[Resveratrol Inhibits T-acute Lymphoblastic Leukemia in Mice by Regulating Notch1 Signaling Pathway].

OBJECTIVE: To observe the effect of resveratrol (Res) on T-acute lymphoblastic leukemia (T-ALL) mice, and further explore its mechanism on Notch1 signaling pathway. METHODS: Twenty-five 6-8 weeks old female C57BL/6 mice were randomly divided into control group, T-ALL group and Res group. Res group was further divided into low-Res, middle-Res and high-Res group. The percentage of leukemia cells in peripheral blood and spleen cell suspension were detected by flow cytometry and Wright-Giemsa staining, pathological morphology of spleen and bone marrow tissues were observed by HE staining, the expression levels of Notch1, Hes-1, c-Myc, miR-19b and PTEN mRNA in spleen tissue were detected by RT-qPCR, and the protein levels of Notch1, Hes-1, c-Myc, p-PTEN and PTEN were detected by Western blot. RESULTS: Compared with control group, the leukemia cells in peripheral blood of mice in T-ALL group were markedly increased, accompanied by diffuse infiltration of leukemia cells in spleen and bone marrow tissues, the mRNA levels of Notch1, Hes-1, c-Myc, miR-19b and the protein levels of Notch1, Hes-1, c-Myc were increased (P <0.01), while the expression of PTEN mRNA and protein were significantly decreased in the spleen tissue of T-ALL mice (P <0.01). The above indicators in the H-Res group were reversed compared with T-ALL group after administration of resveratrol. CONCLUSION: Resveratrol may play a role in anti T-ALL by inhibiting Notch1 signaling pathway in mice.

The Jagged-1/Notch1 Signaling Pathway Promotes the Construction of Tissue-Engineered Heart Valves.

Background: Tissue-engineered heart valves (TEHVs) are promising new heart valve substitutes for valvular heart disease. The Notch signaling pathway plays a critical role in the development of congenital heart valves. Objective: To investigate the role of the Notch signaling pathway in the construction of TEHVs. Methods: The induced endothelial cells, which act as seed cells, were differentiated from adipose-derived stem cells and were treated with Jagged-1 (JAG-1) protein and γ-secretase inhibitor (DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycine t-butyl ester), respectively. Cell phenotypic changes, the expression of proteins relating to the epithelial-mesenchymal transition (EMT), and changes in paxillin expression were detected. Decellularized valve scaffolds were produced from decellularized porcine aortic valves. The seed cells were them inoculated into Matrigel-coated flap scaffolds for complex culture and characterization. Results: JAG-1 significantly reduced apoptosis and promoted the seeded cells' proliferation and migration ability, in contrast to the treatment of DAPT. In addition, the expression of EMT-related proteins, E-cadherin and N-cadherin, was significantly increased after treatment with JAG-1 and was reduced after the application of DAPT. Meanwhile, the adhesive-related expression of paxillin and fibronectin proteins was increased after the activation of Notch1 signaling and vice versa. Of interest, activation of the Notch1 signaling pathway resulted in more closely arranged cells on the valve surface after recellularization. Conclusion: Activation of the JAG-1/Notch1 signaling pathway increased seeded cells' proliferation and migratory ability and promoted the EMT and adhesion of seed cells, which was conducive to binding to the matrix, facilitating accelerated endothelialization of TEHVs.

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.

Dendrobium officinale polysaccharide Converts M2 into M1 Subtype Macrophage Polarization via the STAT6/PPAR-r and JAGGED1/NOTCH1 Signaling Pathways to Inhibit Gastric Cancer.

Dendrobium officinale polysaccharide (DOP) has shown various biological activities. However, the ability of DOP to participate in immune regulation during anti-gastric cancer treatment has remained unclear. In this study, the in vitro results showed that DOP has the potential to polarize THP-1 macrophages from the M2 to the M1 phenotype, downregulate the STAT6/PPAR-r signaling pathway and the protein expression of their down-targeted ARG1 and TGM2, and further decrease the main protein and mRNA expression in the JAGGED1/NOTCH1 signaling pathway. DOP suppressed the migration of gastric cancer cells by decreasing the protein expression of N-cadherin and Vimentin and increasing E-cadherin. In addition, CM-DOP promoted the apoptosis of gastric cancer cells by upregulating Caspase-3 and increasing the ratio of Bax/Bcl-2. In vivo, DOP effectively inhibited the growth of tumors and the expression of Ki-67. In summary, these findings demonstrated that DOP converted the polarization of M2 subtype macrophages into M1 subtypes via the STAT6/PPAR-r and JAGGED1/NOTCH1 signaling pathways in order to reduce apoptosis and prevent migration, thus indicating the potential of DOP as an adjuvant tumor therapy in preclinical and clinical trials.

Salsolinol improves angiotensin II­induced myocardial fibrosis in vitro via inhibition of LSD1 through regulation of the STAT3/Notch­1 signaling pathway.

The clinical incidence of congestive heart failure (CHF) is very high and it poses a significant threat to the health of patients. The traditional Chinese medicine monomer salsolinol is widely used to treat similar symptoms of CHF. However, there have been no reports on the effect of salsolinol for the management of CHF and its effects on myocardial fibrosis. In the present study, salsolinol was used to treat angiotensin II (AngII)-induced human cardiac fibroblasts (HCFs) and cell proliferation and migration were assessed using a CCK-8, EdU staining assay and wound healing assay. Subsequently, immunofluorescence, western blotting and other techniques were used to detect indicators associated with cell fibrosis and relevant kits were used to detect markers of cellular inflammation and reactive oxygen species (ROS) production. Molecular docking analysis was used to predict the relationship between salsolinol and lysine-specific histone demethylase 1A (LSD1). Subsequently, the expression of LSD1 in the serum of CHF patients was detected by reverse transcription-quantitative PCR. Finally, LSD1 was overexpressed in cells to explore the regulatory mechanism of salsolinol in AngII-induced HFCs. Salsolinol reduced the proliferation and migration. Salsolinol reduced the expression of fibrosis marker proteins α-smooth muscle actin, Collagen I and Collagen III in a concentration-dependent manner, thereby reducing cell fibrosis. In addition, salsolinol reduced the levels of TNF-α and IL-6 in the cell supernatant and ROS production following AngII induction. Salsolinol inhibited LSD1 expression and regulated the STAT3/Notch-1 signaling pathway. Upregulation of LSD1 reversed the effects of salsolinol on AngII-induced HCFs. Salsolinol inhibited LSD1 via regulation of the STAT3/Notch-1 signaling pathway to improve Ang II-induced myocardial fibrosis in vitro.

Hypoxia promotes conversion to a stem cell phenotype in prostate cancer cells by activating HIF-1a/Notch1 signaling pathway

Purpose The hypoxic tumor microenvironment and the maintenance of stem cells are relevant to the malignancy of prostate cancer (PCa). However, whether HIF-1α in the hypoxic microenvironment mediates the transformation of prostate cancer to a stem cell phenotype and the mechanism have not been elucidated.Materials and methods Prostate cancer stem cells (PCSCs) from PC-3 cell lines were examined for the expression of CD44, CD133, ALDH1, HIF-1α, Notch1, and HES1. We observed the effect of knockdown HIF-1α in vitro and mice models and evaluated the impact of HIF-1α on the Notch1 pathway as well as stem cell dedifferentiation. The effects on sphere formation, cell proliferation, apoptosis, cell cycle, and invasive metastasis were evaluated. Results In our study, hypoxia upregulated HIF-1α expression and induced a stem cell phenotype through activation of the Notch1 pathway, leading to enhanced proliferation, invasion, and migration of PCa PC-3 cells. The knockdown of HIF-1α significantly inhibited cell dedifferentiation and the ability to proliferate, invade and metastasize. However, the inhibitory effect of knocking down HIF-1α was reversed by Jagged1, an activator of the Notch1 pathway. These findings were further confirmed in vivo, where hypoxia could enhance the tumorigenicity of xenograft tumors by upregulating the expression of HIF-1α to activate the Notch1 pathway. In addition, the expression of HIF-1α and Notch1 was significantly increased in human PCa tissues, and high expression of HIF-1α correlated with the malignancy of PCa. Conclusion In a hypoxic environment, HIF-1α promotes PCa cell dedifferentiation to stem-like cell phenotypes by activating the Notch1 pathway and enhancing the proliferation and invasive capacity of PC-3 cells (AU)

MiR-210 improves postmenopausal osteoporosis in ovariectomized rats through activating VEGF/Notch signaling pathway.

BACKGROUND: To explore the effect and mechanism of action of miR-210 on postmenopausal osteoporosis (PMPO) in ovariectomized rats in vivo. METHODS: An ovariectomized (OVX) rat model was established by ovariectomy. Tail vein injection was performed to overexpress and knock down miR-210 in OVX rats, followed by the collection of blood and femoral tissues from each group of rats. And quantitative real-time polymerase chain reaction (qRT-PCR) was applied to assess the expression level of miR-210 in femoral tissues of each group. Micro computed tomography (Micro CT) was adopted to scan the microstructure of the femoral trabecula in each group to obtain relevant data like bone mineral density (BMD), bone mineral content (BMC), trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), bone surface-to-volume ratio (BS/BV), and trabecular separation (Tb.Sp). ELISA was used for determining the level of bone alkaline phosphatase (BALP), amino-terminal propeptide of type I procollagen (PINP), osteocalcin (OCN), and C-terminal telopeptide of type I collagen (CTX-1) in serum; and Western blot for the protein level of Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and collagen type I alpha 1 (COL1A1) in femoral tissues. RESULTS: MiR-210 expression was significantly decreased in femoral tissues of OVX rats. Overexpression of miR-210 could obviously increase BMD, BMC, BV/TV and Tb.Th, whereas significantly decrease BS/BV and Tb.Sp in femurs of OVX rats. Moreover, miR-210 also downregulated BALP and CTX-1 level, upregulated PINP and OCN level in the serum of OVX rats promoted the expression of osteogenesis-related markers (Runx2, OPN and COL1A1) in the femur of OVX rats. Additionally, further pathway analysis revealed that high expression of miR-210 activated the vascular endothelial growth factor (VEGF)/Notch1 signaling pathway in the femur of OVX rats. CONCLUSION: High expression of miR-210 may improve the micromorphology of bone tissue and modulate bone formation and resorption in OVX rats by activating the VEGF/Notch1 signaling pathway, thereby alleviating osteoporosis. Consequently, miR-210 can serve as a biomarker for the diagnosis and treatment of osteoporosis in postmenopausal rats.

Effect of the Lipoxin Receptor Agonist BML-111 on Cigarette Smoke Extract-Induced Macrophage Polarization and Inflammation in RAW264.7 Cells.

Background: Macrophages are known to play a crucial role in the chronic inflammation associated with Chronic Obstructive Pulmonary Disease (COPD). BML-111, acting as a lipoxin A4 (LXA4) receptor agonist, has shown to be effective in protecting against COPD. However, the precise mechanism by which BML-111 exerts its protective effect remains unclear. Methods: In order to establish a cell model of inflammation, cigarette smoke extract (CSE) was used on the RAW264.7 cell line. Afterwards, an Enzyme-linked immunosorbent assay (ELISA) kit was employed to measure concentrations of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1ß), interleukin-18 (IL-18), and interleukin-10 (IL-10) in the cell supernatants of the RAW264.7 cells.In this study, we examined the markers of macrophage polarization using two methods: quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Additionally, we detected the expression of Notch-1 and Hes-1 through Western blotting. Results: BML-111 effectively suppressed the expression of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-18, as well as inflammasome factors NLRP3 and Caspase-1, while simultaneously up-regulating the expression of the anti-inflammatory cytokine IL-10 induced by CSE. Moreover, BML-111 reduced the expression of iNOS, which is associated with M1 macrophage polarization, and increased the expression of Arg-1, which is associated with M2 phenotype. Additionally, BML-111 downregulated the expression of Hes-1 and the ratio of activated Notch-1 to Notch-1 induced by CSE. The effect of BML-111 on inflammation and macrophage polarization was reversed upon administration of the Notch-1 signaling pathway agonist Jagged1. Conclusion: BML-111 has the potential to suppress inflammation and modulate M1/M2 macrophage polarization in RAW264.7 cells. The underlying mechanism may involve the Notch-1 signaling pathway.

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

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.

Abnormal mechanical stress on bicuspid aortic valve induces valvular calcification and inhibits Notch1/NICD/Runx2 signal.

Background: Bicuspid aortic valve (BAV) is a congenital cardiac deformity, increasing the risk of developing calcific aortic valve disease (CAVD). The disturbance of hemodynamics can induce valvular calcification, but the mechanism has not been fully identified. Methods: We constructed a finite element model (FEM) of the aortic valve based on the computed tomography angiography (CTA) data from BAV patients and tricuspid aortic valve (TAV) individuals. We analyzed the hemodynamic properties based on our model and investigated the characteristics of mechanical stimuli on BAV. Further, we detected the expression of Notch, NICD and Runx2 in valve samples and identified the association between mechanical stress and the Notch1 signaling pathway. Results: Finite element analysis showed that at diastole phase, the equivalent stress on the root of BAV was significantly higher than that on the TAV leaflet. Correspondingly, the expression of Notch1 and NICH decreased and the expression of Runx2 elevated significantly on large BAV leaflet belly, which is associated with equivalent stress on leaflet. Our findings indicated that the root of BAV suffered higher mechanical stress due to the abnormal hemodynamic environment, and the disturbance of the Notch1/NICD/Runx2 signaling pathway caused by mechanical stimuli contributed to valvular calcification.

Decreased NOTCH1 signaling activated autophagy in the mid-secretory endometrium of patients with recurrent implantation failure†.

Recurrent implantation failure severely impairs fertility in females of childbearing age, which poses a great challenge to assisted reproductive technology, and its etiology is still unclear. Several studies have demonstrated that endometrial autophagy takes an important part in human endometrial receptivity, but its role in recurrent implantation failure remains largely unknown. Here, we collected mid-secretory endometrial tissue from recurrent implantation failure patients and fertile controls during menstruation and early pregnancy. Immunohistochemistry, western blotting, and quantitative real-time PCR were performed to compare the expression of microtubule-associated protein 1 light chain 3B, sequestosome 1, NOTCH1 signaling pathway members, and endometrial receptivity markers between recurrent implantation failure and control groups. In addition, to assess endometrial autophagy, transmission electron microscopy was used to observe autophagosomes. By RNA interference, we further investigated the effects of NOTCH1 on autophagy in Ishikawa cells. We found that endometrial autophagy was upregulated in the mid-secretory and decidual phases than in the early-proliferative phase. Compared to the control group, more autophagosomes were observed in the mid-secretory endometrium of recurrent implantation failure patients, which was accompanied by the downregulation of NOTCH1 signaling pathway members and endometrial receptivity markers. Moreover, knockdown of NOTCH1 impaired the receptivity of Ishikawa cells via protein kinase B/mammalian target of rapamycin pathway-mediated autophagy activation. Our data suggested that abnormally elevated autophagy and decreased NOTCH1 signaling pathway activity were observed in the mid-secretory endometrium of patients with recurrent implantation failure, perhaps due to decreased NOTCH1 pathway-mediated autophagy activation in endometrial cells impairing receptivity.

Hypoxia promotes conversion to a stem cell phenotype in prostate cancer cells by activating HIF-1α/Notch1 signaling pathway.

PURPOSE: The hypoxic tumor microenvironment and the maintenance of stem cells are relevant to the malignancy of prostate cancer (PCa). However, whether HIF-1α in the hypoxic microenvironment mediates the transformation of prostate cancer to a stem cell phenotype and the mechanism have not been elucidated. MATERIALS AND METHODS: Prostate cancer stem cells (PCSCs) from PC-3 cell lines were examined for the expression of CD44, CD133, ALDH1, HIF-1α, Notch1, and HES1. We observed the effect of knockdown HIF-1α in vitro and mice models and evaluated the impact of HIF-1α on the Notch1 pathway as well as stem cell dedifferentiation. The effects on sphere formation, cell proliferation, apoptosis, cell cycle, and invasive metastasis were evaluated. RESULTS: In our study, hypoxia upregulated HIF-1α expression and induced a stem cell phenotype through activation of the Notch1 pathway, leading to enhanced proliferation, invasion, and migration of PCa PC-3 cells. The knockdown of HIF-1α significantly inhibited cell dedifferentiation and the ability to proliferate, invade and metastasize. However, the inhibitory effect of knocking down HIF-1α was reversed by Jagged1, an activator of the Notch1 pathway. These findings were further confirmed in vivo, where hypoxia could enhance the tumorigenicity of xenograft tumors by upregulating the expression of HIF-1α to activate the Notch1 pathway. In addition, the expression of HIF-1α and Notch1 was significantly increased in human PCa tissues, and high expression of HIF-1α correlated with the malignancy of PCa. CONCLUSION: In a hypoxic environment, HIF-1α promotes PCa cell dedifferentiation to stem-like cell phenotypes by activating the Notch1 pathway and enhancing the proliferation and invasive capacity of PC-3 cells.

Gastrodin Regulates the Notch-1 Signal Pathway via Renin-Angiotensin System in Activated Microglia.

Notch-1 and renin angiotensin system (RAS) are involved in microglia activation. It has been reported that gastrodin inhibited inflammatory responses mediated by activated microglia. This study explored the possible interaction between this two pathways, and to determine whether gastrodin would exert its effects on both of them. Expression of RAS, Notch-1 signaling and proinflammatory mediators in lipopolysaccharide (LPS) activated BV-2 microglia subjected to various treatments was determined by Western blot and immunofluorescence. The protein expression of RAS, Notch-1 pathway and TNF-α and IL-1ß was significantly increased in activated microglia. Exogenous Ang II markedly enhanced the expression of these biomarkers. Meanwhile, Azilsartan [a specific inhibitor of AT1 (AT1I)] inhibited the expression of Notch-1 pathway and proinflammatory cytokines. When Notch-1 signaling was inhibited with DAPT, ACE and AT1 expression remained unaffected, indicating that RAS can regulate the Notch-1 pathway in activated microglia but not reciprocally. Additionally, we showed here that gastrodin inhibited the RAS, Notch-1 pathway and inflammatory response. Remarkably, gastrodin did not exert any effect on expression of Notch-1 signaling when RAS was blocked by AT1I, suggesting that gastrodin acts on the RAS directly, not through the Notch-1 pathway. Furthermore, TNF-α and IL-1ß expression was significantly increased in activated microglia treated with exogenous Ang II; the expression, however, was suppressed by gastrodin. Of note, expression of proinflammatory cytokines was further decreased in gastrodin and AT1I combination treatment. The results suggest that gastrodin acts via the RAS which regulates the Notch-1 signaling and inflammation in LPS-induced microglia.

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.

MECP2 promotes the migration and invasion of gastric cancer cells by modulating the Notch1/c-Myc/mTOR signaling pathways by suppressing FBXW7 transcription.

Methyl-CpG-binding protein 2 (MECP2), an epigenetic regulatory factor, promotes the carcinogenesis and progression of a number of cancers. However, its role in the migration and invasion of gastric cancer (GC), as well as the underlying molecular mechanisms, remain unclear. In this study, we found that MECP2 promoted the migration, invasion and metastasis of GC cells. Investigation of the molecular mechanism revealed that MECP2 repressed F-box and WD40 domain protein 7 (FBXW7) transcription in GC by binding to the methylated CpG sites in the FBXW7 promoter region. MECP2 expression was markedly negatively correlated with the FBXW7 level in GC tissues. FBXW7 expression was significantly downregulated in GC tissues and cell lines, and low FBXW7 expression was correlated with unfavorable clinicopathologic features. FBXW7 inhibited cell migration and invasion by regulating the Notch1/c-Myc/mTOR signaling pathways, and knockdown of FBXW7 reversed the effects of silencing MECP2. Moreover, MECP2 upregulated the Notch1/c-Myc/mTOR signaling pathways by inhibiting FBXW7 expression at the transcriptional level. This study demonstrates that MECP2 promotes the migration and invasion of GC cells by modulating the Notch1/c-Myc/mTOR signaling pathways via suppression of FBXW7 transcription. These findings suggest that MECP2 may be a novel effective therapeutic target in GC.

IL-7-Treated Periodontal Ligament Cells Regulate Local Immune Homeostasis by Modulating Treg/Th17 Cell Polarization.

Both interleukin (IL)-7 and human periodontal ligament cells (hPDLCs) have immunomodulatory properties. However, their combined effect on CD4+T cells has never been studied. In this study, we aimed to investigate the effect of conditioned medium of hPDLCs treated with rhIL-7 on the differentiation of CD4+T cells into regulatory T cells/T helper 17 cells (Treg/Th17 cells) and observe the effect of IL-7 on the immunomodulatory properties of PDLCs. After hPDLCs were treated with different concentrations of rhIL-7 for 24 h, the collected supernatants were used to incubate CD4+T cells for 3 days. A gamma-secretase inhibitor (DAPT) was used to suppress the activation of the Notch1 signaling pathway. Cell proliferation, apoptosis, and necrosis were determined using the cell counting kit-8 (CCK-8) and flow cytometry (FCM). The expressions of forkhead box P3 (Foxp3) in CD4+T cells and transforming growth factor (TGF-ß) and IL-6 in the supernatants were determined by ELISA. Reverse transcription-quantitative PCR (RT-qPCR), and the Western blot (WB) determined the mRNA levels and protein expression of various target factors. FCM was used to detect the mean fluorescence intensity of PD-L1 in hPDLCs and to analyze the differentiation of Treg/Th17 cells. Our results showed that IL-7 promoted proliferation and inhibited apoptosis in hPDLCs, promoted the expression of TGF-ß, PD-L1, Notch1, Jagged1, and Hes1, and inhibited the levels of hypoxia-inducible factor (HIF)-1α and TCF7, whereas the addition of DAPT effectively reversed these effects. Importantly, we found that the conditioned medium of hPDLCs treated with rhIL-7 promoted the polarization of CD4+T cells into Treg cells but had no significant effect on the differentiation of Th17 cells. Our study indicated that treatment of PDLCs with IL-7 can promote the polarization of CD4+T cells into Treg cells by modulating the expression of inflammatory factors and signaling molecules through activating the Notch1 signaling pathway, thus participating in the regulation of immune homeostasis in the periodontal microenvironment.

Transgelin-2 interacts with CD44 to regulate Notch1 signaling pathway and participates in colorectal cancer proliferation and migration

The abnormal expression of transgelin-2 (TAGLN2) is related to tumor occurrence and progression. However, the underlying molecular mechanism of TAGLN2 in human colorectal cancer (CRC) is still poorly understood. Compared with adjacent tissues, TAGLN2 is overexpressed in CRC tissues. Its expression level is negatively correlated with the overall survival rate of patients with CRC. In addition, knockdown of TAGLN2 inhibited the proliferation and invasion of CRC cells. We also showed that TAGLN2 could interact with CD44 to regulate the Notch-1 signaling pathway. Our findings indicate there is increased TAGLN2 expression in CRC and that it may serve as a promising potential therapeutic target for CRC. (AU)

Intrauterine growth restriction neonates present with increased angiogenesis through the Notch1 signaling pathway.

Intrauterine growth restriction (IUGR) is associated with increased perinatal mortality and morbidity, and plays an important role in the development of adult cardiovascular diseases. This study brings forward a hypothesis that Human umbilical vein endothelial cells (HUVECs) from IUGR newborns present dysfunctions and varying changes of signaling pathways as compared to the Control group. Similar pathways may also be present in pulmonary or systemic vasculatures. HUVECs were derived from newborns. There were three groups according to the different fetal origins: normal newborns (Control), IUGR from poor maternal nutrition (IUGR1), and pregnancy-induced hypertension (IUGR2). We found that IUGR-derived HUVECs showed a proliferative phenotype compared to those from normal subjects. Interestingly, two types IUGR could cause varying degrees of cellular dysfunction. Meanwhile, the Notch1 signaling pathway showed enhanced activation in the two IUGR-induced HUVECs, with subsequent activation of Akt or extracellular signal regulated protein kinases1/2 (ERK1/2). Pharmacological inhibition or gene silencing of Notch1 impeded the proliferative phenotype of IUGR-induced HUVECs and reduced the activation of ERK1/2 and AKT. In summary, elevated Notch1 levels might play a crucial role in IUGR-induced HUVECs disorders through the activation of ERK1/2 and AKT. These pathways could be potential therapeutic targets for prevention of the progression of IUGR associated diseases later in life.

Transgelin-2 interacts with CD44 to regulate Notch1 signaling pathway and participates in colorectal cancer proliferation and migration.

The abnormal expression of transgelin-2 (TAGLN2) is related to tumor occurrence and progression. However, the underlying molecular mechanism of TAGLN2 in human colorectal cancer (CRC) is still poorly understood. Compared with adjacent tissues, TAGLN2 is overexpressed in CRC tissues. Its expression level is negatively correlated with the overall survival rate of patients with CRC. In addition, knockdown of TAGLN2 inhibited the proliferation and invasion of CRC cells. We also showed that TAGLN2 could interact with CD44 to regulate the Notch-1 signaling pathway. Our findings indicate there is increased TAGLN2 expression in CRC and that it may serve as a promising potential therapeutic target for CRC.