Protein Expression Levels Of Notch1 Research Articles

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

Tet1/DLL3/Notch1 signal pathway affects hippocampal neurogenesis and regulates depression-like behaviour in mice

Ten-eleven translocation protein 1 (Tet1) is associated with the regulation of depression-like behaviour in mice. However, the mechanism by which Tet1 affects neurogenesis in mice to regulate depression-like behaviours remains unclear. In this study, the chronic social defeat stress (CSDS) paradigm was constructed by overexpressing Tet1 protein in the mouse hippocampus, and 5-ethynyl-2′-deoxyuridine (EdU, 50 mg/kg) was injected on the seventh day to explore the mechanism of the regulation of the Tet1/Delta-like protein 3 (DLL3)/Notch1 protein pathway in mice hippocampal neurogenesis and its influence on depression-like behaviour. Following CSDS, the expression level of Tet1 decreased significantly. Moreover, due to the downregulation of Tet1 protein, the maintenance of the DNA methylation and demethylation balance was affected, resulting in a significant increase in the methylation levels of Notch1 and DLL3 and a significant decrease in the protein expression levels of DLL3, Notch1, and brain-derived neurotrophic factor (BDNF). At the same time, the proliferation and differentiation of neurones were affected, which was related to a significant decrease in the number of EdU+, doublecortin (DCX)+, and Ki67+ cells in the hippocampus of the CSDS model mice. When the Tet1 protein was overexpressed in the mouse hippocampus, DLL3 and Notch1 protein expression levels were upregulated, promoting hippocampal neurogenesis and alleviating depression-like behaviour in mice. These findings suggest that regulation of the hippocampal Tet1/DLL3/Notch1 protein pathway to influence neurogenesis may be a therapeutic strategy for depression.

Therapeutic effect and mechanism of Yougui Wan in rats with intervertebral disk degeneration

ObjectiveTo explore the potential mechanism of Yougui Wan on deformed lumbar intervertebral disk structure in rats.MethodsThirty male Sprague–Dawley rats were randomly divided into 3 groups, with 10 rats in each group. The animals in the blank control group were healthy rats without specific treatment, and those in the model group and traditional Chinese medicine (TCM) group were used to establish the intervertebral disk degeneration (IDD) model by puncturing the annulus. Four weeks after modeling, rats in the TCM group were administered Yougui Wan by gavage for 2 consecutive weeks. Serum interleukin-6 (IL-10), macrophage migration inhibitory factor (MIF) and tumor necrosis factor alpha (TNF-α) levels were measured by ELISA, and the protein expression levels of collagen II and Notch1 in intervertebral disk tissues were examined by Western blotting. Apoptosis was detected by the TUNEL method.ResultsCompared with those in the blank group, IL-10, MIF and TNF-α levels in the model group and TCM group were increased (P < 0.05), the protein expression levels of collagen II were decreased, and the protein expression levels of Notch1 were increased. Compared with those in the model group, the levels of IL-10 in the TCM group were increased (P < 0.05), the levels of MIF and TNF-α were decreased (P < 0.05), the protein expression levels of collagen II were increased, and the protein expression levels of Notch1 were decreased.ConclusionYougui Wan can inhibit the inflammatory response in IDD rats, reduce the degradation of extracellular matrix, reduce apoptosis in nucleus pulposus cells, and alleviate intervertebral disk degeneration. The mechanism may be related to the regulation of the Notch signaling pathway.

Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification

To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification. Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins. The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01). Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.

Effects of 5-Aza on neurogenesis contribute to learning and memory in the mouse hippocampus

Background5-Aza-2′-deoxycytidine (5-Aza-CdR) is a demethylating agent that has various biological effects related to DNA methylation. DNA methylation plays important roles in learning and memory. We have reported that 5-Aza-CdR improved the performance of mice in the water maze and step-down tests. Some behaviours have been well recognized to be mediated by neurogenesis in the hippocampus. The Notch signalling pathway plays a key role in adult hippocampal neurogenesis. In this study, we examined whether 5-Aza-CdR (DNA methyltransferase inhibitor) affects neurogenesis and Notch1 expression. MethodsThe learning and memory behaviour of mice was evaluated by a conditioned avoidance learning 24 h after 5-Aza-CdR treatment. The mRNA and protein expression levels of Notch1 and HES1 were measured by real-time PCR and Western blotting. The 5-bromo-2′-deoxyuridine (BrdU)-positive cells and the expression of Notch1 in the hippocampal DG were observed through laser confocal microscopy. To further clarify whether 5-Aza-CdR affects behaviour through neurogenesis, the expression level of Notch1, cell viability and cell cycle were analysed using the HT22 cell line. ResultsThe behaviour in conditioned avoidance learning was improved, while neurogenesis and the Notch1 pathway were increased in the hippocampus of mice that were injected with 5-Aza-CdR. In vitro experiments showed that 5-Aza-CdR increased the expression of the Notch1 pathway and upregulated S-phase in the cell cycle and cell viability. ConclusionsOur results suggest that the effect of 5-Aza-CdR on behaviour may be related to an increase in neurogenesis with upregulation of the Notch1 pathway in the hippocampus.

Notch1/Hes1‑PTEN/AKT/IL‑17A feedback loop regulates Th17 cell differentiation in mouse psoriasis‑like skin inflammation.

IL-17A, the effector cytokine of T helper (Th) 17 cells, plays a crucial role in the pathogenesis of psoriasis. The Notch1 and PI3K/AKT signaling pathways are implicated in Th17 cell differentiation and IL-17A production. The present study aimed to evaluate the regulatory effect of the Notch1/hairy and enhancer of split 1 (Hes1)-PTEN/AKT/IL-17A feedback loop on Th17 cell differentiation via the PI3K/AKT inhibitor LY294002 in a mouse model of psoriasis. Mice were randomly divided into 3 groups: a control group, a model group [5% imiquimod (IMQ)-induced group] and an intervention group (5% IMQ-induced plus LY294002-treated group). Skin structural characteristics were recorded and evaluated by hematoxylin and eosin staining. The weights of the spleens and inguinal lymph nodes were measured. Th17 cell percentage, as well as the mRNA and protein expression levels of Notch1, Notch1 intracellular domain (NICD1), Hes1, PTEN, AKT, phosphorylated (p)-AKT, mTOR complex 1 (mTORC1), p-mTORC1, S6 kinase (S6K)1, S6K2 and IL-17A were detected in skin samples of the three experimental groups. Additionally, splenic mononuclear cells from model mice were treated by 10 and 50 µM LY294002 to further evaluate its regulatory effect on Notch1/Hes1-PTEN/AKT/IL-17A feedback loop. Increased Th17 cell percentage, increased expression of Notch1, NICD1, Hes1, AKT, p-AKT, mTORC1, p-mTORC1, S6K1, S6K2 and IL-17A, and decreased PTEN levels were observed in model mice alongside marked psoriasis-like skin inflammation, splenomegaly and lymphadenopathy. LY294002 treatment significantly alleviated the severity of psoriasis-like skin inflammation in the intervention mice, attenuated the degree of epidermal hyperplasia and dermal inflammatory cell infiltration, and mitigated splenomegaly and lymphadenopathy. In addition, LY294002 treatment reversed the increased Th17 cell percentage, as well as the increased expression of Notch1, NICD1, Hes1, AKT, p-AKT, mTORC1, p-mTORC1, S6K1, S6K2 and IL-17A, and the decreased expression of PTEN. In vitro study from 5% IMQ-induced mouse splenic mononuclear cells presented that high dose of LY294002 exerted more obviously regulatory effect on Notch1/Hes1-PTEN/AKT/IL-17A feedback loop. The current findings suggested that the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop regulates Th17 cell differentiation within the disease environment of psoriasis. Blocking the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop may thus be a potential therapeutic method for management of psoriatic inflammation.

Salvia miltiorrhiza-asarum ointment combined with Chinese medical massage alleviates symptoms of osteoarthritis in a rat model through the Notch1/ matrix metalloproteinase-13 signaling pathway.

This study investigated the effect of salvia miltiorrhiza-asarum ointment (SMAO) plus Chinese medical massage on knee osteoarthritis in a rat model. Hulth's method was used to establish a Sprague-Dawley rat model of knee osteoarthritis (OA). The levels of matrix metalloproteinase-13 (MMP-13), collagen-II, aggrecan, interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6 were measured by enzyme-linked immunosorbent assays. The joint space was assessed by a Perlove X-ray system. Histopathology was examined by hematoxylin-eosin and Safranin O staining. The mRNA and protein expression levels of Notch1, MMP-13, collagen-II, and aggrecan were measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. SMAO plus Chinese medical massage significantly decreased the levels of MMP-13, IL-1β, TNF-α, and IL-6, and increased serum collagen-II and aggrecan levels. Pathological injury of the knee joint was improved by SMAO treatment. mRNA and protein expression of Notch1 and MMP-13 was remarkably downregulated, but collagen-II and aggrecan levels were significantly upregulated in cartilage tissues. SMAO combined with Chinese medical massage effectively relieves OA symptoms, which may involve inhibiting inflammation through the Notch1/MMP-13 signaling pathway.

Effects and mechanisms of polycaprolactone-cellulose acetate nanofiber scaffold loaded with rat epidermal stem cells on wound healing of full-thickness skin defects in rats

Objective: To explore the effects and mechanisms of polycaprolactone-cellulose acetate (PCL-CA) nanofiber scaffold loaded with rat epidermal stem cells (ESCs) on wound healing of full-thickness skin defects in rats. Methods: The experiment research method was applied. The primary ESCs were isolated from 1-3 d old Sprague-Dawley (SD) rats (undefined gender) by rapid adherent method and cultured by rapid adherent method. ESCs of the first passage were used for the subsequent experiments after the positive expressions of integrin β1 and cytokeratin 19 (CK19) in primary cells were identified respectively by flow cytometey and immunofluorescence method. PCL-CA nanofiber scaffolds with polycaprolactone and cellulose acetate as components were prepared by electrospinning technique. The topological structure of the nanofiber scaffolds was determined and the diameter of 25 fibers was measured by scanning electron microscope. The constructed PCL-CA nanofiber scaffolds were used as the culture substrate for ESCs, which were cultured in keratinocytes (KCs) medium to construct ESCs-nanofiber scaffold complex (hereinafter referred to as ESCs scaffold). After 3 days of culture, the morphology of ESCs in the scaffold and their relationship was observed by scanning electron microscopy. The ESCs in ESCs scaffold were set as PCL-CA nanofiber scaffold group, and the ESCs cultured with KCs medium in culture dishes coated with type Ⅳ collagen were set as type Ⅳ collagen group. Western blotting was used to detect the protein expression level of CK19 in ESCs in the two groups after 3 days of culture (n=3). The protein expressions of CK19 and proliferating nuclear antigen (PCNA) in ESCs in the two groups were detected by immunofluorescence method after 7 days of culture. A circular full-thickness skin wound of about 2 cm in diameter was prepared on both left and right sides of the back of 15 male SD rats aged 6-8 weeks. The rats were then equally divided into blank control group without implantation, scaffold alone group implanted with PCL-CA nanofiber scaffold, and ESCs scaffold group implanted with ESCs scaffold which were constructed after 3 days of culture according to the random number table. The percentage of wound areas on post injury day (PID) 3, 7, 14, and 21 was calculated (n=5). The new skin tissue at the wound edge was collected on PID 21, the wound healing quality was evaluated by Masson staining, and the protein expression levels of Notch1, Jagged1, and Hes1, which are key proteins of Notch signaling pathway, were detected by Western blotting (n=3). Data were statistically analyzed with one-way analysis of variance, one-way analysis of variance, analysis of variance for repeated measurement, independent sample t test, and Bonferroni correction. Results: The constructed PCL-CA nanofiber scaffolds had a porous, mesh-like, and multilayered three-dimensional structure, in which the surface of the fibers was smooth and non-porous, and the fiber diameter was (383±24) nm. The ESCs in ESCs scaffold showed intact cellular structures and were tightly attached to the scaffold after 3 days of culture. The cells were interconnected and fully extended on the surface of the scaffold to form a membrane. After 3 days of culture, the protein expression level of CK19 of ESCs in PCL-CA nanofiber scaffold group was significantly higher than that in type Ⅳ collagen group (t=24.56, P 0.05). On PID 21, the layer of new skin at the wound edge of rats in each group was intact; compared with that in blank control group or scaffold alone group, the new skin tissue at the wound edge of rats in ESCs scaffold group had more orderly collagen arrangement; the scaffolds in the new skin at the wound edge of rats were completely degraded in ESCs scaffold group and scaffold alone group. On PID 21, the protein expression levels of Notch1, Jagged1, and Hes1 in the new skin tissue at the wound edge of rats in scaffold alone group were similar to those in blank control group (t=1.70, 1.94, 0.18, P>0.05), while the protein expression levels of Notch1, Jagged1, and Hes1 in the new skin tissue at the wound edge of rats in ESCs scaffold group were significantly higher than those in scaffold alone group (t=13.31, 22.07, 20.71, P<0.01). Conclusions: PCL-CA nanofiber scaffolds can inhibit the differentiation of ESCs of rats without affecting their proliferation in vitro. ESCs scaffolds constructed through using PCL-CA nanofiber scaffolds as the carrier to culture ESCs of rats can significantly promote the wound healing of full-thickness skin defects in rats, and the mechanism may be related to the activation of Notch signaling pathway.

Folic Acid-Functionalized Nanomedicine: Folic Acid Conjugated Copolymer and Folate Receptor Interactions Disrupt Receptor Functionality Resulting in Dual Therapeutic Anti-Cancer Potential in Breast and Prostate Cancer

We have previously reported on a functionalized folic acid (FA) conjugated poly(styrene-alt-maleic anhydride) (SMA) via biological linker 2,4-diaminobutyric acid (DABA) (FA-DABA-SMA) copolymer. This biocompatible nanocopolymer self-assembles in a pH-dependent manner, providing stimuli responsiveness, active targeting, and extended release of hydrophobic chemotherapeutic agents and effectively penetrates the inner core of 3-dimensional cancer spheroid models. The empty FA-DABA-SMA decreased tumor spheroid volume, revealing a previously unknown mechanism of action. Here, we investigated the potential mechanism of the small (20 kDa) and large (350 kDa) FA-DABA-SMA empty copolymers affecting the folic acid receptor alpha (FRα) signaling properties in breast and prostate cancer cell lines. Microscopic imaging, immunocytochemistry, flow cytometry, Caspase 3/7 apoptosis assays, Incucyte live cell tracking, the scratch wound assay, the water-soluble tetrazolium salt-1 (WST-1) cell viability assay, morphologic changes, and Western blot for the expression levels of FRα on the cell surface were used on MDA MB-231 and MCF-7 breast and DU-145 prostate cancer cell lines. The findings indicate that FA-DABA-SMA increases FRα expression levels in breast MDA MB-231 cancer cells and then disrupts FR signaling by reducing HES1 and NOTCH1 protein expression levels. Also, FA-DABA-SMA induces apoptosis and further causes a change in the MDA MB-231 cells' morphology and significantly reduces their ability to migrate in a scratch wound assay. Collectively, these findings provide a novel insight into the functionalized FA-DABA-SMA copolymer. The 350 and 20 kDa copolymers actively target FRα to initialize internationalization. However, only the large size and sheet-shaped 350 kDa copolymers disrupt FRα signaling. The significance of these novel findings reveals that the copolymer's intracellular activity is critically dependent on the size and structural shape. This report offers novel therapeutic insight into a dual mechanism of the FA-DABA-SMA copolymer for its therapeutic potential to treat cancer.

SNORA72 Activates the Notch1/c-Myc Pathway to Promote Stemness Transformation of Ovarian Cancer Cells.

Cancer stem cells (CSCs) are responsible for the migration and recurrence of cancer progression. Small nucleolar RNAs (snoRNAs) play important roles in tumor development. However, how snoRNAs contribute to the regulation of the stemness of ovarian CSCs (OCSCs) remains unclear. In the present study, we found that SNORA72 was significantly upregulated in OVCAR-3 spheroids (OS) and CAOV-3 spheroids (CS) with the OCSC characteristics attained by serum-free culture in a suspension of OVCAR-3 (OV) and CAOV-3 (CA) cells. The overexpression of SNORA72 increased self-renewal abilities and migration abilities in OV and CA cells and upregulated the expressions of the stemness markers Nanog, Oct4, and CD133. In addition, the ectopic expression of SNORA72 can elevate the messenger RNA (mRNA) and protein expression levels of Notch1 and c-Myc in parental cells. The opposite results were observed in SNORA72-silenced OCSCs. Moreover, we found that Notch1 knockdown inversed the migration abilities and self-renewal abilities raised by overexpressing SNORA72. In summary, stemness transformation of ovarian cancer cells can be activated by SNORA72 through the Notch1/c-Myc pathway. This study introduces a novel therapeutic strategy for improving the treatment efficiency of ovarian cancer.

Effects of Notch signaling components from breast cancer cells treated in culture with resveratrol

Resveratrol (Res) has an anti-tumor effect. Notch signaling components from breast cancer cells treated in culture with Resveratrol was investigated. MDA-MB-231cells were divided into control group (Res-untreated) and Res-treated groups including six concentrations 0 μM, 10 μM, 20 μM, 40 μM, 80 μM and 160 μM. Cytotoxicity test were evaluated by CCK-8. The mRNA and Protein expression levels of Notch1, Jagged1, Dll4 and Hes-5 were detected by RT-PCR and Western blot. The mRNA expression of Notch1, Jagged1, Hes-5 and Dll4 in the Res administration group decreased significantly (0.01 < p < .05), and Hes-5 and Dll4 were extremely significant (p < .01). Compared with the blank control group, the protein expression of Notch1 and Dll4 decreased significantly in each concentration Res group, but the decrease of protein expression of Jagged1 and Hes-5 was not significant. In conclusion, Res regulates mRNA and protein expression of Notch1, Dll4 of MDA-MB-231 cells via Notch pathway.

Mechanism of Notch1‑saRNA‑1480 reversing androgen sensitivity in human metastatic castration‑resistant prostate cancer.

The aim of the present study was to explore the mechanism by which Notch1-small activating (sa)RNA restored androgen sensitivity in human metastatic castration-resistant prostate cancer (CRPC). After transfection of Notch1-saRNA-1480 in PC3 cells, the expression of Notch1 and androgen receptor (AR) was investigated by reverse transcription quantitative PCR (RT-qPCR) and western blotting. Furthermore, the protein expression level of vascular endothelial growth factor (VEGF) was measured. Then, flow cytometry was used to analyze the cell cycle and apoptosis after transfection. Moreover, the migration and invasion ability of PC3 cells were assessed by transwell assays. Then, angio-genesis experiments were conducted to analyze the abilities of PC3 cells to form blood vessels. Furthermore, in vivo experiments detected the antitumor activity of Notch1-saRNA-1480. The mRNA and protein expression levels of Notch1 were significantly increased after transfection, while the expression levels of AR and VEGF were decreased. After transfection, the cell cycle was arrested at the G0/G1 checkpoint. Notch1-saRNA-1480 significantly increased the proportion of apoptotic cells after transfection. In addition, transwell assay results showed that PC3 cell migration and invasion were inhibited. The total vessel length was significantly decreased based on angiogenesis experiments, which indicated that PC3 cell angiogenesis was inhibited. In vivo experiments showed that Notch1-saRNA-1480 could inhibit tumor growth and volume. The protein expression of Notch1, AR, VEGF receptor 2 (VEGFR2) and VEGF in tumor tissues was consistent with in vitro levels. Notch1-saRNA-1480 could significantly inhibit the proliferation of PC3 cells in vitro and the growth of tumors in vivo, which is associated with the inhibition of the AR and VEGF pathways.

Effect of atorvastatin on pulmonary hypertension rats through regulating notch signaling pathway.

To study the effect of atorvastatin on pulmonary hypertension (PAH) rats through regulating the Notch signaling pathway. The rat model of PAH was established via hypoxic feeding and the Control group (n=10), PAH model group (Model group, n=10) and atorvastatin treatment group (ATO group, n=10) were set up. The right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) in each group were determined, the wet/dry weight (W/D) ratio of lung tissues was determined, and the tumor necrosis factor-α (TNF-α), myeloperoxidase (MPO) and interleukin-6 (IL-6) were detected via enzyme-linked immunosorbent assay (ELISA). Moreover, the pathological changes in lung tissues of rats were detected via hematoxylin-eosin (HE) staining and the apoptosis level was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Finally, the Notch signaling pathway and apoptosis level in tissues were detected via quantitative Polymerase Chain Reaction (qPCR), and the protein expression level of Notch pathway in lung tissues was determined through Western blotting. Both RVSP and RVHI in Model group were significantly higher than those in Control group and ATO group (p<0.05). In Model group, the levels of inflammatory factors MPO, IL-6, and TNF-α were significantly increased, and the W/D ratio was also significantly increased compared with those in Control group (p<0.05). The results of HE staining showed that the lung tissue injury in Model group was severe (p<0.05). According to the TUNEL staining results, the number of apoptotic cells in lung tissues was markedly larger in Model group than that in ATO group (p<0.05), and the expression levels of Caspase-3 and IL-6 in Model group were remarkably higher than those in ATO group (p<0.05), while the expression level of B-cell lymphoma-2 (Bcl-2) in Model group was remarkably lower than that in ATO group (p<0.05). Besides, the gene and protein expression levels of Notch1 in ATO group were evidently lower than those in Model group (p<0.05), indicating that atorvastatin can effectively suppress the expression of Notch. Atorvastatin can inhibit PAH in rats by suppressing the Notch pathway.

Bufei Qingyu Granules Inhibit the Development of Systemic Sclerosis via Notch-1/Jagged-2 Signaling Pathway

Systemic sclerosis (SSc) is a rare chronic autoimmune disorder, mainly characterized by skin sclerosis. In this study, Bufei Qingyu Granules (BQG), a Chinese herbal formula, was used to treat SSc. To better understand the effects and molecular mechanisms of BQG, we successfully established a Bleomycin- (BLM-) induced SSc mouse model, and the mice were treated by BQG. Meanwhile, transcriptomic and bioinformatics analyses were conducted on those samples. As a result, we visually showed that BQG ameliorated the overall health of mice, including body weight, spleen, and thymus index. Thus, it also significantly alleviated inflammation presented by Chemokine (C-X-C motif) ligand 2 (Cxcl2), vasculopathy characterized by α-smooth muscle actin (α-SMA), and fibrotic changes elaborated by not only pathological images, but also the hydroxyproline (HYP) content. After testing by transcriptomic analysis, Cxcl2, Synaptosomal-associated protein 25 (Snap25), and Eukaryotic translation initiation factor 3, and subunit J2 (Eif3j2) which were differentially expressed genes, were verified, so that the data were credible. We further found that BQG could regulate Notch signaling pathway by significantly decreasing both mRNA and protein expression levels of Notch-1 and Jagged-2. Hence, this study demonstrated that BQG could ameliorate the sclerotic skin in mice model involved in inflammation, vascular changes, and fibrosis effects, which was partly mediated by Notch signaling pathway.

MiR-146 regulates the repair and regeneration of intervertebral nucleus pulposus cells via Notch1 pathway.

The aim of this study was to investigate the effects of regulation of micro-ribonucleic acid (miR)-146 expression targeting the Notch1 pathway on the repair and regeneration of intervertebral nucleus pulposus cells, and to explore the possible underlying mechanism. Intervertebral nucleus pulposus cells were harvested from rats and cultured in vitro. All cells were randomly divided into 4 groups, including the normal group, the miR-146 inhibitor group, the miR-146 over-expression group and the negative control group. The cells in the normal group were cultured normally. Meanwhile, the cells in the miR-146 inhibitor group and miR-146 over-expression group were transfected with inhibitor-containing plasmid and miR-146-containing plasmid, respectively. However, the cells in the negative control group were transfected with empty plasmid. 24 h after transfection, the cells were collected for subsequent experiments. The differentiation of nucleus pulposus cells was detected via toluidine blue staining. The relative protein expression levels of Notch1, aggrecan (ACAN) and COL II were detected via Western blotting. Meanwhile, the mRNA expressions of miR-146 and Notch1 were detected via quantitative Polymerase Chain Reaction (qPCR). Furthermore, the apoptosis and proliferation of cells were detected via terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) and Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay, respectively. Compared with the normal group, toluidine blue positive staining was significantly increased in miR-146 overexpression group (p<0.05), whereas was significantly decreased in the miR-146 inhibitor group (p<0.05). The results of Western blotting revealed that compared with normal group, the protein expression of Notch1 was markedly decreased in miR-146 over-expression group (p<0.05), whereas the expression levels of ACAN and COL II were notably increased (p<0.05). However, the miR-146 inhibitor group exhibited significantly increased the protein expression level of Notch1 (p<0.05), as well as markedly decreased the expressions of ACAN and COL II (p<0.05). The results of qPCR showed that compared with the normal group, the expression level of miR-146 was significantly increased in the miR-146 over-expression group. However, the mRNA expression level of Notch1 was remarkably decreased (p<0.05). Similarly, the miR-146 inhibitor group exhibited significantly decreased expression level of miR-146, as well as markedly increased mRNA expression level of Notch1 (p<0.05). Compared with those in the normal group, the cell proliferation rate was markedly increased, whereas cell apoptosis was remarkably decreased (p<0.05) in the miR-146 over-expression group. Furthermore, the cell proliferation rate was significantly decreased, while the cell apoptosis was remarkably increased (p<0.05) in the miR-146 inhibitor group. Regulating miR-146 expression can target the Notch1 signaling pathway, thereby exerting important influences on the repair and regeneration of intervertebral nucleus pulposus cells.

Folic acid deficiency enhanced microglial immune response via the Notch1/nuclear factor kappa B p65 pathway in hippocampus following rat brain I/R injury and BV2 cells.

Recent studies revealed that folic acid deficiency (FD) increased the likelihood of stroke and aggravated brain injury after focal cerebral ischaemia. The microglia‐mediated inflammatory response plays a crucial role in the complicated pathologies that lead to ischaemic brain injury. However, whether FD is involved in the activation of microglia and the neuroinflammation after experimental stroke and the underlying mechanism is still unclear. The aim of the present study was to assess whether FD modulates the Notch1/nuclear factor kappa B (NF‐κB) pathway and enhances microglial immune response in a rat middle cerebral artery occlusion‐reperfusion (MCAO) model and oxygen‐glucose deprivation (OGD)‐treated BV‐2 cells. Our results exhibited that FD worsened neuronal cell death and exaggerated microglia activation in the hippocampal CA1, CA3 and Dentate gyrus (DG) subregions after cerebral ischaemia/reperfusion. The hippocampal CA1 region was more sensitive to ischaemic injury and FD treatment. The protein expressions of proinflammatory cytokines such as tumour necrosis factor‐α, interleukin‐1β and interleukin‐6 were also augmented by FD treatment in microglial cells of the post‐ischaemic hippocampus and in vitro OGD‐stressed microglia model. Moreover, FD not only dramatically enhanced the protein expression levels of Notch1 and NF‐κB p65 but also promoted the phosphorylation of pIkBα and the nuclear translocation of NF‐κB p65. Blocking of Notch1 with N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester partly attenuated the nuclear translocation of NF‐κB p65 and the protein expression of neuroinflammatory cytokines in FD‐treated hypoxic BV‐2 microglia. These results suggested that Notch1/NF‐κB p65 pathway‐mediated microglial immune response may be a molecular mechanism underlying cerebral ischaemia‐reperfusion injury worsened by FD treatment.

Roles of notch signaling pathway and endothelial-mesenchymal transition in vascular endothelial dysfunction and atherosclerosis.

To investigate the role of the Notch signaling pathway on the endothelial-mesenchymal transition (EndMT) during vascular endothelial dysfunction and atherosclerosis. Human coronary artery endothelial cells (HCAEC) were treated with the exogenous Notch homolog 1 (Notch1) factor to activate the Notch1 pathway, and cells were then observed under the microscope for morphologic changes. Changes in the expression of related proteins were detected by Western blot. In vivo experiments were performed using 18 Sprague Dawley® (SD) rats, and GSI factor was used to specifically inhibit Notch pathway activation. Rats were used and randomly divided into three groups: normal diet (ND) group, high-fat diet (HFD) group, and high-fat diet + GSI (HFD+GSI) group, 6 rats in each group. Hematoxylin and eosin (H&E) staining was used to examine the cardiac aortic morphology of the rats in each treatment group. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of Notch1, Hes1, VE-cadherin and α-SMA in the aortic tissues of rats in each group at mRNA and protein levels, respectively. After HCAECs were treated with Notch1, endothelial protein levels of VE-cadherin were significantly decreased and levels of the interstitial protein α-SMA were significantly increased. In the animal model, the rats fed with high-fat diet for two months presented obvious atherosclerosis spots in their aorta, but those fed with the same diet and treated with GSI inhibitor of Notch pathway showed significantly fewer atherosclerosis signs. Compared with ND group, mRNA and protein expression levels of Notch1, Hes1 and α-SMA were significantly increased, and the expression levels of endothelial marker VE-cadherin were significantly decreased in aortas of rats in HFD group. Compared with the rats in HFD group, the rats in HFD+GSI group showed significantly reduced expression levels of Notch1, Hes1 and α-SMA. The activation of Notch signaling pathway can induce the EndMT progression and promote the development of atherosclerotic lesions.

MicroRNA‑455 inhibits cell proliferation and invasion of epithelial ovarian cancer by directly targeting Notch1.

Ovarian cancer (OC) is the fifth leading cause of cancer‑associated mortality among women in developed countries. Numerous microRNAs are aberrantly expressed in epithelial (E)OC, and are involved in EOC formation and progression. As such, microRNAs may be investigated as diagnostic and prognostic molecular biomarkers, and therapeutic targets for patients with EOC. For instance, microRNA‑455 (miR‑455) is abnormally expressed in various types of human cancer. However, details on the expression level, biological roles and underlying mechanism of miR‑455 in EOC remain unclear. In the present study, the expression of miR‑455 in EOC was detected, and its association with clinical characteristics was analysed. The functional roles and underlying mechanisms of miR‑455 in EOC were also investigated. The results revealed that miR‑455 expression in EOC tissues and cell lines was significantly downregulated. Furthermore, miR‑455 downregulation was correlated with tumor size, International Federation of Gynecology and Obstetrics stage and lymph node metastasis of patients with EOC. Restoration of miR‑455 expression efficiently inhibited cell proliferation and invasion invitro. Notch1 was identified as a direct target of miR‑455 in EOC. The mRNA and protein expression levels of Notch1 were higher in EOC tissues compared with in normal ovarian epithelial tissues. Spearman's correlation analysis indicated that miR‑455 expression was negatively correlated with the mRNA level of Notch1 in EOC tissues. Notch1 overexpression was able to restore the EOC cell proliferation and invasion abilities suppressed by miR‑455. The present study provided evidence that the dysregulation of the miR‑455/Notch1 signalling pathway may be essential for EOC occurrence and development. In addition, the results confirmed the tumor‑suppressive roles of miR‑455 in modulating EOC proliferation and invasion through regulation of Notch1 expression. Thus, miR‑455 may be a novel miRNA‑based therapeutic target to treat patients with EOC.

Expression levels and mechanism of microRNA-34a and Notch1 signaling pathway in hippocampi of temporal lobe epilepsy rats

Objective To explore the expression levels of microRNA(miR)-34a in hippocampus of temporal lobe epilepsy rats and the effect of miR-34a on its target signaling pathway Notch1. Methods Rats were divided randomly into experiment group (n=40) and control group (n=40) by adopting random number table method.The status epilepticus model and the temporal lobe epilepsy model were induced by using lithium-pilocarpine for experiment group.The control group rats received an injection of an equal amount of 9 g/L saline as instead of pilocarpine.Racine grading was performed at 24 hours, day 3, day 7, day 15, and 1 month after modeling to evaluate the behavior.Real-time fluorescent quantitative polymerase chain reaction was used to test the mRNA expressions of miR-34a and Notch1.Western blot was performed to explore the protein expression of Notch1. Results The expression levels of miR-34a at post-status epilepticus in 24 hours, day 3, day 7, day 15 were 2.55±0.29, 2.11±0.17, 1.68±0.49 and 1.84±0.42, respectively, which showed statistically significant difference compared with the control group (1.00±0.00) (t=-1.55, -1.11, -0.68, -0.84, all P<0.05). The expression levels of Notch1 mRNA at post-status epilepticus in 24 hours, day 3, day 7, day 15, 1 month were 1.44±0.31, 1.27±0.13, 1.52±0.28, 0.91±0.33, and 0.80±0.09 respectively.There were significant differences at 24 hours, day 7 in Notch1 mRNA expression (t=-0.44, -0.52, all P<0.05) compared with the control group(1.00±0.00). The expression level of Notch1 mRNA on day 15 was significantly lower than 24 hours and day 7 (t=-0.54, -0.62, all P<0.05), and the expression in 1 month was significantly lower than in 24 hours, or day 3 and day 7 (t=-0.64, -0.46, -0.72, all P<0.05). The expression levels of Notch1 protein at post-status epilepticus 24 hours, day 3, day 7, day 15, 1 month were 0.78±0.09, 0.57±0.13, 0.55±0.16, 0.42±0.13, and 0.33±0.09, respectively.There was significantly up-regulated at 24 hours of Notch1 protein expression compared with control group (0.51±0.15)(t=-0.20, P<0.05); and the expression level at day 15 were significantly lower than 24 hours (t=-0.26, P<0.05), while the expression in 1 month was significantly lower than in 24 hours and on day 3 (t=-0.36, -0.24, all P<0.05). Conclusion miR-34a is significantly up-regulated in the post-status epilepticus rat hippocampus, and it may contribute to temporal lobe epilepsy by activating Notch1 signaling pathway. Key words: microRNA-34a; Temporal lobe epilepsy; Notch1

A combination of valproic acid sodium salt, CHIR99021, E-616452, tranylcypromine, and 3-Deazaneplanocin A causes stem cell-like characteristics in cancer cells.

Many studies are based on the hypothesis that recurrence and drug resistance in lung carcinoma are due to a subpopulation of cancer stem-like cells (CSLCs) in solid tumors. Therefore it is crucial to screen for and recognize lung CSLCs. In this study, we stimulated non-small cell lung cancer (NSCLC) A549 cells to display stem cell-like characteristics using a combination of five small molecule compounds. The putative A549 stem cells activated an important CSLC marker, CD133 protein, as well multiple CSLC-related genes including ATP-binding cassette transporter G2 (ABCG2), C-X-C chemokine receptor type 4 (CXCR4), NESTIN, and BMI1. The A549 stem-like cells displayed resistance to the chemotherapeutic drugs etoposide and cisplatin, epithelial-to-mesenchymal transition properties, and increased protein expression levels of NOTCH1 and Hes Family bHLH Transcription Factor 1 (HES1). When A549 cells were pretreated with a NOTCH signaling pathway inhibitor before compound induction, expression of the NOTCH1 target gene HES1 was reduced. This demonstrated that the NOTCH signaling pathway in the putative A549 stem-like cells had been activated. Together, the results of our study showed that a combination of five small molecule agents could transform A549 cells into putative stem-like cells, and that these compounds could also elevate CD133 and ABCG2 protein expression levels in H460 cells. This study provides a convenient method for obtaining lung CSLCs, which may be an effective strategy for developing lung carcinoma treatments.