GSK-3β Expression Research Articles

FBXL16 Promotes Endometrial Progesterone Resistance via PP2AB55α /Cyclin D1 Axis in Ishikawa.

Background F-box proteins are essential components of the E3 ubiquitin ligases which are involved in the regulation of almost all life activities such as cell cycle, proliferation, and apoptosis, which have become an important research and drug target. However, there are few studies on F-box and leucine-rich repeat protein 16 (FBXL16) in endometrial carcinoma. Methods Clinical samples were collected for determining the correlation between FBXL16 and endometrial carcinoma. Cells were screened and established with Ishikawa cells which proved the fundamental role of FBXL16 in regulating cell proliferation and cell cycle. The MPA-resistant endometrial carcinoma cell line Ishikawa/MPA was established. FBXL16, PP2AB55α, and cyclin D1 were analyzed separately in MPA sensitive and resistant Ishikawa cells in vitro and in vivo. Results The high expression of FBXL16 was positively correlated with MPA resistance and poor prognosis of endometrial cancer. MPA tolerance of endometrial cancer cells was inhibited by knockdown of FBXL16 in DNA content assessment, CCK-8, and colony formation. It was confirmed that FBXL16 inhibited the activity of substrate PP2AB55α by binding to PP2A, reduced the phosphorylation level at Thr308 site of AKT1, inhibited the expression of GSK-3β, and thus led to a significant decrease in the phosphorylation level of cyclin D1, which prevented the ubiquitination recognition and degradation of cyclin D1. Conclusion In our experiments, FBXL16 binds PP2A to promote the dephosphorylation of Thr286 site of cyclin D1 via AKT1/GSK3β/cyclin D1 pathway, which is required for resisting the ubiquitination degradation and enhances the MPA resistance of Ishikawa.

Effects of plant-based medicinal food on postoperative recurrence and lung metastasis of gastric cancer regulated by Wnt/β-catenin-EMT signaling pathway and VEGF-C/D-VEGFR-3 cascade in a mouse model

BackgroundThe plant-based medicinal food (PBMF) is a functional compound extracted from 6 medicinal and edible plants: Coix seed, L. edodes, A. officinalis L., H. cordata, Dandelion, and G. frondosa. Our previous studies have confirmed that the PBMF possesses anti-tumor properties in a subcutaneous xenograft model of nude mice. This study aims to further investigate the effects and potential molecular mechanisms of the PBMF on the recurrence and metastasis of gastric cancer (GC).MethodsPostoperative recurrence and metastasis model of GC was successfully established in inbred 615 mice inoculated with mouse forestomach carcinoma (MFC) cells. After tumorectomy, 63 GC mice were randomly divided into five groups and respectively subject to different treatments for 15 days as below: model control group, 5-Fu group, and three doses of PBMF (43.22, 86.44, 172.88 g/kg PBMF in diet respectively). The inhibition rate (IR) of recurrence tumor weights and organ coefficients were calculated. Meanwhile, histopathological changes were examined and the metastasis IR in lungs and lymph node tissues was computed. The mRNA expressions related to the canonical Wnt/β-catenin signaling pathway, epithelial-mesenchymal transition (EMT) and lymphangiogenesis were detected by RT-qPCR in recurrence tumors and/or lung tissues. Protein expressions of β-catenin, p-β-catenin (Ser33/37/Thr41), GSK-3β, p-GSK-3β (Ser9), E-cadherin, and Vimentin in recurrence tumors were determined by Western Blot. LYVE-1, VEGF-C/D, and VEGFR-3 levels in recurrence tumors and/or lung tissues were determined by immunohistochemistry staining.ResultsThe mRNA, as well as protein expression of GSK-3β were up-regulated and the mRNA expression of β-catenin was down-regulated after PBMF treatment. Meanwhile, the ratio of p-β-catenin (Ser33/37/Thr41) to β-catenin protein was increased significantly and the p-GSK-3β (Ser9) protein level was decreased. And PMBF could effectively decrease the mRNA and protein levels of Vimentin while increasing those of E-cadherin. Furthermore, PBMF markedly reduced lymphatic vessel density (LVD) (labeled by LYVE-1) in recurrence tumor tissues, and mRNA levels of VEGF-C/D, VEGFR-2/3 of recurrence tumors were all significantly lower in the high-dose group.ConclusionsPBMF had a significant inhibitory effect on recurrence and lung metastasis of GC. The potential mechanism may involve reversing EMT by inhabiting the Wnt/β-catenin signaling pathway. Lymphatic metastasis was also inhibited by PBMF via down-regulating the activation of the VEGF-C/D-VEGFR-2/3 signaling cascade.

Diacerein attenuate LPS-induced acute lung injury via inhibiting ER stress and apoptosis: Impact on the crosstalk between SphK1/S1P, TLR4/NFκB/STAT3, and NLRP3/IL-1β signaling pathways

AimsAcute lung injury (ALI) is a life-threatening clinical problem with high mortality rate and limited treatments or preventive options that represents a major challenge for clinicians. Diacerein (DIA) is a multi-target anthraquinone derivative with potent anti-inflammatory action. The aim of this study is to assess the protective effect of DIA and its potential molecular targets against lipopolysaccharide (LPS)-induced ALI in rats. Materials and methodsAdult male Sprague-Dawley rats were orally administrated DIA (50 mg/kg) for 5 consecutive days followed by a single intraperitoneal injection of LPS (5mg/kg). Key findingsDIA mitigated oxidative lung injury in LPS-challenged rats via significantly decreasing lung wet/dry (W/D) ratio, inflammatory cells infiltration, and lipid peroxidation, with concomitant elevation in enzymatic and non-enzymatic antioxidant levels in lung tissue. Likewise, DIA alleviated endoplasmic reticulum stress and markedly halted inflammation triggered by LPS challenge in pulmonary tissue by suppressing NLRP3/IL-1β and TLR4/NF-κB signaling with parallel decrease in proinflammatory cytokine levels. Interestingly, DIA down regulated Sphk1/S1P axis, reduced GSK-3β and STAT3 proteins expression, and markedly decreased caspase-3 besides increasing Bcl-2 levels in lung tissue of LPS-challenged animals. These biochemical findings was simultaneously associated with marked improvement in histological alterations of lung tissue. SignificanceThese findings verify the protective effect of DIA against LPS-induced ALI through targeting oxidative stress, endoplasmic reticulum stress, and apoptosis. Importantly, DIA halted the hyperinflammatory state triggered by LPS via multi-faceted inhibitory effect on different signaling pathways, hence DIA could potentially reduce mortality in patients with ALI.

Melatonin alleviates PM2.5 -induced glucose metabolism disorder and lipidome alteration by regulating endoplasmic reticulum stress.

Exposure to fine particulate matter (PM2.5 ) was associated with an increased incidence of liver metabolic disease. Melatonin has been shown to prevent liver glucolipid metabolism disorders. However, whether melatonin could rescue PM2.5 -induced liver metabolic abnormalities remains uncertain. This study was to evaluate the mitigating effect of melatonin on PM2.5 -accelerated hepatic glucose metabolism imbalance in vivo and in vitro. Schiff periodic acid shiffstaining and other results showed that PM2.5 led to a decrease in hepatic glycogen reserve and an increase in glucose content, which was effectively alleviated by melatonin. Targeted lipidomics is used to identify lipid biomarkers associated with this process, including glycerolipids, glycerophospholipids, and sphingolipids. In addition, gene microarray and quantitative polymerase chain reaction analysis of ApoE-/- mice liver suggested that PM2.5 activated the miR-200a-3p and inhibited DNAJB9, and the targeting relationship was verified by luciferase reports for the first time. Further investigation demonstrated that DNAJB9 might motivate endoplasmic reticulum(ER) stress by regulating Ca2+ homeostasis, thus altering the protein expression of GSK3B, FOXO1, and PCK2. Meanwhile, melatonin effectively inhibited miR-200a-3p and glucose metabolism disorder. Knockout of miR-200a-3p in L02 cells revealed that miR-200a-3p is indispensable in the damage of PM2.5 and the therapeutic effect of melatonin. In summary, melatonin alleviated PM2.5 -induced liver metabolic dysregulation by regulating ER stress via miR-200a-3p/DNAJB9 signaling pathway. Our data provide a prospective targeted therapy for air pollution-related liver metabolism disorders.

A network pharmacology approach to identify the mechanisms and molecular targets of curcumin against Alzheimer disease.

Background:Alzheimer disease (AD) is a degenerative brain disease, which may lead to severe memory loss and other cognitive disorders. However, few effective drugs are available in the clinic at present. Curcumin, a major ingredient of traditional Chinese medicine, Curcuma Longa, has various pharmacological activities. Therefore, exploring clinical drugs based on the inhibition of AD pathological features is imperative.Methods:First, we utilized the HERB database and Swisstarget Prediction database to get the related targets of curcumin and intersected with the AD targets. The intersection targets were used to construct the protein-protein interaction network and performed gene ontology and kyoto encyclopedia of genes and genomes analyses. Further, we obtained targets of curcumin against AD-related tau and aβ pathology via the AlzData database. These targets were applied to perform GEO and receiver operating characteristic analyses. Finally, the reliability of the core targets was evaluated using molecular docking technology.Results:We identified 49 targets of curcumin against AD, and kyoto encyclopedia of genes and genomes pathway enrichment analysis demonstrated that the Alzheimer disease pathway (has05010) was significantly enriched. Even more, we obtained 16 targets of curcumin-related Aβ and tau pathology. Among these targets, 8 targets involved the Alzheimer disease pathway and the biological process analyses showed that positive regulation of cytokine production (GO:0001819) was significantly enriched. Bioinformatic analyses indicated that HMOX1, CSF1R, NFKB1, GSK3B, BACE1, AR, or PTGS1 expression was significantly different compared to the control group in the AD patients. Finally, molecular docking studies suggested these genes have a good binding force with curcumin.Conclusions:In this study, we identified curcumin exerted the effect of treating AD by regulating multitargets and multichannels through the method of network pharmacology.

Construction of Fzd6Q152Emice through CRISPR/Cas9 technology and their reproduction and identification.

The CRISPR/Cas9 system is widely used for genome editing in human, rat and mouse cells. In this study, we established Fzd6 mutant mice using CRISPR/Cas9 technology, and obtained Fzd6 homozygous mutant (Fzd6Q152E) mice through breeding. Fzd6 plays a role in depression, but there are few related reports. We used this model to investigate the mechanism of Fzd6 involved in depression, and build a solid foundation for subsequent in-depth studies. The target of Fzd6 mutation was obtained by CRISPR/Cas9 technology and hippocampal tissue was collected for Nissl staining and histological analysis. Blood was collected for enzyme linked immunosorbent assay (ELISA); The gene expression of Fzd6 and the related genes expression in wnt pathway was quantified by quantitative real-time PCR (qRT-PCR), and then expression of Fzd6 and proteins in the Wnt pathway were identified by western blotting. ELISA results showed that the expression levels of brain derived neurotrophic factor (BDNF), 5-hydroxytryptamine (5-HT), and Noradrenaline (NE) in serum were significantly decreased in Fzd6Q152E mice, whereas the mRNA expression of Lrp5, Lrp6, and Dkk2 is increased. The western blotting revealed that the expression of Fzd6 and Lrp6 is decreased, although the expression of Dkk2 and Gsk-3β increased. Our study successfully established homozygous Fzd6 mutant mice model. The relationship between Fzd6-Wnt and depression was preliminarily clarified, which provides an ideal animal model for subsequent research on diseases induced by the Fzd6 mutation.

Identification of Hair Growth Promoting Components in the Kernels of Prunus mira Koehne and Their Mechanism of Action.

The application of the seed oil of Prunus mira Koehne (Tibetan name ཁམབུ།), a plant belonging to the Rosaceae family, for the treatment of alopecia has been recorded in Jingzhu Materia Medica (ཤེལ་གོང་ཤེལ་ཕྲེང་།) (the classic of Tibetan medicine) and Dictionary of Chinese Ethnic Medicine. This study aims to reveal the effective components and mechanism of hair growth promotion in the kernel of Prunus mira Koehne. Network pharmacology was used to predict the mechanism of action and effective components in the treatment of the kernel of Prunus mira Koehne. The contents of amygdalin in 12 batches of the kernel of Prunus mira Koehne were determined by HPLC. An animal model of the depilation of KM mice induced by sodium sulfide was created, and five effective components that promoted hair growth were initially screened. In the study of the effectiveness and mechanism of action, KM and C57BL/6 mice are selected as experimental objects, three screening tests for active components of the kernel of P. mira are performed, and three effective components are screened out from the eight components. HE staining was used to detect the number of hair follicles and the thickness of the dermis. RT-PCR and immunohistochemistry were used to evaluate the influence of the expression of indicators in the Wnt/β-catenin signaling pathway in skin, including β-catenin, GSK-3β, and mRNA and protein expression levels of Cyclin D 1 and LEF 1. The network pharmacology study showed 12 signaling pathways involving 25 targets in the treatment of alopecia by the kernel of Prunus mira Koehne. vitamin E (3.125 mg/cm2/d), β-sitosterol (0.061 mg/cm2/d), and linoleic acid (0.156 mg/cm2/d) in the kernel of Prunus mira Koehne can promote hair growth in mice, and the mechanism of action may be related to the Wnt/β-catenin pathway.

Tricin protects rats from ovariectomized-induced osteoporosis by enhancing Wnt/β-catenin pathway

Purpose: To investigate the effects of tricin on ovariectomized-induced osteoporosis, and unravel theunderling mechanism of action.Methods: An osteoporosis rat model was established by conducting ovariectomy (OVX). Changes in the microstructure of the trabecular bone were visualized using Hematoxylin and eosin (H&E) staining, and a three-point bending test was employed to assess the biomechanical stability of the femurs, after the administration of tricin (20 and 40 mg/kg). Subsequently, bone marrow mesenchymal stem cells (BMSCs) were isolated and treated with tricin (7 and 15 μM). Alizarin red staining was performed to assess mineralization, and Runt-related transcription factor 2 (RUNX2); osteocalcin (OCN) and collagen type I alpha 1 (Col1a1) were quantified using western blot analysis. The Wnt/β-catenin pathway related proteins, i.e., Wnt3a, β-catenin, glycogen synthase kinase-3 β (GSK-3 β) were determined.Results: Ovariectomy induced thinner and discontinuous trabecular bone, with increased marrow cavities, while application of tricin significantly improved the density and regularity meshwork, but reduced marrow cavities. Tricin also enhanced biomechanical competence as seen in the upregulated maximum load, stiffness, young modulus and maximum stress compared with OVX group (p < 0.01). Furthermore, tricin increased calcification in BMSCs, and significantly upregulated the expressions of RUNX2, OCN and COL1A1 when compared with OVX group (p < 0.01). It promoted Wnt/β-catenin signaling by enhancing Wnt3a and β-catenin, while inhibiting GSK3β expression, compared with OVX group (p < 0.05 or p < 0.01).Conclusion: Tricin exerts protective effects against OVX-induced osteoporosis by enhancing Wnt/β-catenin pathway. Thus, tricin is a potential therapeutic agent for the management of osteoporosis.

Swimming Training Mitigates Neurological Impairment of Intracerebral Haemorrhage in Mice via the Serine-Threonine Kinase/Glycogen Synthase Kinase 3β Signalling Pathway

Swimming training (ST) can mitigate functional disorders in neurological diseases, but the effect and mechanism of ST in improving the neurological function of intracerebral haemorrhage (ICH) have not been reported. Our study aimed to explore the protective effect of early ST on ICH mice and its relationship with the serine-threonine kinase (Akt)/glycogen synthase kinase 3β (GSK3β) pathway. Our findings showed that the ICH model mice had poor behavioural manifestations in the Y maze test and open field test compared to the ST group and sham group. The modified neurological severity score was increased in the ICH mice, and 7 days of ST intervention significantly attenuated the neurological deficits. The ratios of myo-inositol/creatine, lactate/creatine and glutamate/creatine were decreased, and the ratios of N-acetylaspartate/creatine and choline/creatine were increased in the ICH mice with ST intervention. ST intervention decreased the expression of Iba1 and GFAP. Seven days of ST significantly increased the expression of p-Akt/Akt compared to that in the ICH mice. Furthermore, the Akt kinase inhibitor GSK690693 exacerbated neurological impairment, increased the expression of Iba1, GFAP and Bax/Bcl-2, and reversed the anti-apoptotic effects and anti-glia activation of ST, which was associated with the inhibition of p-Akt/Akt and p-GSK3β/GSK3β expression. These results indicated that the protective role of ST in ICH was mediated via the Akt/GSK3β pathway. In conclusion, ST displayed neuroprotection by inhibiting apoptosis and glial activation in ICH mice by activating the Akt/GSK3β signalling pathway.

Combination of paeoniflorin and calycosin-7-glucoside alleviates ischaemic stroke injury via the PI3K/AKT signalling pathway

Context Paeoniflorin (PF) and calycosin-7-glucoside (CG, Paeonia lactiflora Pall. extract) have demonstrated protective effects in ischaemic stroke. Objective To investigate the synergistic effects of PF + CG on ischaemia/reperfusion injury in vivo and in vitro. Materials and methods Male Sprague-Dawley rats were subjected to the middle cerebral artery occlusion/reperfusion (MCAO/R). After MCAO/R for 24 h, rats were randomly subdivided into 5 groups: sham, model (MCAO/R), study treatment (PF + CG, 40 + 20 mg/kg), LY294002 (20 mg/kg), and study treatment + LY294002. Males were given via intragastric administration; the duration of the in vivo experiment was 8 days. Neurologic deficits, cerebral infarction, brain edoema, and protein levels were assessed in vivo. Hippocampal neurons (HT22) were refreshed with glucose-free DMEM and placed in an anaerobic chamber for 8 h. Subsequently, HT22 cells were reoxygenated in a 37 °C incubator with 5% CO2 for 6 h. SOD, MDA, ROS, LDH and protein levels were measured in vitro. Results PF + CG significantly reduced neurobehavioral outcomes (21%), cerebral infarct volume (44%), brain edoema (1.6%) compared with the MCAO/R group. Moreover, PF + CG increased p-PI3K/PI3K (4.69%, 7.4%), p-AKT/AKT (6.25%, 60.6%) and Bcl-2/BAX (33%, 49%) expression in vivo and in vitro, and reduced GSK-3β (10.5%, 9.6%) expression. In vitro, PF + CG suppressed apoptosis in HT22 cells and decreased ROS and MDA levels (20%, 50%, respectively). Conclusions PF + CG showed a synergistic protective effect against ischaemic brain injury, potentially being a future treatment for ischaemic stroke.

EPS8L3 promotes pancreatic cancer proliferation and metastasis by activating GSK3B.

We intended to investigate the role and regulatory mechanism of EPS8L3 in increase the development of pancreatic cancer (PC). In order to analyze the relationship between EPS8L3 level and clinicopathological indicators of PC patients, qRT-PCR was used to detect the expression of EPS8L3 in tumor specimens of 40 PC patients. EPS8L3 knockdown models were then constructed in PC cell lines. Furthermore, the effect of EPS8L3 on PC cell function was analyzed by CCK-8 and Transwell assay. Dual luciferase reporter gene assay and recovery assay were used to further investigate the underlying mechanism. qRT-PCR results indicated that EPS8L3 was highly expressed in PC tissues compared with adjacent ones. At the same time, the incidence of distant metastasis was higher in PC patients with high EPS8L3 level. In vitro analysis such as CCK-8 and Transwell experimentations indicated that knockdown of EPS8L3 markedly inhibited the proliferative and metastatic ability. Bio-informatics together with luciferase report assay proposing that EPS8L3 can target GSK3B. Western Blot results revealed that knockdown of EPS8L3 markedly reduced the GSK3B expression in PC cells, and there was a positively associated between the two in PC cells. In addition, the recovery experimentation proved that EPS8L3 and GSK3B have a mutual regulation effect. Overexpression of GSK3B can reversal the prohibitive effect of EPS8L3 knockdown on the malignant development of PC cells, thereby jointly regulating the occurrence and development of PC. EPS8L3 promotes the development of PC by regulating GSK3B, suggesting that EPS8L3 can be used as a biomarker for early diagnosis and treatment of PC.

Effects of pre-moxibustion on expression of phosphorylated Tau protein and related protein kinases in hippocampal CA3 region of AD rats

To observe the effect of pre-moxibustion at "Baihui"(GV20), "Shenshu"(BL23) and "Zusanli"(ST36) on expression of Tau protein and related protein kinases as glycogen synthase kinase-3β (GSK-3β), etc. in the hippocampal CA3 region of Alzheimer's disease (AD) rats, so as to explore its mechanism underlying prevention and treatment of AD cognitive impairment. Male SD rats were randomly divided into 4 groups: normal control, sham operation， model and pre-moxibustion，with 9 rats in each group. Rats of the pre-moxibustion group received moxibustion of GV20, BL23 and ST36 for 15 min, once a day, 6 days a week for 3 weeks. After completion of moxibustion, the AD model was reproduced by injection of amyloid beta-peptide 25-35(Aβ 25-35) aggregation solution 1 μL (5 μg/μL) into the bilateral hippocampus, rats of the sham operation group received injection of the same dose of normal saline into the hippocampus. The spatial learning-memory ability was detected using Morris water maze test, and changes of the ultrastructure of hippocampal neurons were observed using electron microscope, and those of histopathological changes of hippocampus tissue observed using hematoxylin eosin (H.E.) staining. The expression levels of hippocampal GSK-3β, p-Tau, CDK5 and Synapsin I proteins were detected by Western blot and immunohistochemistry, respectively. No significances were found between the normal control and sham groups in all the indexes (P>0.05). Compared with the control group, the escape latency of place navigation test of Morris water maze test, expression of GSK-3β and CDK5 and the immunoactivity of GSK-3β, CDK5 and p-Tau were significantly increased (P<0.01), and the residence time in the platform quadrant and the number of platform crossing of spatial prob test and the expression of Synapsin Ⅰ significantly reduced in the model group (P<0.01). Following the intervention, the increase of escape latency, expression of GSK-3β and CDK5 and the immunoactivity of GSK-3β, CDK5 and p-Tau, and the decrease of residence time in the platform quadrant, number of platform crossing and the expression of Synapsin Ⅰ were reversed in the pre-moxibustion group (P<0.05, P<0.01). Outcomes of ultrastructure and histopathological observations respectively showed edema of hippocampal nerve cells at varying degrees, moderate edema of the cytoplasma, chromatin condensation at the edge of the nucleus, partial mitochondrial vacuole-like degeneration, fracture of tubular crest, edema and expansion of Golgi body, disappearance of polarity, fracture of the rough endoplasmic reticulum, degeneration of ribosome and partial myelin axon and reduced synaptic vesicles in the presynaptic capsule; and reduced number of neurons with shrank body, disappearance of nucleolus and blurred nuclear boundary and vacuole-like degeneration in some of them in the model group, which were relatively milder in the pre-moxibustion group. Pre-moxibustion at GV20, BL23 and ST36 plays a role in slowing down the occurrence and development of cognitive impairment in AD rats, which may be related to its functions in inhibiting tau protein hyperphosphorylation and reducing the expression of some related protein kinases in the hippocampus.

UC-MSCs promote frozen-thawed ovaries angiogenesis via activation of the Wnt/β-catenin pathway in vitro ovarian culture system

BackgroundOvarian tissue cryopreservation and transplantation are novel therapeutic approaches for fertility preservation. However, follicle loss caused by ischemic and hypoxic damage is one of the issues after frozen-thawed ovarian tissue transplantation. Promoting angiogenesis in grafts is the key to restore cryopreserved ovarian function. Mesenchymal stem cells (MSCs) have been reported to facilitate angiogenesis in the cryopreserved ovarian tissue transplantation. However, the risk of embolization, immunogenic effect and tumorigenesis hinders the clinical application of MSCs to human organ transplantation. In this study, we established an in vitro ovarian culture system to restore frozen-thawed ovarian function before transplantation with the application of umbilical cord mesenchymal stem cells (UC-MSCs), and explored the effects of UC-MSCs on frozen-thawed ovaries in vitro ovarian culture system and the mechanisms of UC-MSCs on the angiogenesis of frozen-thawed ovaries.MethodsA simple in vitro three dimensional (3D) ovarian culture system using Matrigel was established to support to an ideal niche, and ovary was alone cultured in the 24-well plate as a control. We also evaluated the effects of UC-MSCs treatment on ovarian function with or without Matrigel support. All thawed ovaries were randomly divided into control group (Matrigel−/UC-MSCs−), Matrigel group (Matrigel+/UC-MSCs−), UC-MSCs group (Matrigel−/UC-MSCs+) and UC-MSCs + Matrigel group (Matrigel+/UC-MSCs+). HE staining was used to detect the histological structure of follicles and TUNEL staining was used to detect cell apoptosis. The number of microvessels was counted to evaluate neovascularization. The mRNA expression of VEGFA, IGF1 and ANGPT2 were detected by RT-PCR. Western blotting was used to measure the expression of GSK-3β, β-catenin and p-β-catenin.ResultsIn the absence of UC-MSCs, 3D culture system supported by Matrigel showed significantly improved follicular development and microvascular number. Additionally, UC-MSCs were also found to effectively improve follicular development and microvascular number regardless of the culture condition used. However, alleviated follicular apoptosis, increased mRNA expression of angiogenesis-related gene and activated Wnt/β-catenin pathway occurred only in the UC-MSCs + Matrigel group. Besides, with the application of IWP-2 in UC-MSCs + Matrigel group, Wnt//β-catenin pathway could be blocked by IWP-2 serving as one of Wnt/β-catenin pathway inhibitors.ConclusionsThis in vitro study showed the beneficial effects of UC-MSCs on thawed ovaries and explored a potential mechanism inducing angiogenesis. In particular, 3D ovarian culture system supported by Matrigel further improved UC-MSCs treatment. The in vitro culture system using Matrigel and UC-MSCs may provide a potential treatment strategy for improving the success rate of thawed ovaries transplantation.

Potential mechanism of Jatropha gossypifolia phenolic derivatives in enhancing insulin-signalling cascades GLUT 4, IRβ and GSK-3β in streptozotocin nicotinamide induced type II diabetic in wistar rat model

ObjectiveDiabetes mellitus is a metabolic syndrome that alters the composition of carbohydrates, proteins and fat content in the body due to decreased insulin secretion. This disrupts the insulin-signaling cascade, resulting in failure in the targeted tissues, such as insulin-stimulated glucose absorption muscle and fat. Natural products such as plants, which are rich in nutrient, offer a wide range of treatments for a number of diseases and infections including diabetes mellitus. Hence, this study investigates the anti-hyperglycemic efficacy of the methanolic leaves extract of Jatropha gossypifolia in experimental diabetic rats. MethodsPhytochemical constitution of J. gossypifolia showed tannin content to be significantly increased in methanolic extract and HPLC analysis also revealed phenolic derivative tannic acid to be main constitution. An intraperitoneal administration of 110 mg/kgbody weight (b.w) nicotinamide and 45 mg/kg b.w. streptozotocin was provided to male Wistar albino rats, thus inducing diabetic. Diabetic induced rats treated with methanolic leaves extract of J. gossypifolia at the concentration between 100 and 50 mg/kg for 35 days. Periodic blood glucose level and body weight were measured. Methanolic extract influence was evaluated with q-PCR and western blot analysis on the cellular targets of insulin signaling cascades. ResultsThe results showed the decrease in blood glucose levels and increased serum insulin level in treated groups was significant (P < 0.05) in oral administration of methanol extract. Methanolic extract exhibited a profound increase in the gene expression of the cellular targets of insulin signaling cascade including IRTK, IRS1, PI3K, GS, GLUT 4 and a basal level expression of GSK-3β. In addition, methanolic extract increased protein expression of IRβ (2.7-fold), GSK-3β (2.5-fold) and GLUT 4 (2.4-fold) translocation as evidenced from western blot analysis with the plasma membrane fractions. ConclusionConclusively, methanolic leaves extract of J. gossypifolia exhibited a profound anti-hyperglycemic activity by modulating the expression of the key targets of insulin signaling pathway.

Circ-GSK3B up-regulates GSK3B to suppress the progression of lung adenocarcinoma.

GSK3B is the mRNA form of glycogen synthase kinase 3 beta (GSK-3β), which is a critical repressor of Wnt/β-catenin signaling pathway and generally inhibited in cancer cells. Plenty of researches have disclosed that circular RNAs, namely circRNAs exert important functions in the progression of various human malignancies including lung adenocarcinoma (LUAD). Therefore, we attempted to explore whether there existed certain circRNAs that could mediate LUAD development by regulating GSK3B expression and Wnt/β-catenin pathway. In the present research, circ-GSK3B (hsa_circ_0066903) was found to be significantly down-regulated in LUAD tissues and cells and it suppressed the proliferation, migration and stemness of LUAD cells. Furthermore, it was discovered that circ-GSK3B competitively sponged miR-3681-3p and miR-3909 to elevate GSK3B expression. Circ-GSK3B could impair the binding ability of FKBP51 to GSK-3β to inhibit the phosphorylation of GSK-3βS9, resulting in the inactivation of Wnt/β-catenin signaling. In addition, the regulatory effect of circ-GSK3B on LUAD tumorigenesis and cell progression was testified through in vitro and in vivo rescue experiments. In conclusion, circ-GSK3B suppressed LUAD development through up-regulating and activating GSK3B.

Wnt/β-catenin signaling in the adrenal glands of rats in various types of experimental hypertension.

Wnt/β-catenin signaling plays a key role in maintaining homeostasis, which is disturbed in hypertension. Taking into account the lack of literature describing changes in the Wnt/β-catenin pathway in the adrenal glands under conditions of elevated arterial pressure, here we compare the expression of WNT4, WNT10A, β-catenin, and GSK-3β in the adrenal glands of hypertensive rats of various etiologies. The studies were carried out on the adrenal glands of rats with spontaneous hypertension (SHR), renalvascular (2K1C), and deoxycorticosterone acetate (DOCA)-salt. Immunohistochemical and PCR methods were used to identify the molecular components of the canonical signaling pathway and to evaluate gene expression. Immunoreactivity and expression of WNT4, WNT10A, β-catenin, and GSK-3β in adrenals of SHR was decreased, compared to control rats. In adrenals of 2K1C rats, intensity of immunohistochemical reaction and expression of WNT4 and β-catenin was lower, while immunoreactivity and expression of WNT10A and GSK-3β were higher, compared to normotensive animals. Significantly stronger immunoreaction and expression of WNT4, β-catenin and GSK-3β but weaker immunoreactivity and expression of WNT10A were noted in adrenals in DOCA-salt rats, compared to control rats. In conclusion, our data provide new molecular information indicating that the canonical WNT pathway is disrupted in the adrenal glands of hypertensive rats. They show that the dysregulation of the WNT pathway depends on the etiology of hypertension.

Comparative LC-LTQ-MS-MS Analysis of the Leaf Extracts of Lantana camara and Lantana montevidensis Growing in Egypt with Insights into Their Antioxidant, Anti-Inflammatory, and Cytotoxic Activities.

Lantana camara L. and Lantana montevidensis Briq. (F. Verbenaceae) are invasive ornamental weeds native to the tropical regions of Africa and America. The leaves of both species have been traditionally used as infusions for treating fever, rheumatism, and cancer. LC–MS–MS-guided profiling of the methanolic extracts of the leaves of L. camara and L. montevidensis growing in Egypt led to the putative identification of 59 compounds belonging to terpenoids, flavonoids, iridoid glycosides, phenolic acids, and their derivatives. The in-vitro antioxidants and anti-inflammatory and anticancer activities of the two extracts were investigated. L. camara and L. montevidensis inhibited DPPH• (IC50 = 34.01 ± 1.32 and 47.43 ± 1.74 µg/mL), ABTS+ (IC50 = 30.73 ± 1.42 and 40.37 ± 1.51 µg/mL), and superoxide anion (IC50 = 1.57 ± 0.19 and 1.31 ± 0.14 μg/mL) free radicals. A potent anti-inflammatory effect was observed for both species through the inhibition of elastase release in fMLF/CB-induced human neutrophils (IC50 = 2.40 ± 0.16 and 1.90 ± 0.07 μg/mL). The extracts showed significant cytotoxic activity against a panel of cancer cell lines with the most potent activity against Caco cells (IC50 = 45.65 ± 1.64 and 40.67 ± 1.52 µg/mL for L. camara and L. montevidensis, respectively). Western blotting supported by FACS analysis revealed that the extracts inhibited cancer cell proliferation, reduced metastasis, and induced apoptosis resulting in cell cycle arrest. This was achieved via increasing mRNA and protein expressions of p53 and GSK-3β as well as decreasing the expression of PI3K, Akt, and cyclin D1.

Lack of TRPV1 Channel Modulates Mouse Gene Expression and Liver Proteome with Glucose Metabolism Changes.

To investigate the role of the transient receptor potential channel vanilloid type 1 (TRPV1) in hepatic glucose metabolism, we analyzed genes related to the clock system and glucose/lipid metabolism and performed glycogen measurements at ZT8 and ZT20 in the liver of C57Bl/6J (WT) and Trpv1 KO mice. To identify molecular clues associated with metabolic changes, we performed proteomics analysis at ZT8. Liver from Trpv1 KO mice exhibited reduced Per1 expression and increased Pparα, Pparγ, Glut2, G6pc1 (G6pase), Pck1 (Pepck), Akt, and Gsk3b expression at ZT8. Liver from Trpv1 KO mice also showed reduced glycogen storage at ZT8 but not at ZT20 and significant proteomics changes consistent with enhanced glycogenolysis, as well as increased gluconeogenesis and inflammatory features. The network propagation approach evidenced that the TRPV1 channel is an intrinsic component of the glucagon signaling pathway, and its loss seems to be associated with increased gluconeogenesis through PKA signaling. In this sense, the differentially identified kinases and phosphatases in WT and Trpv1 KO liver proteomes show that the PP2A phosphatase complex and PKA may be major players in glycogenolysis in Trpv1 KO mice.

LncRNA DHFRL1‑4 knockdown attenuates cerebral ischemia/reperfusion injury by upregulating the levels of angiogenesis‑related genes.

The present study aimed to investigate the effects of long non-coding (lncRNA) dihydrofolate reductase-like 1 (DHFRL1-4) on cerebral ischemia/reperfusion (I/R)-induced injury. For this purpose, mice injected with lentivirus with small interfering RNA targeting DHFRL1-4 or negative control siRNA were used to construct models of cerebral I/R injury. Following the establishment of the model, the infarct size, neurological deficit score, apoptosis and the expression levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), Wnt family member 3a (Wnt3a), glycogen synthase kinase-3β (GSK-3β) and phosphorylated GSK-3β were assessed. The expression of DHFRL1-4 was significantly upregulated in the I/R model. In the control and sham groups, the boundaries between the cortex and gray matter were clear, and no edema or necrosis were observed. The nerve cells were arranged orderly and evenly, and the cell membranes were intact with visible nucleus and nucleolus. In the model group however, the nerve fibers were slightly necrotic and swollen, and the number of nerve cells was reduced. In the mice injected with si-DHFRL1-4 lentivirus, the brain tissues exhibited less liquefaction and degeneration, as well as less edema. Compared with the control and sham groups, the model group had a significantly larger infarct area, a higher apoptotic rate, higher bFGF, VEGF, Wnt3a and GSK-3β expression levels and a greater neurological deficit score. However, the mice injected with si-DHFRL1-4 lentivirus exhibited a significantly reduced infarct area, a lower apoptotic rate, lower Wnt3a and GSK-3β expression levels, a lower neurological deficit score, and significantly upregulated bFGF and VEGF levels.

Silibinin Ameliorates Formaldehyde-Induced Cognitive Impairment by Inhibiting Oxidative Stress.

Silibinin is a flavonoid extracted from the medicinal plant Silybum marianum (milk thistle), traditionally used to treat liver disease. Recent studies have shown that the antioxidative stress and anti-inflammatory effects of milk thistle are used in the treatment of neurological diseases. Silibinin has antioxidative stress and antiapoptotic effects and reduces cognitive impairment in models of Alzheimer's disease (AD). However, the underlying mechanism of silibinin related to improvement of cognition remains poorly understood. In this study, we used the model of lateral ventricle injection of formaldehyde to examine the related mechanism of silibinin in improving cognitive impairment disorders. Oral administration of silibinin for three weeks significantly attenuated the cognitive deficits of formaldehyde-induced mice in a Y-maze test and Morris water maze test. Y-maze results show that silibinin increases the rate of spontaneous response alternation in FA-induced mice. Silibinin increases the target quadrant spending time and decreases escape latency in the Morris water maze test. We examined the effect of silibinin on the NRF2 signaling pathway, and silibinin promoted the nuclear transfer of NRF2 and increased the expression of HO-1 but did not significantly increase the protein expression of NRF2 in the hippocampus. Well, silibinin reduces the content of DHE and decreases the levels of apoptosis of mature neuron cells. We investigated the effect of silibinin on the content of formaldehyde degrading enzymes; biochemical analyses revealed that silibinin increased GSH and ALDH2 in formaldehyde-induced mice. In addition, as one of the pathological changes of AD, TAU protein is also hyperphosphorylated in FA model mice. Silibinin inhibits the expression of GSK-3β in model mice, thereby reducing the phosphorylation of TAU proteins ser396 and ser404 mediated by GSK3β. Based on our findings, we verified that the mechanism of silibinin improving cognitive impairment may be antioxidative stress, and silibinin is one of the potentially promising drugs to prevent formaldehyde-induced cognitive impairment.