Salvianolic acid B enhances tissue repair and regeneration by regulating immune cell migration and Caveolin-1-mediated blastema formation in zebrafish.

INTRODUCTION: Non-healing wounds resulting from trauma, surgery, and chronic diseases annually affect millions of individuals globally, with limited therapeutic strategies available due to the incomplete understanding of the molecular processes governing tissue repair and regeneration. Salvianolic acid B (Sal B) has shown promising bioactivities in promoting angiogenesis and inhibiting inflammation. However, its regulatory mechanisms in tissue regeneration remain unclear. PURPOSE: This study aims to investigate the effects of Sal B on wound healing and regeneration processes, along with its underlying molecular mechanisms, by employing zebrafish as a model organism. METHODS: In this study, we employed a multifaceted approach to evaluate the impact of Sal B on zebrafish tail fin regeneration. We utilized whole-fish immunofluorescence, TUNEL staining, mitochondrial membrane potential (MMP), and Acridine Orange (AO) probes to analyze the tissue repair and regenerative under Sal B treatment. Additionally, we utilized transgenic zebrafish strains to investigate the migration of inflammatory cells during different phases of fin regeneration. To validate the importance of Caveolin-1 (Cav1) in tissue regeneration, we delved into its functional role using molecular docking and Morpholino-based gene knockdown techniques. Additionally, we quantified Cav1 expression levels through the application of in situ hybridization. RESULTS: Our findings demonstrated that Sal B expedites zebrafish tail fin regeneration through a multifaceted mechanism involving the promotion of cell proliferation, suppression of apoptosis, and enhancement of MMP. Furthermore, Sal B was found to exert regulatory control over the dynamic aggregation and subsequent regression of immune cells during tissue regenerative processes. Importantly, we observed that the knockdown of Cav1 significantly compromised tissue regeneration, leading to an excessive infiltration of immune cells and increased levels of apoptosis. Moreover, the knockdown of Cav1 also affects blastema formation, a critical process influenced by Cav1 in tissue regeneration. CONCLUSION: The results of this study showed that Sal B facilitated tissue repair and regeneration through regulating of immune cell migration and Cav1-mediated fibroblast activation, promoting blastema formation and development. This study highlighted the potential pharmacological effects of Sal B in promoting tissue regeneration. These findings contributed to the advancement of regenerative medicine research and the development of novel therapeutic approaches for trauma.

Triglyceride-glucose index predicts the risk of gestational diabetes mellitus: a systematic review and meta-analysis.

AIMS: We aimed to investigate the potential predictive efficacy of triglyceride-glucose (T/Gly) index for gestational diabetes mellitus (GDM) in a systematic review and meta-analysis. MATERIALS AND METHODS: Cohort studies demonstrating the association between T/Gly index measured at the first trimester or before pregnancy and the subsequent incidence of GDM were identified by search of PubMed, Embase, China National Knowledge Infrastructure, and WanFang databases. A random-effect model incorporating the heterogeneity was applied to pool the results. Five cohort studies including 382,213 women were included in this meta-analysis. RESULTS: Compared to those with the lowest category of T/Gly index, women with the highest category of T/Gly index were independently associated with higher risk of subsequent GDM (odds ratio [OR]: 2.52, 95% confidence interval [CI]: 1.33 ∼ 4.67, I2=65%, p=.004) after adjustment of potential confounding factors including age, body mass index, and family history of diabetes. The association was stronger in prospective cohort studies than retrospective cohort studies (p for subgroup difference=.007), and a significant association was detected in Asian women (OR: 3.30, 95% CI: 1.50-7.28, p =.003), but not in non-Asian women (OR: 0.96, 95% CI: 0.35-2.63, p=.94). CONCLUSIONS: Higher T/Gly index may independently predict the risk of GDM in Asian women.

Runx1 regulates zebrafish neutrophil maturation via synergistic interaction with c-Myb.

Neutrophils play an essential role in the innate immune defense system in vertebrates. During hematopoiesis, the full function of neutrophils involves maturation of granules and related enzymes. Yet, transcription regulators that promote neutrophil maturation remain largely undefined. Here, two hematopoiesis-defective zebrafish mutants, runx1w84x and c-mybhkz3, were used to investigate the in vivo roles of Runx1 in cooperation with c-Myb in regulating neutrophil maturation. Loss of runx1 impairs primitive neutrophil development. Additional regulation of c-myb+/- and c-myb-/- induces a more severe phenotypes suggesting a synergistic genetic interaction between c-myb and runx1 in neutrophil maturation. Further studies revealed that the two transcription factors act cooperatively to control neutrophil maturation processes via transactivating a series of neutrophil maturation-related genes. These data reveal the in vivo roles of Runx1 in regulating primitive neutrophil maturation while also indicating a novel genetic and molecular orchestration of Runx1 and c-Myb in myeloid cell development. The study will provide new evidence on the regulation of neutrophil maturation during hematopoiesis.

Dysregulated miRNAs contribute to altered placental glucose metabolism in patients with gestational diabetes via targeting GLUT1 and HK2.

INTRODUCTION: Dysregulated genes in glucose transport and metabolize pathways have been found in patients with Gestational diabetes (GDM), but the underlying mechanisms were still unclear. MATERIALS AND METHODS: Placental villous samples were collected from 31 patients with GDM and 20 healthy controls. The expression of GLUT1, GLUT4, GLUT9 and HK2 was examined by immunoblotting and qRT-PCR. The miRNAs have the potential targeting GLUT1 and HK2 were predicted using online bioinformatics tool: TargetScan. The interaction between miRNAs and target genes were confirmed by dual luciferase assay and immunoblotting. The function of miR-9 and miR-22 on glucose metabolism was examined by glucose uptake assay and lactate secretion assay. RESULTS: GLUT1 and HK2 proteins level was found upregulated in patients with GDM, but the mRNA level was not significantly changed. Predicted by using bioinformatics tools and confirmed by dual luciferase assay and immunoblotting, GLUT1 was identified as a target of miR-9 and miR-22, whereas HK2 was identified as a target of miR-9. MiR-9 and miR-22 level was found reduced in the placenta villous and negatively correlated with the expression of GLUT1 and HK2. Functional studies indicated that miR-9 and miR-22 inhibitors upregulated the expression of GLUT1 and HK2, and then increased the glucose uptake, lactate secretion, cell viability and repressed apoptosis in primary syncytiotrophoblasts (STBs) and HTR8/SVneo cells. DISCUSSION: The upregulation of GLUT1 and HK2 in the placenta, which is induced by miR-9 and miR-22 reduction, contributes to the disordered glucose metabolism in patients with GDM.

Loss of runx1 function results in B cell immunodeficiency but not T cell in adult zebrafish.

Transcription factor RUNX1 holds an integral role in multiple-lineage haematopoiesis and is implicated as a cofactor in V(D)J rearrangements during lymphocyte development. Runx1 deficiencies resulted in immaturity and reduction of lymphocytes in mice. In this study, we found that runx1W84X/W84X mutation led to the reduction and disordering of B cells, as well as the failure of V(D)J rearrangements in B cells but not T cells, resulting in antibody-inadequate-mediated immunodeficiency in adult zebrafish. By contrast, T cell development was not affected. The decreased number of B cells mainly results from excessive apoptosis in immature B cells. Disrupted B cell development results in runx1W84X/W84X mutants displaying a similar phenotype to common variable immunodeficiency-a primary immunodeficiency disease primarily characterized by frequent susceptibility to infection and deficient immune response, with marked reduction of antibody production of IgG, IgA and/or IgM. Our studies demonstrated an evolutionarily conserved function of runx1 in maturation and differentiation of B cells in adult zebrafish, which will serve as a valuable model for the study of immune deficiency diseases and their treatments.

Zebrafish nephrosin helps host defence against Escherichia coli infection.

Neutrophils play important roles in innate immunity and are mainly dependent on various enzyme-containing granules to kill engulfed microorganisms. Zebrafish nephrosin (npsn) is specifically expressed in neutrophils; however, its function is largely unknown. Here, we generated an npsn mutant (npsnsmu5 ) via CRISPR/Cas9 to investigate the in vivo function of Npsn. The overall development and number of neutrophils remained unchanged in npsn-deficient mutants, whereas neutrophil antibacterial function was defective. Upon infection with Escherichia coli, the npsnsmu5 mutants exhibited a lower survival rate and more severe bacterial burden, as well as augmented inflammatory response to challenge with infection when compared with wild-type embryos, whereas npsn-overexpressing zebrafish exhibited enhanced host defence against E. coli infection. These findings demonstrated that zebrafish Npsn promotes host defence against bacterial infection. Furthermore, our findings suggested that npsn-deficient and -overexpressing zebrafish might serve as effective models of in vivo innate immunity.

c-Myb acts in parallel and cooperatively with Cebp1 to regulate neutrophil maturation in zebrafish.

Neutrophils are the key effectors for generating innate immunity in response to pathogenic infection and tissue injury in vertebrates. Dysregulation of neutrophil development and function is known to associate with various human disorders. Yet, the genetic network that orchestrates lineage commitment, differentiation, and maturation of neutrophils remains incompletely defined. Here, we present an in vivo study to delineate the genetic program underlying neutrophil development during zebrafish embryonic myelopoiesis. We show that loss of c-Myb function has no effect on macrophages but severely impairs neutrophil terminal differentiation, resulting in the accumulation of neutrophils with unsegmented nuclei and scant granule. This neutrophilic defect, which resembles the neutrophil-specific granule deficiency (SGD) caused by the mutations in CCAAT/enhancer-binding protein ε (C/EBPε) in humans, is attributed, at least in part, to the downregulation of the granule protein transcription. Likewise, genetic inactivation of Cebp1, the zebrafish functional homolog of mammalian C/EBPε, also leads to a similar SGD-like phenotype in zebrafish. Genetic epistasis and biochemical analysis further reveals that c-Myb and Cebp1 act in parallel and cooperatively to control neutrophil differentiation by directly regulating granule protein gene transcription. Our study indicates that c-MYB is an intrinsic master regulator for neutrophil terminal differentiation and a potential target in SGD patients.

Functions of idh1 and its mutation in the regulation of developmental hematopoiesis in zebrafish.

Isocitrate dehydrogenase 1 mutation (IDH1-R132H) was recently identified in acute myeloid leukemia with normal cytogenetics. The mutant enzyme is thought to convert α-ketoglutarate to the pathogenic 2-hydroxyglutarate (2-HG) that affects DNA methylation via inhibition of ten-eleven translocation 2. However, the role of wild-type IDH1 in normal hematopoiesis and its relevance to acute myeloid leukemia is unknown. Here we showed that zebrafish idh1 (zidh1) knockdown by morpholino and targeted mutagenesis by transcription activator-like effector nuclease might induce blockade in myeloid differentiation, as evident by an increase in pu.1 and decrease in mpo, l-plastin, and mpeg1 expression, and significantly reduce definitive hematopoiesis. Morpholino knockdown of zidh2 also induced a blockade in myeloid differentiation but definitive hematopoiesis was not affected. The hematopoietic phenotype of zidh1 knockdown was not rescuable by zidh2 messenger RNA, suggesting nonredundant functions. Overexpression of human IDH1-R132H or its zebrafish ortholog resulted in 2-HG elevation and expansion of myelopoiesis in zebrafish embryos. A human IDH1-R132H-specific inhibitor (AGI-5198) significantly ameliorated both hematopoietic and 2-HG responses in human but not zebrafish IDH1 mutant expression. The results provided important insights to the role of zidh1 in myelopoiesis and definitive hematopoiesis and of IDH1-R132H in leukemogenesis.

Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium.

Our previous study showed that after being treated with 5-azacytidine, Nkx2.5(+) human cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiomyocytes. Although 5-azacytidine is a classical agent that induces myogenic differentiation in various types of cells, the drug is toxic and unspecific for myogenic differentiation. To investigate the possibility of inducing CPCs to differentiate into cardiomyocytes by a specific and non-toxic method, CPCs of passage 15 and mesenchymal stem cells (MSCs) were treated with cardiac ventricular fibroblast-conditioned medium (CVF-conditioned medium). Following this treatment, the Nkx2.5(+) CPCs underwent cardiomyogenic differentiation. Phase-contrast microscopy showed that the morphology of the treated CPCs gradually changed. Ultrastructural observation confirmed that the cells contained typical sarcomeres. The expression of cardiomyocyte-associated genes, such as alpha-cardiac actin, cardiac troponin T, and beta-myosin heavy chain (MHC), was increased in the CPCs that had undergone cardiomyogenic differentiation compared with untreated cells. In contrast, the MSCs did not exhibit changes in morphology or molecular expression after being treated with CVF-conditioned medium. The results indicated that Nkx2.5(+) CPCs treated with CVF-conditioned medium were capable of differentiating into a cardiac phenotype, whereas treated MSCs did not appear to undergo cardiomyogenic differentiation. Subsequently, following the addition of Dkk1 and the blocking of Wnt signaling pathway, CVF-conditioned medium-induced morphological changes and expression of cardiomyocyte-associated genes of Nkx2.5(+) CPCs were inhibited, which indicates that CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs is associated with Wnt signaling pathway. In addition, we also found that the activation of Wnt signaling pathway was accompanied by higher expression of GATA-4 and the blocking of the pathway inhibited the expression of GATA-4 in CVF-conditioned medium-incubated Nkx2.5(+) CPCs. This finding suggests that Wnt signaling pathway may alter GATA-4 expression and activate the cardiogenic program in the regulation of differentiation. In conclusion, Nkx2.5(+) CPCs have enormous potential for cardiomyogenic differentiation and the CVF-conditioned medium specifically induces CPCs to differentiate into a cardiac phenotype. Wnt signaling pathway is involved in CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs.

Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.

BACKGROUND: The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined. METHODS: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H2O2, high glucose/U0126 or normal glucose/H2O2/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions. RESULTS: We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H2O2 stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H2O2 conditions. CONCLUSIONS: These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions.

Effects of krill oil intake on plasma cholesterol and glucose levels in rats fed a high-cholesterol diet.

BACKGROUND: In this study, whole krill oil (WKO) and phospholipid-type krill oil (PKO) with different lipid composition were prepared. The effects of KO intake on plasma cholesterol and glucose levels in Wistar rats fed a high-cholesterol diet (HCD) were investigated. RESULTS: WKO contained 37.63% triglycerides, 48.37% phospholipids, 13.54% free fatty acids and 0.66% cholesterol, whereas the corresponding values for PKO were 0.59, 69.80, 28.53 and 1.09% respectively. Meanwhile, PKO contained much more polyunsaturated fatty acids (PUFA, 37.76%) than WKO (28.36%). After 4 weeks of HCD consumption, plasma levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and glucose increased significantly, but that of high-density lipoprotein cholesterol (HDL-C) decreased significantly. The intake of PKO and WKO for 4 weeks caused a significant reduction in body weight gain and plasma levels of TC and LDL-C in HCD-fed rats. Compared with WKO, PKO was more effective in decreasing plasma TC and LDL-C levels. CONCLUSION: PKO showed better overall cholesterol-lowering effects than WKO, which may be due to its higher n-3 PUFA levels.

Large-scale forward genetic screening analysis of development of hematopoiesis in zebrafish.

Zebrafish is a powerful model for the investigation of hematopoiesis. In order to isolate novel mutants with hematopoietic defects, large-scale mutagenesis screening of zebrafish was performed. By scoring specific hematopoietic markers, 52 mutants were identified and then classified into four types based on specific phenotypic traits. Each mutant represented a putative mutation of a gene regulating the relevant aspect of hematopoiesis, including early macrophage development, early granulopoiesis, embryonic myelopoiesis, and definitive erythropoiesis/lymphopoiesis. Our method should be applicable for other types of genetic screening in zebrafish. In addition, further study of the mutants we identified may help to unveil the molecular basis of hematopoiesis.

Endothelin-induced differentiation of Nkx2.5⁺ cardiac progenitor cells into pacemaking cells.

The mechanisms governing the development of cardiac pacemaking and conduction system are not well understood. In order to provide evidence for the derivation of pacemaking cells and the signal that induce and maintain the cells in the developing heart, Nkx2.5(+) cardiac progenitor cells (CPCs) were isolated from embryonic heart tubes of rats. Endothelin-1 was subsequently added to the CPCs to induce differentiation of them towards cardiac pacemaking cells. After the treatment, Nkx2.5(+) CPCs displayed spontaneous beating and spontaneously electrical activity as what we have previously described. Furthermore, RT-PCR and immunofluorescence staining demonstrated that Tbx3 expression was increased and Nkx2.5 expression was decreased in the induced cells 4 days after ET-1 treatment. And the significantly increased expression of Hcn4 and connexin-45 were detected in the induced cells 10 days after the treatment. In addition, Nkx2.5(+) CPCs were transfected with pGCsi-Tbx3 4 days after ET-1 treatment in an attempt to determine the transcription regulatory factor governing the differentiation of the cells into cardiac pacemaking cells. The results showed that silencing of Tbx3 decreased the pacemaking activity and led to down-regulation of pacemaker genes in the induced cells. These results confirmed that Nkx2.5(+) CPCs differentiated into cardiac pacemaking cells after being treated with ET-1 and suggested that an ET-1-Tbx3 molecular pathway govern/mediate this process. In conclusion, our study support the notion that pacemaking cells originate from Nkx2.5(+) CPCs present in embryonic heart tubes and endothelin-1 might be involved in diversification of cardiomyogenic progenitors toward the cells.