[mRNA Expression Profile Changes in Angiotensin-â ¡-Induced Atrial Myocardial Fibrosis in Rats].

Objective: To study the differences between the mRNA expression profile in angiotensin Ⅱ (Ang Ⅱ)-induced fibrotic cardiomyocytes and that of normal cardiomyocytes and the relevant signaling pathways. Methods: Six 8-week-old male Sprague-Dawley (SD) rats were randomly assigned to a control group and an Ang Ⅱ group, with 3 rats in each group. Rats in the control group were injected via caudal vein with 0.9% normal saline at 2 mg/kg per day, while rats in the Ang Ⅱ group were injected with Ang Ⅱ via caudal vein at 2 mg/kg per day. The medications were continuously administered in the two groups for 14 days. The degree of myocardial fibrosis was determined by Masson's Trichrome staining and the content of collagen Ⅰ was determined by immunohistochemistry. High throughput sequencing was performed to measure the mRNA expression of rat cardiomyocytes in the two groups and to screen for differentially-expressed mRNAs. The differentially-expressed mRNAs were analyzed by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results: Compared with those of the control group, the degree of myocardial fibrosis and the content of collagen Ⅰ in Ang Ⅱ group were significantly higher ( P<0.05). Through sequencing, 313 differentially-expressed mRNAs were identified, with 201 being up-regulated and 112 being down-regulated. Go and KEGG analyses showed that these differentially-expressed mRNA were involved in a variety of biological regulatory functions and pathways of myocardial fibrosis. Conclusion: Ang Ⅱ can cause myocardial fibrosis in rats. There are significant differences in mRNA expression between fibrotic cardiomyocytes and normal cardiomyocytes. The differentially expressed mRNAs may play an important role in biological processes, including immune response, cell remodeling, and extracellular matrix deposition.

Screening of cell-virus, cell-cell, gene-gene crosstalk among animal kingdom at single cell resolution.

BACKGROUND: The exact animal origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains obscure and understanding its host range is vital for preventing interspecies transmission. METHODS: Herein, we applied single-cell sequencing to multiple tissues of 20 species (30 data sets) and integrated them with public resources (45 data sets covering 26 species) to expand the virus receptor distribution investigation. While the binding affinity between virus and receptor is essential for viral infectivity, understanding the receptor distribution could predict the permissive organs and tissues when infection occurs. RESULTS: Based on the transcriptomic data, the expression profiles of receptor or associated entry factors for viruses capable of causing respiratory, blood, and brain diseases were described in detail. Conserved cellular connectomes and regulomes were also identified, revealing fundamental cell-cell and gene-gene cross-talks from reptiles to humans. CONCLUSIONS: Overall, our study provides a resource of the single-cell atlas of the animal kingdom which could help to identify the potential host range and tissue tropism of viruses and reveal the host-virus co-evolution.

Endothelial cell heterogeneity and microglia regulons revealed by a pig cell landscape at single-cell level.

Pigs are valuable large animal models for biomedical and genetic research, but insights into the tissue- and cell-type-specific transcriptome and heterogeneity remain limited. By leveraging single-cell RNA sequencing, we generate a multiple-organ single-cell transcriptomic map containing over 200,000 pig cells from 20 tissues/organs. We comprehensively characterize the heterogeneity of cells in tissues and identify 234 cell clusters, representing 58 major cell types. In-depth integrative analysis of endothelial cells reveals a high degree of heterogeneity. We identify several functionally distinct endothelial cell phenotypes, including an endothelial to mesenchymal transition subtype in adipose tissues. Intercellular communication analysis predicts tissue- and cell type-specific crosstalk between endothelial cells and other cell types through the VEGF, PDGF, TGF-ß, and BMP pathways. Regulon analysis of single-cell transcriptome of microglia in pig and 12 other species further identifies MEF2C as an evolutionally conserved regulon in the microglia. Our work describes the landscape of single-cell transcriptomes within diverse pig organs and identifies the heterogeneity of endothelial cells and evolutionally conserved regulon in microglia.

Molecular mechanisms governing circulating immune cell heterogeneity across different species revealed by single-cell sequencing.

BACKGROUND: Immune cells play important roles in mediating immune response and host defense against invading pathogens. However, insights into the molecular mechanisms governing circulating immune cell diversity among multiple species are limited. METHODS: In this study, we compared the single-cell transcriptomes of immune cells from 12 species. Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells. RESULTS: Our data revealed the heterogeneity and compositions of circulating immune cells among 12 different species. Additionally, we explored the conserved and divergent cellular crosstalks and genetic regulatory networks among vertebrate immune cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells. CONCLUSIONS: This study is the first to provide a comprehensive analysis of the cross-species single-cell transcriptome atlas for peripheral blood mononuclear cells (PBMCs). This research should advance our understanding of the cellular taxonomy and fundamental functions of PBMCs, with important implications in evolutionary biology, developmental biology, and immune system disorders.

VThunter: a database for single-cell screening of virus target cells in the animal kingdom.

Viral infectious diseases are a devastating and continuing threat to human and animal health. Receptor binding is the key step for viral entry into host cells. Therefore, recognizing viral receptors is fundamental for understanding the potential tissue tropism or host range of these pathogens. The rapid advancement of single-cell RNA sequencing (scRNA-seq) technology has paved the way for studying the expression of viral receptors in different tissues of animal species at single-cell resolution, resulting in huge scRNA-seq datasets. However, effectively integrating or sharing these datasets among the research community is challenging, especially for laboratory scientists. In this study, we manually curated up-to-date datasets generated in animal scRNA-seq studies, analyzed them using a unified processing pipeline, and comprehensively annotated 107 viral receptors in 142 viruses and obtained accurate expression signatures in 2 100 962 cells from 47 animal species. Thus, the VThunter database provides a user-friendly interface for the research community to explore the expression signatures of viral receptors. VThunter offers an informative and convenient resource for scientists to better understand the interactions between viral receptors and animal viruses and to assess viral pathogenesis and transmission in species. Database URL: https://db.cngb.org/VThunter/.

miR-451-3p alleviates myocardial ischemia/reperfusion injury by inhibiting MAP1LC3B-mediated autophagy.

OBJECTIVE AND DESIGN: We aim to explore the molecular mechanism of myocardial ischemia-reperfusion injury (MIRI). METHODS: The H9C2 cells were cultured under hypoxia/reoxygenation (H/R) condition to induce myocardial injury in vitro. The expression of miR-451-3p and MAP1LC3B was detected by RT-qPCR. Dual-luciferase reporter assay and RNA pull-down assay were performed to examine the relationship between microRNA (miR)-451-3p and MAP1LC3B. CCK8 was used to test cell viability. The level of LDH and CK was evaluated via ELISA. Immunofluorescence assay and flow cytometry were applied to detect autophagy and apoptosis, respectively. Autophagy-related protein expressions were determined by western blotting. Furthermore, an in vivo rat model of MIRI was established by subjection to 30 min ischemia and subsequently 24 h reperfusion for validation of the role of miR-451-3p in regulating MIRI in vivo. RESULTS: miR-451-3p was down-regulated in MIRI, and miR-451-3p mimics transfection alleviated autophagy and apoptosis induced by MIRI. miR-451-3p could target MAP1LC3B directly. Co-transfection miR-451-3p mimics and pcDNA 3.1 MAP1LC3B curbed the protected effects of miR-451-3p mimics on MIRI. CONCLUSIONS: miR-451-3p played a protective role in MIRI via inhibiting MAP1LC3B-mediated autophagy, which may provide new molecular targets for the treatment of MIRI and further improves the clinical outcomes of heart diseases.

6-Gingerol relieves myocardial ischaemia/reperfusion injury by regulating lncRNA H19/miR-143/ATG7 signaling axis-mediated autophagy.

Myocardial ischemia/reperfusion injury (MIRI) causes severe damage in cardiac tissue, thereby resulting in a high rate of mortality. 6-Gingerol (6-G) is reported to play an essential role in alleviating MIRI. However, the underlying mechanism remains obscure. This study was intended to explore the potential mechanism by which 6-G functions. Q-PCR was employed to quantify the relative RNA levels of long noncoding RNA (lncRNA) H19 (H19), miR-143, and ATG7, an enzyme essential for autophagy, in HL-1 cells. Western blotting, immunofluorescence, and immunohistochemistry were employed for protein evaluation in cultured cells or mouse tissues. Cell viability, cytotoxicity, and apoptosis were analysed by CCK-8, LDH, and flow cytometry assays, respectively. The binding sites for miR-143 were predicted using starBase software and experimentally validated through a dual-luciferase reporter system. Here, we found that 6-G elevated cellular H19 expression in hypoxia/reoxygenation (H/R)-treated HL-1 cells. Moreover, 6-G increased Bcl-2 expression but reduced cleaved caspase 3 and caspase 9 protein levels. Mechanistically, H19 directly interacted with miR-143 and lowered its cellular abundance by acting as a molecular sponge. Importantly, ATG7 was validated as a regulated gene of miR-143, and the depletion of miR-143 by H19 caused an increased in ATG7 expression, which in turn promoted the autophagy process. Last, mouse experiments highly supported our in vitro findings that 6-G relieves MIRI by enhancing autophagy. The H19/miR-143/ATG7 axis was shown to be critical for the function of 6-G in relieving MIRI.

[6-Gingerol Pretreatment Alleviates Hypoxia/Reoxygenation-induced H9C2 Cardiomyocyte Injury by Inhibiting Oxidative Stress and Inflammation].

OBJETIVE: To observe the effect of 6-gingerol (6-G) pretreatment on hypoxia/reoxygenation (H/R) induced injury in H9C2 myocardial cell and investigate its related mechanism. METHODS: The H/R in vitro model of cardiomyocytes was prepared by conventional methods. In detail, H9C2 cells were added with the nitrogen-saturated hypoxic liquid, and placed in an incubator, mixed with gas (1% O 2, 5% CO 2, 94% N 2) applying for 15 min. After culturing for 3 h, the cells were taken out and placed in an incubator (37℃, 5% CO 2) for 1 h. Before establishing the cell model, the cells were pretreated with 6-G, and the cell viability was measured by MTT method to observe the protective effect of different concentrations of 6-G on H/R-induced cell damage. The 6-G mass concentration for pretreatment that led to the highest cell viability was used for follow-up experiments. DCFH-DA fluorescent probe was used to detect the effect of 6-G pretreatment on H9C2 oxidative stress level, and the intracellular oxidative stress was observed with fluorescence microscope and flow cytometry. Western blot method was used to detect the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in H/R-induced cell inflammatory responses. RESULTS: Compared with the H/R group, the cell viability of the 6-G+H/R group began to increase when the concentration of 6-G promoted to50 µg/mL. The cell viability was the highest after pretreated with 200 µg/mL 6-G. Therefore, 200 µg/mL was considered as the best 6-G intervention concentration for subsequent experiment. The content of reactive oxygen species (ROS) in the 200 µg/mL 6-G group had no significant changes compared with the control group (P>0.05), and the ROS fluorescence peak did not migrate significantly. However the ROS content in the H/R group increased significantly compared with the control (P<0.05), and the ROS fluorescence peak shifted to the right. Compared with the H/R group, the ROS content of the 6-G+H/R group decreased (P<0.05), and the ROS fluorescence peak shifted to the left. Compared with the control group, the expressions of TNF-α, IL-6, IL-1ß in the 6-G group had no significant changes (P>0.05); the expressions of TNF-α, IL-6, IL-1ß in the H/R group increased (P<0.05). Compared with H/R group, the expressions of TNF-α, IL-6 and IL-1ß in 6-G+H/R group decreased (P<0.05). CONCLUSION: 6-G pretreatment can alleviate H/R-induced H9C2 myocardial injury, which may be related to the inhibition of oxidative stress and inflammatory responses.

JWH133 inhibits MPP+-induced inflammatory response and iron influx in astrocytes.

BACKGROUND: We investigated the anti- inflammatory effect of type II cannabinoid receptor (CB2 receptor) activation and their relationship to iron influx on 1-methyl-4-phenylpyridinium (MPP+) treated astrocytes. METHODS AND RESULTS: By western blots, real-time PCR and ELISA, the expressions of CB2 receptor, divalent metal transporter-1 (DMT1), cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), interleukin-1ß (IL-1ß) and tumor necrosis factor- α (TNF-α) were measured. Iron influx into astrocytes was determined by the quenching of calcein fluorescence. We found that pre-treatment with JWH133, a selective CB2 receptor agonist, significantly suppressed the MPP+-induced up-regulation of COX-2, iNOS, IL- 1ß and TNF-α in astrocytes. In addition, JWH133 significantly inhibited the MPP+-induced up- regulation of DMT1. Further studies indicated that JWH133 suppressed the MPP+-accelerated iron influx into astrocytes. These effects were blocked by co-treatment with AM630, the CB2 receptor antagonist. CONCLUSIONS: These results suggest that activation of CB2 receptor inhibit MPP +-induced inflammatory response and iron influx in astrocytes.