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.

Involvement of NOTCH1-mediated Microglia Activation in Neuromodulation of Chronic Prostatitis-related Pain.

BACKGROUND/AIM: This study aimed to investigate the role of NOTCH receptor 1 (NOTCH1)-mediated activation of microglia in the L5-S2 spinal dorsal horn in chronic prostatitis pain. MATERIALS AND METHODS: Rats were divided into chronic prostatitis (CP) group and control group. Complete Freund's adjuvant was injected into the prostate, and prostate pathology and pain-related behavior were monitored to assess the successful establishment of the CP-related pain model. The dorsal horn of the L5-S2 spinal cord was collected for the detection of ionized calcium-binding adapter molecule 1 (IBA-1) and NOTCH1 expression by quantitative real time polymerase chain reaction and the detection of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) by enzyme-linked immunosorbent assay. Electrical excitability was assessed with whole-cell patch clamp. In addition, NOTCH1 receptor inhibitor or inhibitor of microglial cell activation was injected into the subarachnoid space, and the pro-inflammatory cytokines in the spinal cord were detected. RESULTS: In the CP group, the expression of NOTCH1, IBA-1, TNF-α and IL-1ß began to increase at 4 days, peaked at 12 days, and began to decline at 24 days, and it was significantly higher than in the control group (p<0.01). Inhibition of microglia or NOTCH1 receptor markedly reduced the content of TNF-α and IL-1ß in the spinal cord (p<0.05). At 4, 12 and 24 days, the amplitude and frequency of neuronal action potential increased and the threshold decreased markedly as compared to the control group (p<0.05), and spontaneous action potential was noted. CONCLUSION: NOTCH1 mediates the activation of microglia in the L5-S2 spinal cord, leading to the secretion of inflammatory factors and enhanced electrical excitability of neurons, which is related to persistent and refractory chronic prostatitis-related pain.

N6-Methyladenosine Reader YTHDF1 Promotes Stemness and Therapeutic Resistance in Hepatocellular Carcinoma by Enhancing NOTCH1 Expression.

N6-methyladenosine (m6A) RNA modification is the most common and conserved epigenetic modification in mRNA and has been shown to play important roles in cancer biology. As the m6A reader YTHDF1 has been reported to promote progression of hepatocellular carcinoma (HCC), it represents a potential therapeutic target. In this study, we evaluated the clinical significance of YTHDF1 using human HCC samples and found that YTHDF1 was significantly upregulated in HCCs with high stemness scores and was positively associated with recurrence and poor prognosis. Analysis of HCC spheroids revealed that YTHDF1 was highly expressed in liver cancer stem cells (CSC). Stem cell-specific conditional Ythdf1 knockin (CKI) mice treated with diethylnitrosamine showed elevated tumor burden as compared with wild-type mice. YTHDF1 promoted CSCs renewal and resistance to the multiple tyrosine kinase inhibitors lenvatinib and sorafenib in patient-derived organoids and HCC cell lines, which could be abolished by catalytically inactive mutant YTHDF1. Multiomic analysis, including RNA immunoprecipitation sequencing, m6A methylated RNA immunoprecipitation sequencing, ribosome profiling, and RNA sequencing identified NOTCH1 as a direct downstream of YTHDF1. YTHDF1 bound to m6A modified NOTCH1 mRNA to enhance its stability and translation, which led to increased NOTCH1 target genes expression. NOTCH1 overexpression rescued HCC stemness in YTHDF1-deficient cells in vitro and in vivo. Lipid nanoparticles targeting YTHDF1 significantly enhanced the efficacy of lenvatinib and sorafenib in HCC in vivo. Taken together, YTHDF1 drives HCC stemness and drug resistance through an YTHDF1-m6A-NOTCH1 epitranscriptomic axis, and YTHDF1 is a potential therapeutic target for treating HCC. SIGNIFICANCE: Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.

Dan-Deng-Tong-Nao softgel capsule promotes angiogenesis of cerebral microvasculature to protect cerebral ischemia reperfusion injury via activating HIF-1α-VEGFA-Notch1 signaling pathway.

BACKGROUND: A proprietary Chinese herbal product called Dan-Deng-Tong-Nao softgel capsule (DDTNC) is used to treat ischemic stroke. However, the preventive mechanisms of DDTNC against cerebral ischemia reperfusion injury (CIRI) haven not been characterized. OBJECTIVE: To explore the mechanisms of protective effects of DDTNC against CIRI from both internal and external levels. METHODS: Chemical characterization was performed using UPLC. The potential protective mechanisms of DDTNC against CIRI were predicted using network pharmacology. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in rats. An model of brain microvascular endothelial cells (BMECs) induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was also established. We evaluated neurological deficits, cerebral infarct volume, cortical neuron damage, and mitochondrial swelling in vivo. We evaluated the expression of VEGFR2, VEGFA, HIF-1α, CD31, and CD34 in ischemic cortex, and VEGF, bFGF, BDNF, angiostatin, and endostatin in serum of rats and in BMEC supernatants. We also evaluated cell viability, cytotoxicity, intracellular ROS, apoptosis, and migration ability in vitro. RESULTS: Seven components were detected in DDTNC. KEGG enrichment analysis showed that DDTNC may modulate angiogenesis via the HIF-1 signaling pathway. DDTNC treatment reduced neurological score and infarct volume, and improved cell morphology of damaged neurons. Transmission electron microscopy showed that DDTNC reduced mitochondria swelling in cortical neurons. Furthermore, DDTNC reduced intracellular ROS and inhibited apoptosis. DDTNC boosted the expression of CD31, CD34, VEGFR2, VEGFA and HIF-1α, highlighting its involvement in angiogenesis, according to immunofluorescence studies. Furthermore, DDTNC enhanced tube formation and migration of BMECs in vitro. ELISA and western blotting indicated that DDTNCCSF induced the expression of VEGF, BDNF and bFGF, reduced the level of angiostatin and endostatin, increased the protein expression of VEGFA, Notch1 and HIF-1α in vitro and in vivo. CONCLUSIONS: DDTNC promoted angiogenesis to protect brain tissue against MCAO/R, and exerted protective effects against OGD/R in BMECs via activating HIF-1α-VEGFA-NOTCH1 signal transduction pathway.

Melatonin modulates endometrial decidualization via NOTCH1-NRF2-FOXO1-GSH pathway†.

Melatonin is important for oocyte maturation, fertilization, early embryonic development, and embryo implantation, but less knowledge is available regarding its role in decidualization. The present study found that melatonin did not alter the proliferation of human endometrial stromal cells (ESCs), as well as cell cycle progress, but suppressed stromal differentiation after binding to the melatonin receptor 1B (MTNR1B), which was visualized in decidualizing ESCs. Further analysis evidenced that application of melatonin resulted in the diminishment for NOTCH1 and RBPJ expression. Supplementation of recombinant NOTCH1 protein (rNOTCH1) counteracted the impairment of stromal differentiation conferred by melatonin, while the addition of the NOTCH signaling pathway inhibitor DAPT aggravated the differentiation progress. Meanwhile, melatonin might restrain the expression and transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2), whose blockage accelerated the fault of stromal differentiation under the context of melatonin, but this restraint was subsequently ameliorated by rNOTCH1. Forkhead box O 1 (FOXO1) was identified as a downstream target of melatonin in decidualization. Repression of NRF2 antagonized the retrieval of rNOTCH1 due to aberrant FOXO1 expression elicited by melatonin. Moreover, melatonin brought about the occurrence of oxidative stress accompanied by an obvious accumulation of intracellular reactive oxygen species and a significant reduction in glutathione (GSH) content, as well as enzymatic activities of glutathione peroxidase and glutathione reductase, whereas supplementation of rNOTCH1 improved the above-mentioned effects. Nevertheless, this improvement was disrupted by the blockage of NRF2 and FOXO1. Furthermore, addition of GSH rescued the defect of stromal differentiation by melatonin. Collectively, melatonin might impair endometrial decidualization by restraining the differentiation of ESCs dependent on NOTCH1-NRF2-FOXO1-GSH pathway after binding to the MTNR1B receptor.

Xue-Jie-San prevents the early development of colitis-associated intestinal fibrosis by blocking Notch1 and FGL1 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Xue-Jie-San (XJS), as a traditional Chinese herb prescription, has satisfactory effects on improving clinical symptoms and facilitating the healing of intestinal ulcers in patients with Crohn's disease (CD). This motivates the application of XJS on CD-associated complications. AIM OF THE STUDY: Intestinal fibrosis is a debilitating complication of CD. Currently, there is no effective medication available for preventing or reversing CD-related intestinal fibrosis. This study aimed to assess the efficacy and underlying mechanisms of XJS in the treatment of colitis-associated intestinal fibrosis. MATERIALS AND METHODS: A rat model of CD-related intestinal fibrosis was induced by 2,4,6-trinitrobenzene sulfonic acid administration and treated with XJS. The pathological changes of intestinal fibrosis were evaluated using Masson staining. Collagen deposition and epithelial-to-mesenchymal transition (EMT) were verified by immunohistochemical staining and Western blot analysis. Endothelial-to-mesenchymal transition (EndoMT) was assessed with immunofluorescence and immunohistochemical staining as well as Western blot analysis. Transmission electron microscopy was utilized to observe autophagosomes. The levels of autophagy-related proteins were detected via immunofluorescence staining and Western blot. Finally, the mTOR/ULK1 signaling pathway regulated by Notch1 or FGL1 was analyzed by Western blot. RESULTS: The results found that XJS ameliorated intestinal fibrosis through reducing the deposition of collagens such as Collagen 1 and Collagen 3. XJS inhibited the EMT process by increasing E-cadherin levels and decreasing the expressions of N-cadherin, Vimentin and Snail, which played a crucial role in collagen secretion and intestinal fibrosis. In addition, XJS also repressed the EndoMT process as reflected by the upregulation of CD31 and VE-cadherin levels and the downregulation of FSP1 and α-SMA expressions. Autophagy was activated following XJS treatment via suppression of the mTOR/ULK1 signaling pathway. Furthermore, XJS acted as an inhibitor of Notch1 and FGL1 signals, both of which regulated the mTOR signaling. CONCLUSIONS: Our findings validated that XJS prevented the early development of CD-related intestinal fibrosis by blocking the Notch1 and FGL1 signaling pathways to activate autophagy and thereby inhibit EMT and EndoMT.

Short-chain fatty acids ameliorate necrotizing enterocolitis-like intestinal injury through enhancing Notch1-mediated single immunoglobulin interleukin-1-related receptor, toll-interacting protein, and A20 induction.

Single immunoglobulin interleukin-1-related receptor (SIGIRR), toll-interacting protein (TOLLIP), and A20 are major inhibitors of toll-like receptor (TLR) signaling induced postnatally in the neonatal intestine. Short-chain fatty acids (SCFAs), fermentation products of indigestible carbohydrates produced by symbiotic bacteria, inhibit intestinal inflammation. Herein, we investigated the mechanisms by which SCFAs regulate SIGIRR, A20, and TOLLIP expression and mitigate experimental necrotizing enterocolitis (NEC). Butyrate induced NOTCH activation by repressing sirtuin 1 (SIRT1)-mediated deacetylation of the Notch intracellular domain (NICD) in human intestinal epithelial cells (HIECs). Overexpression of NICD induced SIGIRR, A20, and TOLLIP expression. Chromatin immunoprecipitation revealed that butyrate-induced NICD binds to the SIGIRR, A20, and TOLLIP gene promoters. Notch1-shRNA suppressed butyrate-induced SIGIRR/A20 upregulation in mouse enteroids and HIEC. Flagellin (TLR5 agonist)-induced inflammation in HIEC was inhibited by butyrate in a SIGIRR-dependent manner. Neonatal mice fed butyrate had increased NICD, A20, SIGIRR, and TOLLIP expression in the ileal epithelium. Butyrate inhibited experimental NEC-induced intestinal apoptosis, cytokine expression, and histological injury. Our data suggest that SCFAs can regulate the expression of the major negative regulators of TLR signaling in the neonatal intestine through Notch1 and ameliorate experimental NEC. Enteral SCFAs supplementation in preterm infants provides a promising bacteria-free, therapeutic option for NEC.NEW & NOTEWORTHY Short-chain fatty acids (SCFAs), such as propionate and butyrate, metabolites produced by symbiotic gut bacteria are known to be anti-inflammatory, but the mechanisms by which they protect against NEC are not fully understood. In this study, we reveal that SCFAs regulate intestinal inflammation by inducing the key TLR and IL1R inhibitors, SIGIRR and A20, through activation of the pluripotent transcriptional factor NOTCH1. Butyrate-mediated SIGIRR and A20 induction represses experimental NEC in the neonatal intestine.

SIRT1/Notch1 signal axis involves in the promoting effect of Segetalin B on bone formation.

Phytoestrogens are a class of potential natural medicines for treating postmenopausal osteoporosis (PMOP). Segetalin B (SB) is a cyclic peptide compound showing estrogenic activity. This study reports the effect of SB on bone formation among ovariectomized (OVX) rats. The bone marrow mesenchymal stem cells (BMSCs) from OVX rats were cultured in vitro. Alizarin Red staining was utilized to observe the effect of SB on the mineralization of BMSCs. The levels of alkaline phosphatase (ALP), osteocalcin, bone morphogenetic protein (BMP-2), and Sirtuin 1 (SIRT1) activities were detected. The OVX rats were treated with SB in vivo. Micro-CT was utilized for imaging analysis. Urine calcium and phosphorus, and ALP activity in bone marrow were assayed. Western blot analysis and immunofluorescence were incorporated to detect protein expressions in vitro and in vivo. The results showed that SB dose-dependently promoted mineralization of OVX rat-derived BMSCs in vitro increased the level of Osteocalcin, BMP-2, ALP, and SIRT1 activity. Moreover, it upregulated expressions of Runx2, Osterix, and SIRT1, downregulated expressions of Notch intracellular domain (NICD), acetyl-NICD, and hairy and enhancer of split 1 (Hes1). In addition, SB treatment significantly decreased bone loss, inhibited calcium and phosphorus loss, elevated ALP activity, upregulated Runx2, Osterix, and SIRT1, and downregulated NICD and Hes1 in OVX rats in vivo. However, EX527, a SIRT1-selective inhibitor, could reverse the above effects of SB in vitro or in vivo. These results indicate that SB is a potential natural medicine to improve PMOP. Thus, its mechanism of promoting bone formation involves the SIRT1/Notch1 signaling axis.

[Effect of "nape seven needles" on expression of hypoxia inducible factor-1α and Notch1 in cervical intervertebral disc degeneration rats].

OBJECTIVE: To observe the effect of "nape seven needles" on the expressions of hypoxia inducible factor-1α (HIF-1α) and Notch1 in cervical intervertebral disc of cervical disc degeneration (CDD) rats, so as to reveal its underlying mecha-nisms in improving CDD. METHODS: SD male rats were randomly divided into normal, model, non-acupoint and nape seven needles groups, with 10 rats in each group. Staticdynamic imbalance method was used to establish CDD model. Rats in the nape se-ven needles group were treated with acupuncture at "Fengfu"(GV16), and bilateral "Fengchi"(GB20), "Wangu"(GB12) and "Tianzhu"(BL10), and rats in the non-acupoint group received acupuncture at the sham acupoints at the caudal tip and armpit, both for 20 min, 6 days a week for 4 weeks. After intervention, tilted plane test and spiral CT were used to assess the neck movement function and cervical degeneration degree of rats; immunohistochemistry was used to observe the protein expression levels of HIF-1α and Notch1 in cervical discs tissue; Western blot and real-time quantitative PCR were used to detect the protein and mRNA expression levels of HIF-1α and Notch1 in cervical discs tissue, respectively. RESULTS: After modeling, the cervical curvature was straightened, with narrowed intervertebral space, rough and hardened articular surface, osteophytes, and blurred articular space and articular process, which was relatively milder in the nape seven needles group. Compared with the normal group, the angle of tilted plane was significantly reduced (P<0.05), while cervical scores, HIF-1α mRNA expression level in cervical intervertebral disc tissue were significantly increased (P<0.05) in the model group. Compared with the model and the non-acupoint groups, cervical scores were significantly reduced (P<0.05), while the angle of tilted plane, HIF-1α and Notch1 positive expressions, HIF-1α mRNA expression level, Notch1 protein and mRNA expression levels in cervical intervertebral disc tissue were significantly increased (P<0.05) in the nape seven needles group. CONCLUSION: Acupuncture of "nape seven needles" can reduce the degree of cervical degeneration in rats, which was possibly associated with its effects in up-regulating the expressions of HIF-1α and Notch1 in cervical intervertebral disc tissue, promoting the proliferation and recovery of endogenous cells in nucleus pulposus.

The licorice flavonoid isoliquiritigenin attenuates Mycobacterium tuberculosis-induced inflammation through Notch1/NF-κB and MAPK signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Glycyrrhiza is a small perennial herb that has been traditionally used to treat many diseases across the world. Licorice (Gancao in Chinese) is the dried root and rhizome of G. glabra, G. uralensis or G. inflata. Licorice plays an important role in traditional Chinese medicine (TCM), and is the most frequently used in Chinese herbal formulas. Isoliquiritigenin (ISL) is a flavonoid extracted from licorice, and has been evaluated for its various biological activities, including anti-inflammatory, anti-tumor and anti-oxidant activities. Excessive and persistent inflammation in the Mycobacterium tuberculosis (Mtb) infection is not conducive to the elimination of Mtb, but contributes to serious pulmonary dysfunction. AIM OF THE STUDY: This study aimed to examine the anti-inflammatory effects of ISL in the Mtb infection. METHODS: In vitro models of Mtb-infected macrophages were established. Murine macrophage Raw 264.7 cells and primary peritoneal macrophages were used in this study. Cell viability was determined by the cell counting kit-8 (CCK-8) assay. The effects of ISL on the secretion levels of interleukin -1ß (IL-1ß), tumor necrosis factor -α (TNF-α), and interleukin -6 (IL-6) were detected by the enzyme-linked immunosorbent assay (ELISA). The expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were measured by the real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. Western blot was used to assess the effects of ISL on the activation of NLRP3 inflammasome and Notch1/NF-κB and MAPK signaling pathways. Immunofluorescence assays was used to detected the translocation of phosphorylation of p65 subunit of NF-κB. RESULTS: It was revealed that ISL inhibited the secretion of IL-1ß and the activation of pore-forming protein (gasdermin D, GSDMD) by suppressing the activation of NLPR3 inflammasome induced by Mtb infection. ISL was also shown to have promising inhibitory effects on inflammatory factors, such as TNF-α, IL-6, iNOS and COX2. Regarding the anti-inflammatory mechanism of ISL, it was found that ISL exerted its anti-inflammatory effects by inhibiting the activation of Notch1/NF-κB and MAPK signaling pathways. CONCLUSION: ISL reduced Mtb-induced inflammation through the Notch1/NF-κB and MAPK signaling pathways. ISL might be used as a potential adjuvant drug to treat tuberculosis by adjusting host immune responses.

Hydroxysafflor Yellow A Alleviates Ischemic Stroke in Rats via HIF-1[Formula: see text], BNIP3, and Notch1-Mediated Inhibition of Autophagy.

Stroke has become a major cause of death and disability worldwide. The cellular recycling pathway autophagy has been implicated in ischemia-induced neuronal changes, but whether autophagy plays a beneficial or detrimental role is controversial. Hydroxysafflor Yellow A (HSYA), a popular herbal medicine, is an extract of Carthamus tinctorius and is used to treat ischemic stroke (IS) in China. HSYA has been shown to prevent cardiovascular and cerebral ischemia/reperfusion injury in animal models. However, the specific active ingredients and molecular mechanisms of HSYA in IS remain unclear. Here, we investigated the effect of HSYA treatment on autophagy in a rat model of IS. IS was induced in rats by middle cerebral artery occlusion. Rats were treated once daily for 3 days with saline, HYSA, or the neuroprotective agent Edaravone. Neurobehavioral testing was performed on days 1, 2, and 3 post-surgery. Brains were removed on day 3 post-surgery for histological evaluation of infarct area, morphology, and for qRT-PCR and western blot analysis of the expression of the autophagy factor LC3 and the signaling molecules HIF-1[Formula: see text], BNIP3, and Notch1. Molecular docking studies were performed in silico to predict potential interactions between HSYA and LC3, HIF-1[Formula: see text], BNIP3, and Notch1 proteins. The result showed that HSYA treatment markedly alleviated IS-induced neurobehavioral deficits and reduced brain infarct area and tissue damage. HSYA also significantly reduced hippocampal expression levels of LC3, HIF-1[Formula: see text], BNIP3, and Notch1. The beneficial effect of HSYA was generally superior to that of Edaravone. Molecular modeling suggested that HSYA may bind strongly to HIF-1[Formula: see text], BNIP3, and Notch1 but weakly to LC3. In conclusion, HSYA inhibits post-IS autophagy induction in the brain, possibly by suppressing HIF-1[Formula: see text], BNIP3 and Notch1. HSYA may have utility as a post-IS neuroprotective agent.

Keap-NRF2 signaling contributes to the Notch1 protected heart against ischemic reperfusion injury via regulating mitochondrial ROS generation and bioenergetics.

Myocardial ischemia/reperfusion (I/R) injury is recognized as the leading cause of death worldwide. However, the molecular mechanisms involved in this process are still not fully understood. We previously reported that the combined action of Notch1 and Keap1-NRF2 signaling pathway can significantly increase the activity of cardiomyocytes, inhibit the apoptosis of cardiomyocytes, reduce the formation of reactive oxygen species, and improve the antioxidant activity in neonate rat myocardial cells. However, the regulatory mechanism of Notch1 signaling pathway on the NRF2 signaling pathway and its actual role on I/R injury are still unclear. Herein, we found that Keap-NRF2 signaling is activated by Notch1 in RBP-Jκ dependent manner, thus protects the heart against I/R injury via inhibiting the mitochondrial ROS generation and improves the mitochondrial bioenergetics in vitro and in vivo. These results suggest that Keap-NRF2 signaling might become a promising therapeutic strategy for treating myocardial I/R injury.

DT7 peptide-modified lecithin nanoparticles co-loaded with γ-secretase inhibitor and dexamethasone efficiently inhibit T-cell acute lymphoblastic leukemia and reduce gastrointestinal toxicity.

T-cell acute lymphoblastic leukemia (T-ALL) is a serious hematologic malignancy and glucocorticoid resistance is the main recurrent cause for a high relapsed and death rate. Here, we proposed an effective therapeutic regimen of combining gamma-secretase inhibitors (GSIs) with dexamethasone (DEX) to overcome glucocorticoid resistance. Moreover, the bone marrow targeting DT7 peptide-modified lecithin nanoparticles co-loaded with DEX and GSI (TLnp/D&G) were developed to enhance T-ALL cells recognition and endocytosis. In vitro cytotoxicity studies showed that TLnp/D&G significantly inhibited cell survival and promoted apoptosis of T-ALL cells. Mechanically, we found that GSIs promoted DEX-induced cell apoptosis by two main synergetic mechanisms: 1) GSIs significantly upregulated glucocorticoid receptor (GR) expression in T-ALL and restored the glucocorticoid-induced pro-apoptotic response. 2) Both DEX and GSI synergistically inhibited BCL2 and suppressed the survival of T-ALL cells. Furthermore, in vivo studies demonstrated that TLnp/D&G showed high bone marrow accumulation and better antileukemic efficacy both in leukemia bearing models and in systemic Notch1-induced T-ALL models, with excellent biosafety and reduced gastrointestinal toxicity. Overall, our study provides new strategies for the treatment of T-ALL and promising bone marrow targeting systems with high transformation potential.

Basic fibroblast growth factor inhibits aortic valvular interstitial cells calcification via Notch1 pathway.

Calcific aortic valve disease (CAVD) is an active pathological process mediated by abnormal activation and transdifferentiation of valvular interstitial cells (VICs). The present study aims to investigate the function and underlying mechanism of the basic fibroblast growth factor (BFGF) on osteogenic differentiation of VICs. Porcine VICs cultured with osteogenic induction medium are supplemented with or without BFGF. Morphology of VICs is identified by fluorescein isothiocyanate-labeled phalloidin, the cell viability is assessed by the cell counting kit-8 method, and protein and mRNA expression level of osteogenic differentiation markers, including Runx2, osteopontin, and Sp7, are verified by western blot analysis and quantitative real-time PCR, respectively. RNA sequencing is used to identify changes in gene profiles. Alizarin Red S staining is used to measure calcium deposition. The results demonstrate that the content of calcium deposition and the expression level of osteogenic markers are downregulated by supplementing BFGF. Notch1 signaling pathway is extracted as a candidate target after bioinformatics analysis by RNA sequencing. The transfection of si-Notch1 abolishes the calcification inhibitory effect of BFGF. Taken together, our findings shed the light on the mechanism and potential therapeutics of BFGF for CAVD.

Drynaria fortunei improves lipid profiles of elderly patients with postmenopausal osteoporosis via regulation of Notch1-NLRP3 inflammasome-mediated inflammation.

BACKGROUND: Dyslipidemia is a common comorbidity in elderly patients with postmenopausal osteoporosis (PMOP). Drynaria fortunei (Rhizoma drynariae) is well-known in traditional Chinese medicine for its ability to improve bone mineral density (BMD). However, whether and how Drynaria fortunei improves plasma lipid profiles in elderly PMOP patients remains unclear. METHODS: Eighty elderly female patients with concurrent PMOP and hyperlipemia were randomly assigned to Drynaria fortunei 2(n = 40) or control (n = 40) groups. The clinical efficacies of Drynaria fortunei were evaluated. At 0, 3-, 6-, 9-, and 12-month of follow-up, plasma levels of IL-1ß, IL-18, TNF-α, IL-6, IL-8, and IL-10 were measured using ELISA, whereas PBMC levels of NLRP3, ASC, caspase-1, NF-κB, SIRT1, and Notch1 were measured using RT-qPCR. PBMC isolated from PMOP patients were cultured and treated with Drynaria fortunei to determine its influence on NLRP3 inflammasome and associated cytokines. RESULTS: Drynaria fortunei effectively improved patients' BMD and lipid profiles. IL-1ß, IL-18, TNF-α, IL-6, IL-8 levels, as well as inflammasome-molecules of NLRP3, ASC, caspase-1, and NF-κB increased over time in the control group, but were significantly attenuated with Drynaria fortunei administration. In vitro, Drynaria fortunei suppressed NLRP3 inflammasome and associated cytokines by increasing SIRT1 or decreasing Notch1. Drynaria fortunei had inhibitory effects on NLRP3 inflammasome and Notch1 even when SIRT1 expression was suppressed. CONCLUSIONS: Drynaria fortunei has been demonstrated to significantly improve lipid profiles for elderly PMOP patients. Drynaria fortunei may down-regulate Notch1 independently of SIRT1 to suppress NLRP3 inflammasome-mediated inflammation, thus improving plasma lipid profile.

Triptonide effectively inhibits triple-negative breast cancer metastasis through concurrent degradation of Twist1 and Notch1 oncoproteins.

BACKGROUND: Triple-negative breast cancer (TNBC) is highly metastatic and lethal. Due to a lack of druggable targets for this disease, there are no effective therapies in the clinic. METHODS: We used TNBC cells and xenografted mice as models to explore triptonide-mediated inhibition of TNBC metastasis and tumor growth. Colony formation assay was used to quantify the tumorigenesis of TNBC cells. Wound-healing and cell trans-well assays were utilized to measure cell migration and invasion. Tube formation assay was applied to access tumor cell-mediated vasculogenic mimicry. Western blot, quantitative-PCR, immunofluorescence imaging, and immunohistochemical staining were used to measure the expression levels of various tumorigenic genes in TNBC cells. RESULTS: Here, we showed that triptonide, a small molecule from the traditional Chinese medicinal herb Tripterygium wilfordii Hook F, potently inhibited TNBC cell migration, invasion, and vasculogenic mimicry, and effectively suppressed TNBC tumor growth and lung metastasis in xenografted mice with no observable toxicity. Molecular mechanistic studies revealed that triptonide strongly triggered the degradation of master epithelial-mesenchymal transition (EMT)-inducing protein Twist1 through the lysosomal system and reduced Notch1 expression and NF-κB phosphorylation, which consequently diminished the expression of pro-metastatic and angiogenic genes N-cadherin, VE-cadherin, and vascular endothelial cell growth factor receptor 2 (VEGFR2). CONCLUSIONS: Triptonide effectively suppressed TNBC cell tumorigenesis, vasculogenic mimicry, and strongly inhibited the metastasis of TNBC via degradation of Twist1 and Notch1 oncoproteins, downregulation of metastatic and angiogenic gene expression, and reduction of NF-κB signaling pathway. Our findings provide a new strategy for treating highly lethal TNBC and offer a potential new drug candidate for combatting this aggressive disease.

Salvia miltiorrhiza-asarum ointment combined with Chinese medical massage alleviates symptoms of osteoarthritis in a rat model through the Notch1/ matrix metalloproteinase-13 signaling pathway.

OBJECTIVE: This study investigated the effect of salvia miltiorrhiza-asarum ointment (SMAO) plus Chinese medical massage on knee osteoarthritis in a rat model. METHODS: Hulth's method was used to establish a Sprague-Dawley rat model of knee osteoarthritis (OA). The levels of matrix metalloproteinase-13 (MMP-13), collagen-II, aggrecan, interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α), and IL-6 were measured by enzyme-linked immunosorbent assays. The joint space was assessed by a Perlove X-ray system. Histopathology was examined by hematoxylin-eosin and Safranin O staining. The mRNA and protein expression levels of Notch1, MMP-13, collagen-II, and aggrecan were measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: SMAO plus Chinese medical massage significantly decreased the levels of MMP-13, IL-1ß, TNF-α, and IL-6, and increased serum collagen-II and aggrecan levels. Pathological injury of the knee joint was improved by SMAO treatment. mRNA and protein expression of Notch1 and MMP-13 was remarkably downregulated, but collagen-II and aggrecan levels were significantly upregulated in cartilage tissues. CONCLUSION: SMAO combined with Chinese medical massage effectively relieves OA symptoms, which may involve inhibiting inflammation through the Notch1/MMP-13 signaling pathway.

Selective suppression of M1 macrophages is involved in zinc inhibition of liver fibrosis in mice.

Zinc deficiency is common in the liver of patients with chronic liver disease. Zinc supplementation suppresses the progression of liver fibrosis induced by bile duct ligation (BDL) in mice. The present study was undertaken to specifically investigate a possible mechanism by which zinc plays this role in the liver. Kunming mice were subjected to BDL for 4 weeks to induce liver fibrosis, and concomitantly treated with zinc sulfite or saline as control by gavage once a day. The results showed that zinc supplementation significantly suppressed liver fibrosis and inflammation along with inhibition of hepatic stellate cells activation induced by BDL. These inhibitory effects were accompanied by the reduction of collagen deposition and a significant reduction of macrophage infiltration affected livers. Importantly, zinc selectively inhibited M1 macrophage polarization and M1-related inflammatory cytokines. This inhibitory effect was further confirmed by the reduction of relevant biomarkers of M1 macrophages including inducible NO synthase (iNOS), monocyte chemotactic protein-1 (MCP-1/CCL2), and tumor necrosis factor-α in the zinc supplemented BDL livers. In addition, zinc inhibition of M1 macrophages was associated with a decrease of Notch1 expression. Taken together, these data indicated that zinc supplementation inhibited liver inflammation and fibrosis in BDL mice through selective suppression of M1 macrophages, which is associated with inhibition of Notch1 pathway in M1 macrophage polarization.

Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures.

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models. Our datasets reveal common mechanisms of GSI resistance, including a distinct kinase signature that involves protein kinase C delta. We demonstrate that the PKC inhibitor sotrastaurin enhances the anti-leukemic activity of GSI in PDX models and completely abrogates the development of acquired GSI resistance in vitro. Overall, we highlight the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer.

A lineage-specific requirement for YY1 Polycomb Group protein function in early T cell development.

Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group protein (PcG) with important functions for regulating lymphocyte development and stem cell self-renewal. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that result in histone modifications. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Here, we demonstrate that a conditional knockout of Yy1 in the hematopoietic system results in an early T cell developmental blockage at the double negative (DN) 1 stage with reduced Notch1 signaling. There is a lineage-specific requirement for YY1 PcG function. YY1 PcG domain is required for T and B cell development but not necessary for myeloid cells. YY1 functions in early T cell development are multicomponent and involve both PcG-dependent and -independent regulations. Although YY1 promotes early T cell survival through its PcG function, its function to promote the DN1-to-DN2 transition and Notch1 expression and signaling is independent of its PcG function. Our results reveal how a ubiquitously expressed PcG protein mediates lineage-specific and context-specific functions to control early T cell development.