The effect of macrophages and their exosomes in ischemic heart disease.

Ischemic heart disease (IHD) is a leading cause of disability and death worldwide, with immune regulation playing a crucial role in its pathogenesis. Various immune cells are involved, and as one of the key immune cells residing in the heart, macrophages play an indispensable role in the inflammatory and reparative processes during cardiac ischemia. Exosomes, extracellular vesicles containing lipids, nucleic acids, proteins, and other bioactive molecules, have emerged as important mediators in the regulatory functions of macrophages and hold promise as a novel therapeutic target for IHD. This review summarizes the regulatory mechanisms of different subsets of macrophages and their secreted exosomes during cardiac ischemia over the past five years. It also discusses the current status of clinical research utilizing macrophages and their exosomes, as well as strategies to enhance their therapeutic efficacy through biotechnology. The aim is to provide valuable insights for the treatment of IHD.

Studies Related to the Involvement of EsA in Improving Intestinal Inflammation in Acute Pancreatitis via the NF-κB Pathway.

Background: Acute pancreatitis (AP) is a clinically frequent acute abdominal condition, which refers to an inflammatory response syndrome of edema, bleeding, and even necrosis caused by abnormal activation of the pancreas's own digestive enzymes. Intestinal damage can occur early in the course of AP and is manifested by impaired intestinal mucosal barrier function, and inflammatory reactions of the intestinal mucosa, among other factors. It can cause translocation of intestinal bacteria and endotoxins, further aggravating the condition of AP. Therefore, actively protecting the intestinal mucosal barrier, controlling the progression of intestinal inflammation, and improving intestinal dynamics in the early stages of AP play an important role in enhancing the prognosis of AP. Methods: The viability and apoptosis of RAW264.7 cells treated with Esculentoside A (EsA) and/or lipopolysaccharide were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. The expression of apoptosis-related proteins and NF-κB signaling pathway-related proteins were detected by western blot (WB). An enzyme-linked immunosorbent assay was used to measure TNF-α and IL-6 secretion. Results: In vitro experiments demonstrated that EsA not only promoted the apoptosis of inflammatory cells but also reduced the secretion of TNF-α and IL-6 in a dose-dependent manner. Additionally, it inhibited the activation of the NF-κB signaling pathway by decreasing the expression of phosphorylated-p65(p-p65) and elevating the expression of IκBα. Similarly, in vivo experiments using a rat AP model showed that EsA inhibited the expression of p-p65 elevating the expression of IκBα in the intestinal tissues of the rat AP model and promoting the apoptosis of inflammatory cells in the intestinal mucosa in vivo experiments, while improving the pathological outcome of the pancreatic and intestinal tissues. Conclusion: Our results suggest that EsA can reduce intestinal inflammation in the rat AP model and that EsA may be a candidate for treating intestinal inflammation in AP and further arresting AP progression.

Macrophage CREBZF Orchestrates Inflammatory Response to Potentiate Insulin Resistance and Type 2 Diabetes.

Chronic adipose tissue inflammation accompanied by macrophage accumulation and activation is implicated in the pathogenesis of insulin resistance and type 2 diabetes in humans. The transcriptional coregulator CREBZF is a key factor in hepatic metabolism, yet its role in modulating adipose tissue inflammation and type 2 diabetes remains elusive. The present study demonstrates that overnutrition-induced CREBZF links adipose tissue macrophage (ATM) proinflammatory activation to insulin resistance. CREBZF deficiency in macrophages, not in neutrophils, attenuates macrophage infiltration in adipose, proinflammatory activation, and hyperglycemia in diet-induced insulin-resistant mice. The coculture assays show that macrophage CREBZF deficiency improves insulin sensitivity in primary adipocytes and adipose tissue. Mechanistically, CREBZF competitively inhibits the binding of IκBα to p65, resulting in enhanced NF-κB activity. In addition, bromocriptine is identified as a small molecule inhibitor of CREBZF in macrophages, which suppresses the proinflammatory phenotype and improves metabolic dysfunction. Furthermore, CREBZF is highly expressed in ATM of obese humans and mice, which is positively correlated with proinflammatory genes and insulin resistance in humans. This study identifies a previously unknown role of CREBZF coupling ATM activation to systemic insulin resistance and type 2 diabetes.

Nano-modulators with the function of disrupting mitochondrial Ca2+ homeostasis and photothermal conversion for synergistic breast cancer therapy.

Breast cancer treatment has been a global puzzle, and apoptosis strategies based on mitochondrial Ca2+ overload have attracted extensive attention. However, various limitations of current Ca2+ nanogenerators make it difficult to maintain effective Ca2+ overload concentrations. Here, we constructed a multimodal Ca2+ nano-modulator that, for the first time, combined photothermal therapy (PTT) and mitochondrial Ca2+ overload strategies to inhibit tumor development. By crosslinking sodium alginate (SA) on the surface of calcium carbonate (CaCO3) nanoparticles encapsulating with Cur and ICG, we prepared a synergistic Ca2+ nano-regulator SA/Cur@CaCO3-ICG (SCCI). In vitro studies have shown that SCCI further enhanced photostability while preserving the optical properties of ICG. After uptake by tumor cells, SCCI can reduce mitochondrial membrane potential and down-regulate ATP production by producing large amounts of Ca2+ at low pH. Near-infrared light radiation (NIR) laser irradiation made the tumor cells heat up sharply, which not only accelerated the decomposition of CaCO3, but also produced large amounts of reactive oxygen species (ROS) followed by cell apoptosis. In vivo studies have revealed that the Ca2+ nano-regulators had excellent targeting, biocompatibility, and anti-tumor effects, which can significantly inhibit the proliferation of tumor cells and play a direct killing effect. These findings indicated that therapeutic strategies based on ionic interference and PTT had great therapeutic potential, providing new insights into antitumor therapy.

BDH1-mediated ßOHB metabolism ameliorates diabetic kidney disease by activation of NRF2-mediated antioxidative pathway.

A ketogenic diet (KD) and ß-hydroxybutyrate (ßOHB) have been widely reported as effective therapies for metabolic diseases. ß-Hydroxybutyrate dehydrogenase 1 (BDH1) is the rate-limiting enzyme in ketone metabolism. In this study, we examined the BDH1-mediated ßOHB metabolic pathway in the pathogenesis of diabetic kidney disease (DKD). We found that BDH1 is downregulated in the kidneys in DKD mouse models, patients with diabetes, and high glucose- or palmitic acid-induced human renal tubular epithelial (HK-2) cells. BDH1 overexpression or ßOHB treatment protects HK-2 cells from glucotoxicity and lipotoxicity by inhibiting reactive oxygen species overproduction. Mechanistically, BDH1-mediated ßOHB metabolism activates NRF2 by enhancing the metabolic flux of ßOHB-acetoacetate-succinate-fumarate. Moreover, in vivo studies showed that adeno-associated virus 9-mediated BDH1 renal expression successfully reverses fibrosis, inflammation, and apoptosis in the kidneys of C57 BKS db/db mice. Either ßOHB supplementation or KD feeding could elevate the renal expression of BDH1 and reverse the progression of DKD. Our results revealed a BDH1-mediated molecular mechanism in the pathogenesis of DKD and identified BDH1 as a potential therapeutic target for DKD.

Evaluation of white matter microstructural alterations in premature infants with necrotizing enterocolitis.

Background: Preterm infants with necrotizing enterocolitis (NEC) are at high risk of adverse neurodevelopmental outcomes. The aim of this study was to explore the value of diffusion tensor imaging (DTI) combined with serum C-reactive protein (CRP) and procalcitonin (PCT) in evaluating alterations of white matter (WM) microstructure in preterm infants with NEC. Methods: A retrospective cross-sectional study was conducted in which all participants were consecutively enrolled at The Third Affiliated Hospital of Zhengzhou University from June 2017 and October 2021. Data from 30 preterm infants with NEC [mean gestational age at birth 31.41±1.15 weeks; mean age at magnetic resonance imaging (MRI) 37.53±3.08 weeks] and 40 healthy preterm infants with no NEC were recorded (mean gestational age at birth 32.27±2.09 weeks; mean age at MRI 37.15±3.23 weeks). WM was used to obtain the fractional anisotropy (FA) and mean diffusivity (MD) values of the regions of interest (ROIs). Additionally, serum levels of CRP and PCT were determined. Spearman correlation analysis was performed between the WM-derived parameters, CRP level, and the PCT serum index. Results: Preterm infants with NEC had reduced FA values and elevated MD values in WM regions [posterior limbs of the internal capsule (PLIC), lentiform nucleus (LN), frontal white matter (FWM)] compared to the control group (P<0.05). Additionally, the FA of the PLIC was negatively correlated with serum CRP (r=-0.846; P<0.05) and PCT (r=-0.843; P<0.05). Meanwhile, the MD of PLIC was positively correlated with serum CRP (r=0.743; P<0.05) and PCT (r=0.743; P<0.05, respectively). The area under the curve (AUC) of FA and MD combined with CRP and PCT in the diagnosis of WM microstructure alterations with NEC was 0.968, representing a considerable improvement in predicted efficacy over single indicators, including FA [AUC: 0.938; 95% confidence interval (CI): 0.840-0.950], MD (AUC: 0.807; 95% CI: 0.722-0.838), CRP (AUC: 0.867; 95% CI: 0.822-0.889), and PCT (AUC: 0.706; 95% CI: 0.701-0.758). Conclusions: WM can noninvasively and quantitatively assess the WM microstructure alterations in preterm infants with NEC. WM combined with serum CRP and PCT demonstrated superior performance in detecting and evaluating WM microstructure alterations in preterm infants with NEC.

L-type calcium ion channel-mediated activation of autophagy in vascular smooth muscle cells via thonningianin A (TA) alleviates vascular calcification in type 2 diabetes mellitus.

Vascular calcification (VC) is associated with increased morbidity and mortality, especially among people with type 2 diabetes mellitus (T2DM). The pathogenesis of vascular calcification is incompletely understood, and until now, there have been no effective therapeutics for vascular calcification. The L-type calcium ion channel in the cell membrane is vital for Ca2+ influx. The effect of L-type calcium ion channels on autophagy remains to be elucidated. Here, the natural compound thonningianin A (TA) was found to ameliorate vascular calcification in T2DM via the activation of L-type calcium ion channels. The results showed that TA had a concentration-dependent ability to decrease the transcriptional and translational expression of the calcification-related proteins runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteopontin (OPN) (P < 0.01) via ATG7-dependent autophagy in ß-glycerophosphate (ß-GP)- and high glucose (HG)-stimulated primary mouse aortic smooth muscle cells (MASMCs) and alleviate aortic vascular calcification in VitD3-stimulated T2DM mice. However, nifedipine, an inhibitor of L-type calcium ion channels, reversed TA-induced autophagy and Ca2+ influx in MASMCs. Molecular docking analysis revealed that TA was located in the hydrophobic pocket of Cav1.2 α1C and was mainly composed of the residues Ile, Phe, Ala and Met, which confirmed the efficacy of TA in targeting the L-type calcium channel of Cav1.2 on the cell membrane. Moreover, in an in vivo model of vascular calcification in T2DM mice, nifedipine reversed the protective effects of TA on aortic calcification and the expression of the calcification-related proteins RUNX2, BMP2 and OPN (P < 0.01). Collectively, the present results reveal that the activation of cell membrane L-type calcium ion channels can induce autophagy and ameliorate vascular calcification in T2DM. Thonningianin A (TA) can target and act as a potent activator of L-type calcium ion channels. Thus, this research revealed a novel mechanism for autophagy induction via L-type calcium ion channels and provided a potential therapeutic for vascular calcification in T2DM.

Advances in the study of exosomes derived from mesenchymal stem cells and cardiac cells for the treatment of myocardial infarction.

Acute myocardial infarction has long been the leading cause of death in coronary heart disease, which is characterized by irreversible cardiomyocyte death and restricted blood supply. Conventional reperfusion therapy can further aggravate myocardial injury. Stem cell therapy, especially with mesenchymal stem cells (MSCs), has emerged as a promising approach to promote cardiac repair and improve cardiac function. MSCs may induce these effects by secreting exosomes containing therapeutically active RNA, proteins and lipids. Notably, normal cardiac function depends on intracardiac paracrine signaling via exosomes, and exosomes secreted by cardiac cells can partially reflect changes in the heart during disease, so analyzing these vesicles may provide valuable insights into the pathology of myocardial infarction as well as guide the development of new treatments. The present review examines how exosomes produced by MSCs and cardiac cells may influence injury after myocardial infarction and serve as therapies against such injury. Video Abstract.

The effects and mechanism of LncRNA NORAD on doxorubicin-induced cardiotoxicity.

In recent years, the role and mechanism of long non-coding RNA (LncRNA) in cardiovascular diseases have received increasing attention. The chemotherapy agent, doxorubicin (DOX), is one of the most effective drugs for various cancers, but its efficacy is limited by its cardiotoxicity. Therefore, further exploration is required for the molecular mechanism of DOX-induced cardiotoxicity. This study intended to investigate the role of LncRNA Non-coding RNA activated by DNA damage (NORAD) in DOX-induced cardiotoxicity, for which we adopted the AC16 human cardiomyocyte cell line for the exploration. The results showed that LncRNA NORAD knockdown could increase DOX-induced cardiomyocyte apoptosis and mitochondrial ROS level. LncRNA NORAD overexpression obtained reverse results, which further validated its role in DOX-induced cardiomyocyte apoptosis and mitochondrial ROS level. Moreover, cardiotoxicity was induced in both LncRNA NORAD-knockout and wild-type mice with DOX, showing that gene knockout aggravated pathologic lesions in the myocardial tissues of mice. Taken together, LncRNA NORAD affected DOX-induced cardiotoxicity via mitochondrial apoptosis, fission (PUM-MFF), and autophagy (p53-Parkin) pathways both in vivo and in vitro.

Understanding sorafenib-induced ferroptosis and resistance mechanisms: Implications for cancer therapy.

Sorafenib is an important first-line treatment option for liver cancer due to its well-characterized safety profile. While novel first-line drugs may have better efficacy than Sorafenib, they also have limitations such as worse safety and cost-effectiveness. In addition to inducing apoptosis, Sorafenib can also trigger ferroptosis, which has recently been recognized as an immunogenic cell death, unleashing new possibilities for cancer treatment. However, resistance to Sorafenib-induced ferroptosis remains a major challenge. To overcome this resistance and augment the efficacy of Sorafenib, a wide range of nanomedicines has been developed to amplify its pro-ferroptotic effects. This review highlights the mechanisms underlying Sorafenib-triggered ferroptosis and its resistance, and outlines innovative strategies, particularly nanomedicines, to overcome ferroptosis resistance. Moreover, we summarize molecular biomarkers that signify resistance to Sorafenib-mediated ferroptosis, which can assist in predicting therapeutic outcomes.

Respiratory Fluid Mechanics of the Effect of Mouth Breathing on High-Arched Palate: Computational Fluid Dynamics Analyses.

Computational fluid dynamics (CFD) was introduced into the study of palate growth and development to explain the mechanisms by which mouth breathing affects palate descent from an aerodynamic perspective. Cone beam computed tomography (CBCT) data were used to reconstruct a 3-dimensional model during natural mouth breathing of a volunteer. The model was imported into CFX 19.0 for numerical simulation of nasal breathing, mouth-nasal breathing, and mouth breathing. The pressure in the oronasal cavity was analyzed, and the pressure difference between the oral and nasal surfaces of hard palate under different breathing patterns was calculated. CFD can be used to simulate the stress on the oral and nasal surfaces of the palate under different breathing patterns. The pressure differences and resultant force between the oral and nasal surfaces of the hard palate during nasal inspiration, nasal expiration, mouth-nasal inspiration, mouth-nasal expiration, mouth inspiration, and mouth expiration were 0 Pa, 4 Pa (upward), 9 Pa (upward), 3 Pa (downward), 474 Pa (upward), 263 Pa (downward), respectively, and 87.99 N (upward), 88.03 N (upward), 88.01 N (upward), 88.01 N (upward), 88.05 N (upward), 87.94 N (upward), respectively. Therefore, CFD can be used to investigate the growth and development of the palate. When the volunteer opened his mouth, the pressure difference between the oral and nasal surfaces of the hard palate was about 88 N upward regardless of whether there was airflow in the mouth. The reversal of the direction of the force on the hard palate may be one of the factors affecting its descent of it.

Integrating network pharmacology and experimental verification to explore the pharmacological mechanisms of asparagus against polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder in women of reproductive age that still lacks effective treatment. Inflammation is one of the important features of PCOS. Asparagus (ASP) has anti-inflammatory, antioxidant, and anti-aging pharmacological effects, and its anti-tumor effects have been demonstrated in a variety of tumors. However, the role and mechanism of ASP in PCOS remain unclear. METHODS: The active components of ASP and the key therapeutic targets for PCOS were obtained by network pharmacology. Molecular docking was used to simulate the binding of PRKCA to the active components of ASP. The effects of ASP on inflammatory and oxidative stress pathways in PCOS, and the regulation of PRKCA were examined by KGN, a human derived granulosa cell line. PCOS mouse model validated the results of in vivo experiments. RESULTS: Network pharmacology identified 9 major active ingredients of ASP with 73 therapeutic targets for PCOS. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment yielded 101 PCOS-related signaling pathways. The hub gene PRKCA was obtained after taking the gene intersection of the top 4 pathways. Molecular docking showed the binding of PRKCA to the 7 active components in ASP. In vitro and in vivo experiments showed that ASP alleviated the course of PCOS through antioxidant, anti-inflammatory effects. ASP can partially restore the low expression of PRKCA in the PCOS models. CONCLUSION: The therapeutic effect of ASP on PCOS is mainly achieved by targeting PRKCA through the 7 active components of ASP. Mechanistically, ASP alleviated the course of PCOS through antioxidant, anti-inflammatory effects, and PRKCA was its potential target.

Hypoxia-responsive lncRNA G077640 promotes ESCC tumorigenesis via the H2AX-HIF1α-glycolysis axis.

Long noncoding RNAs (lncRNAs) contribute to esophageal squamous cell carcinoma (ESCC) progression, but the underlying mechanisms remain elusive. In this study, we verified a hitherto uncharacterized hypoxia-responsive lncRNA, G077640, which is upregulated in human ESCC cells and tissues, supporting the proliferation and migration of ESCC cells. Mechanistically, G077640 prevented hypoxia-inducible factor-1α (HIF1α) from being degraded by directly interacting with histone H2AX and further modulated the interaction of HIF1α and H2AX. In addition, G077640 reprogrammed glycolytic metabolism by regulating the expression of glucose transporter 4 (GLUT4), hexokinase 2 (HK2) and pyruvate dehydrogenase kinase 1 (PDK1) for ESCC proliferation and migration. Clinically, G077640 was associated with poor prognosis in ESCC patients. Taken together, our findings identified a hypoxia-responsive lncRNA that contributes to ESCC cells proliferation and migration, and targeting G077640 and its pathway might be a potential therapeutic strategy for ESCC.

Micelle encapsulation zinc-doped copper oxide nanocomposites reverse Olaparib resistance in ovarian cancer by disrupting homologous recombination repair.

Micelle Encapsulation Zinc-doped copper oxide nanocomposites (MEnZn-CuO NPs) is a novel doped metal nanomaterial prepared by our group based on Zinc doped copper oxide nanocomposites (Zn-CuO NPs) using non-micellar beam. Compared with Zn-CuO NPs, MEnZn-CuO NPs have uniform nanoproperties and high stability. In this study, we explored the anticancer effects of MEnZn-CuO NPs on human ovarian cancer cells. In addition to affecting cell proliferation, migration, apoptosis and autophagy, MEnZn-CuO NPs have a greater potential for clinical application by inducing HR repair defects in ovarian cancer cells in combination with poly (ADP-ribose) polymerase inhibitors for lethal effects.

The mechanism of rh-endostatin-induced cardiotoxicity and its protection by dihydromyricetin[in vivo/in vitro, C57BL/6 mice, AC16 and hiPSC-CMs].

Recombinant human endostatin (rh-endostatin) is an anti-angiogenic drug, which is used for the treatment of advanced non-small-cell lung cancer (NSCLC) and other cancers. However, its side effects, especially the cardiotoxicity with unclear mechanisms limit its wide application in clinical practice. In this study, human cardiomyocyte cell line AC16 and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) treated with different doses of rh-endostatin were used to analyze its effect on cardiac cell toxicity. The results revealed that rh-endostatin dose-dependently enhanced cardiomyocyte apoptosis through Apaf-1 apoptotic factor and apoptosis-related proteins such as p53. rh-endostatin-induced changes of mitochondrial function and mitophagy were involved in rh-endostatin-mediated cardiac cell toxicity. Rh-endostatin-induced cardiotoxicity was further verified in vivo in mice. Interestingly, Rh-endostatin-induced cardiotoxicity was inhibited by dihydromyricetin (DHM) both in cultured cells in vitro and in mouse hearts in vivo. The study provides new inside into rh-endostatin-induced cardiotoxicity and identified a novel potential medication DHM to overcome the serious adverse effect.

The Application of Ethnomedicine in Modulating Megakaryocyte Differentiation and Platelet Counts.

Megakaryocytes (MKs), a kind of functional hematopoietic stem cell, form platelets to maintain platelet balance through cell differentiation and maturation. In recent years, the incidence of blood diseases such as thrombocytopenia has increased, but these diseases cannot be fundamentally solved. The platelets produced by MKs can treat thrombocytopenia-associated diseases in the body, and myeloid differentiation induced by MKs has the potential to improve myelosuppression and erythroleukemia. Currently, ethnomedicine is extensively used in the clinical treatment of blood diseases, and the recent literature has reported that many phytomedicines can improve the disease status through MK differentiation. This paper reviewed the effects of botanical drugs on megakaryocytic differentiation covering the period 1994-2022, and information was obtained from PubMed, Web of Science and Google Scholar. In conclusions, we summarized the role and molecular mechanism of many typical botanical drugs in promoting megakaryocyte differentiation in vivo, providing evidence as much as possible for botanical drugs treating thrombocytopenia and other related diseases in the future.

Ferredoxin 1 regulates granulosa cell apoptosis and autophagy in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), a common reproductive endocrine disorder in women of reproductive age, causes anovulatory infertility. Increased apoptosis of granulosa cells has been identified as one of the key factors contributing to abnormal follicular development. Ferredoxin 1 (FDX1) encodes a small ferredoxin that is involved in the reduction in mitochondrial cytochromes and the synthesis of various steroid hormones and has the potential to influence the function of granulosa cells. In the present study, we aimed to determine the relationship between FDX1 and follicular granulosa cell function. To this end, we investigated the difference between FDX1 expression in the granulosa cells of 50 patients with PCOS and that of the controls. Furthermore, we sought to elucidate the role and mechanism of FDX1 in PCOS granulosa cells by establishing a mouse PCOS model with dehydroepiandrosterone and KGN (a steroidogenic human granulosa cell-like tumor cell line). The results indicated significant up-regulation of FDX1 in the granulosa cells after androgen stimulation. Knockdown of FDX1 promoted the proliferation of KGN and inhibited apoptosis. Moreover, FDX1 could regulate autophagy by influencing the autophagy proteins ATG3 and ATG7. Our results demonstrated that FDX1 plays a critical role in female folliculogenesis by mediating apoptosis, autophagy, and proliferation. Therefore, FDX1 may be a potential prognostic factor for female infertility.

Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings.

BACKGROUND: Non-peptide thrombopoietin receptor (TPOR) agonists are promising therapies for the mitigation and treatment of thrombocytopenia. However, only few agents are available as safe and effective for stimulating platelet production for thrombocytopenic patients in the clinic. PURPOSE: This study aimed to develop a novel small molecule TPOR agonist and investigate its underlying regulation of function in megakaryocytes (MKs) differentiation and thrombopoiesis. METHODS: A potential active compound that promotes MKs differentiation and thrombopoiesis was obtained by machine learning (ML). Meanwhile, the effect was verified in zebrafish model, HEL and Meg-01 cells. Next, the key regulatory target was identified by Drug Affinity Responsive Target Stabilization Assay (DARTS), Cellular Thermal Shift Assay (CETSA), and molecular simulation experiments. After that, RNA-sequencing (RNA-seq) was used to further confirm the associated pathways and evaluate the gene expression induced during MK differentiation. In vivo, irradiation (IR) mice, C57BL/6N-TPORem1cyagen (Tpor-/-) mice were constructed by CRISPR/Cas9 technology to examine the therapeutic effect of TMEA on thrombocytopenia. RESULTS: A natural chemical-structure small molecule TMEA was predicted to be a potential active compound based on ML. Obvious phenotypes of MKs differentiation were observed by TMEA induction in zebrafish model and TMEA could increase co-expression of CD41/CD42b, DNA content, and promote polyploidization and maturation of MKs in HEL and Meg-01 cells. Mechanically, TMEA could bind with TPOR protein and further regulate the PI3K/AKT/mTOR/P70S6K and MEK/ERK signal pathways. In vivo, TMEA evidently promoted platelet regeneration in mice with radiation-induced thrombocytopenia but had no effect on Tpor-/- and C57BL/6 (WT) mice. CONCLUSION: TMEA could serve as a novel TPOR agonist to promote MKs differentiation and thrombopoiesis via mTOR and ERK signaling and could potentially be created as a promising new drug to treat thrombocytopenia.

MALAT1 promotes platelet activity and thrombus formation through PI3k/Akt/GSK-3ß signalling pathway.

BACKGROUND: Ischaemic stroke and other cardiovascular illnesses are characterised by abnormalities in the processes of thrombosis and haemostasis, which rely on platelet activity. In platelets, a wide variety of microRNAs (long non-coding RNA, lncRNAs) is found. Due to the absence of nuclear DNA in platelets, lncRNAs may serve as critical post-transcriptional regulators of platelet activities. However, research into the roles of lncRNAs in platelets is limited. OBJECTIVE: The purpose of this study is to learn more about the molecular mechanism by which MALAT1 affects platelet activity and thrombus formation. METHODS/RESULTS: The CD34+ megakaryocytes used in this research as an in vitro model for human megakaryocytes and platelets. Cell adhesion and spreading are enhanced in the absence and presence of agonists in CD34+ megakaryocytes subjected to MALAT1 knockdown (KD). The adhesion and activity of platelet-like particles produced by MALAT1 KD cells are significantly enhanced at rest and after thrombin activation. Thrombus development on a collagen matrix is also greatly enhanced in the microfluidic whole-blood perfusion model: platelets lacking MALAT1 exhibit elevated accumulation, distributing area and activity. In addition, MALAT1-deficient mice bleed less and form a stable occlusive thrombus more quickly than wild-type mice. PTEN and PDK1 regulated the activity of MALAT1 in platelets to carry out its PI3k/Akt/GSK-3ß signalling pathway-related function. CONCLUSION: The suppression of MALAT1 expression significantly increases platelet adhesion, spreading, platelet activity, and thrombus formation. lncRNAs may constitute a unique class of platelet function modulators.

Targeting a thrombopoietin-independent strategy in the discovery of a novel inducer of megakaryocytopoiesis, DMAG, for the treatment of thrombocytopenia.

Thrombocytopenia is a thrombopoietin (TPO)-related disorder with very limited treatment options, and can be lifethreatening. There are major problems with typical thrombopoietic agents targeting TPO signaling, so it is urgent to discover a novel TPO-independent mechanism involving thrombopoiesis and potential druggable targets. We developed a drug screening model by the multi-grained cascade forest (gcForest) algorithm and found that 3,8-di-O-methylellagic acid 2- O-glucoside (DMAG) (10, 20 and 40 µM) promoted megakaryocyte differentiation in vitro. Subsequent investigations revealed that DMAG (40 mM) activated ERK1/2, HIF-1b and NF-E2. Inhibition of ERK1/2 blocked megakaryocyte differentiation and attenuated the upregulation of HIF-1b and NF-E2 induced by DMAG. Megakaryocyte differentiation induced by DMAG was inhibited via knockdown of NF-E2. In vivo studies showed that DMAG (5 mg/kg) accelerated platelet recovery and megakaryocyte differentiation in mice with thrombocytopenia. The platelet count of the DMAG-treated group recovered to almost 72% and 96% of the count in the control group at day 10 and 14, respectively. The platelet counts in the DMAG-treated group were almost 1.5- and 1.3-fold higher compared with those of the irradiated group at day 10 and 14, respectively. Moreover, DMAG (10, 25 and 50 mM) stimulated thrombopoiesis in zebrafish. DMAG (5 mg/kg) could also increase platelet levels in c-MPL knockout (c-MPL-/-) mice. In summary, we established a drug screening model through gcForest and demonstrated that DMAG promotes megakaryocyte differentiation via the ERK/HIF1/NF-E2 pathway which, importantly, is independent of the classical TPO/c-MPL pathway. The present study may provide new insights into drug discovery for thrombopoiesis and TPO-independent regulation of thrombopoiesis, as well as a promising avenue for thrombocytopenia treatment.