Didymin ameliorates ulcerative colitis-associated secondary liver damage by facilitating Notch1 degradation.

BACKGROUND: Didymin is a dietary flavonoid originally discovered by our group as a potent anti-ulcerative colitis (UC) agent. However, whether didymin plays a protective role in UC-associated inflammatory liver injury is still unclear. PURPOSE: This study aimed to evaluate the therapeutic potential of didymin on UC-associated inflammatory liver injury and explore the underlying mechanism. STUDY DESIGN AND METHODS: Colitis model was established in C57BL/6 mice by exposure to DSS, and didymin was administrated intragastrically for consecutive 10 days. The inflammatory liver injury was assessed by levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum and histopathological damage in the liver. In vitro Kupffer cells and RAW264.7 cells challenged with lipopolysaccharides (LPS) were used to explore the modulatory activity of didymin on pro-inflammatory cytokines secretion and Notch1 signaling pathway activation. RESULTS: Didymin significantly mitigated liver coefficiency, ALT and AST levels in serum, and the hepatic histopathological damage caused by DSS-induced acute and chronic colitis. The mRNA expressions of pro-inflammatory factors including Tnf, Il1, and Il6 in liver tissues, Kupffer cells, and RAW264.7 cells stimulated by the influx of LPS was significantly deprived after didymin treatment. Mechanistically, didymin obstructed the protein expression, nuclear translocation of notch intracellular domain 1 (Notch1-ICD) and mRNA expression of hairy and enhancer of split 1 (Hes1). Further, the inhibitory mechanism of the Notch1-Hes1 pathway was dependent on c-Cbl-mediated Notch1-ICD lysosomal degradation. CONCLUSION: Our study verified for the first time that didymin could prevent UC-associated diseases, such as inflammatory liver injury, and the mechanism was related to facilitating Notch1 lysosomal degradation rather than proteasome degradation via promoting protein expression of c-Cbl in macrophages. Our findings that the inhibition of Notch1 signaling transduction helps to alleviate UC-associated liver injury provides possible therapeutics for the treatment of colitis and also furnishes a research paradigm for the study of flavonoids with similar structures.

[ORY-1001 inhibits glioblastoma cell growth by downregulating the Notch/HES1 pathway via suppressing lysine-specific demethylase 1 expression].

OBJECTIVE: To explore the inhibitory effect ORY-1001, a lysine-specific histone demethylase 1 (LSD1) inhibitor, on growth of glioblastoma (GBM) and the underlying mechanism. METHODS: We analyzed LSD1 expressions in GBM and normal brain tissues based on data from TCGA and HPA databases. Female BALB/c mouse models bearing xenografts derived from U87 cells or cells with lentivirus-mediated LSD1 silencing or Notch overexpression were treated with saline or 400 µg/kg ORY-1001 by gavage every 7 days, and GBM formation and survival time of the mice were recorded. The effect of ORY-1001 on GBM cell viability was assessed, and its effect on LSD1 expression was analyzed with Western blotting. The genes and pathways associated with LSD1 were analyzed using bioinformatics methods. Western blotting and qRT-PCR were used to detect Notch/HES1 pathway expression after LSD1 silencing and ORY-1001 treatment. The impact of ORY-1001 on viability of U87 cells with Notch1 silencing or overexpression was assessed, and the regulatory effects of ORY-1001 on Notch/HES1 pathway were analyzed using chromatin immunoprecipitation assay. RESULTS: A high expression of LSD1 in GBM was negatively correlated with patient survival (P < 0.001). ORY-1001 and LSD1 silencing obviously reduced tumor burden and prolonged the survival time of GBM-bearing mice. ORY-1001 treatment significantly inhibited the viability and dose-dependently decreased LSD1 expression in GBM cells, and such inhibitory effect of ORY-1001 was attenuated by LSD1 silencing. The Notch pathway enriched the differential genes related to LSD1, and Notch/HES1 pathway expression was significantly down-regulated after LSD1 silencing and ORY-1001 treatment. Notch1 overexpression significantly attenuated the anti-tumor effect of ORY-1001 on GBM. Mechanistically, ORY-1001 disrupted the interaction between LSD1 and the Notch pathway target genes including Notch3, HES1 and CR2. CONCLUSION: ORY-1001 down-regulates the Notch/HES1 pathway by inhibiting LSD1 expression to suppress the growth of GBM in mice.

MicroRNA-124a promoted the differentiation of bone marrow mesenchymal stem cells into neurons through Notch signal pathway.

This study investigated the possible mechanisms of microRNA-124a on the differentiation of bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanism. ß-Thiol ethanol induced Notch1 mRNA expression, microRNA-124a inhibitor reduced the effects of ß-thiol ethanol on Notch1 mRNA expression in BMSCs. Baicalin induced Hes1 mRNA expression, and microRNA-124a inhibitor reduced the effects of baicalin on Hes1 mRNA expression in BMSCs. Si-Notch1 suppressed Hes1 mRNA expression in BMSCs. Baicalin increased the effects of Notch1 on Hes1 mRNA expression in BMSCs. Si-Notch1 increased cell growth of BMSCs. Baicalin reduced the effects of si-Notch1 on cell growth and the differentiation of BMSCs. We demonstrated that microRNA-124a promoted the differentiation of BMSCs into neurons through Notch/Hes1 signal pathway.

Unravelling differential Hes1 dynamics during axis elongation of mouse embryos through single-cell tracking.

The intricate dynamics of Hes expression across diverse cell types in the developing vertebrate embryonic tail have remained elusive. To address this, we have developed an endogenously tagged Hes1-Achilles mouse line, enabling precise quantification of dynamics at the single-cell resolution across various tissues. Our findings reveal striking disparities in Hes1 dynamics between presomitic mesoderm (PSM) and preneural tube (pre-NT) cells. While pre-NT cells display variable, low-amplitude oscillations, PSM cells exhibit synchronized, high-amplitude oscillations. Upon the induction of differentiation, the oscillation amplitude increases in pre-NT cells. Additionally, our study of Notch inhibition on Hes1 oscillations unveils distinct responses in PSM and pre-NT cells, corresponding to differential Notch ligand expression dynamics. These findings suggest the involvement of separate mechanisms driving Hes1 oscillations. Thus, Hes1 demonstrates dynamic behaviour across adjacent tissues of the embryonic tail, yet the varying oscillation parameters imply differences in the information that can be transmitted by these dynamics.

The impact of NUMB on chicken abdominal adipogenesis: A comprehensive analysis.

Excessive abdominal fat deposition negatively impacts poultry meat production and carcass yield. Identification of novel adipogenesis regulators may help improve production performance declines caused by excessive fat deposition. NUMB Endocytic Adaptor Protein (NUMB) typically functions as a cell fate determinant and plays a significant role in cell development and various diseases. Here, we found that NUMB is abundantly expressed in chicken abdominal fat depots and is induced in cultured adipocytes following adipogenic treatment. The gain- and loss-of-function experiments demonstrated that NUMB promotes the proliferation and G1/S transition of chicken adipocytes, enhances adipocyte differentiation, and increases the expression of PPARγ1 transcript. Through mRNA-seq analysis and molecular experiments, we further confirmed that NUMB inhibits the transcriptional activation of the NOTCH1 pathway and the expression of the downstream transcription factor HES1 by inducing NOTCH1 degradation. Nevertheless, the inhibition of the NOTCH1/HES1 axis alone cannot fully explain NUMB's role in adipogenesis, as NUMB also regulates the expression of multiple adipogenic transcription factors such as E2F1, EGR2, and NR4A3. Our data suggest that NUMB is a potent activator of adipogenesis and enhances our understanding of its regulatory mechanisms in chicken abdominal fat deposition.

Indoxyl Sulfate Inhibits Osteogenesis in Bone Marrow Mesenchymal Stem Cells through the AhR/Hes1 Pathway.

Uremic toxins cause bone disorders in patients with chronic kidney disease (CKD). These disorders are characterized by low turnover osteodystrophy and impaired bone formation in the early stages of CKD. Evidence indicates that the aryl hydrocarbon receptor (AhR) mediates signals that suppress early osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). However, whether the AhR mediates the effects of indoxyl sulfate (IS), a uremic toxin, on BMSC osteogenesis remains unclear. We investigated whether IS affects osteogenesis through the AhR/Hes1 pathway. Expression levels of osteogenesis genes (Runx2, Bmp2, Alp, and Oc), AhR, and Hes1 were measured in mouse BMSCs (D1 cells). At concentrations of 2-50 µM, IS significantly reduced mineralization, particularly in the early stages of BMSC osteogenesis. Furthermore, IS significantly downregulated the expression of Runx2, Bmp2, Oc, and Alp. Notably, this downregulation could be prevented using an AhR antagonist and through Ahr knockdown. Mechanistically, IS induced the expression of Hes1 through AhR signaling, thereby suppressing the transcription of Runx2 and Bmp2. Our findings suggest that IS inhibits early osteogenesis of BMSCs through the AhR/Hes1 pathway, thus suppressing the transcription of Runx2 and Bmp2. Our findings may guide new therapeutic strategies against CKD-related bone disorders.

HES1 is required for mouse fetal hematopoiesis.

BACKGROUND: Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. METHODS: In this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. RESULTS: We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. CONCLUSION: Together, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.

Dibenzazepine, a γ-Secretase Enzyme Inhibitor, Protects Against Doxorubicin-Induced Cardiotoxicity by Suppressing NF-κB, iNOS, and Hes1/Hey1 Expression.

Doxorubicin (DOX) is an effective chemotherapeutic drug; however, its cardiotoxicity and resistance compromise its therapeutic index. The Notch pathway was reported to contribute to DOX cancer resistance. The role of Notch pathway in DOX cardiotoxicity has not been identified yet. Notch receptors are characterized by their extracellular (NECD) and intracellular (NICD) domains (NICD). The γ-secretase enzyme helps in the release of NICD. Dibenzazepine (DBZ) is a γ-secretase inhibitor. The present study investigated the effect of Notch pathway inhibition on DOX cardiotoxicity. Twenty-four male Wistar rats were divided into four groups: control group, DOX group, acute cardiotoxicity was induced by a single dose of DOX (20 mg/kg) i.p., DOX (20 mg/kg) plus DBZ group, and DBZ group. The third and fourth groups received i.p. injection of DBZ daily for 14 days at 2 mg/kg dose. DOX cardiotoxicity increased the level of serum creatine kinase-MB and cardiac troponin I, and it was confirmed by the histopathological examination. Moreover, the antioxidants glutathione peroxidase and superoxide dismutase levels were markedly decreased, and the inflammatory markers, inducible nitric oxide synthase, nuclear factor-kB, and tumor necrosis factor-α were markedly increased. Furthermore, DOX increased BAX protein and downregulated BCL-2. In addition, DOX upregulated Notch pathway-related parameters: Hes1 and Hey1 mRNA levels, and increased Hes1 protein levels. DBZ ameliorated DOX-induced cardiotoxicity, evidenced by reducing the cardiac injury biomarkers, improving cardiac histopathological changes, correcting antioxidant levels, and reducing inflammatory and apoptotic proteins. Our study indicates the protective effect of Notch inhibitor against DOX-induced cardiotoxicity.

Based on NF-κB and Notch1/Hes1 Signaling Pathways, the Mechanism of Artesunate on Inflammation in Osteoporosis in Ovariectomized Rats was Investigated.

BACKGROUND: Artesunate (ART) has the potential to modulate the nuclear factor kappa B (NF-κB) and Notch1/Hes1 signaling pathways, which play crucial roles in the pathogenesis of osteoporosis. This study aims to explore whether ART participates in the progression of osteoporosis by regulating these signaling pathways. METHODS: In the in vitro experiments, we treated bone marrow mesenchymal stem cells (BMSCs) with different concentrations of ART (0, 3, 6, 12 µM) and evaluated osteogenic differentiation using alkaline phosphatase staining (ALP) and alizarin red S staining (ARS) staining. The expression levels of osteocalcin (OCN), RUNT-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), and receptor activator of the nuclear factor kappa ligand (RANKL) were detected by real-time quantitative PCR (RT-qPCR). The effects of ART on NF-κB p65 and Notch1 protein expression were analyzed by Western blot (WB) and immunofluorescence (IF). In the in vivo experiments, a postmenopausal osteoporosis rat model was established via ovariectomy. Bone tissue pathological injury was evaluated using hematoxylin eosin (HE) staining. Serum ALP levels were measured using a kit, bone density was determined by dual-energy X-ray absorptiometry, and serum levels of bone gla protein (BGP), OPG, RANKL, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and IL-1ß were measured by enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of NF-κB p65 and Notch1 in tissues was assessed by immunohistochemistry. RESULTS: In vitro experiments revealed that compared to the control group, ART dose-dependently promoted BMSCs proliferation and enhanced their osteogenic differentiation capability. The expression of OCN, RUNX2, and OPG significantly increased in the ART-treated group, while RANKL expression decreased significantly (p < 0.05). ART significantly inhibited the expression of NF-κB p65 and Notch1/Hes1 signaling pathway proteins (p < 0.05). Compared to ART treatment alone, combined treatment with ART and phorbol myristate acetate (PMA) or valproic acid (VPA) resulted in increased expression of NF-κB p65 and Notch1 proteins and decreased osteogenic differentiation capability (p < 0.05). In vivo experiments showed that in rats treated with ART, bone damage was significantly reduced, bone density and mineral content were restored considerably, and the expression of inflammatory factors (TNF-α, IL-6, IL-1ß) decreased significantly (p < 0.05). Additionally, ART treatment significantly reduced the expression of NF-κB p65 and Notch1 proteins, increased OPG expression, and decreased BGP and RANKL levels (p < 0.05). CONCLUSION: In summary, ART facilitates the osteogenic differentiation of BMSCs by inhibiting the NF-κB and Notch1/Hes1 signaling pathways, thereby exerting significant protective effects against osteoporosis.

Uncovering the mechanisms of host mitochondrial cardiolipin release in syphilis: Insights from human microvascular endothelial cells.

The release of host mitochondrial cardiolipin is believed to be the main factor that contributes to the production of anti-cardiolipin antibodies in syphilis. However, the precise mechanism by which mitochondria release cardiolipin in this context remains elusive. This study aimed to elucidate the mechanisms underlying mitochondrial cardiolipin release in syphilis. We conducted a cardiolipin quantitative assay and immunofluorescence analysis to detect mitochondrial cardiolipin release in human microvascular endothelial cells (HMEC-1), with and without Treponema pallidum (Tp) infection. Furthermore, we explored apoptosis, a key mechanism for mitochondrial cardiolipin release. The potential mediator molecules were then analyzed through RNA-sequence and subsequently validated using in vitro knockout techniques mediated by CRISPR-Cas9 and pathway-specific inhibitors. Our findings confirm that live-Tp is capable of initiating the release of mitochondrial cardiolipin, whereas inactivated-Tp does not exhibit this capability. Additionally, apoptosis detection further supports the notion that the release of mitochondrial cardiolipin occurs independently of apoptosis. The RNA-sequencing results indicated that microtubule-associated protein2 (MAP2), an axonogenesis and dendrite development gene, was up-regulated in HMEC-1 treated with Tp, which was further confirmed in syphilitic lesions by immunofluorescence. Notably, genetic knockout of MAP2 inhibited Tp-induced mitochondrial cardiolipin release in HMEC-1. Mechanically, Tp-infection regulated MAP2 expression via the MEK-ERK-HES1 pathway, and MEK/ERK phosphorylation inhibitors effectively block Tp-induced mitochondrial cardiolipin release. This study demonstrated that the infection of live-Tp enhanced the expression of MAP2 via the MEK-ERK-HES1 pathway, thereby contributing to our understanding of the role of anti-cardiolipin antibodies in the diagnosis of syphilis.

The Neurog2-Tbr2 axis forms a continuous transition to the neurogenic gene expression state in neural stem cells.

Neural stem cells (NSCs) differentiate into neuron-fated intermediate progenitor cells (IPCs) via cell division. Although differentiation from NSCs to IPCs is a discrete process, recent transcriptome analyses identified a continuous transcriptional trajectory during this process, raising the question of how to reconcile these contradictory observations. In mouse NSCs, Hes1 expression oscillates, regulating the oscillatory expression of the proneural gene Neurog2, while Hes1 expression disappears in IPCs. Thus, the transition from Hes1 oscillation to suppression is involved in the differentiation of NSCs to IPCs. Here, we found that Neurog2 oscillations induce the accumulation of Tbr2, which suppresses Hes1 expression, generating an IPC-like gene expression state in NSCs. In the absence of Tbr2, Hes1 expression is up-regulated, decreasing the formation of IPCs. These results indicate that the Neurog2-Tbr2 axis forms a continuous transcriptional trajectory to an IPC-like neurogenic state in NSCs, which then differentiate into IPCs via cell division.

Effects of KEAP1 Silencing on NRF2 and NOTCH Pathways in SCLC Cell Lines.

The KEAP1/NRF2 pathway is a master regulator of several redox-sensitive genes implicated in the resistance of tumor cells against therapeutic drugs. The dysfunction of the KEAP1/NRF2 system has been correlated with neoplastic patients' outcomes and responses to conventional therapies. In lung tumors, the growth and the progression of cancer cells may also involve the intersection between the molecular NRF2/KEAP1 axis and other pathways, including NOTCH, with implications for antioxidant protection, survival of cancer cells, and drug resistance to therapies. At present, the data concerning the mechanism of aberrant NRF2/NOTCH crosstalk as well as its genetic and epigenetic basis in SCLC are incomplete. To better clarify this point and elucidate the contribution of NRF2/NOTCH crosstalk deregulation in tumorigenesis of SCLC, we investigated genetic and epigenetic dysfunctions of the KEAP1 gene in a subset of SCLC cell lines. Moreover, we assessed its impact on SCLC cells' response to conventional chemotherapies (etoposide, cisplatin, and their combination) and NOTCH inhibitor treatments using DAPT, a γ-secretase inhibitor (GSI). We demonstrated that the KEAP1/NRF2 axis is epigenetically controlled in SCLC cell lines and that silencing of KEAP1 by siRNA induced the upregulation of NRF2 with a consequent increase in SCLC cells' chemoresistance under cisplatin and etoposide treatment. Moreover, KEAP1 modulation also interfered with NOTCH1, HES1, and DLL3 transcription. Our preliminary data provide new insights about the downstream effects of KEAP1 dysfunction on NRF2 and NOTCH deregulation in this type of tumor and corroborate the hypothesis of a cooperation of these two pathways in the tumorigenesis of SCLC.

RIP140 regulates transcription factor HES1 oscillatory expression and mitogenic activity in colon cancer cells.

The transcription factor receptor-interacting protein 140 (RIP140) regulates intestinal homeostasis and tumorigenesis through Wnt signaling. In this study, we investigated its effect on the Notch/HES1 signaling pathway. In colorectal cancer (CRC) cell lines, RIP140 positively regulated HES1 gene expression at the transcriptional level via a recombining binding protein suppressor of hairless (RBPJ)/neurogenic locus notch homolog protein 1 (NICD)-mediated mechanism. In support of these in vitro data, RIP140 and HES1 expression significantly correlated in mouse intestine and in a cohort of CRC samples, thus supporting the positive regulation of HES1 gene expression by RIP140. Interestingly, when the Notch pathway is fully activated, RIP140 exerted a strong inhibition of HES1 gene transcription controlled by the level of HES1 itself. Moreover, RIP140 directly interacts with HES1 and reversed its mitogenic activity in human CRC cells. In line with this observation, HES1 levels were associated with a better patient survival only when tumors expressed high levels of RIP140. Our data identify RIP140 as a key regulator of the Notch/HES1 signaling pathway, with a dual effect on HES1 gene expression at the transcriptional level and a strong impact on colon cancer cell proliferation.

HES1 induces ITPR1-mediated autophagy to exert anti-metastatic effects in pituitary adenomas.

Background: Pituitary adenomas (PAs) are prevalent intracranial tumors necessitating a comprehensive exploration of their molecular intricacies. This study delved into the molecular interactions among HES1 (hairy and enhancer of split 1), ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1), and autophagy to elucidate their contributions to PA progression. Methods: Our in-depth bioinformatics analysis identified ITPR1 as a central hub gene in the PA-associated dataset. It exhibited reduced expression in PA and held significant clinical diagnostic relevance. Motivated by this discovery, we investigated the consequences of ITPR1 overexpression, as well as the use of autophagy inhibitors 3-Methyladenine (3-MA) and Baf A1, while considering the transcriptional influence of HES1. Results: In vitro experiments utilizing PA cell lines revealed that ITPR1 overexpression significantly hindered tumorigenic activities. In contrast, both 3-MA and Baf A1 exacerbated these tumorigenic properties, confirmed by a decreased LC3 II/LC3 I ratio, indicative of autophagy inhibition. Intriguingly, the concurrent introduction of ITPR1 and these inhibitors mitigated these intensified effects, implying a tumor-suppressive role for ITPR1. Further investigations pinpoint HES1 as a potential upstream regulator of ITPR1 transcription. Silencing HES1 lead to reduced ITPR1 promoter activity, weakening the impact of ITPR1 overexpression on autophagy. This neutralized the ITPR1-mediated suppressions on PA cell activities, including proliferation, invasion, and migration. Conclusions: In summary, our research uncovered a complex regulatory interplay among HES1, ITPR1, and autophagy in the context of PA progression. These findings opened up promising avenues for novel therapeutic interventions targeting this intricate network to enhance PA treatment.

GDNF facilitates the differentiation of ADSCs to Schwann cells and enhances nerve regeneration through GDNF/MTA1/Hes1 axis.

Adipose tissue-derived stem cells (ADSCs) are a kind of stem cells with multi-directional differentiation potential, which mainly restore tissue repair function and promote cell regeneration. It can be directionally differentiated into Schwann-like cells to promote the repair of peripheral nerve injury. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in the repair of nerve injury, but the underlying mechanism remains unclear, which seriously limits its further application.The study aimed to identify the molecular mechanism by which overexpression of glial cell line-derived neurotrophic factor (GDNF) facilitates the differentiation of ADSCs into Schwann cells, enhancing nerve regeneration after injury. In vitro, ADSCs overexpressing GDNF for 48 h exhibited changes in their morphology, with 80% of the cells having two or more prominences. Compared with that of ADSCs, GDNF-ADSCs exhibited increased expression of the Schwann cell marker S100, nerve damage repair-related factors.ADSC cells in normal culture and ADSC cells were overexpressing GDNF(GDNF-ADSCs) were analysed using TMT-Based Proteomic Analysis and revealed a significantly higher expression of MTA1 in GDNF-ADSCs than in control ADSCs. Hes1 expression was significantly higher in GDNF-ADSCs than in ADSCs and decreased by MTA1 silencing, along with a simultaneous decrease in the expression of S100 and nerve damage repair factors. These findings indicate that GDNF promotes the differentiation of ADSCs into Schwann cells and induces factors that accelerate peripheral nerve damage repair.

Astragalus Polysaccharide Promotes Neuronal Injury Repair via the Notch1/NFκB Signaling Axis in the Ventroposterior Thalamic Nucleus in Rats with Focal Cerebral Ischemia.

BACKGROUND: Ischemic stroke is the most common form of stroke and the second most common cause of death and incapacity worldwide. Its pathogenesis and treatment have been the focus of considerable research. In traditional Chinese medicine, the root of Mongolian astragalus has been important in the treatment of stroke since ancient times. Astragalus polysaccharide (APS) is a key active ingredient of astragalus and offers therapeutic potential for conditions affecting the neurological system, the heart, cancer, and other disorders. However, it is not yet known how APS works to protect against ischemic stroke. METHODS: Rats were subjected to middle cerebral artery occlusion (MCAO) to imitate localized cerebral ischemia. Each of four experimental groups (normal, sham, MCAO, and MCAO+APS) contained 12 adult male Sprague-Dawley (SD) rats selected randomly from a total of 48 rats. Following successful establishment of the model, rats in the MCAO+APS group received intraperitoneal injection of APS (50 mg/kg) once daily for 14 days, whereas all other groups received no APS. The Bederson nerve function score and the forelimb placement test were used to detect motor and sensory function defects, while Nissl staining was used to investigate pathological defects in the ventroposterior thalamic nucleus (VPN). Immunohistochemical staining and Western blot were used to evaluate the expression of Neurogenic locus notch homolog protein 1 (Notch1), hairy and enhancer of split 1 (Hes1), phospho-nuclear factor-κB p65 (p-NFκB p65), and nuclear factor-κB p65 (NFκB p65) proteins in the VPN on the ischemic side of MCAO rats. RESULTS: APS promoted the recovery of sensory and motor function, enhanced neuronal morphology, increased the number of neurons, and inhibited the expression of Notch1/NFκB signaling pathway proteins in the VPN of rats with cerebral ischemia. CONCLUSION: After cerebral ischemia, APS can alleviate symptoms of secondary damage to the VPN, which may be attributed to the suppression of the Notch1/NFκB pathway.

NOTCH1 as a Negative Regulator of Avian Adipocyte Differentiation: Implications for Fat Deposition.

The NOTCH signaling pathway plays a pivotal role in diverse developmental processes, including cell proliferation and differentiation. In this study, we investigated whether this signaling molecules also contribute to avian adipogenesis. Using previous mRNA-seq datasets, we examined the expression of 11 signaling members during avian adipocyte differentiation. We found most members are down-regulated throughout differentiation (p < 0.05). As a representative, NOTCH1 was decreased in cultured chicken abdominal adipocytes during adipogenesis at mRNA and protein levels (p < 0.05). Moreover, using an overexpression plasmid for NOTCH1's intracellular domain (NICD1), as well as siRNA and DAPT to activate or deplete NOTCH1 in cells, we investigated the role of NOTCH1 in avian adipogenesis. Our findings illuminate that NOTCH1 activates the expression of HES1 and SOCS3 while it decreases NR2F2 and NUMB (p < 0.05), as well as inhibits oleic acid-induced adipocyte differentiation (p < 0.01). We further demonstrate that HES1, a downstream transcription factor activated by NOTCH1, also significantly inhibits adipogenesis by suppressing PPARγ and C/EBPα (p < 0.01). Collectively, these findings establish NOTCH1 as a negative regulator of avian adipocyte differentiation, unveiling NOTCH signaling as a potential target for regulating avian fat deposition.

Neural stem cell-derived exosomes regulate cell proliferation, migration, and cell death of brain microvascular endothelial cells via the miR-9/Hes1 axis under hypoxia.

BACKGROUND: Our previous study found that mouse embryonic neural stem cell (NSC)-derived exosomes (EXOs) regulated NSC differentiation via the miR-9/Hes1 axis. However, the effects of EXOs on brain microvascular endothelial cell (BMEC) dysfunction via the miR-9/Hes1 axis remain unknown. Therefore, the current study aimed to determine the effects of EXOs on BMEC proliferation, migration, and death via the miR-9/Hes1 axis. METHODS: Immunofluorescence, quantitative real-time polymerase chain reaction, cell counting kit-8 assay, wound healing assay, calcein-acetoxymethyl/propidium iodide staining, and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs. RESULTS: EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions. The overexpression of miR-9 promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions. Moreover, miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death. Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death. Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice. Meanwhile, EXO treatment improved cerebrovascular alterations. CONCLUSION: NSC-derived EXOs can promote BMEC proliferation and migration and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions. Therefore, EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.

bHLH transcription factors Hes1, Ascl1 and Oligo2 exhibit different expression patterns in the process of physiological electric fields-induced neuronal differentiation.

BACKGROUND: Recent studies have shown that the expression of bHLH transcription factors Hes1, Ascl1, and Oligo2 has an oscillating balance in neural stem cells (NSCs) to maintain their self-proliferation and multi-directional differentiation potential. This balance can be disrupted by exogenous stimulation. Our previous work has identified that electrical stimulation could induce neuronal differentiation of mouse NSCs. METHODS: To further evaluate if physiological electric fields (EFs)-induced neuronal differentiation is related to the expression patterns of bHLH transcription factors Hes1, Ascl1, and Oligo2, mouse embryonic brain NSCs were used to investigate the expression changes of Ascl1, Hes1 and Oligo2 in mRNA and protein levels during EF-induced neuronal differentiation. RESULTS: Our results showed that NSCs expressed high level of Hes1, while expression of Ascl1 and Oligo2 stayed at very low levels. When NSCs exited proliferation, the expression of Hes1 in differentiated cells began to decrease and oscillated at the low expression level. Oligo2 showed irregular changes in low expression level. EF-stimulation significantly increased the expression of Ascl1 at mRNA and protein levels accompanied by an increased percentage of neuronal differentiation. What's more, over-expression of Hes1 inhibited the neuronal differentiation induced by EFs. CONCLUSION: EF-stimulation directed neuronal differentiation of NSCs by promoting the continuous accumulation of Ascl1 expression and decreasing the expression of Hes1.

Endothelial cell flow-mediated quiescence is temporally regulated and utilizes the cell cycle inhibitor p27.

Background: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. Methods: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time to cell cycle re-entry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA seq analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. Results: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous-flow exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. Conclusions: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence mis-regulation that leads to vascular dysfunction and disease.