mRNA Expression Levels Of Notch1 Research Articles

Role of Notch 1 signaling and glycolysis in the pathogenic mechanism of adenomyosis

To investigate the expressions of glycolysis-related factors and changes in Notch1 signaling in endometrial tissues of adenomyosis (AM) and Ishikawa cells to explore the pathogenesis of AM. Eutopic endometrial tissues were collected from 8 patients with AM and 8 patients with uterine fibroids matched for clinical characteristics (control group). The expressions of Notch1 signaling proteins and glycolysis-related factors in the collected tissues were detected using qRT-PCR and Western blotting, and the levels of glucose and lactic acid were determined. An Ishikawa cell model with lentivirus-mediated stable Notch1 overexpression was established for assessing cell survival rate with CCK-8 assay, cell migration and invasion abilities with Transwell migration and invasion assays, and glycolytic capacity by determining the extracellular acidification rate. Compared with those in the control group, the endometrial tissues in AM group showed significantly increased expression level of carbohydrate antigen 125 (CA125), increased mRNA expression levels of Notch1, HK2 and PDHA and protein expressions of Notch1, GLUT1, HK2, PKM and PDHA, lowered glucose level and increased lactate level. The Ishikawa cell models with stable Notch1 overexpression exhibited significantly increased cell survival rate with attenuated cell migration and invasion abilities and decreased glycolysis capacity and reserve. The Notch1 signaling pathway participates in the pathogenesis of AM possibly by regulating the proliferation, migration, invasion and glycolysis of endometrial cells.

Aflatoxin B1 exposure deteriorates immune abnormalities in a BTBR T+ Itpr3tf/J mouse model of autism by increasing inflammatory mediators' production in CD19-expressing cells

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by deficiencies in communication, repetitive and stereotyped behavioral patterns, and difficulties in reciprocal social engagement. The presence of immunological dysfunction in ASD has been well established. Aflatoxin B1 (AFB1) is a prevalent mycotoxin found in food and feed, causing immune toxicity and hepatotoxicity. AFB1 is significantly elevated in several regions around the globe. Existing research indicates that prolonged exposure to AFB1 results in neurological problems. The BTBR T+ Itpr3tf/J (BTBR) mice, which were used as an autism model, exhibit the primary behavioral traits that define ASD, such as repeated, stereotyped behaviors and impaired social interactions. The main objective of this work was to assess the toxic impact of AFB1 in BTBR mice. This work aimed to examine the effects of AFB1 on the expression of Notch-1, IL-6, MCP-1, iNOS, GM-CSF, and NF-κB p65 by CD19+ B cells in the spleen of the BTBR using flow cytometry. We also verified the impact of AFB1 exposure on the mRNA expression levels of Notch-1, IL-6, MCP-1, iNOS, GM-CSF, and NF-κB p65 in the brain of BTBR mice using real-time PCR. The findings of our study showed that the mice treated with AFB1 in the BTBR group exhibited a substantial increase in the presence of CD19+Notch-1+, CD19+IL-6+, CD19+MCP-1+, CD19+iNOS+, CD19+GM-CSF+, and CD19+NF-κB p65+ compared to the mice in the BTBR group that were treated with saline. Our findings also confirmed that administering AFB1 to BTBR mice leads to elevated mRNA expression levels of Notch-1, IL-6, MCP-1, iNOS, GM-CSF, and NF-κB p65 in the brain, in comparison to BTBR mice treated with saline. The data highlight that exposure to AFB1 worsens immunological abnormalities by increasing the expression of inflammatory mediators in BTBR mice.

Increased Expression of NOTCH-1 and T Helper Cell Transcription Factors in Patients with Acquired Aplastic Anemia.

Acquired aplastic anemia is an autoimmune disease in which auto-aggressive T cells destroy hematopoietic progenitors. T-cell differentiation is controlled by transcription factors that interact with NOTCH-1, which influences the respective T-cell lineages. Notch signaling also regulates the BM microenvironment. The present study aimed to assess the gene expressions of NOTCH-1 and T helper cell transcription factors in the acquired aplastic anemia patients. Using quantitative real-time PCR, we studied the mRNA expression level for NOTCH-1, its ligands (DLL-1 and JAG-1), and T helper cell transcription factors (T-BET, GATA-3, and ROR-γt) in both PB and BM of aAA patients and healthy controls. Further, patients of aplastic anemia were stratified by their disease severity as per the standard criteria. The mRNA expression level of NOTCH-1, T-BET, GATA-3, and ROR-γT genes increased in aAA patients compared to healthy controls. There was no significant difference in the mRNA expression of Notch ligands between patients and controls. The mRNA expression level of the above-mentioned genes was found to be higher in SAA and VSAA than NSAA patients. In addition, NOTCH-1 and T helper cell-specific transcription factors enhanced in aAA. We also observed a significant correlation between the genes and hematological parameters in patients. The interaction between NOTCH-1, T-BET, GATA-3, and ROR-γT might lead to the activation, proliferation, and polarization of T helper cells and subsequent BM destruction. The mRNA expression levels of genes varied with disease severity, which may contribute to pathogenesis of aAA.

Tibetan medicine Qi-Sai-Er-Sang-Dang-Song Decoction inhibits TNF-α-induced rheumatoid arthritis in human fibroblast-like synoviocytes via regulating NOTCH1/NF-κB/NLRP3 pathway.

Qi-Sai-Er-Sang-Dang-Song Decoction (QSD, ཆུ་སེར་སེང་ལྡེང་སུམ་ཐང་།), a Tibetan classical herbal formula, is commonly used in Tibetan hospital preparation for the treatment of rheumatoid arthritis (RA). Its efficacy is to relieve inflammation, dispel cold, remove dampness, and alleviate pain. However, its anti-RA mechanism is still unclear. This study aimed to investigate the effect of QSD on rheumatoid arthritis and explore its anti-inflammatory mechanism against human fibroblast-like synoviocytes (HFLSs) by regulating the notch family of receptors (NOTCH1)/Nuclear factor-κB (NF-κB)/nucleotide-binding (NLRP3) pathway. We used ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC-Q-TOF-MS) to identify the chemical composition of QSD. Then, HFLSs were exposed to drug-containing serum. The effect of QSD drug-containing serum on HFLS viability was detected using the cell counting kit-8 (CCK-8) assay. Next, we explored the anti-inflammatory effect of QSD using enzyme-linked immunosorbent assay (ELISA) for inflammatory factors, such as interleukin-18 (IL-18), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The expression of NOTCH-related proteins, a member of the NOTCH1, Cleaved NOTCH1, hairy and enhancer of split-1 (HES-1), NF-κB p65, NF-κB pp65, NLRP3, and delta-like 1 (DLL-1), was examined using western blotting. Furthermore, the relative mRNA expression levels of NOTCH1, NF-κB p65, NLRP3, DLL-1, and HES-1 were detected using real-time quantitative (RT-qPCR). To explore the mechanism underlying the anti-RA effect of QSD, we the used the NOTCH signaling pathway inhibitor LY411575 and transfection with a NOTCH1 siRNA. In addition, we employed immunofluorescence to determine the expression of HES-1 and NF-κB p65 in vitro. Our results revealed that QSD ameliorated inflammation in HFLSs. Compared with the model group, the QSD drug-containing serum group had obviously down-regulated levels of IL-18, IL-1β, and IL-6. Consistently, the CCK-8 results showed that the QSD drug-containing serum had no obvious toxicity towards HFLSs. Moreover, both LY411575 and siNOTCH1, QSD could reduce NOTCH1, NLRP3, and HES-1 protein expression levels, and LY411575 could significantly inhibit the expression levels of NF-κB p65, NF-κB pp65, and Cleaved NOTCH1 (p<0.05). siNOTCH1 could also suppress the expression of DLL-1. The RT-qPCR results indicated that QSD could downregulate the relative mRNA expression levels of NOTCH1, NF-κB p65, NLRP3, DLL-1, and HES-1 in HFLSs (p<0.05). In the immunofluorescence experiment, the fluorescence intensities of HES-1 and NF-κB p65 in HFLSs were found to decrease after exposure to QSD drug-containing serum (p<0.05). Ultimately, 44 chemical components were detected in QSD using UPLC-Q-TOF-MS. This study reveals that the QSD can markedly ameliorate inflammation induced by TNF-α on HFLS. The effect of QSD on HFLS may be exerted by inhibition of the NOTCH1/NF-κB/NLRP3 signaling pathway.

The potential value of Notch1 and DLL1 in the diagnosis and prognosis of patients with active TB.

The Notch signaling pathway has been implicated in the pathogenesis of active tuberculosis (TB), and Th1-type cell-mediated immunity is essential for effective control of mycobacterial infection. However, it remains unclear whether Notch signaling molecules (Notch1, DLL1, and Hes1) and Th1-type factors (T-bet and IFN-γ) can serve as biomarkers for tracking the progression of active TB at different stages along with peripheral blood white blood cell (WBC) parameters. A total of 60 participants were enrolled in the study, including 37 confirmed TB patients (mild (n=17), moderate/severe (n=20)) and 23 healthy controls. The mRNA expression of Notch1, DLL1, Hes1, T-bet and IFN-γ in the peripheral blood mononuclear cells (PBMCs) of the subjects was measured by RT-qPCR, then analyzed for differences. Receiver Operating Characteristic curve (ROC) was used to assess the effectiveness of each factor as a biomarker in identifying lung injury. We found that mRNA expression levels of Notch1, DLL1, and Hes1 were upregulated in active TB patients, with higher levels observed in those with moderate/severe TB than those with mild TB or without TB. In contrast, mRNA levels of T-bet and IFN-γ were downregulated and significantly lower in mild and moderate/severe cases. Furthermore, the combiROC analysis of IFN-γ and the percentage of lymphocytes (L%) among WBC parameters showed superior discriminatory ability compared to other factors for identifying individuals with active TB versus healthy individuals. Notably, Notch pathway molecules were more effective than Th1-type factors and WBC parameters in differentiating mild and moderate/severe cases of active TB, particularly in the combiROC model that included Notch1 and Hes1. Our study demonstrated that Notch1, Hes1, IFN-γ, and L% can be used as biomarkers to identify different stages of active TB patients and to monitor the effectiveness of treatment.

Alteration of N6-methyladenosine epitranscriptome profiles in bilateral ureteral obstruction-induced obstructive nephropathy in juvenile rats.

Urinary tract obstruction is a common cause of renal failure in children and infants, and the pathophysiological mechanisms of obstructive nephropathy are largely unclear. It has been reported that m6A modulation is involved in renal injury. However, whether m6A RNA modulation is associated with obstructive nephropathy has not yet been reported. The aim of this study was to investigate the m6A epitranscriptome profiles in the kidneys of bilateral ureteral obstruction (BUO) in young rats. The total level of m6A in the kidneys was measured by liquid chromatography-tandem mass spectrometry. The mRNAs of related genes were detected by real-time PCR. Methylated RNA immunoprecipitation sequencing was performed to map the epitranscriptome-wide m6A profile. Global m6A levels were increased after BUO, and the mRNA expression levels of m6A methyltransferases and demethylases were significantly decreased in BUO group rat kidneys; the expression levels of EGFR and Brcal were significantly upregulated, while the mRNA expression levels of Notch1 were downregulated (P < 0.05). A total of 154 genes associated with 163 m6A peaks were identified. The m6A epitranscriptome was significantly altered in BUO rat kidneys, which is potentially implicated in the pathophysiological processes of obstructive nephropathy. The m6A RNA modification was associated with the process of renal injury in ureteral obstructive nephropathy by participating in multiple dimensions. The dysregulation of m6A methyltransferases and demethylases may be related to the pathophysiological changes of BUO-induced obstructive nephropathy. The m6A RNA modulation of the genes EGFR, Brca1, and Notch1 that were related to the regulation of aquaporin2 might be the potential mechanism for the polyuresis after ureteral obstruction.

Investigation of the Anticancer Effect of S-Allyl-L Cysteine on the C6 Rat Glioma Cell Line in vitro in 2D- and 3D-Cell Culture Models.

In our study, we aimed to investigate the effects of SAC on C6 glioblastoma cells and its antiproliferative and apoptotic effects on two- and three-dimensional cell culture systems. The rat glioma cell line C6 was separated into 2D-Control, 2D-SAC, 3D-CMC-Control and 3D-CMC-SAC groups. While control cells were incubated under standard culture conditions, cells in the SAC groups were incubated with the IC50 dose (50 µM for both the 2D-SAC C6 and 3D-CMC-SAC groups) of SAC in culture medium for 24 and 48 hours. Thereafter, all groups were stained with antibodies recognizing NOTCH1 and JAGGED1, and the mRNA expression levels of NOTCH1 and JAGGED1 were evaluated by qRT-PCR. Increasing doses of SAC were administered for 24 hours in the C6 glioma cell line. The concentration of 50 μM was chosen as the most suitable dose for administration. The gene expression profiles were different in the two forms of culture. NOTCH1 receptor mRNA expression levels were significantly decreased in cells treated with 50 μM SAC for 24 hours compared to control cells in both the two-dimensional (2D) and three-dimensional (3D) cell cultures. The immunoreactivities of both of these markers (JAGGED1 and NOTCH1) were significantly decreased in the glioma cells in the SAC group compared to the control group. This study shows that SAC has potential as a drug for human use, as indicated by its nontoxic nature. The present study confirmed the anticancer activity of SAC by modulating NOTCH1 and JAGGED1 signaling in glioma cancer cells.

Resveratrol impairs cellular mechanisms associated with the pathogenesis of endometriosis

Research questionDoes resveratrol exert a potent inhibitory effect on the development of endometriosis by interfering with some pivotal processes? DesignIn-vitro cultures of primary endometriotic stromal cells, immortalized endometrial stromal (St-T1b) and endometriotic epithelial (12Z) cells were used to assess the effects of resveratrol on endometrial cell mechanisms. The effects of resveratrol on 12Z and St-T1b cell viability were assessed by MTT assay, apoptosis by FITC Annexin V assay and cleaved caspase-3 levels and cell migration by wound healing assay. The effect of resveratrol on the expression of genes related to cell migration, angiogenesis and cell stemness was evaluated by qRT-PCR. ResultsResveratrol significantly decreased cell viability (P= 0.0065 to P = 0.0180), cell migration (P < 0.001 to P = 0.0225) and increased the number of apoptotic cells (P = 0.0031 to P = 0.0432) in both cell lines. In cell lines and primary culture, the treatment reduced MMP-2/TIMP-1 (P < 0.001 to P = 0.0180), VEGF (P = 0.0052 to P = 0.0243) and Ang-1 mRNA (P < 0.001 to P = 0.0382) expression. Among the stem cell phenotype markers, resveratrol 100 µM increased mRNA expression levels of Notch-1 (P < 0.001 to P = 0.0018), KLF-4 (P = 0.0011 to P = 0.0137), SOX-2 (P < 0.001 to P = 0.0070) and TERT (P < 0.001 to P = 0.0193) in both cell lines and primary cultures. The mRNA expression level of Snail-1 increased in the cell lines (P < 0.001 to P = 0.0087), whereas OCT-4 mRNA expression increased in St-T1b (P = 0.0396) and primary cultures (P = 0.0148). Vimentin mRNA expression showed a significant upregulation in primary cultures (P < 0.001). The expression of Msi-1 (P = 0.0145) and NANOG (P = 0.0080) decreased only in St-T1b cells. ConclusionResveratrol showed inhibitory effects on cell behaviour related to the development of endometriosis by differentially affecting growth, apoptosis, migration and stem cell phenotype of endometrial and endometriotic cells in vitro.

MiR-499b-5p inhibits cervical cancer cell proliferation and induces apoptosis by targeting the Notch1 signaling pathway.

We investigated the effect of miR-499b-5p on the tumorigenesis and development of cervical cancer by targeting the Notch1 signaling pathway to identify a new potential clinical target of cervical cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to determine the mRNA expression levels of Notch1 and miR-499b-5p in cervical cancer tissues/cell lines. Cell counting kit-8 (CCK-8) assay, transwell assay, and flow cytometry were conducted to detect cell viability, cell migration, and cell apoptosis abilities. A Dual-Luciferase reporter assay was performed to test the binding site between miR-499b-5p and Notch1. An in vivo experiment was carried out using nude mice, and xenograft tumor models were established. OD450 of the SiHa and HeLa cells of the miR-499b-5p agomir group was lower than that of the miR-499b-5p agomir-NC group. More apoptotic cells and fewer invasive cells were found in the former than in the latter. MiR-499b-5p inhibited the viability and migration of cervical cancer cells and promoted their apoptosis. Further detection of the Luciferase reporter gene confirmed the binding site of miR-499b-5p to Notch1. Western blot results showed that miR-499b-5p inhibited the expression of Notch1 and activated the expression of ChK2 and p-p38MAPK. Notch1 knockdown also inhibited the viability and migration of cervical cancer cells and promoted their apoptosis. MiR-499b-5p overexpression prevented the tumorigenesis and development of cervical cancer in xenograft tumor models. MiR-499b-5p inhibits the proliferation of cervical cancer cells and induces their apoptosis by targeting the Notch1 signaling pathway.

Palbociclib induces cell senescence and apoptosis of gastric cancer cells by inhibiting the Notch pathway.

Palbociclib (PD0332991), a selective cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has been reported to exert anticancer activity in some cancers, including gastric cancer (GC). However, the role of palbociclib in GC remains largely unknown. The present study aimed to investigate the effects of palbociclib on the progression of GC and the potential mechanisms underlying its effects. The colony formation, proliferation, senescence, as well as apoptosis and cell cycle progression of AGS and HGC-27 cells following treatment with palbociclib were analyzed using colony formation assays, MTT assays, senescence-associated β-galactosidase (SA-β-gal) staining and flow cytometry, respectively. The protein expression levels of Bax, Caspase-3, Bcl-2, p16, p21, p53, Notch1, Notch2 and hairy and enhancer of split 1 (Hes1) were measured in AGS and HGC-27 cells using western blotting. Moreover, the mRNA expression levels of Notch1, Notch2 and Hes1 in AGS and HGC-27 cells were determined by reverse transcription-quantitative PCR. In the present study, palbociclib significantly inhibited cell proliferation and induced cell senescence, cell cycle arrest and apoptosis in both cell lines in a dose-dependent manner. Additionally, palbociclib significantly increased the expression levels of Bax, Caspase-3, p16, p21 and p53, whilst decreasing the expression of Bcl-2, Notch1, Notch2 and Hes1 in AGS and HGC-27 cells. Furthermore, the Notch pathway activator Jagged-1/FC reversed the effects of palbociclib on cell proliferation, apoptosis, senescence and cell cycle progression. These findings demonstrated that palbociclib could inhibit proliferation and induce senescence, cell cycle arrest and apoptosis in GC cells by inhibiting the Notch pathway.

FLI-06 Intercepts Notch Signaling And Suppresses The Proliferation And Self-renewal Of Tongue Cancer Cells.

PurposeThe Notch signaling pathway plays an oncogenic role in tongue squamous cell carcinoma. The aim of this study was to inhibit the proliferation and self-renewal of tongue cancer cells by applying Notch signaling pathway inhibitor FLI-06 (Selleck, USA) and to lay a foundation for the clinically targeted treatment of tongue cancer for the future.MethodsThe mRNA expression level of Notch1 and the overall survival rate of patients with tongue cancer were examined by analyzing the TCGA database. Tongue cancer cells were treated with FLI-06. Cell proliferation, apoptosis, and stem cell self-renewal ability were tested in appropriate ways. A xenograft mouse model was established to observe tumor growth.ResultsFrom the TCGA data, we demonstrated that patients with high expression of Notch1 had a poor prognosis. We observed that the Notch signaling pathway inhibitor FLI-06 can restrain the activation of the Notch signaling pathway, decrease cell proliferation and induce cell apoptosis in vitro. The xenograft experiment indicated that intraperitoneal injection of FLI-06 inhibited tumor growth and increased cell apoptosis. FLI-06 suppressed both the mRNA and protein expression of Notch receptor and Notch targeted genes. We also observed that FLI-06 suppressed the proliferation of tongue cancer stem cells.ConclusionFLI-06 can block the proliferation and self-renewal of tongue cancer cells. It is inferred that this compound, which inhibits the Notch signaling pathway, may serve as a potential targeted drug for the treatment of tongue cancer in the clinic.

Effects of Yiqi Jiedu prescription drug serum on the apoptosis of human renal cell carcinoma ACHN cells and Notch1 gene

Objective To observe the effects of Yiqi Jiedu prescription drug serum on the apoptosis of human renal cell carcinoma ACHN cells and Notch1 gene in Notch signal pathway. Methods The healthy SD female rats were selected, which were divided into 4 groups, 8 rats in each group: the rats were fed with the normal saline 10 ml·kg-1·d-1 (the blank serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 50 g·kg-1·d-1 (the high-concentration of Yiqi Jiedu prescription serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 25 g·kg-1·d-1 (the medium-concentration of Yiqi Jiedu prescription serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 12.5 g·kg-1·d-1 (the low-concentration medicated of Yiqi Jiedu prescription serum group); then the serum would be extracted 10 days later in each group. ACHN cells at exponential phase were cultured in the above 4 groups. The proliferation of ACHN cells in each group was observed by using CCK-8 method. The apoptosis of ACHN cells was detected by using flow cytometry (FCM). The expression levels of Notch1 mRNA of ACHN cells in each groups were detected by using real time fluorescence quantitative polymerase chain reaction (RT-PCR). Results The inhibition rates of ACHN cells in the high-concentration group, the medium-concentration group, the low-concentration group of Yiqi Jiedu prescription and the blank serum group 24 h later were (12.34±4.25)%, (7.47±1.40)%, (2.52±0.62)%, (1.05±0.31)%, respectively (F=15.04, P < 0.01); after 48 h, the inhibition rates were (24.20±2.41)%, (14.23±1.56)%, (5.08±1.34)%, (1.16±0.14)%, respectively (F=227.36, P < 0.01); after 72 h, the inhibition rates were (32.16±2.45)%, (25.05±3.69)%, (10.29±2.76)%, (1.07±0.71)%, respectively (F=110.51, P < 0.01). The results showed that Yiqi Jiedu prescription drug serum could significantly inhibit the proliferation of ACHN cells in a dose-dependent manner. Besides, the inhibition rate differences at all time points of the high-concentration serum group (F=31.44, P < 0.01), the medium-concentration serum group (F=49.61, P < 0.01) and the low-concentration serum group (F=68.78, P < 0.01) were all statistically significant, and they were in a time-dependent manner. The apoptosis rate of cells in the high-concentration group, the medium-concentration group, the low-concentration group of Yiqi Jiedu prescription and the control serum group was (34.5±1.5)%, (24.4±1.5)% and (13.1±0.5)%, (5.2±0.3)%, respectively, and the differences were statistically significant (F=1 153.36, P < 0.01). The relative expression level of Notch1 mRNA of cells in the high-concentration group, the medium-concentration group, the low-concentration serum group of Yiqi Jiedu prescription and the control serum group was 0.213±0.032, 0.432±0.049, 0.781±0.018, 1.013±0.047, respectively, and the differences were statistically significant (F=270.60, P < 0.01). Conclusion Yiqi Jiedu prescription drug serum can induce apoptosis of ACHN cells, and its mechanism may be related to the down-regulation of the expression level of Notch1 receptors. Key words: Kidney neoplasms; Yiqi Jiedu prescription; Apoptosis; Drug serum; Notch1 receptors

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.

Effect of \u03b3-secretase inhibitor on Th17 cell differentiation and function of mouse psoriasis-like skin inflammation

BackgroundTh17 cells and its effective cytokine IL-17A play an important role in the pathogenesis of abnormal immune responses in psoriasis. Notch1 signaling has been implicated in Th17 cell differentiation and function. In this study, our aim was to evaluate the possible inhibitory effect of Notch1 signaling inhibitor, γ-secretase inhibitor DAPT, on psoriatic Th17 cell differentiation and function in a mouse model of psoriasis-like skin inflammation.MethodsMouse psoriasis-like skin inflammation model was established by topical 5% imiquimod (IMQ) application, and experimental mice were divided into control group, IMQ-treated group and IM + DAPT-treated group. DAPT and the equivalent amount of Dimethyl sulfoxide was intraperitoneally injected in IMQ + DAPT-treated group and the other two experimental groups respectively. Skin tissues of the three experimental groups were acquired and stained with haematoxylin and eosin (HE). Splenic single-cells and serum were collected to detect the percentage of Th17 cells, the mRNA expression levels of Notch1 and its target gene Hes-1, Th17-specific transcription factor RORγt and its effective cytokines IL-17A, as well as IL-17A serum concentration. In addition, splenic CD4+ T cells from IMQ-treated mice were isolated and treated by DAPT to further measure the inhibitory effect of DAPT on the Th17 cell differentiation and IL-17A secretion in vitro.ResultsDAPT treatment alleviated the severity of IMQ-induced mouse psoriasis-like skin inflammation and decreased the scores of erythema, scaling and thickening. HE stain reveals obviously reduced epidermal hyperplasia and dermal inflammatory cells infiltration in IMQ + DAPT-treated mice. The increased expression of splenic Th17 cell percentage, along with Notch1, Hes-1, RORγt and IL-17A mRNA and IL-17A serum concentration in IMQ-treated mice were significantly decreased when experimental mice were treated by IMQ and DAPT combinedly. Data obtained from in vitro study in IMQ-treated mice also demonstrated that blocking Notch1 signaling by DAPT can result in a dose-dependent decrease of Th17 cell proportion, mRNA expression of Notch1, Hes-1, RORγt and IL-17A as well as IL-17A secretion in splenic CD4+ T cells.ConclusionThese data suggest that Notch1 inhibition by DAPT can effectively alleviate the severity of mouse psoriasis-like skin inflammation by regulating the differentiation and function of Th17 cells, indicating that DAPT might be a potential therapeutic candidate for the treatment of psoriatic inflammation.

Longitudinal study of esophageal mucosal damage after esophagectomy and gastric interposition: relationship between reflux-related mucosal injury and Notch signaling.

Esophagectomy with gastric interposition could serve as a good human reflux model to study the molecular pathogenesis of esophageal mucosal damage induced by gastroesophageal reflux. This study was to investigate the role of Notch signaling in reflux injury of esophageal mucosa. Patients undergoing Ivor-Lewis esophagectomy for early stage esophageal squamous cell carcinoma were included. Follow-ups were scheduled at 6, 18, 36 and 48 months postoperatively, including reflux symptom assessment, endoscopic and histological evaluation of esophageal mucosal damage. The expressions of Notch1 and its downstream target gene Hes1 were evaluated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). Forty-four out of 48 patients completed four follow-ups. Injuries of esophageal remnant confirmed by endoscopical and histological examinations were both more often with a longer postoperative period (P<0.05). The mRNA expression levels of Notch1 and Hes1 were decreased in a time-dependent manner after operation (P<0.001). Notch1 and Hes1 mRNA levels were significantly higher in normal squamous mucosa than in esophagitis, and higher in esophagitis than in metaplasia (P<0.05). Immunohistochemical study also demonstrated a similar protein expression pattern. Samples with endoscopic evidence of mucosal damage exhibited lower expression of Notch1 mRNA levels as compared to biopsies without visualized damage (P=0.035). This is the first longitudinal study on Notch signaling in human esophagectomy model, our preliminary findings suggest decreased Notch signaling might be involved in the development of mucosa damage caused by gastroesophageal reflux.

Effects of curcumin photodynamic therapy on notch signaling pathway in cervical cancer cells

Objective To explore the mechanism of notch signaling pathway in the treatment of cervical cancer with notch receptor blocker(DAPT). Methods After treatment of Me180 cells with different concentrations of DAPT combined with curcumin-photodynamic therapy(PDT), 3-(4, 5 Dimethylthiazlo-yl)-2, 5 Dimethylthiazol-2-yl)-2, 5 dipheny tetrazo lium bromide(MTT)assay was used to detect the effect on the proliferation of Me180 cells, annexin V-FITC/PI combined with flow cytometry was used to detect the effect on apoptosis, and real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of Notch1 gene respectively. Results Both DAPT(1.0 μmol/L)and curcumin-PDT groups could inhibit the proliferation and induce apoptosis of cervical cancer cell line Me180, and the synergistic effect of the two groups was significant(P<0.05 or<0.01). DAPT and curcumin-PDT group reduced the mRNA expression level of Notch1 in cervical cancer Me180 cells with inhibition ratio of 40.0% and 32.3% respectively.DAPT and curcumin-PDT group inhibited the protein expressions of Notch1, nuclear factor-kappa B(NF-kB), vascular endothelial growth factor(VEGF), and the synergistic effect of the two groups was statistically significant. Conclusions Both DAPT and curcumin-PDT may effectively block the conduction of Notch signaling pathway, which is associated with down-regulation of the expression of Notch1 and NF-kappa B. Notch signaling pathway may be one of the targets of curcumin photodynamic therapy. Key words: Curcumin; Photodynamic therapy; Apoptosis; Uterine cervical neoplasms

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

The ratio of 1/3 linoleic acid to alpha linolenic acid is optimal for oligodendrogenesis of embryonic neural stem cells

During neural development, embryonic neural stem cells (eNSCs) differentiate toward glial, oligodendrocytic, and neuronal cells. Dysregulation of polyunsaturated fatty acids (PUFAs) induce a wide range of neurological and developmental disorders. In this study, we investigated the effect of various concentrations and ratios of linoleic acid (LA) and alpha linolenic acid (ALA), which belong respectively to omega-6 and omega-3 PUFAs, on the proliferation and differentiation of eNSCs.Results showed that low (25 and 50μM) or high (100 and 200μM) concentrations of ALA, but not LA, and the ratio of 1:3 of LA/ALA significantly increased neurospheres size, frequency and cell numbers, in comparison to controls. Moreover, low or high concentrations of ALA, but not LA, and different ratios of LA/ALA resulted in a significant increase in mRNA expression levels of Notch1, Hes1 and Ki-67, and the differentiation of eNSCs toward astrocytes (GFAP) and oligodendrocytes (MBP), but not neurons (β-III Tubulin), with the highest increase being for LA/ALA ratio of 1:3, in comparison to controls. These results demonstrate the importance of higher concentrations of ALA in enhancing proliferation and differentiation of eNSCs, which could be used in diet to help preventing neurodevelopmental syndromes, cognitive decline during aging, and various psychiatric disorders.

627 - Oxidized products of α-linolenic acid negatively affect cell survival and motility of breast cancer cells

During neural development, embryonic neural stem cells (eNSCs) differentiate toward glial, oligodendrocytic, and neuronal cells. Dysregulation of polyunsaturated fatty acids (PUFAs) induce a wide range of neurological and developmental disorders. In this study, we investigated the effect of various concentrations and ratios of linoleic acid (LA) and alpha linolenic acid (ALA), which belong respectively to omega-6 and omega-3 PUFAs, on the proliferation and differentiation of eNSCs.Results showed that low (25 and 50 μM) or high (100 and 200 μM) concentrations of ALA, but not LA, and the ratio of 1:3 of LA/ALA significantly increased neurospheres size, frequency and cell numbers, in comparison to controls. Moreover, low or high concentrations of ALA, but not LA, and different ratios of LA/ALA resulted in a significant increase in mRNA expression levels of Notch1, Hes1 and Ki-67, and the differentiation of eNSCs toward astrocytes (GFAP) and oligodendrocytes (MBP), but not neurons (β-III Tubulin), with the highest increase being for LA/ALA ratio of 1:3, in comparison to controls. These results demonstrate the importance of higher concentrations of ALA in enhancing proliferation and differentiation of eNSCs, which could be used in diet to help preventing neurodevelopmental syndromes, cognitive decline during aging, and various psychiatric disorders.

Aberrant Expression Notch1 and Asb2 mRNA in Bone Marrow from Patients with P210(+) Chronic Myeloid Leukemia

To explore the mRNA expression level of Notch1 and ASB2 in bone marrow mononuclear cells of patients with P210(+) chronic myeloid leukemia and the correlation between Notch signaling pathway and ubiquitination in chronic myeloid leukemia, so as to provide the valuable information for investigating the pathogenesis of chronic myeloid leukemia and clinical treatment. Bone marrow was collected from 32 patients with newly diagnosed chronic myeloid leukemia and 34 non-hematopathic and healthy individuals (control group), respectively. The mRNA expression levels of Notch1 and ASB2 were detected by real-time quantitative PCR. The expression of Notch1 mRNA in CML patients were significantly different from that of healthy individuals group (t=36.3, P<0.01), which was 337.8 times of the healthy individuals. Moreover, the expression level of ASB2 mRNA in CML group was significantly higher than that in healthy individuals (t=19.4, P<0.01). The mRNA expression of Notch1 and ASB2 gene was positive correlation (r=0.504, P<0.01). The aberrant expression of Notch1 and Asb2 exists in patients with P210 positive CML, which may be involved in incidence and development of CML.