Inhibition of Notch 1 signaling in the subacute stage after stroke promotes striatal astrocyte-derived neurogenesis

Inhibition of Notch1 signaling has been shown to promote astrocyte-derived neurogenesis after stroke.To investigate the regulatory role of Notch1 signaling in this process,in this study,we used a rat model of stroke based on middle cerebral artery occlusion and assessed the behavior of reactive astrocytes post-stroke.We used theγ-secretase inhibitor N-[N-(3,5-diuorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester(DAPT)to block Notch1 signaling at 1,4,and 7 days after injury.Our results showed that only administration of DAPT at 4 days after stroke promoted astrocyte-derived neurogenesis,as manifested by recovery of white matter fiber bundle integrity on magnetic resonance imaging,which is consistent with recovery of neurologic function.These findings suggest that inhibition of Notch1 signaling at the subacute stage post-stroke mediates neural repair by promoting astrocyte-derived neurogenesis.

Buyang Huanwu decoction up-regulates Notch1 gene expression in injured spinal cord

Expression of genes in the Notch signaling pathway is altered in the injured spinal cord, which indicates that Notch participates in repair after spinal cord injury. Buyang Huanwu decoction, a traditional Chinese herbal preparation, can promote the growth of nerve cells and nerve fibers; however, it is unclear whether Buyang Huanwu decoction affects the Notch signaling pathway in injured spinal cord. In this study, a rat model was established by injuring the T10 spinal cord. At 2 days after injury, rats were intragastrically administered 2 m L of 0.8 g/m L Buyang Huanwu decoction daily until sacrifice. Real-time reverse transcription polymerase chain reaction analysis demonstrated that at 7, 14 and 28 days after injury, the expression of Notch1 was increased in the Buyang Huanwu decoction group compared with controls. These findings confirm that Buyang Huanwu decoction can promote the expression of Notch1 in rats with incomplete spinal cord injury, and may indicate a mechanism to promote the repair of spinal cord injury.

Baicalin Antagonizes Prostate Cancer Stemness via Inhibiting Notch1/NF-κB Signaling Pathway

Objective To investigate the molecular mechanisms underlying the effect of baicalin on prostate cancer(PCa)progression both in vivo and in vitro.Methods The in situ PCa stem cells(PCSCs)-injected xenograft tumor models were established in BALB/c nude mice.Tumor volume and weight were respectively checked after baicalin(100 mg/kg)treatment.Hematoxylin-eosin(HE)staining was used to observe the growth arrest and cell necrosis.mRNA expression levels of acetaldehyde dehydrogenase 1(ALDH1),CD44,CD133 and Notch1 were determined by reverse transcription-polymerase chain reaction.Protein expression levels of ALDH1,CD44,CD133,Notch1,nuclear factorκB(NF-κB)P65 and NF-κB p-P65 were detected by Western blot.Expression and subcellular location of ALDH1,CD44,CD133,Notch1 and NF-κB p65 were detected by immunofluorescence analysis.In vitro,cell cycle distribution and cell apoptosis of PC3 PCSCs was assessed by flow cytometry after baicalin(125µmol/L)treatment.The migration and invasion abilities of PCSCs were assessed using Transwell assays.Transmission electron microscopy scanning was utilized to observe the structure and autophagosome formation of baicalin-treated PCSCs.In addition,PCSCs were infected with lentiviruses expressing human Notch1.Results Compared with the control group,the tumor volume and weight were notably reduced in mice treated with 100 mg/kg baicalin(P<0.05 or P<0.01).Histopathological analysis showed that baicalin treatment significantly inhibited cell proliferation and promoted cell apoptosis.Furthermore,baicalin treatment reduced mRNA and protein expression levels of CD44,CD133,ALDH1,and Notch1 as well as the protein expression of NF-κB p-P65 in the xenograft tumor(P<0.01).In vitro,the cell proliferation of PCSCs was significantly attenuated after treatment with 125µmol/L baicalin for 72 h(P<0.01).The cell migration and invasion rates were decreased following treatment with baicalin for 48 and 72 h(P<0.01).Baicalin notably induced cell apoptosis and seriously damaged the structure of PCSCs.The mRNA and protein expressions of CD133,CD44,ALDH1 and Notch1 in PCSCs were significantly downregulated following baicalin treatment(P<0.01).Importantly,the inhibitory effects of baicalin on PCa progression and stemness were reversed by Notch1 overexpression(P<0.05 or P<0.01).Conclusion Mechanistically,baicalin exhibited a potential therapeutic effect on PCa via inhibiting the Notch1/NF-κB signaling pathway and its mediated cancer stemness.

Activated Notch1 reduces myocardial ischemia reperfusion injury in vitro during ischemic postconditioning by crosstalk with the RISK signaling pathway

Background Ischemic postconditioning （IPost）,able to significantly attenuate myocardial ischemia reperfusion injury,is dependent on RISK signaling.Studies have shown that Notch signaling repairs damaged myocardium,and this study aimed to investigate the effect of Notch signaling in myocardial IPost.Methods We used H9c2 cells to establish the myocardial IPost and Hypoxia/Reoxygenation （H/R） model in vitro,which were randomly divided into control,H/R,IPost,Hepatocyte growth factor （HGF）＋IPost and DAPT＋IPost,N1ICD＋IPost,miRNA＋lPost,and Mock treatment groups.The myocardial cell viability was assessed by MTT,the cell apoptosis was detected using Annexin V/PI double staining and flow cytometry analyses.The expression of N1ICD,Hes1,PTEN Phospho-Akt/Akt,Phospho-GSK-3β/GSK-3β were detected by Western blotting.Finally,we assessed the changes in Ψm using the potential-sensitive dye JC-1 and measured using flow cytometry analyses.Results The Notch1 signaling is activated by HGF and ectopic expression of N1ICD during myocardial IPost,which increased myocardial cell viability,prevented cardiomyocyte apoptosis,and reduced loss of the mitochondrial membrane potential.However,myocardial ischemia reperfusion injury was increased in IPost when Notch1 signaling was inhibited using DAPT or with knockdown by Notch1-miRNA.Western blotting found that PTEN was down-regulated by Hes1 when Notch1 was activated,which consequently promoted Akt and GSK-3β phosphorylation.Conclusions Notch1 crosstalk with RISK signaling may be dependent on PTEN,which plays a cardioprotective role during IPost.This mechanism could provide a promising therapeutic target for the treatment of ischemic heart disease.

Brucine Inhibits Bone Metastasis of Breast Cancer Cells by Suppressing Jagged1/Notch1 Signaling Pathways

Objective： To examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand（RANKL）-induced osteoclastogenesis. Methods： The osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL（50 ng/m L） and macrophage-colony stimulating factor（50 ng/m L） were added to this system, followed by treatment with brucine（0.02, 0.04 and 0.08 mmol/L）, or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1（TGF-β1）, nuclear factor-kappa B（NF-κB） and Hes1 were determined by enzyme-linked immunosorbent assay. Results： Compared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells（P 〈0.01）. Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1（P〈0.05 or P〈0.01）. Conclusion： Brucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.

APPL2 Negatively Regulates Olfactory Functions by Switching Fate Commitments of Neural Stem Cells in Adult Olfactory Bulb via Interaction with Notch1 Signaling

Adult olfactory neurogenesis plays critical roles in maintaining olfactory functions.Newly-generated neurons in the subventricular zone migrate to the olfactory bulb(OB) and determine olfactory discrimination,but the mechanisms underlying the regulation of olfactory neurogenesis remain unclear.Our previous study indicated the potential of APPL2(adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 2) as a modulating factor for neurogenesis in the adult olfactory system.In the present study,we report how APPL2 affects neurogenesis in the OB and thereby mediates olfactory discrimination by using both in vitro neural stem cells(NSCs) and an in vivo animal model-APPL2 transgenic(Tg) mice.In the in vitro study,we found that APPL2 overexpression resulted in NSCs switching from neuronal differentiation to gliogenesis while APPL2 knockdown promoted neurogenesis.In the in vivo study,APPL2 Tg mice had a higher population of glial cells and dampened neuronal production in the olfactory system,including the corpus callosum,OB,and rostral migratory stream.Adult APPL2 Tg mice displayed impaired performance in olfactory discrimination tests compared with wild-type mice.Furthermore,we found that an interaction of APPL2 with Notch1 contributed to the roles of APPL2 in modulating the neurogenic lineage-switching and olfactory behaviors.In conclusion,APPL2 controls olfactory discrimination by switching the fate choice of NSCs via interaction with Notch1 signaling.