZ-induced type 2 diabetes (T2D)-related cognitive impairment in high fat/high fructose fed rats. The efficacy of low doses of caffeine, CSZ, and CSZ plus caffeine against abnormal behavioral, biochemical, histological, or genetic changes of animal models of AD was examined. Eight weeks treatment with CSZ plus caffeine was more effective than CSZ or caffeine in improving impaired behavioral tests for cognition and memory. Histological examination exhibited a significant augmentation in the efficacy of CSZ by caffeine in protecting neurons from damage in T2D rats. Importantly, CSZ and caffeine normalized the accumulation of Amyloid beta (Aβ-42) and phosphorylated tau protein (p-tau) positive cells in the brain of T2D rats. CSZ or CSZ plus caffeine reversed low glutamate gene expression, elevated cholinesterase level, and elevated caspase-3 activity in T2D rats. Furthermore, CSZ plus caffeine was significantly more effective than CSZ or caffeine in inhibiting the increase in malondialdehyde (MDA) level, total oxidative stress, pro-inflammatory cytokines and glucogen synthase kinase-3 beta (GSK-3β) in the hippocampus of T2D rats. Also, CSZ plus caffeine was more effective than CSZ or caffeine in alleviating insulin resistance and hypercholesterolemia in T2D rats. Our findings suggest the possibility of effective treatment of AD by enhancing the therapeutic potential of CSZ through combined treatment with lower doses of caffeine. The enhancement of CSZ effect by caffeine is attributed to the increased inhibitory effect of CSZ on insulin resistance, GSK-3β activity, hypercholesterolemia, oxidative stress and pro-inflammatory cytokines.

Eriodictyol (ERD) is a natural flavonoid that exists in many vegetables and fruits, especially citrus fruits. It has been proven to have many pharmacological effects, such as antioxidative, anti-inflammatory and neuroprotective effects. Our previous study showed that eriodictyol could inhibit the proliferation and induce the apoptosis of glioblastoma cells by downregulating the PI3K/Akt/NF-κB pathway and restraining its migration and invasion. However, the mechanism by which eriodictyol prevents glioblastoma metastasis is still unknown. Epithelial-mesenchymal transition (EMT) is a key process for many cancer metastases; it also confers locomotivity to tumor cells, including glioblastoma. In this study, we found that eriodictyol can suppress the migration and invasion of glioblastoma A172 and U87 MG cell lines by suppressing the EMT markers - N-cadherin and E-cadherin through Wound healing and Transwell assays, Western blot, RT-qPCR, immunofluorescence and immunohistochemistry. Further research revealed that the mechanism could be connected with downregulation of the P38 MAPK/GSK-3β/ZEB1 signaling pathway. These findings can provide a new idea for the treatment of glioblastoma.

Perioperative neurocognitive disorder (PND) is a common complication of elderly patients after surgery and lacks effective prevention and treatment measures. We investigated the effect and mechanism of gastrodin (GAS), a natural plant ingredient, on postoperative cognition induced by laparotomy in aged mice. Male aged (18 months) mice were subjected to laparotomy and orally treated with GAS (25, 50, and 100 mg/kg) 3 weeks before surgery and 1 week after surgery. In addition, some male aged (18 months) mice were subjected to viral vector or GSK-3β expression virus injection followed by laparotomy with or without 100 mg/kg GAS treatment. GAS improved learning and memory in aged mice after surgery. Surgery increased the levels of pro-inflammatory factors (TNF-α, IL-1β and IL-6) and decreased the level of an anti-inflammatory factor (IL-10) in the mouse hippocampus, and these changes were reversed by GAS treatment. GAS also suppressed the activation of microglia. GAS inhibited the phosphorylation of GSK-3β and Tau. Furthermore, surgery induced more serious cognitive dysfunction, inflammatory factors, activation of microglia, and phosphorylation of GSK-3β and Tau in GSK-3β overexpressing aged mice. The improvement of learning and memory, the reduction of inflammation and microglia activation, and the suppression of GSK-3β and Tau phosphorylation by GAS were prevented when GSK-3β was overexpressed in aged mice subjected to surgery. Our finding suggested that GAS exerts neuroprotective effects in aged mice subjected to laparotomy by suppressing neuroinflammation and GSK-3β and Tau phosphorylation. Thus, these findings suggest that GAS may be a promising agent for PND.

Increased O-Linked β-N-acetylglucosamine (O-GlcNAc) is observed in several pathologies, and unbalanced O-GlcNAcylation levels favor endothelial dysfunction. Whether augmented O-GlcNAc impacts the uterine artery (UA) function and how it affects the UA during pregnancy remains to be elucidated. We hypothesized that glucosamine treatment increases O-GlcNAc, leading to uterine artery dysfunction and this effect is prevented by pregnancy. Pregnant (P) and non-pregnant (NP) Wistar rats were treated with glucosamine (300 mg/kg; i.p.) for 21 days. Concentration response-curves (CRC) to acetylcholine (in the presence or absence of L-NAME) and sodium nitroprusside were performed in UAs. In NP rats, glucosamine treatment increased O-GlcNAc expression in UAs accompanied by decreased endothelium-dependent relaxation, which was abolished by L-NAME. Endothelial nitric oxide synthase (eNOS) activity and total Akt expression were decreased by glucosamine-treatment in NP rats. Further, NP rats treated with glucosamine displayed increased glycogen synthase kinase 3 beta (GSK3β) activation and O-GlcNAc-transferase (OGT) expression in the UA. P rats treated with glucosamine displayed decreased O-GlcNAc in UAs and it was accompanied by improved relaxation to acetylcholine, whereas eNOS and GSK3β activity and total Akt and OGT expression were unchanged. Sodium nitroprusside-induced relaxation was not changed in all groups, indicating that glucosamine treatment led to endothelial dysfunction in NP rats. The underlying mechanism is, at least in part, dependent on Akt/GSK3β/OGT modulation. We speculate that during pregnancy, hormonal alterations play a protective role in preventing O-GlcNAcylation-induced endothelial dysfunction in the UAs.

The anticancer prodrug butyroyloxymethyl diethylphosphate (AN-7), upon metabolic hydrolysis, releases the histone deacetylase inhibitor butyric acid and imparts histone hyperacetylation. We have shown previously that AN-7 increases doxorubicin-induced cancer cell death and reduces doxorubicin toxicity and hypoxic damage to the heart and cardiomyocytes. The cardiofibroblasts remain unprotected against both insults. Herein we examined the selective effect of AN-7 on hypoxic cardiomyocytes and cardiofibroblasts and investigated mechanisms underlying the cell specific response. Hypoxic cardiomyocytes and cardiofibroblasts or H2O2-treated H9c2 cardiomyoblasts, were treated with AN-7 and cell damage and death were evaluated as well as cell signaling pathways and the expression levels of heme oxygenase-1 (HO-1). AN-7 diminished hypoxia-induced mitochondrial damage and cell death in hypoxic cardiomyocytes and reduced hydrogen peroxide damage in H9c2 cells while increasing cell injury and death in hypoxic cardiofibroblasts. In the cell line, AN-7 induced Akt and ERK survival pathway activation in a kinase-specific manner including phosphorylation of the respective downstream targets, GSK-3β and BAD. Hypoxic cardiomyocytes responded to AN-7 treatment by enhanced phosphorylation of Akt, ERK, GSK-3β and BAD and a significant 6-fold elevation in HO-1 levels. In hypoxic cardiofibroblasts, AN-7 did not activate Akt and ERK beyond the effect of hypoxia alone and induced a limited (~1.5-fold) increase in HO-1. The cell specific differences in kinase activation and in heme oxygenase-1 upregulation may explain, at least in part, the disparate outcome of AN-7 treatment in hypoxic cardiomyocytes and hypoxic cardiofibroblasts.

Dipeptidyl Peptidase-4 (DPP-4) is a specific enzyme hydrolyzing the incretin hormone glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) to reduce insulin secretion, meanwhile DPP-4 inhibitors play an important role in diabetic therapy. In present study, 14 potential inhibitors were screened with an inhibition over 50% on DPP-4 activity from Xiao-Ke-An formula (XKA) and 12 of them exhibited a dose-dependently inhibitory effect at concentrations of 5–50 μmol/l. We found 10 DPP-4 inhibitors restrained differentiation of 3T3-L1 pre-adipocytes as well as reducing the triglycerides and total cholesterol content in 3T3-L1 adipocytes. Furthermore, 7 DPP-4 inhibitors promoted the glucose consumption in insulin-resistance BNL CL.2 cells. Thereinto, ginsenoside Rk1 up-regulated the protein kinase B (AKT) and glycogen synthase kinase-3 (GSK-3β) phosphorylation expression, while kukoamine B and coptisine hydrochloride obviously increased the phosphorylation of AKT protein and columbamine, panaxadiol, ginsenoside Ro, timosaponin AI significantly promoted the phosphorylation of GSK-3β protein. It's our first effort to confirm those seven compounds could serve as DPP-4 inhibitors to attenuate DPP-4 activities, accompanied with the ability to adjust glucolipid metabolism. Moreover, activating the AKT/GSK-3β signaling pathway to ameliorate insulin resistant may be the anti-diabetic mechanism of XKA.

Chronic low-grade inflammation plays a major role in the development of insulin resistance. The potential role and underlying mechanism of vitamin C, an antioxidant and anti-inflammatory agent, was investigated in tumor necrosis factor-α (TNF-α)-induced insulin resistance. Gulonolactone oxidase knockout (Gulo-/-) mice genetically unable to synthesize vitamin C were used to induce insulin resistance by continuously pumping small doses of TNF-α for seven days, and human liver hepatocellular carcinoma cells (HepG2 cells) were used to induce insulin resistance by treatment with TNF-α. Vitamin C deficiency aggravated TNF-α-induced insulin resistance in Gulo-/- mice, resulting in worse glucose tolerance test (GTT) results, higher fasting plasma insulin level, and the inactivation of the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK3β) pathway in the liver. Vitamin C deficiency also worsened liver lipid accumulation and inflammation in TNF-α-treated Gulo-/- mice. In HepG2 cells, vitamin C reversed the TNF-α-induced reduction of glucose uptake and glycogen synthesis, which were mediated by increasing GLUT2 levels and the activation of the insulin receptor substrate (IRS-1)/AKT/GSK3β pathway. Furthermore, vitamin C inhibited the TNF-α-induced activation of not only the mitogen-activated protein kinase (MAPKs), but also nuclear factor-kappa B (NF-κB) signaling. Taken together, vitamin C is essential for preventing and improving insulin resistance, and the supplementing with vitamin C may be an effective therapeutic intervention for metabolic disorders.

Cardiotoxicity is a critical side-effect of nilotinib during treatment for cancer, such as chronic myeloid leukemia, while the potential signaling mechanisms remain unclear. The role of and the relationship between endoplasmic reticulum (ER) stress and mitochondrial dysfunction was investigated in nilotinib-induced cardiac H9C2 injury as a suitable cell model. Our results showed that ER stress was persistently induced in nilotinib-treated cells, evidenced by increase of GRP78, CHOP, ATF4 and XBP1 as well as phospho-PERKThr980. The results from 4-phenylbutyrate (PBA, an ER stress inhibitor) and SC79 (a specific Akt activator) suggested that ER stress increased activity of glycogen synthase kinase-3 beta (GSK3β) that is reflected by decrease of phospho-GSK3βSer9, through downregulation of phospho-AktSer473, and that prolonged ER stress and activated GSK3β involved nilotinib-induced apoptosis. In addition, the data from JNK inhibition using SP600125 showed that over-activated JNK was responsible for Akt de-phosphorylation. Moreover, the abundance of NADPH oxidase (Nox4) was significantly increased following nilotinib treatment, which was prevented by SB216763 (a specific GSK3β inhibitor). Additionally, mitochondrial dysfunction was indicated by reduced mitochondrial membrane potential (MMP) level and increased reactive oxygen species level. In nilotinib-treated cells, knockdown of Nox4 preserved MMP level, abrogated reactive oxygen species production, and decreased apoptosis. Accordingly, our data demonstrated that inhibition of ER stress may protect cardiomyocytes against nilotinib toxicity potentially through inactivation of Akt-GSK3β-Nox4 signaling. These findings may provide an attractive therapeutic target for treatment of nilotinib-related cardiotoxicity.

Protein tyrosine phosphatase 1 B (PTP1B) is one of main causes involved in type 2 diabetes, it dephosphorylates insulin receptor substrate (IRS) and dysregulates insulin signaling pathway, thus inducing insulin resistance. Our previous work first reported that FYGL, a neutral hyperbranched proteoglycan ingredient extracted from Ganoderma lucidum, has hypoglycemic activity in vivo and inhibitory potency on PTP1B in vitro, but the underlying mechanism was still unclear. In this study, we sought to investigate effects of FYGL on insulin signaling pathway involved with PTP1B as the targeting point in hepatocytes. We found that FYGL inhibited overexpression of PTP1B in liver tissues of ob/ob mice and HepG2 cells, significantly improved the phosphorylation of IRS1 on tyrosine residues, activated phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) cascades and increased phosphorylation of glycogen synthesis kinase-3β (GSK3β), finally enhanced insulin-stimulated glycogen synthesis in HepG2 cells and decreased blood glucose in insulin resistance model mice. Our study clearly illustrated the hypoglycemic mechanism of a novel proteoglycan possibly used in type 2 diabetes management in vivo.

The role of glycosaminoglycan sulfation patterns, particularly in regard to scar formation and inhibition of neuritogenesis, has been mainly studied in cell culture with a focus on chondroitin 4-sulfate. In this study, we investigated chondroitin 6-sulfate (C6S) and found that it also inhibits neurite outgrowth of mouse cerebellar granule neurons in vitro. To examine whether the inhibitory activity of C6S could be neutralized, seven previously characterized high-affinity C6S-binding peptides were tested, among which three peptides neutralized the inhibitory functions of C6S. We further investigated these peptides in a mouse model of spinal cord injury, since upregulation of C6S expression in the glial scar following injury has been associated with reduced axonal regrowth and functional recovery. We here subjected mice to severe compression injury at thoracic levels T7-T9, immediately followed by inserting gelfoam patches soaked in C6S-binding peptides or in a control peptide. Application of C6S-binding peptides led to functional recovery after injury and prevented fibrotic glial scar formation, as seen by decreased activation of astrocytes and microglia/macrophages. Decreased expression of several lecticans and deposition of fibronectin at the site of injury were also observed. Application of C6S-binding peptides led to axonal regrowth and inhibited the C6S-mediated activation of RhoA/ROCK and decrease of PI3K-Akt-mTOR signaling pathways. Taken together, these results indicate that treatment with C6S-binding peptides improves functional recovery in a mouse model of spinal cord injury.

Despite the years of research, epilepsy remains uncontrolled in one-third of afflicted individuals and poses a health and economic burden on society. Currently available anti-epileptic drugs mainly target the excitatory-inhibitory imbalance despite targeting the underlying pathophysiology of the disease. Recent research focuses on understanding the pathophysiologic mechanisms that lead to seizure generation and on possible new treatment avenues for preventing epilepsy after a brain injury. Various signaling pathways, including the mechanistic target of rapamycin (mTOR) pathway, mitogen-activated protein kinase (MAP-ERK) pathway, JAK-STAT pathway, wnt/β-catenin signaling, cAMP pathway, and jun kinase pathway, have been suggested to play an essential role in this regard. Recent work suggests that the mTOR pathway intervenes epileptogenesis and proposes that mTOR inhibitors may have antiepileptogenic properties for epilepsy. In the same way, several animal studies have indicated the involvement of the Wnt signaling pathway in neurogenesis and neuronal death induced by seizures in different phases (acute and chronic) of seizure development. Various studies have also documented the activation of JAK-STAT signaling in epilepsy and cAMP involvement in epileptogenesis through CREB (cAMP response element-binding protein). Although studies are there, the mechanism for how components of these pathways mediate epileptogenesis requires further investigation. This review summarises the current role of various signaling pathways involved in epileptogenesis and the crosstalk among them. Furthermore, we will also discuss the mechanical base for the interaction between these pathways and how these interactions could be a new emerging promising target for future epilepsy therapies.

Aim Mangiferin (MF), a xanthonoid from Mangifera indica, possesses anti-inflammatory, immunomodulatory, and potent antioxidant effects; however, its protective effect against mesenteric ischemia/reperfusion (I/R)-induced liver injury has not been fully clarified. The study was designed to assess the possible mechanism of action of MF against mesenteric I/R model. Main methods Male Wister rats were treated with MF (20 mg/kg, i.p) or the vehicle for 3 days before I/R, which was induced by clamping the superior mesenteric artery for 30 min followed by declamping for 60 min. Key findings The mechanistic studies revealed that MF protected the 2 organs studied, viz., liver and intestine partly via increasing the content of β-catenin and PPAR-γ along with decreasing that of GSK-3β and the phosphorylated NF-қB-p65. MF antioxidant effect was evidenced by increasing contents of total antioxidant capacity and GST, besides normalizing that of MDA. Regarding the anti-inflammatory effect, MF reduced IL-1β and IL-6, effects that were mirrored on the tissue content of MPO. Moreover, MF possessed anti-apoptotic character evidenced by elevating Bcl-2 content and reducing that of caspase-3. In the serum, intestinal I/R increased the activity of ALT, AST, and creatine kinase. Significance The intimated protective mechanisms of MF against mesenteric I/R are mediated, partially, by modulation of oxidative stress, inflammation, and apoptosis possibly via the involvement of Wnt/β-catenin/NF-қβ/ PPAR-γ signaling pathways.

Salidroside (Sal) is a natural antioxidant that elicits cardioprotective and neuroprotective effects in vivo and in vitro; however, its impact on hepatic ischemia/reperfusion (I/R) injury remains unclear. The purpose of this study was to investigate the hepatoprotective effects of salidroside against segmental (70%) warm hepatic I/R injury in rats. Animals were randomized into Sham, Sham+salidroside pretreatment (Sal), Sham+Sal+carboxyatractyloside (CATR), Sham+CATR, I/R, I/R+Sal, I/R+Sal+CATR and I/R+CATR groups. The hepatic artery, left portal vein and median liver lobes were occluded for 60min and then unclamped to allow reperfusion. Pretreatment with salidroside (20mg/kg/day for 7 days, intraperitoneally) significantly decreased serum alanine aminotransferase (sALT) and serum aspartate aminotransferase (sAST) levels after 6h and 24h of reperfusion and protected the liver against I/R-induced injury. However, this protective effect could be reversed by CATR, a mitochondrial permeability transition pore (MPTP) opener (5mg/kg 30min before I/R insult, intraperitoneally). Mechanistic studies have revealed that salidroside inhibits glycogen synthase kinase-3 beta (GSK-3β) activity and enhances the NF-E2-related factor (Nrf2)-dependent antioxidant response by activating the Akt signaling pathway, thereby reducing mitochondrial reactive oxygen species generation, increasing MPTP resistance and preventing apoptosis by suppressing cytochrome c release and caspase activation during reperfusion. Therefore, salidroside ameliorates hepatocyte death and apoptosis through activation of the GSK-3β/Nrf2-dependent antioxidant response and subsequent MPTP inhibition. These results provide experimental evidence supporting the clinical use of salidroside for hepatoprotection in surgical settings.

Colorectal cancer (CRC) is common worldwide, and most treatments for CRC have undesirable side effects. Many researchers have demonstrated that inositol hexaphosphate (IP6) has potent anticarcinogenic activity against CRC and no apparent toxicity to normal cells. However, the underlying mechanism is still unclear. In this study, we investigated the anticancer and anti-proliferative properties of IP6 in CRC and its possible mechanisms during this chemopreventive process. We examined the expression of genes related to the PI3K/Akt and Wnt pathways at the transcriptional and translational levels in a DMH-induced rat CRC model following IP6 administration. In addition, we also conducted cell proliferation analysis. The results demonstrated that IP6 could inhibit tumors, in terms of tumor incidence, number, weight and volume in DMH-induced rats. Additionally, Akt and c-Myc mRNA levels were significantly decreased. IP6 was also shown to downregulate Akt, pAkt, pGSK-3β, and c-Myc protein expression and upregulate pβ-catenin protein expression. Furthermore, tumor tissues from IP6-treated rats showed decreased proliferation. In conclusion, the anti-proliferative effect of IP6 may be related to crosstalk between the PI3K/Akt and Wnt pathways, revealing a potential mechanism of CRC inhibition by IP6 in our model.

Remote limb ischemic post conditioning (RIPOC) has been reported to attenuate cerebral ischemic reperfusion (I/R) injury, while the molecular mechanisms remain elusive. Various studies have highlighted the involvement of glycogen synthase kinase (GSK-3β) in cerebral I/R injury and cognitive disorders. Hence, the present study was designed to explore the role of GSK-3β and its downstream regulators in RIPOC mediated neuroprotection against cerebral I/R injury and associated cognitive impairment. Male Wistar rats are randomly assigned into four groups: Sham, bilateral common carotid arteries occlusion (BCCAO), RIPOC and BCCAO+RIPOC. BCCAO was achieved by transient occlusion of bilateral common carotid arteries for 20min, followed by reperfusion. Non-invasive RIPOC was induced by 3 cycles each of 10min occlusion and reperfusion of both femoral arteries by using tourniquets, during early reperfusion phase. A battery of behavioral and cognitive tests were performed. Biochemical estimation of oxidative markers, anti-oxidants and pro-inflammatory markers were estimated. Levels of GSK-3β, CREB and BDNF were estimated to confirm the molecular mechanism. Hippocampal structural abnormalities were confirmed by H and E staining. The neurobehavioral analysis revealed that neurological and cognitive deficits caused by BCCAO, were reduced by RIPOC intervention. Meanwhile, the results of biochemical tests suggested that RIPOC attenuates the BCCAO induced oxidative damage, neuroinflammation and hippocampal structural abnormalities. Further, RIPOC prevented the elevation of BCCAO induced GSK-3β. RIPOC exerts neuroprotective effect against I/R injury, putatively by attenuating GSK-3β expression and upregulating the levels of CREB and BDNF.

Leukotriene B

Inhibition of Aβ aggregation and neurotoxicity has been developed as an attractive therapeutic strategy to combat Alzheimer’s disease (AD). Bis(propyl)-cognitin (B3C) is a multifunctional dimer derived from tacrine. Herein, the anti-aggregation and disassembly effects of B3C on Aβ, together with the neuroprotective effects and underlying mechanisms of B3C against Aβ-induced neurotoxicity were investigated in silico, in vitro and in vivo. Data from Thioflavin-T fluorescence and atomic force microscopy assays indicated that B3C (1-10 μM), but not its monomer tacrine, greatly inhibited the formation of Aβ fibrils and disaggregated pre-formed mature Aβ fibrils. Comparative molecular dynamics simulation results revealed a possible binding mode that prevented Aβ fibrils formation, showing that B3C favorably bound to Aβ via hydrophobic interactions. Additionally, B3C was able to block the neurotoxicity caused by Aβ fibrils in cultured PC12 cells. Very encouragingly, B3C (0.3 and 0.45 mg/kg) markedly alleviated the cognitive impairments in rats insulted by intra-hippocampal injection of Aβ1-42 fibrils, more potently than tacrine (1 and 2 mg/kg). Furthermore, mechanistic studies demonstrated that B3C reversed the inhibition of phospho-GSK3β at Ser9 site in vitro and in vivo caused by Aβ, suggesting the neuroprotection of B3C was achieved through the inhibition of GSK3β pathway. These findings indicate that B3C could serve as an effective inhibitor of Aβ aggregation and neurotoxicity, and provide novel molecular insights into the potential application of B3C in AD prevention and treatment.

Neferine has long been recognized as a medicinal herbal ingredient with various physiological and pharmacological activities. Although previous studies have reported its antithrombotic effect, the underlying mechanisms have not been thoroughly investigated. Since platelets play a key role in thrombosis, we investigated the effects of neferine on human platelet function and the potential mechanisms. Platelet aggregation, adhesion and spreading were performed to investigate the effect of neferine on inhibition of platelet function. Flow cytometry was used to determine platelet alpha granule secretion and integrin IIb/IIIa activation, as detected by CD62P (P-selectin) expression, PAC-1 and fibrinogen binding. Western blotting was utilized to investigate the effect of neferine on intracellular signaling of activated platelet. We found that neferine significantly suppressed platelet aggregation and remarkably promoted the dissociation of platelet aggregates induced by collagen, thrombin, U46619, ADP and adrenaline in a dose-dependent manner. Flow cytometry analysis showed that neferine inhibited thrombin-induced platelet P-selectin expression, PAC-1 and fibrinogen binding. In addition, neferine reduced the adhesion of human platelets on coated collagen under both static and shearing condition at an arterial shear rate of 40 dyne/cm2. Neferine also inhibited the spreading of human platelets on immobilized fibrinogen. Western blot analysis showed that neferine inhibited PI3K activation, and decreased the levels of phosphorylation of Akt, GSK3β and p38 MAPK in platelets. In summary, neferine has the potential to be an antiplatelet and antithrombotic agent by inhibiting the PI3K-Akt-GSK3β/p38 MAPK signaling pathway.

Nobiletin (3',4',5,6,7,8-hexamethoxyflavone), a dietary polymethoxylated flavonoid found in Citrus fruits, was reported to exhibit protective activity against ischemia/reperfusion (I/R). However, the role of nobiletin in myocardial I/R injury remains unclear. This study was designed to examine the cardioprotective effect of nobiletin from myocardial hypoxia/reoxygenation (H/R) injury in vitro, and to explore the potential molecular mechanisms. Our results showed that nobiletin improved cell viability in H9c2 cells after H/R treatment. In addition, nobiletin significantly inhibited the production of reactive oxygen species and malondialdehyde (MDA), cell apoptosis, as well as suppressed the levels of pro-inflammatory factors in H/R-stimulated H9c2 cells. Furthermore, we observed that pretreatment with nobiletin significantly activated the Akt/GSK-3β signaling pathway in H/R-stimulated H9c2 cells. Taken together, these findings demonstrated that nobiletin attenuates myocardial I/R injury via the activation of Akt/GSK-3β pathway in H9c2 cardiomyocytes. Thus, nobiletin may be regarded as a promising drug for the prevention of myocardial I/R injury and ischemic heart disease.

CAPE-pNO

P2X7 receptor (P2X7R) plays an important role in regulating the growth of tumor cells. However, the role of P2X7R in colorectal cancer (CRC) has remained poorly understood. Therefore, in this study, in vivo and in vitro experiments were performed to investigate the effect of P2X7R on the proliferation of CRC. The results showed that P2X7R was expressed in CRC cell lines (SW620 and HCT116). ATP and BzATP increased the expression of P2X7R in CRC cells, while the application of P2X7R antagonist A438079 and AZD9056 decreased the P2X7R expression induced by BzATP. Moreover, ATP and BzATP induced the activation of P2X7R to promote the proliferation, migration and invasion of CRC cells. Conversely, A438079, AZD9056 or siRNA transfection targeting P2X7R (siP2X7R) knockdown P2X7R expression inhibited the proliferation and migration of CRC cells. TGF-β1 promoted the migration and invasion of CRC cells, while the application of P2X7R antagonist could inhibit TGF-β1 induced migration of CRC cells. Furthermore, activation of P2X7R increased the expression of Vimentin, Snail, Fibronectin and decreased the expression of E-cadherin. While reducing the expression of P2X7R could inhibit these genes expression. In addition, ATP and BzATP increased the expression of p-Akt, p-GSK-3beta and β-catenin via P2X7R. P13/Akt pathway inhibitor LY294002 inhibited the proliferation of CRC cells, and the P13/Akt signaling was required for BzATP induced the proliferation of CRC cells. Our conclusion is that P2X7R mediated the PI3K/Akt/GSK-3beta signaling to promote the proliferation and EMT of CRC, indicating that P2X7R may be used as a potential therapeutic target for CRC.

Gastric cancer (GC), known for high morbidity and mortality, is poorly prognosed with traditional chemotherapy and biological agents. Current studies have found that over-activation of AKT is a common molecular characteristic in GC. Although the development of this targeted inhibitor has entered clinical phases, limited success is reported because of its compensatory signaling pathways. Here, we found that GC cell lines with high phosphorylation of AKT show different sensitivity to AKT inhibitors (AKTis), but a reduction of p-GSK3β related sensitivity of AKTis in GC cells. Besides, we revealed that Ceritinib exerted a strongly synergistic antitumor effect with AKT inhibitors both in vitro and in vivo. Obviously, Ceritinib improved the sensitivity of Capivasertib (AZD5363, AKTs) and Afuresertib (GSK2110183, AKTis) in gastric cancer cells, as illustrated by a significant reduction in the GC cell proliferation and enhanced apoptosis. The drug combination showed tumor regression in BALB/c (nu/nu) mouse MKN45 (Gastric cancer), tumor model. Also, the combination strategy indicated significantly low p-AKT levels due to AKTis compensation and reduced the levels of p-GSK3β in both GC cell lines and GC patient-derived cells. These findings may provide a novel combination strategy for gastric cancer treatment.

We tested the hypothesis that ranolazine (Ran) is cardioprotective in a model of ischemia /reperfusion and we elucidated the intracellular mechanism. Anesthetized rabbits were subjected to is chemia and reperfusion and were divided into 5 groups: 1) Control, 2) Preconditioning (PreC), 3) Postconditioning (PostC), 4) RanA and 5) RanB, respectively treated with intravenous ranolazine, either 10min before or during index ischemia. Ranolazine was initially given over 60s and then from the beginning and throughout the whole reperfusion period. The infarcted to the risk ratio was calculated (%I/R). In a second series consisting of respective to the first series groups, the animals were subjected to the same interventions up to the 10th min of reperfusion where tissue samples were taken for immunoblotting of Akt, eNOS, ERK½ and GSK3β (RISK pathway). In a third series, RanA+Wort, RanB+Wort and Wort groups were treated with ranolazine as RanA and RanB groups but with the addition of the PI3 inhibitor Wortmaninn (Wort) and %I/R calculated. Ranolazine reduced the % I/R in RanA and RanB compared to the Control (23.1±1.7%, 17.6±2.0% vs 47.6±1.0%, P0.05). %I/R reduction achieved in the RanA and RanB groups was comparable to that observed in PreC and PostC (16.3±2.1%, 26.2±2.1%, respectively P0.05 vs Control). Phosphorylation of Akt, ERK½, eNOS and GSK3β were higher in PreC, PostC and in both ranolazine treated groups. Wortmannin abrogated ranolazine's %I/R reduction (RanA+Wort 31.4±1.7%, RanB+Wort 32.4±2.4%). Ranolazine reduces %I/R and triggers cardioprotection with a similar to conditioning mechanism which upregulates the RISK pathway.

Proliferation and migration of vascular smooth muscle cells (VSMCs) play crucial roles in the development of vascular restenosis. Our previous study showed that CCN4, namely Wnt1 inducible signaling pathway protein 1 (WISP1), significantly promotes proliferation and migration of rat VSMCs, but its mechanism remains unclear. This study aims to investigate whether and how WISP1 stimulates proliferation and migration of human VSMCs. Western blot analysis showed that FBS treatment increased WISP1 protein levels in human VSMCs in a dose-dependent manner. Overexpression of WISP1 using adenovirus encoding WISP1 (AD-WISP1) significantly increased proliferation rate of human VSMCs by 2.98-fold compared with empty virus (EV)-transfected cells, shown by EdU incorporation assay. Additionally, Scratch-induced wound healing assay revealed that adenovirus-mediated overexpression of WISP1 significantly increased cell migration compared with EV-transfected cells from 6h (4.56±1.14% vs. 11.23±2.25%, P

Berberine chloride (BBR) is an isoquinoline derivative alkaloid isolated from medicinal herbs, including Coptis chinensis and Berberis aristate. This compound plays significant roles in the treatment of osteoarthritis (OA). The purpose of this study was to investigate the effects of BBR on the proliferation of sodium nitroprusside (SNP)-stimulated chondrocytes in vitro, the articular cartilage in a rat OA model, as well as to discuss the molecular mechanisms underlying these effects. In vitro, we demonstrated that BBR led to cell proliferation, increased the cell population in S-phase and decreased that in G0/G1-phase; moreover, the F-actin remodeling in SNP-stimulated chondrocytes were prevented. In addition, BBR markedly up-regulated β-catenin, c-Myc, and cyclin D1 expression of genes and proteins, and down-regulated glycogen synthase kinase-3β (GSK-3β) and matrix metalloproteinase-7 (MMP-7) expression. Notably, inhibition of the Wnt/β-catenin pathway by XAV939 partially blocked these effects. The in vivo results suggested that BBR promoted β-catenin protein level and enhanced proliferating cell nuclear antigen (PCNA) expression in osteoarthritic rat cartilage. In conclusion, these findings indicate that BBR promotes SNP-stimulated chondrocyte proliferation by promoting G1/S phase transition and synthesis of PCNA in cartilage through activation of Wnt/β-catenin signaling pathway.

Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy.

Neurofibrillary tangles aggregated from hyperphosphorylated tau protein are the main pathological feature of Alzheimer's disease (AD). Complement C3 (or C3a) is the core component of the complement system and is associated with AD pathological processes. However, it remains unclear whether C3a or the C3a receptor has any effect on tau phosphorylation. In this study, we found that exposure of SH-SY5Y cells to okadaic acid (OA) decreased cell viabilities and induced tau hyperphosphorylation. These effects were alleviated by C3a receptor antagonist SB290157 and were further validated by C3a receptor siRNA in OA-treated SH-SY5Y cells. In addition, our results demonstrated that SB290157 markedly inhibited the activities of glycogen synthase kinase 3β (GSK3β), but had no effect on protein phosphatase 2A C subunit (PP2Ac) and cyclin-dependent kinases 5 (CDK5). Our findings here indicate the unique role of the C3a receptor in regulating tau phosphorylation via GSK3β signaling pathways and suggest that the C3a receptor may be a viable target for treating AD.

Tripartite motif 66 (TRIM66) protein, a member of the tripartite motif (TRIM) protein superfamily, has emerged as an oncogenic protein that is closely related to carcinogenesis in multiple cancers. However, whether TRIM66 plays a role in the progression of hepatocellular carcinoma (HCC) remains unknown. This study was aimed to investigate TRIM66 expression and its potential biological function in HCC cell lines. Here we showed that TRIM66 expression was significantly upregulated in HCC cell lines compared with normal control cells. Loss-of-function experiments by RNA interfering knockdown of TRIM66 showed that TRIM66 inhibition significantly reduced the proliferation, colony formation, and invasion of HCC cells, whereas gain-of-function by overexpression of TRIM66 exhibited the opposite effect. Further investigation showed that TRIM66 was involved in regulating glycogen synthase kinase-3β (GSK-3β) phosphorylation and β-catenin expression. Knockdown of TRIM66 impeded the activation of Wnt signaling, while overexpression of TRIM66 promoted Wnt signaling activation. Moreover, inhibition of GSK-3β by specific inhibitor partially reversed TRIM66 inhibition-mediated antitumor effect, while knockdown of β-catenin blocked the oncogenic effect of TRIM66 overexpression in HCC cells. Additionally, in vivo experiments using a xenograft tumor model showed that TRIM66 knockdown blunted the tumorigenicity of HCC cells associated with downregulation of β-catenin expression. Overall, our results showed that TRIM66 functioned as an oncogenic protein in HCC by promoting the activation of Wnt/β-catenin signaling. Our study suggests that TRIM66 is a potential target for HCC treatment.

Our previous studies showed that the effect of resveratrol preventing mitochondrial permeability transition pore (mPTP) opening in myocardial ischemia/reperfusion injury was achieved by regulating voltage-dependent anion channel 1 (VDAC1). However, the underlying mechanism remains unclear. Previous studies demonstrated that the activity and function of VDAC1 are highly regulated by post-translational modification. In present study, we investigated whether resveratrol modulates VDAC1 phosphorylation to achieve cardioprotection and explored the signaling pathways involved. Our findings demonstrated that anoxia/reoxygenation (A/R) treatment, an ischemia/reperfusion model in vitro, enhanced VDAC1 phosphorylation in cardiomyocytes. Moreover, we found phosphorylated VDAC1 showed increased affinity to Bax, whereas interaction with hexokinase 2 (HK2) was reduced. Accordingly, the generation of reactive oxygen species increased, the mitochondrial membrane potential collapsed, mPTP opening increased and cytochrome c released into cytoplasm, thereby leading to increased apoptosis. Moreover, our data showed that pretreatment with resveratrol prior to A/R injury inhibited VDAC1 phosphorylation. Dephosphorylated VDAC1 using pretreated resveratrol promoted dissociation with Bax and binding to HK2, which subsequently protected cardiomyocytes against A/R injury. In addition, Akt and its downstream glycogen synthase kinase 3 β (GSK3β) were phosphorylated by the action of resveratrol. Akt inhibitor IV abrogated Akt-GSK3β phosphorylation and thereby abolished the dephosphorylation activity of resveratrol on VDAC1. Moreover, all resveratrol-mediated protective effects on A/R injured cardiomyocytes were abolished by Akt inhibitor IV. Taken together, our data indicated that A/R injury enhanced VDAC1 phosphorylation in cardiomyocytes, whereas pretreatment with resveratrol dephosphorylated VDAC1 through the Akt-GSK3β pathway, thereby protecting cardiomyocytes against A/R injury.