Effect of Buzhong Yiqitang on Th17/Treg Immune Imbalance and Notch1 Signaling Pathway in AIT Mice / 中国实验方剂学杂志

ObjectiveTo explore the effect of Buzhong Yiqitang on the immune imbalance of helper T cell 17 （Th17）/regulatory T cell （Treg） and Notch1 signaling pathway in mice with autoimmune thyroiditis （AIT）. MethodA total of 60 8-week-old NOD.H-2h4 mice were randomly divided into the normal group， model group， western medicine group （selenium yeast tablet， 32.5 mg·kg-1）， and low-dose （4.78 g·kg-1·d-1）， middle-dose （9.56 g·kg-1·d-1）， and high-dose （19 g·kg-1·d-1） Buzhong Yiqitang groups， with 10 mice in each group. The normal group was fed with distilled water， and the other groups were fed with water containing 0.05% sodium iodide for eight weeks. After the animal model of AIT was formed spontaneously， the mice were killed under anesthesia after intragastric administration for eight weeks. Serum anti-thyroglobulin antibodies （TGAb）， thyroid-stimulating hormone （TSH）， free triiodothyronine （FT3）， and free thyroid hormone （FT4） were detected by enzyme-linked immunosorbent assay （ELISA）， and thyroid tissue changes were observed by hematoxylin-eosin （HE） staining. The mRNA and protein expressions of retinoid-related orphan receptor-γt （RORγt）， interleukin （IL）-17， forkhead box P3 （FoxP3）， IL-10， Notch1， and hair division-related enhancer 1 （Hes1） in thyroid tissue were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the normal group， the thyroid structure of the model group was severely damaged， and lymphocytes were infiltrated obviously. The levels of serum TGAb， FT3， and FT4 contents were significantly increased， and TSH content was significantly decreased （P<0.01）. The mRNA and protein expression levels of RORγt， IL-17， Notch1， and Hes1 were significantly increased， while those of FoxP3 and IL10 were significantly decreased in the model group （P<0.01）. Compared with the model group， thyroid structural damage and lymphocyte infiltration were improved in the treatment groups， and serum TGAb， FT3， and FT4 contents were significantly decreased. TSH content was increased， and mRNA and protein expression levels of RORγt， IL-17， Notch1， and Hes1 were decreased. mRNA and protein expression levels of FoxP3 and IL-10 were increased to different degrees （P<0.05， P<0.01）， and the middle-dose Buzhong Yiqitang group had the most significant intervention effect. ConclusionBuzhong Yiqitang can alleviate the thyroid structural damage in AIT mice， and its mechanism may be related to improving the abnormal differentiation of Th17/Treg immune cells and inhibiting the activation of the Notch1 signaling pathway.

A molecular approach to triple-negative breast cancer: targeting the Notch signaling pathway

ABSTRACT Introduction Triple-negative breast cancer is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. This phenotype renders triple-negative breast cancer cells refractory to conventional therapies, resulting in poor clinical outcomes and an urgent need for novel therapeutic approaches. Recent studies have implicated dysregulation of the Notch receptor signaling pathway in the development and progression of triple-negative breast cancer. Objective This study aimed to conduct a comprehensive literature review to identify potential therapeutic targets of the Notch pathway. Our analysis focused on the upstream and downstream components of this pathway to identify potential therapeutic targets. Results Modulating the Notch signaling pathway may represent a promising therapeutic strategy to treat triple-negative breast cancer. Several potential therapeutic targets within this pathway are in the early stages of development, including upstream (such as Notch ligands) and downstream (including specific molecules involved in triple-negative breast cancer growth). These targets represent potential avenues for therapeutic intervention in triple-negative breast cancer. Comments Additional research specifically addressing issues related to toxicity and improving drug delivery methods is critical for the successful translation of these potential therapeutic targets into effective treatments for patients with triple-negative breast cancer.

Baicalin Antagonizes Prostate Cancer Stemness via Inhibiting Notch1/NF-κB Signaling Pathway / 中国结合医学杂志

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 factor κB (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 (125 µmol/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 125 µmol/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.

Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1 / 中国医学科学院学报

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.

Anti-inflammatory Mechanism of Modified Erchentang on Chronic Obstructive Pulmonary Disease Through Jagged1/Notch1/Hes1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of modified Erchentang on the expression of key molecules in the Jagged1/Notch1/Hes1 signaling pathway in lung tissues of rats with chronic obstructive pulmonary disease （COPD） and explore its anti-inflammatory effect and molecular mechanism on COPD through the Jagged1/Notch1/Hes1 signaling pathway. MethodSixty SD rats were randomly divided into normal group， model group， low-， medium-， and high-dose modified Erchentang groups （5， 10， 20 g·kg-1）， and γ-secretase inhibitor DAPT group （0.02 g·kg-1）， with 10 rats in each group. The COPD model was induced in rats by cigarette smoking combined with intratracheal instillation of lipopolysaccharide （LPS）. Rats were treated with corresponding drugs by gavage， while those in the normal group and the model group were treated with the same amount of normal saline by gavage. The serum levels of Notch1， soluble intercellular adhesion molecule-1 （sICAM-1）， activated leukocyte cell adhesion molecule （ALCAM）， and soluble vascular adhesion molecule-1 （sVCAM-1） were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression of Jagged1， Notch1， and Hes1 was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression of Jagged1， Notch1， Notch1 intracellular domain （NICD1）， and Hes1 in lung tissues of rats was detected by immunohistochemistry （IHC）. ResultCompared with the normal group， the model group showed increased serum content of Notch1， sICAM-1， ALCAM， and sVCAM-1 （P<0.01）， increased mRNA expression of Jagged1， Notch1， and Hes1 in lung tissues （P<0.01）， and increased protein expression of Jagged1， Notch1， NICD1， and Hes1 （P<0.01）. Compared with the model group， the medium- and high-dose modified Erchentang groups and the DAPT group showed decreased serum content of Notch1， sICAM-1， ALCAM， and sVCAM-1 （P<0.05， P<0.05）， down-regulated mRNA expression of Jagged1， Notch1， and Hes1 （P<0.05， P<0.01）， and reduced protein expression of Jagged1， Notch1， NICD1， and Hes1（P<0.05， P<0.01）. ConclusionModified Erchentang may inhibit the inflammatory response in the lung of COPD rats， and its mechanism may be related to the resistance of inflammatory injury in the lung by decreasing the mRNA expression of Jagged1， Notch1， and Hes1 and inhibiting the release of Notch1， sICAM-1， ALCAM， and sVCAM-1.

Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification / 中国骨伤

OBJECTIVE@#To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification.@*METHODS@#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.@*RESULTS@#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).@*CONCLUSION@#Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.

Expressions and Clinical Significance of Notch1 and Hes1 in Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the expressions of Notch1 and Hes1 in diffuse large B-cell lymphoma (DLBCL), and their correlations with clinical features.@*METHODS@#Immunohistochemistry (IHC) was performed on DLBCL samples (54 cases) and lymphadenitis tissues (20 cases) to evaluate the expressions of Notch1 and Hes1, and analyze their correlations with clinical characteristics of patients. Based on Oncomine database, the expressions of Notch1 and Hes1 mRNA and DNA were also explored.@*RESULTS@#IHC result showed that the positive expression rates of Notch1 and Hes1 in DLBCL patients were significantly higher than those in the control group (P <0.05). In DLBCL patients, the expression of Notch1 was closely associated with B symptoms, Ann Arbor stage, lymphocyte count and the level of lactate dehydrogenase (P <0.05), while the expression level of Hes1 was significantly higher in patients with B symptoms (P <0.05). Notch+/Hes1+ expression was found in 21 DLBCL tissues (38.9%), and there was a correlation between Notch1 and Hes1 expression (r =0.296, P <0.05). Bioinformatics analysis (Oncomine database) showed that the mRNA expressions of Notch1 and Hes1 in the Brune dataset were significantly higher than those in the control tissues (P <0.05).@*CONCLUSION@#The expressions of Notch1 and Hes1 in DLBCL are significantly higher than those in lymphadenitis, and correlated with B symptoms and Ann Arbor stage, suggesting that Notch1 and Hes1 play important roles in the occurrence and development of DLBCL.

Regulation of Notch1 and Foxp1 by MiR-34a in psoriasis vulgaris / Regulación de Notch1 y Foxp1 por MiR-34ª en psoriasis vulgaris

SUMMARY: This study is to investigate the regulation of Notch1 and Foxp1 by miR-34a in the development of psoriasis vulgaris. RT-PCR was used to compare the levels of miR-34a in the skin lesions of 20 patients with psoriasis vulgaris and 20 normal skin tissues. Immunohistochemistry was used to detect the expression of Notch1 and Foxp1 in 51 patients with psoriasis vulgaris, which were further compared with that in 29 normal control tissues. In addition, in HaCaT cells, we used miR-34a mimics and inhibitors to overexpress and inhibit miR-34a, respectively, and detected the mRNA and protein levels of miR-34a, Notch1, and Foxp1. The level of miR-34a in the skin lesions of patients with psoriasis vulgaris was significantly higher than that in normal skin tissues (t=2.192, P<0.05). The positive rate of Notch1 in the skin lesions of patients with psoriasis vulgaris was 76.47 %, which was significantly higher than that in normal skin tissues (13.79 %) (t=29.215, P<0.01). The positive rate of FOXP1 in the psoriasis vulgaris group was 92.16 %, which was also significantly higher than that in the normal skin group (65.52 %) (t=9.087, P<0.01). In addition, overexpression of miR-34a significantly promoted the expression of Notch1 and Foxp1. However, inhibition of miR-34a significantly reduced Notch1 and Foxp1 levels. miR- 34a is highly expressed in the skin tissues of patients with psoriasis vulgaris, and may participate in the development of psoriasis vulgaris by regulating Notch1 and Foxp1.

RESUMEN: El objetivo de este estudio fue investigar la regulación de Notch1 y Foxp1 por miR-34a en el desarrollo de la psoriasis vulgar. Se utilizó RT-PCR con el fin de comparar los niveles de miR-34a en las lesiones cutáneas de 20 pacientes con psoriasis vulgar y 20 tejidos de piel normales. Se utilizó inmunohistoquímica para detectar la expresión de Notch1 y Foxp1 en 51 pacientes con psoriasis vulgar, que se compararon además con la de 29 tejidos normales control. Además, en las células HaCaT, usamos miméticos e inhibidores de miR-34a para sobreexpresar e inhibir miR-34a, respectivamente, y detectamos los niveles de ARNm y proteína de miR-34a, Notch1 y Foxp1. El nivel de miR- 34a en las lesiones cutáneas de pacientes con psoriasis vulgar fue significativamente mayor que en los tejidos normales de la piel (t=2,192, P<0,05). La tasa de positividad de Notch1 en las lesiones cutáneas de pacientes con psoriasis vulgar fue del 76,47 %, que fue significativamente mayor que la de los tejidos normales de la piel (13,79 %) (t=29,215, P<0,01). La tasa positiva de FOXP1 en el grupo de psoriasis vulgar fue del 92,16 %, que también fue significativamente mayor que la del grupo de piel normal (65,52 %) (t=9,087, P<0,01). Además, la sobreexpresión de miR-34a promovió significativamente la expresión de Notch1 y Foxp1. Sin embargo, la inhibición de miR-34a redujo de manera importante los niveles de Notch1 y Foxp1. miR-34a se expresa en gran medida en los tejidos de la piel en pacientes con psoriasis vulgar y puede participar en el desarrollo de la psoriasis vulgar mediante la regulación de Notch1 y Foxp1.

Prognostic value of NOTCH1 and OCT4 in gastric carcinoma

Background: NOTCH1 pathway activation has been recently described to be a key player in gastric carcinogenesis, enhance the survival and proliferation of cancer stem cells (CSCs) and mediate chemoresistance in several malignancies. Aim: This study investigated the correlation between NOTCH1 and CSC marker OCT4 (octamer binding transcription factor-4) expression and the clinicopathological properties, survival and treatment outcome in patients with gastric carcinoma (GC) receiving adjuvant chemotherapy. Materials and Methods: NOTCH1 and OCT4 were immunohistochemically detected in 50 post-operated specimens of GC. Patients' data regarding disease-free survival (DFS), overall survival (OS), and the response to the chemotherapy was statistically analyzed. Results: NOTCH1 and OCT4 overexpression was detected in 60% and 52% of GC tissues, respectively, and that was significantly higher than the rates in adjacent non-neoplastic gastric mucosa (P < 0.05). A significant correlation was detected between overexpression of NOTCH1 and OCT4 in GC and aggressive clinicopathological features; poor differentiation (P = 0.021, P = 0.037, respectively), depth of tumor invasion (P < 0.001 for both), TNM stage (P < 0.001 for both), lymph node metastasis (P = 0.002, P = 0.003, respectively) and distant metastasis (P < 0.001 for both). NOTCH1 was positively correlated with OCT4 (P = 0.002). Survival analysis disclosed that upregulation of NOTCH1 and OCT4 was associated with worse DFS (P = 0.013, P < 0.001, respectively) and OS (P < 0.001 for both). Overexpression of NOTCH1 and OCT4 correlated with poor response to chemotherapy (P = 0.013, P = 0.005, respectively) and worse clinical outcome. Conclusion: Combined detection of these proteins might disclose even better predictive value for shorter survival and resistance to chemotherapy.

Effects of Notch1 signaling on regulatory T cells and coronary artery lesions in childhood Kawasaki disease / 中华风湿病学杂志

Objective:To explore the effect of Notch1 signaling on regulatory T cells and its roles in vascular damage in patients with Kawasaki disease (KD).Methods:A total of 42 children with KD were enrolled in the present study from March 2019 to June 2020, as 32 age-matched healthy children were recruited as control. The proportions of CD4 +CD25 hiFoxp 3+ regulatory T cells (Treg) and expressions of transcription factor forkhead box protein 3 (Foxp3), cytotoxic T lymphocyte associated antigen-4 (CTLA4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and Notch1 protein were evaluated by flow cytometry. Chromatin immunoprecipitation was conducted to detect acetylation level of histone H4 (H4Ac) associated with the promoter of Foxp3 gene and its binding abilities of Notch1 intracellular domain 1 (NICD1), recombination signal binding protein for immunoglobulin kappa J region (RBP-J) and p300 in CD4 + T cells. Transcription levels of Foxp3, presenilin 1 (PSEN1), mastermind like transcriptional coactivator 1 (MAML1), and RBP-J in CD4 + T cells were determined by real-time polymerase chain reaction (PCR). Concentrations of interleukin (IL)-10 and transforming growth factor-β (TGF-β) in plasma and culture supernatant stimulated with Jagged1 were measured by enzyme linked immunosorbent assay. Independent-sample t-test, Pearson correlation analysis was used as the statistical method in this study. Results:① The frequencies of Treg in acute KD patients decreased significantly [(4.3±1.5)% vs (7.9±2.9)%; t=6.41, P<0.001], as protein levels of Foxp3, CTLA4 and GITR and concentrations of IL-10 and TGF-β in plasma reduced remarkably in acute KD patients ( t=6.87, P<0.001; t=4.26, P<0.001; t=7.88, P<0.001; t=8.42, P<0.001; t=13.01, P<0.001). All parameters afore-mentioned in patients combined with coronary artery lesions (CAL) were lower than those of patients without coronary artery lesions (NCAL) ( t=5.83, P<0.001; t=3.83, P<0.001; t=3.28, P=0.002; t=5.05, P<0.001; t=5.96, P<0.001; t=5.17, P<0.001), and increased after therapy ( t=7.13, P<0.001; t=6.10, P<0.001; t=4.31, P<0.001; t=6.55, P<0.001; t=7.40, P<0.001; t=7.84, P<0.001). ② H4Ac associated with promoter of Foxp3 gene and the binding abilities of NICD1 and p300 in acute KD patients were lower than those of the controls ( t=10.25, P<0.001; t=6.93, P<0.001; t=6.75, P<0.001), and increased remarkably after therapy ( t=7.72, P<0.001; t=4.16, P<0.001; t=5.76, P<0.001). Meanwhile, the three items in CAL group were found to be less than those of NCAL group ( t=6.08, P<0.001; t=2.66, P=0.011; t=6.02, P<0.001). Pearson correlation analysis showed a positive correlation between H4Ac associated with Foxp3 promoter and its mRNA level in acute KD patients ( r=0.47, P<0.001). No statistical significant difference about the binding ability of RBP-J with Foxp3 promoter were found among the groups ( t=0.57, P>0.05; t=0.61, P>0.05; t=1.20, P>0.05). ③ Protein level of Notch1 and the expressions of PSEN1, MAML1 and RBP-J mRNA in CD4 + T cells from acute KD patients were down-regulated remarkably ( t=5.28, P<0.001; t=6.31, P<0.001; t=11.78, P<0.001; t=8.06, P<0.001), and restored after therapy ( t=4.77, P<0.001; t=6.43, P<0.001; t=11.95, P<0.001; t=7.79, P<0.001). In parallel, the four indexes aforementioned of CAL group were lower than those of NCAL group ( t=3.16, P=0.003; t=4.13, P<0.001; t=5.42, P<0.001; t=4.05, P<0.001). Upon rhJagged1 stimulation for 48 hours, H4Ac level of Foxp3 promoter and its binding abilities with NICD1 and p300 in CD4 + T cells in KD patients and control group was significantly higher than those of untreated group [(KD: t=15.36, P<0.001; t=7.25, P<0.001; t=14.29, P<0.001), (Ctrl: t=7.87, P<0.001; t=5.71, P<0.001; t=8.74, P<0.001)], as the binding ability of RBP-J with Foxp3 promoter increased slightly without statistically significant difference (KD: t=1.11, P>0.05; Ctrl: t=1.37, P>0.05). Simultaneously, H4Ac level of Foxp3 promoter and its binding abilities with NICD1 and p300 in KD group were still lower than those of the control group after stimulation ( t=3.86, P<0.001; t=3.42, P=0.001; t=2.85, P=0.006). ④ After incubation of PBMC from heathy children with KD serum, the proportion of Treg cells, protein level of Foxp3 and expressions of Notch1 and RBP-J in CD4 + T cells in the group treated with IVIG increased significantly compared with the untreated group ( t=7.10, P<0.001; t=10.16, P<0.001; t=8.06, P<0.001; t=9.77, P<0.001), as well as H4Ac level of Foxp3 promoter and its binding abilities with NICD1 in the group treat with IVIG were also higher than the latter ( t=7.24, P<0.001; t=8.24, P<0.001). Conclusion:Insufficiency and impaired function of Treg caused by aberrant Notch1 signaling may be the important factor contributing to immune dysfunction and vascular damage in KD.

Inhibitory effect of gamma-secretase inhibitor on retinal neovascularization and regulative mechanism of Notch1 signal pathway / 中华实验眼科杂志

Objective:To investigate the inhibitory effect and underlying mechanism of gamma-secretase inhibitor blocking Notch1 signaling on retinal neovascularization caused by oxygen-induced retinopathy (OIR) in mice.Methods:To establish the OIR model, 7-day-old pups of C57BL/6J mice were exposed to 75% oxygen together with their mother until postnatal day (P)12.On P12, the mice were transferred to room air.All the mice were randomly divided into three groups, OIR group as control group, OIR+ DAPT group and OIR+ DMSO group receiving 1 μl intravitreal injection of gamma-secretase inhibitor (DAPT, 10 mmol/L) and 1∶20 DMSO dilution respectively.The right eye was taken as experimental eye.The mice were euthanized on P17 and the eyes were harvested to obtain retinas for further investigation.The total proteins were extracted from the retinas.The relative expression levels of Notch1 signal pathway and its downstream Hes1, the markers of M1 phenotype inducible nitric oxide synthase (iNOS) and M2 phenotype arginase-1 (Arg-1) microglia were measured by western blot.Retinal flat mounts were made and the retinal vessels were stained with isolectin B4 (IB4) to investigate the relative retinal neovascularization areas which was calculated as the ratio of neovascularization area/retinal area.The mumber of the neovascular endothelium cells beyond the inner limiting membrane was observed by hematoxylin-eosin staining.The use and care of animals complied with ARVO statement.This study protocol was approved by the Animal Ethics Committee of Guangdong Provincial People's Hospital (No.KY-Z-2021-2015-01).Results:The relative protein expression levels of Notch1 and Hes1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.68±0.06 and 0.70±0.08, 1.00±0.00 and 1.00±0.00, 1.03±0.08 and 1.02±0.07, respectively, with statistically significant differences among them ( F=70.62, 53.65; both at P<0.01). Compared with the OIR group and OIR+ DMSO group, the expressions of Notch1 and Hes1 were significantly reduced in OIR+ DAPT group (all at P<0.01). The relative protein expression levels of iNOS and Arg-1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.74±0.07 and 1.49±0.12, 1.00±0.00 and 1.00±0.00, 1.04±0.10 and 0.94±0.07, respectively, showing statistically significant differences ( F=31.63, 89.32; both at P<0.01). Compared with OIR group and OIR+ DMSO group, the expression of iNOS in OIR+ DAPT group was significantly reduced, and the expression of Arg-1 was significantly increased (all at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells beyond the inner limiting membrane in OIR+ DAPT group, OIR group, and OIR+ DMSO group were (8.82±2.71)% and 38.17±3.29, (22.32±5.34)% and 60.83±5.11, (20.27±3.36)% and 58.67±4.75, respectively, showing statistically significant differences ( F=33.72, 39.44; both at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells in OIR+ DAPT group were significantly reduced in comparison with OIR group and OIR+ DMSO group (all at P<0.01). Conclusions:Intravitreal injection of DAPT can inhibit the retinal neovascularization in OIR mice through blocking Notch1 signaling activation and promoting retinal microglia polarization from M1 to M2 phenotype.

" Multi-hits" based SD rat models of cognitive dysfunction and research on related mechanisms / 中华实用儿科临床杂志

Objective:To establish Sprague-Dawley (SD) rat models of cognitive impairment through repeated stimulation of lipopolysaccharide (LPS) in the early brain development, and to inquire into the effect of " multi-hits" mediated by inflammatory response on the histology and behavior of SD rat models and related molecular mechanisms.Methods:This study adopted a group design for experiments.The " multi-hits" SD rat models were established by intraperitoneal injection of LPS.According to the random number table method, 24 pregnant rats were randomly divided into 4 groups: control group, LPS1 group, LPS2 group and LPS3 group, 6 rats in each group.In the control group, saline was intraperitoneally injected into rats with gestational age of 18 days and 20-day-old neonatal rats.Rats with gestational age of 18 days were intraperitoneally injected with saline in the LPS1 group, 0.05 mg/kg LPS in the LPS2 group, and 0.1 mg/kg LPS in the LPS3 group.The pups in LPS1-3 groups were all injected intraperitoneally with 1 mg/kg LPS at the postnatal age of 20 days.The motor and cognitive function of the pups were evaluated overall by behavioral experiments such as forelimb suspension tests, grid tests and water maze tests.The relative expression of glial fibrillary acidic protein (GFAP), Notch1 and Jagged1 in brain tissue of pups was mainly detected by Western blot (WB) and histological experiments.One-way ANOVA analysis of variance and independent samples t- test were used to compare data among groups and between groups, respectively. Results:(1) Behavioral experiments: compared with the control group, LPS1-3 groups showed progressive decrease in forelimb suspension time [(34.81±5.66) s, (22.47±4.35) s, and (13.20±4.25) s vs.(43.88 ± 4.85) s], and the number of missteps in the grid experiment increased progressively (16.13±2.90, 20.75±3.10, 25.13±4.45 vs.9.00±2.72). The differences were statistically significant ( F=69.77, 35.59, all P<0.001). Both the escape latency and total distance in Morri′s water maze test increased progressively ( P<0.05). (2) WB experiment: the relative expression levels of GFAP, Notch1 and Jagged1 proteins in LPS1-3 groups were significantly higher than that in the control group ( P<0.05). (3) Hematoxylin-eosin (HE) staining and electron microscope pathology: compared with the control group, LPS1-3 groups had more loosely arranged frontal cortices and more obvious cell pyknosis.Under the electron microscope, the cytoplasm was swelling to varying degrees, mitochondrial cristae were broken, and part of the nuclear membrane was damaged. Conclusions:In the " multi-hits" cognitive impairment model, the damage to the brain tissue structure and behavioral changes of pups may be related to the up-regulation of Notch1/Jagged1 pathway mediated by repeated exposure to LPS.

Mechanism study on regulation of glioblastoma cell proliferation and apoptosis by sciadopitysin combined with CX-4945 through Notch1 pathway / 国际肿瘤学杂志

Objective:To investigate the effects and mechanism of sciadopitysin combined with CK2 inhibitor CX-4945 on proliferation and apoptosis of glioblastoma U87 cells.Methods:Glioblastoma U87 cells were cultured in vitro, and treated with 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin respectively. U87 cells were treated with 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945. U87 cells were divided into control group (without any treatment), sciadopitysin group (100.00 μmol/L of sciadopitysin), CX-4945 group (5.00 μmol/L of CX-4945), sciadopitysin combined with CX-4945 group (100.00 μmol/L of sciadopitysin plus 5.00 μmol/L of CX-4945). MTT method was used to detect cell viability, Caspase3/7 activity assay and Annexin Ⅴ/ PI double staining were used to detect cell apoptosis, and Western blotting was used to detect the expressions of Notch1 pathway related proteins ICN1, HES1 and DLL3. Results:The cell viabilities of U87 cells treated with 0, 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin were (100.00±6.30) %, (112.02±7.63) %, (140.84±6.73) %, (113.92±7.92) %, (102.60±7.12) % and (73.16±2.74) % respectively, and there was a statistically significant difference ( F=55.21, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 0.01, 0.10, 1.00, 100.00 μmol/L of sciadopitysin treatment ( P=0.009; P<0.001; P=0.003; P<0.001). The cell viability of U87 cells was inhibited by 100.00 μmol/L of sciadopitysin, while sciadopitysin at other low concentrations manifested as enhancement or no obvious effect. The cell viabilities of U87 cells treated with 0, 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945 were (100.00±5.53) %, (108.70±10.24) %, (93.14±2.82) %, (81.46±4.92) %, (56.92±3.99) % and (31.24±2.67) % respectively, and there was a statistically significant difference ( F=135.18, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 1.25, 5.00, 10.00, 20.00 μmol/L of CX-4945 treatment ( P=0.022; P<0.001; P<0.001; P<0.001). Low concentration (1.25 μmol/L) of CX-4945 enhanced the cell viability of U87 cells, however higher concentrations (5.00, 10.00, 20.00 μmol/L) of CX-4945 shown inhibitory effect. The cell viabilities of U87 cells in the control group, sciadopitysin group, CX-4945 group and sciadopitysin combined with CX-4945 group were (100.00±5.53) %, (71.96±2.10) %, (77.66±4.12) % and (42.56±4.22) % respectively, and there was a statistically significant difference ( F=160.56, P<0.001). There were statistically significant differences between the control group and each treatment groups (all P<0.001). There were statistically significant differences between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (both P<0.001). The Caspase3/7 activities of U87 cells in the above four groups were 2.34±0.47, 4.02±0.22, 3.67±0.32 and 5.85±0.28 respectively, and there was a statistically significant difference ( F=55.80, P<0.001). The apoptosis rates of each groups were (0.40±0.10) %, (17.37±0.57) %, (3.00±0.66) % and (33.47±0.87) % respectively, and there was a statistically significant difference ( F=1 822.18, P<0.001). Further pairwise comparison showed that there were statistically significant differences in Caspase3/7 activities and apoptosis rates between the control group and each treatment groups ( P<0.001, P=0.001, P<0.001; P<0.001, P=0.001, P<0.001). There were statistically significant differences in Caspase3/7 activities and apoptosis rates between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (all P<0.001). The protein expression levels of Notch 1 pathway related proteins ICN1 (0.55±0.07 vs. 1.01±0.09), HES1 (0.66±0.08 vs. 1.00±0.06) and DLL3 (0.74±0.04 vs. 1.01±0.09) in U87 cells decreased significantly after treatment with 100.00 μmol/L of sciadopitysin ( t=5.94, P=0.004; t=5.15, P=0.007; t=4.00, P=0.016) . Conclusion:Sciadopitysin can synergize with CK2 inhibitor CX-4945 to inhibit the proliferation and promote apoptosis of glioblastoma U87 cells by inhibiting Notch1 signaling pathway.

LncRNA Gm13568 regulates the activation of A1 astrocytes and affects the EAE process in mice / 中华微生物学和免疫学杂志

Objective:To investigate the effects of long non-coding RNA (lncRNA) Gm13568 on the activation of A1 astrocytes and the progress of experimental autoimmune encephalomyelitis (EAE) in mice.Methods:A recombinant lentiviral vector (LV-Inhibit-Gm13568) carrying astrocyte-specific promoter of glial fibrillary acidic protein (GFAP) was established to inhibit the function of endogenous Gm13568. A control vector (LV-ctrl) was established as well. The recombinant vectors were packaged. C57BL/6 mice were injected with 1×10 7 transforming units of viral suspension via the tail vein and 7 d after the injection, myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) was used to establish the mouse model of EAE. Four groups, PBS group, EAE group, LV-ctrl+ EAE group and LV-Inhibit-Gm13568+ EAE group, were included in this study. Clinical signs of the mice were monitored daily in a double-blinded manner. The mice were sacrificed 23 d after the EAE model was established and the spinal cord tissues were collected. The expression of Serping 1, C3, Srgn and H2-T23 at mRNA level was detected by real-time PCR. Changes in the expression of IL-6, TNF-α, macrophage chemotactic protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10) were measured. Western blot was used to investigate the expression of GFAP and Notch1 in spinal cord tissues and the phosphorylation of signal transduction and transcription activator 3 (STAT3). The expression of Notch1 intracellular domain (NICD) and GFAP in spinal cord tissues was detected by immunofluorescence. Furthermore, the infiltration of inflammatory cells and the demyelination of spinal cord were observed using HE and Luxol fast blue (LFB) staining methods. Results:Compared with PBS group, A1 astrocytes were activated and Notch1 expression was significantly up-regulated in EAE group and LV-ctrl+ EAE group. The clinical score of mice in LV-Inhibit-Gm13568+ EAE group was decreased from an average score of 3.5 to less than 1 on 23 d after antigen induction and the clinical symptoms were alleviated as compared with the mice in LV-ctrl+ EAE group. Meanwhile, the activation of A1 astrocytes was down-regulated, and the production of inflammatory cytokines and chemokines was also reduced. The expression of Notch1, GFAP and NICD at protein level and the phosphorylation of STAT3 were significantly reduced. Moreover, the infiltration of inflammatory cells and demyelination of spinal cord tissues were alleviated significantly.Conclusions:LncRNA Gm13568 might regulate the activation of A1 astrocytes via the Notch1/STAT3 pathway, thus affecting the production of inflammatory cytokines and chemokines and participating in the process of EAE.

Effect of achyranthes bidentata polysaccharides on the differentiation of bone mesenchymal stem cells into nucleus nulposus-like cells via Notch1 pathway / 西安交通大学学报(医学版)

【Objective】 To investigate the effect and mechanism of achyranthes bidentata polysaccharides （ABPS） on the differentiation of bone mesenchymal stem cells （BMSCs） into nucleus pulposus-like cells. 【Methods】 Five 4-week-old SD rats were sacrificed and their BMSCs were isolated and purified by repeated adherent method. The third-generation BMSCs were treated with different concentrations of ABPS for 48 h. The cell viability was detected by MTT method. The highest concentration of ABPS was selected for subsequent experiments. According to different treatment methods， cells were divided into control group （conventional culture）， ABPS group （200 mg/L of ABPS）， Notch1 group （transfected with Notch1）， Notch1 negative control group （transfected with Notch1 negative control）， and ABPS+Notch1 group （200 mg/L of ABPS was added after transfection with Notch1）. After 14 days of induction， the cell survival rate was measured by MTT method. CollagenⅡ was detected by immunohistochemical staining. The mRNA and protein expression levels of collagenⅡ， aggrecan， Notch1 and Hes1 were detected by qPCR and Western Blotting respectively. The glucosaminoglycan （GAG） was detected by alcian blue method on the 1st， 4th， 7th， 10th and 14th day of cell culture. 【Results】 After BMSCs were treated with 400 mg/L of ABPS for 48 h， the cell survival rate was significantly lower than that in the control group （P<0.05）. When the concentration was ≤200 mg/L， ABPS had no significant toxic effect on the growth of BMSCs. After 14 days of induction， compared with the control group， A value， GAG content in the medium， the expressions of collagen II and aggrecan mRNA and protein in the ABPS group were increased， and the expressions of Notch1 and Hes1 mRNA and protein were decreased （P<0.05）. The relative cell viability， GAG content in the medium， the expressions of collagen II and aggrecan mRNA and protein in the Notch1 group were decreased， and the expressions of Notch1 and Hes1 mRNA and protein were increased （P<0.05）. ABPS+Notch1 could partially reverse the inhibitory effect of Notch1 overexpression on the differentiation of BMSCs into nucleus pulposus-like cells. 【Conclusion】 ABPS can promote the differentiation of BMSCs into nucleus pulposus-like cells， and the mechanism may be related to the inhibition of Notch1 pathway.

Chidamide inhibits the NOTCH1-MYC signaling axis in T-cell acute lymphoblastic leukemia / 医学前沿

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most dangerous hematological malignancies, with high tumor heterogeneity and poor prognosis. More than 60% of T-ALL patients carry NOTCH1 gene mutations, leading to abnormal expression of downstream target genes and aberrant activation of various signaling pathways. We found that chidamide, an HDAC inhibitor, exerts an antitumor effect on T-ALL cell lines and primary cells including an anti-NOTCH1 activity. In particular, chidamide inhibits the NOTCH1-MYC signaling axis by down-regulating the level of the intracellular form of NOTCH1 (NICD1) as well as MYC, partly through their ubiquitination and degradation by the proteasome pathway. We also report here the preliminary results of our clinical trial supporting that a treatment by chidamide reduces minimal residual disease (MRD) in patients and is well tolerated. Our results highlight the effectiveness and safety of chidamide in the treatment of T-ALL patients, including those with NOTCH1 mutations and open the way to a new therapeutic strategy for these patients.

Study on the improvement of Compound danshen tablets on blood lipid levels and the mechanism of protecting renal functions in hyperlipidemia model rats / 中国药房

OBJECTIVE To investigate the effect of Compound danshen tablet s o n improving the blood lipid levels and the mechanism of protecting renal functions in hyperlipidemia model rats. METHODS Sixty male SD rats were divided into normal group，model group ，simvastatin combined with （2S）-N-[N-（3，5-difluorophenylacetyl）-L-alanyl]-2-phenylglycine tert butyl （DAPT）group and low-dose ，medium-dose and high-dose groups of Compound danshen tablets ，with 10 rats in each group. Rats in the normal group received routine diet. The other 5 groups were intraperitoneally injected with 75％ yolk emulsion 10 mL/kg， fasting and drinking freely. After 16 h，they were fed high-fat diet for 4 weeks. Simvastatin combined with DAPT group was given simvastatin 0.002 g/kg and DAPT 0.012 g/kg at the same time of modeling. The low-dose ，medium-dose and high-dose groups of Compound danshen tablets were given Compound danshen tablets 0.25，0.5 and 1 g/kg respectively at the same time of modeling ， the normal group and model group were given equal volume of distilled water ，once a day ，for 4 weeks. The serum levels of total cholesterol（TC），triglyceride（TG），creatinine（Cr）and urea nitrogen （BUN）in serum were detected by biochemical method ； kidney coefficient of rats was calculated ；histopathological changes of rat kidney were observed by HE staining ，and the renal injury was scored according to the degree of renal tubular injury and glomerular sclerosis in renal cortex ；expression levels of Notch signal receptor 1（Notch 1），Notch signal ligand 1（Jagged1）and hairy division associated enhancer 1（Hes1）in kidney were detected by immunohistochemistry ；mRNA expressions of Notch 1，Jagged1 and Hes 1 in renal tissue were detected by real-time fluorescence quantitative polymerase chain reaction. RESULTS Compared with normal group ，the serum levels of TG ， TC，Cr and BUN were increased significantly in model group （P＜0.05）；renal coefficient increased significantly （P＜0.05）； pathological changes occurred in renal tissue ，and the scores of renal tubular injury and glomerular sclerosis increased significantly （P＜0.05）；protein and mRNA expressions of Notch1， Jagged1， Hes1 in renal tissue were increased significantly（P＜0.05）. Compared with model group ，serum levels of TG ，TC，Cr and BUN ，renal coefficient ，the scores of renal tubular injury and glomerular sclerosis ，protein and mRNA expression of Notch 1，Jagged1 and Hes 1 in renal tissue were all decreased in low-dose ，medium-dose and high-dose groups of Compound danshen tablets （P＜0.05），and most indexes showed a dose-dependent trend ；the degree of renal lesions was reduced. CONCLUSIONS Compound danshen tablets possess obvious hypolipidemic effect ，and can protect the renal function of hyperlipidemia model rats by down-regulating Notch 1/Jagged1 signal pathway.

Relationship between the Expression of miRNA181a-5p and the Imbalance of Treg/Th17 in Primary Immune Thrombocytopenia / 中国实验血液学杂志

OBJECTIVE@#To investigate the role of relationship between the expression of miRNA181a-5p and imbalance of Treg/Th17 in the pathogenesis of primary immune thrombocytopenia(ITP), which contributes to clarify the mechanism of T cell immune imbalance in ITP patients.@*METHODS@#Peripheral blood was collected from 37 ITP patients, concluding 21 untreated patients and 16 effectively treated patients, and 19 healthy controls; Peripheral blood mononuclear cells (PBMC) were isolated and the expression of miRNA181a-5p and Notch1 was analyzed by RT-PCR. The proportion of Th17 subsets and Treg cells in the peripheral circulation was detected by flow cytometer (FCM). Clinical data of ITP group was collected, including age, platelet count and disease course.@*RESULTS@#The expression of miR-181a-5p was significantly decreased in ITP group than that of healthy control group (P＜0.01). After effective treatment, the expression of miR-181a-5p was significantly higher than that of ITP group (P＜0.05), but still significantly lower than that of healthy control group (P＜0.01); The expression of Notch1 was significantly increased in ITP group and effectively treated group than that of healthy control group (P＜0.01). There was no significant difference in proportion of Treg cells in ITP group, effectively treated group and healthy control group (P＞0.05). The proportion of Th17 subsets in ITP group was significantly increased than that of healthy control group (P＜0.05), while the ratio of Treg/Th17 was significantly decreased (P＜0.05). There was a positive correlation between the expression of miR-181a-5p and ratio of Treg/Th17 in ITP group (r=0.555).@*CONCLUSION@#The expression of miR-181a-5p is significantly decreased in ITP patients, which is closely related to the imbalance of Treg/Th17 cells. After effective treatment, the expression of miR-181a-5p can be significantly corrected, but still failed to reach the level of healthy people. While the expression of Notch1 is significantly increased in ITP patients, and could not reach the level of healthy people after effective treatment.

Mechanism of compound Fufangteng mixture-containing serum in promoting proliferation of bone marrow mesenchymal stem cell / 器官移植

Objective To investigate the effect of compound Fufangteng mixture-containing serum on the proliferation of bone marrow mesenchymal stem cell (BMSC) and its mechanism. Methods Rat BMSC were isolated, cultured and purified in vitro by direct adherence method. Cell morphology was observed. Surface markers were identified by flow cytometry. The rats were treated with compound Fufangteng mixture at a dose of 3 mL/(kg·d) by gavage for 14 d, and then the drug-containing serum was collected. BMSC were divided into the blank control group, drug-containing serum group, Notch1 small interfering ribonucleic acid (siRNA) group and Notch1 siRNA+drug-containing serum group. The proliferation rate of BMSC was detected and the relative expression levels of Notch1 signaling pathway-associated messenger ribonucleic acid (mRNA) and proteins were measured in each group. Results Microscopic observation showed that the first generation BMSC were seen in the long spindle shape, and grown in the parallel or spiral pattern. The third generation BMSC positively expressed CD90 and CD44, whereas were negative for CD45. Compared with the blank control group, the proliferation rate of BMSC in the drug-containing serum group and Notch1 siRNA+ drug-containing serum group was significantly increased, whereas that of BMSC was significantly decreased in the Notch1 siRNA group (all P < 0.05). Compared with the Notch1 siRNA group, the proliferation rate of BMSC was significantly increased in the Notch1 siRNA+drug-containing serum group (P < 0.05). Compared with the blank control group, the relative expression levels of Hey1 and Delta-like ligand (DLL)1 mRNA and proteins were significantly up-regulated in the drug-containing serum group, whereas those were significantly down-regulated in the Notch1 siRNA group and Notch1 siRNA+drug-containing serum group (all P < 0.05). Compared with the Notch1 siRNA group, the relative expression levels of Hey1 and DLL1 mRNA and proteins were significantly up-regulated in the Notch1 siRNA+drug-containing serum group (all P < 0.05). Conclusions Compound Fufangteng mixture-containing serum may promote the proliferation of rat BMSC, and its mechanism is probably associated with the activation of Notch1 signaling pathway.

Effects of Notch1 signaling on histone acetylation of Foxp3 gene in children with B-cell precursor acute lymphoblastic leukemia / 中华微生物学和免疫学杂志

Objective:To investigate the effects of Notch1 signaling on histone acetylation of Foxp3 gene and its roles in regulating regulatory T (Treg) cells in children with acute B-cell precursor lymphoblastic leukemia (BCP-ALL).Methods:Blood samples were collected form 38 children with BCP-ALL before treatment and 15 age-matched healthy children (control group). Flow cytometry was performed to detect the proportion of peripheral blood CD4 + CD25 hiFoxp3 + Treg cells and the expression of Foxp3, cytotoxic lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumor necrosis factor receptor (GITR), CD39 and Notch1 at protein level. Histone 4 acetylation (H4Ac) at Foxp3 gene promoter and the binding abilities of Foxp3 gene promoter to NICD1 and p300 in CD4 + T cells were measured by chromatin immunoprecipitation. Quantitative real-time PCR was performed to detect the expression of Foxp3, presenilin 1 (PSEN1), mastermind-like transcriptional coactivator 1 (MAML1), SKI-interacting protein (SKIP), F-box and WD40 domain protein 7 (FBXW7), glycogen synthase kinase-3 beta (GSK3β) and IKAROS at mRNA level in CD4 + T cells. The concentrations of TGF-β and IL-10 in plasma were evaluated by ELISA. Results:(1) The proportion of peripheral blood CD4 + CD25 hiFoxp3 + Treg cells, the expression of differentiation- and function-associated molecules (Foxp3, CTLA4, GITR and CD39) and the concentrations of TGF-β and IL-10 in plasma were higher in the BCP-ALL group than in the control group ( P<0.05). (2) In children with acute BCP-ALL, H4Ac at Foxp3 promoter and the binding abilities of Foxp3 gene promoter to NICD1 and p300 were significantly increased as compared with those in control group( P<0.05). The binding abilities of Foxp3 gene promoter to NICD1 and p300 were positively correlated with the expression of Foxp3 at mRNA level ( r=0.58 and 0.46, both P<0.05). After competitive inhibition, the three aforementioned indexes in the acute BCP-ALL group were significantly lower than those in untreated group ( P<0.05); the binding ability of Foxp3 gene promoter to NICD1 in the control group was also significantly lower than that in untreated control group ( P<0.05), but no statistical differences in the other two indexes were found between the control groups with or without treatment ( P>0.05). ⑶ Compared with the control group, the expression of Notch1, PSEN1, MAML1 and SKIP in CD4 + T cells were elevated significantly ( P<0.05), while the transcription level of negative regulatory factor FBXW7 was decreased remarkably in children with acute BCP-ALL ( P<0.05). No statistical differences in the expression of GSK3β or IKAROS were found between the two groups ( P>0.05). Conclusions:Overactivation of Notch1 signaling caused by low expression of FBXW7 might be the key factor resulting in histone 4 hyperacetylation at foxp3 gene promoter and Treg cell dysfunction in children with acute BCP-ALL.