Hes1 Protein Expression Research Articles

Mechanism of electroacupuncture on hippocampal neuron regeneration in depression rats based on Wnt/β-catenin and Notch signaling pathway

To observe the effect of electroacupuncture (EA) on the proliferation of hippocampal neurons and Wnt/β-catenin and Notch signaling pathways in depression like behavior rats, so as to explore its mechanism underlying improvement of depression. SD rats were randomly divided into blank, model, medication and EA groups, with 12 rats in each group. The depression model was established by chronic unpredictable mild stimulation combined with solitary raising for 35 consecutive days. After 14 days modeling, EA was applied to "Baihui"(GV20) and "Taichong"(LR3) for 15 min once daily for 3 weeks. For rats of the medication group, clomipramine (2.5 mg/kg) was given (i.p.) once a day for 3 weeks. The depression-like behavior was evaluated by open field test and forced swimming test. The ultrastructure of hippocampal neurons was observed by transmission electron microscope (TEM). The positive expression of 5-bromo-2'-deoxyuridine/neuron-specific nucleoprotein (BrdU/NeuN) in hippocampal dentate gyrus of rats was detected by immunofluorescence double labeling method. Western blot was used to detect the relative expression of Wnt/β-catenin and Notch pathway key proteins β-catenin, G1/S-specific cyclin-D1 (Cyclin D1), NICD and Hes1 in hippocampus of rats. Compared with the blank group, the total distance and average speed of open field movement were decreased (P<0.01), and the immobility time of forced swimming was increased (P<0.01). The positive expression of BrdU/NeuN in hippocampal dentate gyrus was decreased (P<0.01). The relative expression of β-catenin and Cyclin D1 protein in hippocampus were decreased (P<0.01), while the relative expression of NICD and Hes1 protein increased (P<0.01) in the model group. Following the treatment and compared with the model group, the total distance and average speed of movement in the open field were increased (P<0.05), and the immobility time of forced swimming was decreased (P<0.05) in both medication and EA groups. The positive expression of BrdU/NeuN in hippocampal dentate gyrus was increased (P<0.01). The relative expression of β-catenin and Cyclin D1 protein in hippocampus were increased (P<0.01), while the relative expression of NICD and Hes1 protein were decreased (P<0.01, P<0.05) in both medication and EA groups. TEM microscope observation showed irregularity of hippocampal neurons, rupture of nuclear membranes, disruption of mitochondrial structure, and dilation of endoplasmic reticulum in the model group, which was relatively milder in both medication and EA groups. EA can improve the depressive state of depression model rats, which may be associated with its functions in regulating Wnt and Notch signaling pathways, thereby promoting the regeneration of hippocampal neurons.

Berberine ameliorates septic cardiomyopathy through protecting mitochondria and upregulating Notch1 signaling in cardiomyocytes.

Septic cardiomyopathy (SCM) arises as a consequence of sepsis-associated cardiovascular dysfunction, for which there is currently no specific targeted therapy available. Previous studies have demonstrated the beneficial therapeutic effect of berberine (BBR) on SCM; however, the underlying mechanisms of action remain unclear. The objective of this is to elucidate how BBR alleviates SCM. Septic cardiomyopathy rat model was established by performing cecal ligation and puncture (CLP), while a cardiomyocyte injury model was provoked in H9C2 cells using lipopolysaccharide (LPS). Cardiac function was assessed through echocardiography, and myocardial histopathology was examined with hematoxylin-eosin (HE) staining. Cardiomyocyte viability was determined through Cell Counting Kit-8 (CCK8) assay, and measurement of ATP levels was done with an ATP assay kit. Mitochondrial ultrastructure was observed using transmission electron microscopy. Real-time polymerase chain reaction (RT-PCR) and Western blotting were employed to analyze the expression of Notch1 signaling pathway components and downstream molecules in myocardial tissues and cells. In vivo, BBR markedly improved symptoms and cardiac function in SCM rats, leading to enhanced ATP content, and ameliorated mitochondrial structure. Additionally, BBR increased Notch1 protein expression in myocardial tissue of the rats. In vitro, BBR elevated the survival rates of H9C2 cell, improved mitochondrial morphology, and raised ATP levels. The mRNA expression of Notch1, Hes1, and Hes2, and Notch1 protein expression was upregulated by BBR. While these effects were reversed upon inhibiting the Notch1 signaling pathway. BBR improves septic cardiomyopathy by modulating Notch1 signaling to protect myocardial mitochondria.

Protective Effects of Gastrodin Against Gentamicin-Induced Vestibular Damage by the Notch Signaling Pathway.

Gentamicin is a broad-spectrum antibiotic commonly used in clinical practice. However, the drug causes side effects of ototoxicity, leading to disruption in balance functionality. This study investigated the effect of gastrodin, a prominent compound present in Gastrodia, and the underlying mechanism on the development of gentamicin-induced vestibular dysfunction. Wild-type C57BL/6 mice were randomly assigned to three groups: control, gentamicin, and gentamicin + gastrodin groups. The extent of gentamicin-induced vestibular impairment was assessed through a series of tests including the swimming test, contact righting reflex test, and air-righting reflex. Alterations in vestibular hair cells were monitored through immunofluorescence assay, and cellular apoptosis was observed using TUNEL staining. The mRNA and protein expression of Notch1, Jagged1, and Hes1 was quantified through qRT-PCR, immunofluorescence, and western blot analyses. Gentamicin treatment led to pronounced deficits in vestibular function and otolith organ hair cells in mice. Nevertheless, pretreatment with gastrodin significantly alleviated these impairments. Additionally, the Notch signaling pathway was activated by gentamicin in the utricle, contributing to a notable increase in the expression levels of apoptosis-associated proteins. By contrast, gastrodin treatment effectively suppressed the Notch signaling pathway, thereby mitigating the occurrence of apoptosis. Collectively, these findings underscore the crucial role of gastrodin in safeguarding against gentamicin-induced vestibular dysfunction through the modulation of the Notch signaling pathway. This study suggests the potential of gastrodin as a promising therapeutic agent for preventing vestibular injuries.

ADAM10 Alleviates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Activating the Notch Signaling Pathway.

The occurrence of myocardial ischemia/reperfusion injury is commonly observed during cardiac surgery; however, there remains a dearth of effective therapeutic strategies to mitigate this injury. The a disintegrin and metallopeptidase domain 10 (ADAM10) is a transmembrane protein anchored on the cell membrane surface, and its precise mechanism of action in myocardial ischemia/reperfusion injury remains incompletely understood. This study aims to investigate the impact of ADAM10 on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R) and elucidate the underlying mechanisms. The ADAM10 overexpression plasmid was transfected into H9c2 cells, which were subsequently treated with the Notch signaling pathway inhibitor DAPT and cultured under H/R conditions. Cell proliferation activity was assessed using the CCK-8 assay. The levels of LDH, SOD, and MDA were quantified through colorimetric analysis. The levels of ROS and the rate of apoptosis were measured using flow cytometry. The morphological changes in the nucleus of H9c2 cells were observed by employing Hoechst 33258 staining. The mRNA expression levels of ADAM10, Notch1, NICD, and Hes1 in H9c2 cells were determined using qRT-PCR. The expressions of Notch signaling pathway and apoptosis-related proteins were analyzed by Western blot. Overexpression of ADAM10 provided protection to H9c2 cells against injury induced by H/R, leading to an increase in SOD levels and alleviation of oxidative stress caused by the accumulation of ROS and the decrease of SOD activity. Meanwhile, overexpression of ADAM10 inhibited apoptosis in H9c2 cells exposed to H/R by regulating the expression of apoptosis-related proteins, such as Bax, Bcl-2 and Cleaved-caspase-3. Additionally, overexpression of ADAM10 facilitated the activation of the Notch1 signaling pathway in H9c2 cells exposed to H/R by upregulating the protein expression of Notch1, NICD, and Hes1. However, the protective effect of ADAM10 on H/R-induced H9c2 cells was partially reversed by DAPT. Our findings demonstrate that ADAM10 exerts protective effects in H/R-induced H9c2 cells by suppressing oxidative stress and apoptosis via the activation of the Notch signaling pathway.

Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role.

Analysis of Primary Chronic Lymphocytic Leukemia Cells' Signaling Pathways.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by a specific expansion of mature B-cell clones. We hypothesized that the disease has a heterogeneous clinical outcome that depends on the genes and signaling pathways active in the malignant clone of the individual patient. It was found that several signaling pathways are active in CLL, namely, NOTCH1, the Ikaros family genes, BCL2, and NF-κB, all of which contribute to cell survival and the proliferation of the leukemic clone. Therefore, we analyzed primary CLL cells for the gene and protein expression of NOTCH1, DELTEX1, HES1, and AIOLOS in both peripheral blood lymphocytes (PBLs) and the bone marrow (BM) of patients, as well as the expression of BCL2 and miRNAs to see if they correlate with any of these genes. BCL2 and AIOLOS were highly expressed in all CLL samples as previously described, but we show here for the first time that AIOLOS expression was higher in the PBLs than in the BM. On the other hand, NOTCH1 activation was higher in the BM. In addition, miR-15a, miR-181, and miR-146 were decreased and miR-155 had increased expression in most samples. The activation of the NOTCH pathway in vitro increases the susceptibility of primary CLL cells to apoptosis despite high BCL2 expression.

Weaning causes imbalanced T lymphocyte distribution and impaired intestinal immune barrier function in piglets

Context The immune system of piglets is not fully developed at weaning. The immature adaptive immune system along with the change in the feed types and the living conditions make piglets susceptible to pathogenic infections, and result in diarrhoea and decreased growth. However, the underlying mechanisms require further research. Aims This study was conducted to investigate the effects of weaning on the growth performance and intestinal immune function of piglets. Methods In total, 40 piglets with similar bodyweights were selected in pairs at 21 days old and divided into the suckling group (SG, breastfed by their mothers) and weaning group (WG, weaned at 21 days old). Eight piglets from each group were randomly selected and sacrificed at 24 days (SG3 and WG3) and 28 days of age (SG7 and WG7). The growth performance, T lymphocyte subpopulations, the concentration of cytokines and immunoglobulins, and the expression of Notch2 signalling proteins were determined. Key results The weaning caused a decrease in bodyweight (P &lt; 0.01) and the ratio of CD3+CD4+/CD3+CD8+ T cells in the thymus (P &lt; 0.05). Compared with SG3, the concentration of secretory immunoglobulin A (sIgA) in the jejunum was decreased, and that of interleukin 2 (IL-2) in serum and ileum, IL-1β and IL-2 in jejunum were upregulated (P &lt; 0.01), while IL-10 in the small intestine was downregulated (P &lt; 0.05) in WG3. Weaning downregulated gene expression of IL-4 and upregulated gene expression of IL-1β, IL-12, and interferon γ (IFN-γ) in small intestine (P &lt; 0.05). Weaning downregulated protein expression of Notch2 and Hes1 but upregulated Jagged1 expression in small intestine of piglets (P &lt; 0.05). Conclusions Weaning caused an imbalance in T lymphocyte distribution, thus impairing the intestinal immune function of piglets, which might be associated with the Notch2 signalling. Furthermore, weaning caused adverse effects on piglets especially on the first 3 days, which were partly recovered after 3 days, but still existed at 7 days after weaning. Implications The results of this study may guide the pig-raising industry, especially for the management of piglets shortly after weaning.

P038 Notch pathway in fibrosis: a new anti-fibrotic therapy in Crohn's disease?

Abstract Background Fibrosis represent the main complications related to Crohn's disease (CD). Notch signalling mediate fibrogenic process, including epithelial-mesenchymal transition (EMT) and fibroblast senescence but its role in CD fibrosis is currently unknown. Previous studies have shown a high expression of NOTCH4, NOTCH3, DLL3 and DLL4 in CD fibrotic tissue. DLL4 mainly activates transcription factors involved in EMT, and macrophages could act as a possible source of DLL4 (Edo, et al., 2022. JCC (i200)).The general aim of the present study is to determine the possible potential of Notch pathway as a therapeutic target in intestinal fibrosis associated with CD. Specifically, we pretend: to analyze the localization of NOTCH3/4 receptors in the intestinal tissue of patients with CD complicated; to study the relevance of NOTCH3/4 receptors in the EMT; to study the relevance of Notch pathway in the senescence of intestinal fibroblast. Methods We have analyzed in intestinal samples from CD patients with complicated lesions: the localization of NOTCH3/4 receptors by IH and the protein expression of senescence markers (BCL2 and P53) by WB. We carry out in vitro studies and analyze: the protein expression of HES1 (effector Notch pathway) and EMT markers in DLL4-HT29 treated cells transfected with miNOTCH3 or miNOTCH4; and the protein expression of senescence proteins in HSIF fibroblasts treated with DLL4 or DLL3. Results NOTCH3 was located preferentially in muscular areas -muscularis mucosa, endothelium and muscularis externa- with a striking staining of infiltrated cells in the mucosa of the unaffected area. NOTCH4 is found more specifically in the crypts of the mucosa, as well as in cells of the lamina propria of the unaffected mucosa. The expression of senescence proteins in the tissue showed elevated levels of BCL2 and P53, compared to the unaffected tissue of the same patient. DLL4 increased the protein expression of EMT markers in HT29 cells, and NOTCH4 silencing significantly reverted the expression of these EMT markers. NOTCH3 silencing produced no significant changes after DLL4-HT29 treatment. DLL3, and not DLL4, produced in intestinal fibroblasts a significant increase in the protein expression levels of BCL2, and P53, compared to the vehicle (Figure). Conclusion NOTCH pathway is involved in the regulation of key cellular functions and processes essential for the pathogenesis of intestinal fibrosis in CD patients. DLL4-NOTCH4 interaction triggers transcription factors involved in mesenchymal epithelial transition in colonic epithelial cells, while DLL3 seems to have a more relevant role in activation of senescence in fibroblasts.

Astragaloside IV inhibits the proliferation and migration of lens epithelial cells induced by TGF-β through the Jagged-1/Notch pathway

Aberrant proliferation and migration of residual lens epithelial cells (LECs) is the main cause of posterior capsule opacification (PCO) after cataract surgery. The purpose of this study was to confirm the protective effects of Astragaloside IV (AST) in transforming growth factor beta (TGF-β)-induced proliferation and migration of LECs. Human LECs, SRA01/04, were used in this study. MTT assay and EdU assay were used to perform cell viability and proliferation, respectively. The cell cycle was determined using flow cytometry. Cell invasion and migration were determined using transwell assay and wound healing assay, respectively. Protein expression was examined using western blotting. Type I and IV collagen (Col I and IV) and fibronectin (FN) in cells were also detected using immunofluorescence (IF). Cell viability, proliferation, migration and invasion were suppressed by AST in a concentration-dependent manner in TGF-β-treated LECs. AST stopped the cell cycle at G2 phase in a concentration-dependent manner. Expression of Cyclin B1 and CDK1 was suppressed by AST in TGF-β-treated LECs. Protein expression of FN, Col I, Col IV, α-SMA (α-smooth muscle actin), Snail and Slug was downregulated by AST in a concentration-dependent manner. AST inhibited the protein expression of Jagged-1, Notch1, Notch2, Notch3, Hes-1 and Hey-1. Taken together, data from this study demonstrated that AST inhibited LEC viability, proliferation, migration and invasion, as well as fibroblastic differentiation and epithelial-mesenchymal transition process through inhibition of Jagged-1/Notch pathway, providing a potential supplemental treatment for PCO.

Ezrin accelerates breast cancer liver metastasis through promoting furin-like convertase-mediated cleavage of Notch1.

Ezrin, known as a crosslinker between the plasma membrane and actin cytoskeleton, is closely associated with breast cancer (BC) progression. Here, we explored a novel role of ezrin in breast cancer liver metastasis (BCLM). The clinical relevance of ezrin was evaluated using in silico tools and confirmed in BC specimens. The effect of ezrin on proliferation, migration and invasion was examined in vitro and in vivo using murine primary liver-metastatic breast cancer cells (mLM). The molecular mechanism involved in ezrin-mediated activation of the Notch1 signaling pathway was elucidated using in vitro models. Data-mining demonstrated that ezrin mRNA and protein expression is up-regulated in breast cancer cohorts and has prognostic significance. Ezrin overexpression promotes cell proliferation, migration and invasion in vitro and in vivo. Hairy and enhancer of split-1 (Hes1) is one of the most significantly enriched candidates of differentially expressed genes in ezrin overexpression and control mLM cells. Ezrin can positively regulate Hes1 mRNA and protein expression, and their coexpression was associated with poor prognosis in BC patients. Ezrin promoted BC cell proliferation in a Hes1-dependent manner without directly interacting with Hes1. The functional link between ezrin and Hes1 is dependent on Notch1 activation through promotion of furin-like convertase cleavage. Our results demonstrated that ezrin drives BCLM through activation of the Notch signaling pathway via furin-like convertase. These findings provide a better understanding of the mechanism of ezrin in breast cancer progression, with the goal of discovering a novel target for the treatment of BCLM in the future.

Fluoxetine shows neuroprotective effects against LPS-induced neuroinflammation via the Notch signaling pathway.

To determine the neuroprotective effects of fluoxetine on depression-like and motor behaviors in rats treated with lipopolysaccharide (LPS) and the mechanisms involved. A rat model of depression in Parkinson's disease (dPD) was established by administering LPS (0.5mg/kg, i.p.) for 4days. The sucrose preference test (SPT), open field test (OFT), and rotarod test evaluated depression-like and motor behaviors. White matter fiber integrity and intrinsic activity in the brain were assessed using magnetic resonance imaging. For pathological and molecular expression detection, hematoxylin-eosin staining, immunohistochemistry, Luminex technology, western blotting, and quantitative real-time PCR were used. Fluoxetine increased the sucrose preference in the SPT, the horizontal and center distances in the OFT, and the standing time in the rotarod test. Fluoxetine also improved intrinsic activities and white matter fiber damage in the brain, increased c-Fos expression, reduced Iba-1 expression in the prefrontal cortex, hippocampus, and substantia nigra, and increased TH expression in the substantia nigra. Fluoxetine reduced the concentration of inflammatory cytokines (IL-1α, IL-6, TNF-α, and IFN-γ). The gene and protein expression of Notch1, Jagged1, Hes1, and Hes5 were significantly lower than the LPS group after treatment with fluoxetine. Fluoxetine plays neuroprotective effects in relieving LPS-induced depression-like and motor behaviors. The underlying mechanisms may be related to inhibiting microglial activation, regulating the Notch signaling pathway, and inhibiting the inflammatory response.

Effects of Hypoxia-Inducible Factor 1 (HIF-1) Signaling Pathway on Acute Ischemic Stroke.

Background Epidemiological surveys show that a large number of cerebrovascular diseases occur in China every year, and among these cerebrovascular diseases, ischemic diseases are predominant. Ischemia leads to irreversible degenerative necrosis of a large number of brain neurons and severe neurological deficits. Aims This study is aimed at exploring the mechanism of the major regulatory effect of hypoxia-inducible factor 1 (HIF-1) pathway on proangiogenesis and providing new ideas for the treatment of ischemic stroke. Materials and Methods The rats were randomly divided into normal and ischemic control groups, and the ischemic control group was subjected to the middle cerebral artery occlusion (MCAO) cerebral ischemia model by the wire embolization method, and the rats were executed in batches at 6 h, 1 d, and 3 d after ischemia-reperfusion, and the brain tissue specimens were taken for examination to investigate the effect of hypoxia-inducible factor 1 (HIF-l) signaling pathway on acute ischemic stroke. Results At 3 d, the number of VEGFR2 positive cells increased significantly, and there was a significant difference compared with the control group (P < 0.05). At 3 d, the number of HIF-1α-positive cells increased significantly, and there was a significant difference compared with the control group (P < 0.05). The number of Hes1+factor VIII positive cells in the ischemic cortex increased significantly on the 1st and 3rd day, and there was a significant difference compared with the control group (P < 0.05). The expression of Hes1 protein was significantly lower than the normal level after 6 h of ischemia, and the protein expression was significantly increased at 1 d and 3 d after ischemia (P < 0.05). Conclusion By detecting the expression changes of Hesl+factor VII in the ischemic area, the results show that ischemia and hypoxia activate the HIF-1, making the HIF-l the main regulatory pathway in the process of angiogenesis after ischemia.

EP14.02-007 Hes1 Protein Expression and Its Significance in Resected Small Cell Lung Cancers

Small cell lung cancer (SCLC) is a highly aggressive malignancy prone to early recurrence and metastasis. Preclinical studies have found that Hes1, as a transcription repressor of the basic helix-loop-helix (BHLH) family, can not only prevent the proliferation and migration of SCLC tumor cells, but also inhibit the neuroendocrine transcription factor ASCL1. However, the prognostic implication of Hes1 protein on surgically resected SCLCs remains unclear. The current study aims to analyze the expression pattern and prognostic value of Hes1 protein in SCLC.

30P Hes1 protein expression and its significance in resected small cell lung cancers

Small cell lung cancer (SCLC) is a highly aggressive malignancy prone to early recurrence and metastasis. Preclinical studies have found that Hes1, as a transcription repressor of the basic helix-loop-helix (BHLH) family, can not only prevent the proliferation and migration of SCLC tumor cells, but also inhibit the neuroendocrine transcription factor ASCL1. However, the prognostic implication of Hes1 protein on surgically resected SCLCs remains unclear. The current study aims to analyze the expression pattern and prognostic value of Hes1 protein in SCLC.

Ischemic preconditioning/ischemic postconditioning alleviates anoxia/reoxygenation injury via the Notch1/Hes1/VDAC1 axis.

Ischemic preconditioning (IPC), and ischemic postconditioning (IPost) have a significant protective effect on myocardial ischemia/reperfusion (MI/R) injury by alleviating oxidative stress and mitochondrial disturbances, although the underlying molecular mechanisms are unclear. The study was to demonstrate that cardioprotection against anoxia/reoxygenation (A/R) injury is transduced via the Notch1/Hes1/VDAC1 signaling pathway. Using mass spectrometry and tandem affinity purification (TAP), to screen for differentially expressed proteins associated with Hes1, followed by standard bioinformatics analysis. The co-immunoprecipitation (Co-IP) assay confirmed an interaction between Hes1 and VDAC1 proteins. H9c2 cells were transfected with Hes1 adenoviral N-terminal TAP vector (AD-NTAP/Hes1) and Hes1-short hairpin RNA adenoviral vector (AD-Hes1-shRNA) to establish A/R injury, IPC, and IPost models, respectively. The expression of Hes1 and VDAC1 proteins were measured by western blot analysis, while the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and apoptosis were evaluated by flow cytometry. AD-NTAP/Hes1 can activate the exogenous protein expression of Hes1, thus decreasing creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activity and promoting cell viability. The study found that VDAC1 was a potential target protein for Hes1 and the overexpression of Hes1 protein expression downregulated protein expression levels of VDAC1, reduced ROS production, stabilized ΔΨm, and inhibited apoptosis in H9c2 cells. Additionally, downregulation of Hes1 protein expression also upregulated VDAC1 protein expression, increased ROS production, imbalanced ΔΨm, promoted cell apoptosis, and attenuated the cardioprotection afforded by IPC and IPost. The Notch1/Hes1 signaling pathway activated by IPC/IPost can directly downregulate the protein expression of VDAC1 and consequently relieve A/R injury.

Bufei Yishen formula attenuates cigarette smoke extract-induced airway mucus hypersecretion by regulating Notch signaling pathway

To explore the mechanism of Bufei Yishen formula (BYF) on attenuating cigarette smoke extract (CSE)-induced airway mucus hypersecretion by regulating Notch signaling pathway. The human airway epithelial cell 16HBE was cultured in vitro, and the cells in logarithmic growth phase were used for the experiments. (1) Intervention condition screening experiment: the 16HBE cells were grouped, methylthiazolyldiphenyl-tetrazolium (MTT) method and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of different concentrations of CSE (2.5%, 5%, 10%, 20%, 40%), different concentrations of BYF drug-containing serum (5%, 10%, 20%, 40%), and different concentrations of Notch signal pathway blocker DAPT (5, 10, 20, 40 μmol/L) on cell activity and secretion of mucin 5AC (MUC5AC) levels. In addition, a blank control group was set up to screen out the best conditions for preparing CSE-induced cell mucus hypersecretion model and BYF and DAPT intervention. (2) Intervention experiment: the 16HBE cells were divided into four groups. The blank control group was not given any treatment; the 16HBE cells were induced by 10% CSE for 24 hours to prepare mucus hypersecretion model in the CSE model group; the cells in the CSE+BYF group and CSE+DAPT group were given 10% BYF or 20 μmol/L DAPT, respectively, for intervention at the same time for 24 hours. Real-time fluorescent quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expressions of MUC5AC, Notch3 and hairy and enhancer of split 1 (HES1) in the cells. Western blotting was used to detect the protein expressions of Notch3 and HES1 in the cells. (1) Results of the screening experiment of intervention conditions: compared with the blank control group, 10% CSE induction for 24 hours was the best condition for establishing cell mucus hypersecretion model that neither affected cell viability nor increased the secretion of MUC5AC; while 10% BYF and 20 μmol/L DAPT was the optimal intervention condition. (2) Intervention experiment results: compared with the blank control group, the mRNA expressions of MUC5AC, Notch3, and HES1 and the protein expressions of Notch3 and HES1 in the CSE model group were significantly increased, indicating that CSE activated Notch3 and HES1 signal activation and induced 16HBE cells to secrete mucus protein. Compared with the CSE model group, BYF and DAPT could significantly down-regulate the mRNA and protein expressions of MUC5AC, Notch3, and HES1 in cells [MUC5AC mRNA (2-ΔΔCT): 1.03±0.13, 0.96±0.05 vs. 1.35±0.07, Notch3 mRNA (2-ΔΔCT): 1.10±0.14, 1.10±0.02 vs. 1.31±0.15, HES1 mRNA (2-ΔΔCT): 1.26±0.10, 1.14±0.15 vs. 1.45±0.08, Notch3 protein (Notch3/GAPDH): 0.10±0.03, 0.16±0.03 vs. 0.31±0.09, HES1 protein (HES1/GAPDH): 0.37±0.06, 0.34±0.08 vs. 0.50±0.05, all P < 0.05]. The mechanism of BYF attenuating mucus hypersecretion of 16HBE cells induced by CSE was associated with the inhibition of Notch signaling pathway activation.

JNK and Jag1/Notch2 co-regulate CXCL16 to facilitate cypermethrin-induced kidney damage

Cypermethrin (CYP), a widely-used composite pyrethroid pesticide, has underlying nephrotoxic effects. To elucidate potential roles of the MAPK pathway, the Jag/Notch pathway, and miRNAs in CYP-mediated kidney lesion, Sprague-Dawley rats and glomerular mesangial cells were used in this work. Results displayed that β-CYP abnormally altered renal histomorphology and ultrastructures, induced renal DNA damage, and impaired renal functions, as evidenced by the increase in plasma levels of Cys-C and β2-Mg. β-CYP activated the JNK/c-Jun pathway by inducing ROS and oxidative stress. Meanwhile, β-CYP changed the miRNA expression profile, miR-21–5p showing the most significant increase. Moreover, the Jag1/Notch2/Hes1 pathway was directly targeted by miR-21–5p, the mRNA and protein expression of Jag1, Notch2, and Hes1 being declined in vivo and in vitro. The chemokine CXCL16 was induced by β-CYP, accompanied by the inflammatory factor production and inflammatory cell infiltration in kidneys. The specific JNK inhibitor, Jag1 overexpression, Hes1 overexpression, bidirectional Co-IP, ChIP, and CXCL16 silencing demonstrated that CXCL16 co-regulated by the JNK/c-Jun and Jag1/Notch2/Hes1 pathways elicited renal inflammation. Collectively, our findings indicate that β-CYP is of nephrotoxicity and it not only directly changes renal histomorphology and ultrastructures, but induces CXCL16 to trigger renal inflammation via the JNK/c-Jun and Jag1/Notch2/Hes1 pathways, finally synergistically contributing to kidney damage.

Microencapsulated islet transplantation alleviates podocyte injury in diabetic nephropathy via inhibiting Notch-1 signaling

ObjectivePodocyte injury has a critical role in the pathogenesis of diabetic nephropathy (DN). Microencapsulated islet transplantation (MIT) is identified as an effective method for improving the clinical condition of DN. This study aimed to explore the role and mechanism of MIT in alleviating podocyte injury in DN. MethodsA mouse model of DN was constructed using streptozotocin (STZ). Mice were divided into 3 groups: the untreated diabetic nephropathy group (DN group), the microencapsulated islet transplantation-treated group (MIT group) and the control group. The mice were raised for 6 weeks posterior to islet transplantation to identify the role of MIT. Renal function and structure of glomerular filtration barrier were assessed by urine analysis, histopathological examination, and transmission electron microscopy. The expression levels of several proteins including Caspase-3, Bcl2/Bax, β-galactosidase, Ki-67, synaptopodin, WT-1, Jagged-1, Notch-1, and Hes-1 in renal tissues were identified via immunohistochemistry (IHC), immunofluorescence (IF), and western blotting techniques. ResultsCompared with the DN group, the MIT group presented decreased levels of blood glucose, urinary albumin/creatinine, urea nitrogen, and serum creatinine while their body weight gradually increased. Glomerular injury in the MIT group was significantly better than that in the DN group. The MIT group indicated significantly decreased expression of Caspase-3, β-galactosidase, Bax/Bcl-2, and Ki-67 when compared with DN group, while the proportion of synaptopodin- and WT-1-positive cells was significantly increased (P < 0.05). The protein expression of Jagged-1, Notch-1, and Hes-1 in the glomerulus of the MIT group was significantly lower than that in the DN group (P < 0.05). ConclusionMIT alleviates podocyte injury induced by DN by inhibiting Notch-1 signaling. The identification of signaling pathways influencing podocyte restoration can help evaluate personalized medicine efficacy for patients treated with islet transplantation.

P106 Macrophages as a source of NOTCH Ligands in Crohn’s disease: implications in fibrosis

Abstract Background Fibrosis constitute the main complications associated to Crohn′s disease (CD). NOTCH signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of NOTCH ligands derived from macrophages in the complications of CD. Methods The aim of the present study is to analyze the role of NOTCH ligands derived from macrophages in the complications of CD. We have analyzed: the protein expression of NOTCH ligands and receptors in CD patients with fistulizing (B3) and stenting pattern (B2), the protein expression of NOTCH ligands in macrophages treated with the main cytokines present in CD patients (IFNγ-, IL10-, IL4, TNFα-U937 treated cells), the protein expression of HES1 and fibrosis markers in DLL4-HT29 and DLL3-HT29 treated cells. Results are expressed as fold induction (mean±SEM). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test or t-tet. Results The expression of DLL4 and NOTCH4 were significantly higher in intestinal samples from B3 CD patients (3,2 ± 0,6 N=4* and 3,8 ± 0,6 N=8*, respectively) than in B2 patients (1,6 ± 0,2 N=4 and 1,7 ± 0,3 N=8, respectively) and controls (1,0 ± 0,1N=3 and 1,0 ± 0,1 N=8, respectively). IFNγ-U937 treated cells increased significantly the protein expression of DLL3 and DLL4 (1,6 ± 0,09 N=6* and 1,3 ± 0,1 N=7*, respectively) respect vehicle; IL4 increased significantly the expression of DLL4 (1,4 ± 0,1 N=7*) and TNFα increased significantly the expression of DLL3 (1,4 ± 0,1 N=5*), respect vehicle. DLL4-HT29 treated cells increased significantly fibrosis markers (VIMENTIN: 1,7 ± 0,1 N=3*; SNAIL: 2,2 ± 0,2 N=3*) and HES1 (1,4 ± 0,06 N=3*), respect vehicle (1,0 ± 0,07 N=6; 1,0 ± 0,05 N=6; and 1,0 ± 0,05 N=6, respectively). DLL3-HT29 treated cells only produced a reduction in the protein expression of ECADHERIN (0,4 ± 0,1 N=3*), respect vehicle (1,0 ± 0,09 N=6). Conclusion Macrophages may act as a source of NOTCH ligands who could act as fibrosis mediators in CD patients with a fistulizing (B3) behavior. The microenvironment rich in IFNγ could activate the fibrosis process in epithelial cells by favoring the expression of DLL4 and DLL3 in macrophages. DLL4 mainly activates transcription factors involved in mesenchymal epithelial transition in colonic epithelial cells (SNAIL), while DLL3 seems to have a more relevant role in cell-cell junction modification (ECADHERIN).

Role of oleanolic acid in relieving psoriasis and its underlying mechanism of action

The present study aimed to determine whether oleanolic acid (Ole) could be used to treat psoriasis and its related underlying mechanism of action via in vitro analysis. HaCaT cells were stimulated with IL-22 to established an in vitro psoriasis cell model. MTT, flow cytometry and TUNEL assays, respectively. Transmission electron microscopy was used to observe the cell ultrastructure. LC3B protein expression levels were analyzed using immunofluorescence, other protein expression levels were determined using western blotting. Cell viability was significantly increased, while the apoptotic rate was significantly decreased in the model group (P < 0.001). In addition, Notch1, Hes1, beclin 1 and LC3B protein expression levels were significantly downregulated, while P62 protein expression levels were significantly upregulated in the model group compared with the control group (P < 0.001). Supplementation of Ole, the increased levels of proliferation were significantly suppressed, while cell apoptosis was significantly increased (P < 0.05) in a dose-dependent manner, which was discovered to occur via Notch1 upregulation. Notably, the transfection with small interfering RNA-Notch1 significantly reversed the effect of Ole treatment (P < 0.001) and the levels of autophagy were also decreased. In conclusion, the findings of the current study suggested that Ole may relieve psoriasis via upregulating Notch1, which subsequently regulates cell autophagy.