Guili Lian

Background: Pulmonary arterial hypertension (PAH) is a progressive vascular disorder characterized by immune dysregulation and vascular remodeling. Cellular senescence modulates immune responses, but its role in PAH, particularly in monocytes/macrophages, remains unclear. Here, we integrated single-cell and bulk transcriptomic data to identify senescence-associated candidate genes associated with monocytes/macrophages in PAH. Methods: Publicly available single-cell RNA-seq data from PAH and control pulmonary vascular tissues were analyzed. A senescence-associated score was calculated for each cell based on MSigDB gene sets. Senescence-associated genes in monocytes/macrophages were identified through correlation and differential expression screening, and further examined in bulk transcriptomic datasets. Bulk transcriptomic discovery used GSE113439, GSE117261, and GSE254617. A sample-title audit identified 68 shared PHBI identifiers between GSE117261 and GSE254617; therefore, the merged data represented 257 expression profiles from 189 unique subjects. LASSO, Random Forest, and XGBoost were used as complementary feature-selection approaches, and genes jointly retained by the three algorithms were defined as candidates for evaluation in independent cohorts. Results: Single-cell RNA-seq identified 14 cell types in PAH pulmonary vascular tissues, with dendritic cells, T cells, and monocytes/macrophages exhibiting the highest senescence-associated scores. Integrative screening identified 405 senescence-associated genes enriched in monocytes/macrophages. Multi-algorithm screening prioritized 10 candidate genes for subsequent evaluation. After within-cohort multiple-testing correction, external cohort analyses reproduced the discovery-stage expression directions for RGS1 , PHLDA1 , MAFB , and RNF13 in lung tissue cohorts, and for MAFB in a PBMC cohort. With nominal P values, RGS1 was significantly upregulated in GSE48149 and GSE53408 (both lung), and PHLDA1 was upregulated in GSE48149 (lung). MAFB was significantly downregulated in GSE33463 (PBMC) and GSE48149 (lung), whereas RNF13 was downregulated in GSE15197 and GSE48149 (both lung). Functional analysis revealed significant enrichment of immune-inflammatory and phagocytosis-related pathways among the candidate genes. Consensus clustering based on the candidate genes classified PAH patients into two exploratory transcriptomic groups with distinct expression and pathway activity profiles. In vivo evaluation in an MCT-induced PAH rat model showed seven significant direction-consistent changes, two direction-consistent nonsignificant trends, and one discordant gene ( PDE4B ), supporting tissue-level expression concordance. Conclusion: These analyses identify a Mono.Macs-associated senescence-related candidate gene set in PAH. Independent expression analyses provide additional support for RGS1 , PHLDA1 , MAFB , and RNF13 as candidate genes for subsequent mechanistic and functional validation.

The gut microbiota is associated with cardiometabolic disorders (including coronary artery disease, type 2 diabetes, stroke, heart failure, and hypertension) through the gut–heart or gut–brain axis, among which metabolic processes play crucial roles. However, the exact causal mechanisms remain unknown. Our study sought to uncover the causal relationships between the gut microbiota, circulating metabolites and cardiometabolic diseases via Mendelian randomization (MR) analysis. Forward MR was utilized to investigate the causal effects of the gut microbiota and circulating metabolites on the risk of cardiometabolic diseases. Reverse MR was subsequently performed to analyze the significant gut microbiota and circulating metabolites. Two-step MR was employed to examine the impact of circulating metabolites on the relationship between the gut microbiota and cardiometabolic diseases and to determine the mediated fractions. Our results revealed a nominal causal relationship with 23, 23, 22, 20, and 19 gut microbiota constituents for coronary artery disease, type 2 diabetes, stroke, heart failure, and hypertension, respectively. We also identified 2 significant associations and 13 suggestive associations between circulating metabolites and these cardiometabolic diseases. Reverse MR analysis revealed that genetically predicted type 2 diabetes was suggestively associated with 3 circulating metabolites, whereas stroke demonstrated a suggestive association with 3 distinct gut microbiota. Further screening identified 4 circulating metabolites as potential mediators in the pathway from the gut microbiota to cardiometabolic diseases. Our study revealed a causal link between gut microbiome components and cardiometabolic diseases and that circulating metabolites potentially act as intermediaries in this association.

Background: Muscle atrophy is a severe complication of diabetes, with autophagy playing a critical role in its progression. Zinc has been shown to alleviate hyperglycaemia and several diabetes‐related complications, but its direct role in mediating diabetic muscle atrophy remains unclear. This study explores the potential role of zinc in the pathogenesis of diabetic muscle atrophy. Methods: In vivo, C57BL/6J mice were induced with diabetes by streptozotocin (STZ) and treated with ZnSO₄ (25 mg/kg/day) for six weeks. Gastrocnemius muscles were collected for histological analysis, including transmission electron microscopy (TEM). Serum zinc levels were measured by ICP‐MS. Protein expression was evaluated using immunofluorescence (IF), immunohistochemistry (IHC) and Western blotting (WB). Bioinformatics analysis was used to identify key genes associated with muscle atrophy. In vitro, a high‐glucose‐induced diabetic C2C12 cell model was established and received ZnSO₄, rapamycin, SRT1720, TC‐G‐1008, or GPR39‐CRISPR Cas9 intervention. Autophagy was observed by TEM, and protein expression was assessed by IF and WB. Intracellular zinc concentrations were measured using fluorescence resonance energy transfer (FRET). Results: In vivo, muscle atrophy, autophagy activation, and upregulation of SIRT1 and FoxO1, along with downregulation of GPR39, were confirmed in the T1D group. ZnSO₄ protected against muscle atrophy and inhibited autophagy (T1D + ZnSO₄ vs. T1D, all p < 0.0001), as evidenced by increased grip strength (212.40 ± 11.08 vs. 163.90 ± 10.95 gf), gastrocnemius muscle index (10.67 ± 0.44 vs. 8.80 ± 0.72 mg/g), muscle fibre cross‐sectional area (978.20 ± 144.00 vs. 580.20 ± 103.30 μm ² ), and serum zinc levels (0.2335 ± 0.0227 vs. 0.1561 ± 0.0123 mg/L). ZnSO₄ down‐regulated the expression of Atrogin‐1 and MuRF1, and decreased the formation of autophagosomes in the gastrocnemius muscle of T1D mice (all p < 0.0001). RNA‐seq analysis indicated activation of the SIRT1/FoxO1 signalling pathway in diabetic mice. ZnSO₄ down‐regulated LC3B, SIRT1 and FoxO1, while upregulating P62 and GPR39 (all p < 0.05). In vitro, muscle atrophy, autophagy activation, and down‐regulation of GPR39 were confirmed in the diabetic cell model (all p < 0.05). Both ZnSO₄ and TC‐G‐1008 down‐regulated Atrogin‐1, LC3B, SIRT1, and FoxO1, and up‐regulated P62 and GPR39, inhibiting autophagy and improving muscle atrophy (all p < 0.05). The beneficial anti‐atrophic effects of ZnSO₄ are diminished following treatment with SRT1720 or RAPA. Upon GPR39 knockout, SIRT1, FoxO1, and Atrogin‐1 were upregulated, while P62 was downregulated. Intracellular zinc concentrations in ZnSO₄‐treated group remained unchanged ( p > 0.05), indicating that zinc supplementation did not affect zinc ion entry but acted through the cell surface receptor GPR39. Conclusion: ZnSO ₄ inhibits excessive autophagy in skeletal muscle and alleviates muscle atrophy in diabetic mice via the GPR39‐SIRT1/FoxO1 axis. These findings suggest that zinc supplementation may offer a potential therapeutic strategy for managing diabetic muscle atrophy.

Septic Cardiomyopathy (SCM) is a syndrome of acute cardiac dysfunction in septic patients, unrelated to cardiac ischemia. Multiomics studies including transcriptomics and proteomics have provided new insights into the mechanisms of SCM. In here, a rat model of SCM was established by intraperitoneal injection of lipopolysaccharide (LPS). Biomarkers of SCM were characterized via a multi-omics analysis. The differentially expressed (DE) mRNAs predominantly appeared in pathways linked to the immune response, inflammatory response, and the complement and coagulation cascades, while DE proteins were mainly enriched in pathways associated with the complement and coagulation cascades. On this basis, the integrated analysis was performed between transcriptome and proteome. The potential biomarkers were further verified by RT-qPCR and WB. The current proteotranscriptomic research has furnished a valuable dataset and fresh perspectives that will enhance our comprehension of the development of SCM. This, in turn, is expected to expedite the formulation of novel approaches for the prevention and management of SCM in patients.

Septic cardiomyopathy (SCM), a complication initiated by sepsis, presents a significant clinical challenge, leading to increased mortality rates. However, the mechanisms of SCM have not been fully uncovered. Our study involved analyzing RNA sequencing (RNA-seq) data from rat heart tissue, along with utilizing molecular docking and molecular dynamics (MD) simulations, to discover key targets and potential pharmacological actions of the calcitonin gene-related peptide (CGRP) against SCM. A lipopolysaccharide-induced SCM model was established in rats (LPS 10 mg/kg, intraperitoneal (i.p.)). Thereafter, the myocardial tissues from the three groups of rats (Ctrl group, LPS group, and CGRP group) (n = 5) were extracted and underwent RNA-seq, followed by bioinformatics analyses. The qPCR-validated hub targets potentially interacting with CGRP were identified. Following this, homology modeling was utilized to obtain the 3D structure of hub targets, and molecular docking was conducted to evaluate the interaction between CGRP and hub targets. MD simulations (300 ns) were performed to confirm the findings further. Our findings demonstrated that CGRP significantly lowered mortality in SCM rats. 633 DEGs were affected by LPS, contrasted with the Ctrl group. 96 DEGs were affected by CGRP compared to the LPS group. In total, ten fully annotated CGRP-triggered hub genes were obtained. The molecular docking and MD simulations indicate that the relationship between CGRP and eight hub genes is extremely strong. This research offers a thorough examination of the possible objectives and fundamental molecular processes of CGRP in combating SCM, laying the groundwork for investigating the potential protective mechanisms of CGRP against SCM.

Septic cardiomyopathy (SCM) is a common and severe complication of sepsis, characterized by left ventricular dilation and reduced ejection fraction leading to heart failure. The pathogenesis of SCM remains unclear. Understanding the SCM pathogenesis is essential in the search for effective therapeutic agents for SCM. This study was to investigate the pathophysiology of SCM and explore new therapeutic drugs by bioinformatics. An SCM rat model was established by injection of 10 mg/kg lipopolysaccharide (LPS) for 24 h, and the myocardial tissues were collected for RNA sequencing. The differentially expressed genes (DEGs) between LPS rats and control (Ctrl) with the thresholds of |log2^{fold change}|≥ 1 and P < 0.05. A protein–protein interaction (PPI) network was constructed based on the DEGs. The hub genes were identified using five algorithms of Cytoscape in the PPI networks and validated in the GSE185754 dataset and by RT-qPCR. The hub genes were analyzed by Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as Gene set enrichment analyses (GSEA). In addition, the miRNAs of hub genes were predicted through miRWalk, and the candidate therapeutic drugs were identified using the Connectivity Map (CMAP) database. This study revealed the identified hub genes (Itgb1, Il1b, Rac2, Vegfa) and key miRNAs (rno-miR-541-5p, rno-miR-487b-3p, rno-miR-1224, rno-miR-378a-5p, rno-miR-6334, and rno-miR-466b-5p), which were potential biological targets and biomarkers of SCM. Anomalies in cytokine-cytokine receptor interactions, complement and coagulation cascades, chemokine signaling pathways, and MAPK signaling pathways also played vital roles in SCM pathogenesis. Two high-confidence candidate compounds (KU-0063794 and dasatinib) were identified from the CMAP database as new therapeutic drugs for SCM. In summary, these four identified hub genes and enrichment pathways may hold promise for diagnosing and treating SCM.

Metal responsive transcription factor 1 (MTF-1) is a zinc-dependent transcription factor involved in the development of pulmonary arterial hypertension (PAH), which is a life-threatening disease characterized by elevated pulmonary artery pressure and pulmonary vascular remodeling. However, little is known about the role and regulatory signaling of MTF-1 in PAH. This study aimed to investigate the effect and mechanism of MTF-1 on the proliferation of pulmonary arterial smooth muscle cells (PASMCs). Several techniques including intracellular-free zinc detected by fluorescent indicator-fluozinc-3-AM, western blot, luciferase reporter, and cell proliferation assay were conducted to perform a comprehensive analysis of MTF-1 in proliferation of PASMCs in PAH. Increased cytosolic zinc was shown in monocrotaline (MCT)-PASMCs and ZnSO₄-treated PASMCs, which led to overexpression and overactivation of MTF-1, followed by the up-regulation of placental growth factor (PlGF). Elevated MTF-1 and PlGF were observed in western blot, and high transcriptional activity of MTF-1 was confirmed by luciferase reporter in ZnSO₄-treated cells. Further investigation of cell proliferation revealed a favorable impact of zinc ions on PASMCs proliferation, with the deletion of Mtf-1/Plgf attenuating ZnSO₄-induced proliferation. Flow cytometry analysis showed that blockade of PKC signaling inhibited the cell cycle of MCT-PASMCs and ZnSO4-treated PASMCs. The Zinc/PKC/MTF-1/PlGF pathway is involved in the up-regulatory effect on the PASMCs proliferation in the process of PAH. This study provided novel insight into zinc homeostasis in the pathogenesis of PAHs, and the regulation of MTF-1 might be a potential target for therapeutic intervention in PAH.

Sarcopenia has become a leading cause of disability and mortality in the elderly. It has been reported that programmed cell death (PCD) is associated with the development of sarcopenia that is characterized by reduction of muscle fiber size and number. TNF-α is also validated to play a prominent role in sarcopenia through its complex signaling pathways including cell death signaling. However, it is still unclear whether TNF-α contributes to sarcopenia by mediating pyroptosis, one type of PCD. Here, we first established naturally aged mice with sarcopenia model and confirmed an inflammatory state represented by TNF-α in aged mice. Evidence of GSDME-mediated pyroptosis and activation of apoptotic caspase-8/-3 were also found in skeletal muscle cells of aged mice with sarcopenia. We demonstrated that TNF-α triggered GSDME-mediated pyroptosis in myotubes through activating caspase-8 and caspase-3 by using caspase-8 and caspase-3 inhibitors. Comparing the activation of caspase-8 and GSDME expression between TNF Complex IIa and TNF Complex IIb, TNF-α was found to be more inclined to assemble TNF Complex IIb in activating caspase-8 and triggering pyroptosis. Moreover, pyroptotic myotubes were validated to result in decreased expression of MHC1 and finally loss of myotubes by knockdown of GSDME. Our work reveals a novel mechanism that TNF-ɑ/caspase-8/caspase-3/GSDME signaling-mediated pyroptosis contributes to the development of sarcopenia. Caspase-3/GSDME signaling-mediated pyroptosis may be a promising therapeutic target for sarcopenia.

Background: Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and pulmonary arterial pressure, with complex etiology, difficult treatment and poor prognosis. The objective of this study was to investigate the potential biomarkers for PAH based on bioinformatics analysis. Methods: The GSE117261 datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified by screening PAH patients and controls. Then the DEGs were analyzed using a Weighted Gene Co-expression Network Analysis (WGCNA) and the key modules were determined, and to further explore their potential biological functions via Gene Ontology analysis (GO), Kyoto Encyclopedia of Genes and Genomes Pathway analysis (KEGG), and Gene Set Enrichment Analysis (GSEA). Moreover, Protein–protein interaction (PPI) networks were constructed to identify hub gene candidates in the key modules. Finally, real-time quantitative polymerase chain reaction was supplied to detect the expressions of hub genes in human pulmonary arterial smooth cells treated with cobalt chloride (COCl₂) which was used to mimic hypoxia. Results: There were 2299 DEGs identified. WGCNA indicated that yellow module was the key one correlated with PAH. GO and KEGG analysis demonstrated that genes in the yellow module were mainly enriched in ‘Pathways in cancer’. GSEA revealed that ‘HALLMARK_MYC_TARGETS_V1’ was remarkably enriched in PAH. Based on the PPI network, vascular endothelial growth factor A, proto-oncogene receptor tyrosine kinase (KIT), PNN interacting serine and arginine rich protein (PNISR) and heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) were identified as the hub genes. Additionally, the PCR indicated that the elevated expressions of PNISR and HNRNPH1 were in line with the bioinformatics analysis. ROC analysis determined that PNISR and HNRNPH1 may be potential biomarkers to provide better diagnosis of PAH. Conclusion: PNISR and HNRNPH1 were potential biomarkers to diagnosis PAH. In summary, the identified DEGs, modules, pathways, and hub genes provide clues and shed light on the potential molecular mechanisms of PAH.

Inflammation and immunity play a causal role in the pathogenesis of pulmonary vascular remodelling and pulmonary arterial hypertension (PAH). However, the pathways and mechanisms by which inflammation and immunity contribute to pulmonary vascular remodelling remain unknown. RNA sequencing was used to analyse the transcriptome in control and rats injected with monocrotaline (MCT) for various weeks. Using the transcriptional profiling of MCT‐induced PAH coupled with bioinformatics analysis, we clustered the differentially expressed genes (DEGs) and chose the increased expression patterns associated with inflammatory and immune response. We found the enrichment of Toll‐like receptor (TLR) and Nod‐like receptor (NLR) pathways and identified NF‐κB‐mediated inflammatory and immune profiling in MCT‐induced PAH. Pathway‐based data integration and visualization showed the dysregulated TLR and NLR pathways, including increased expression of TLR2 and NLRP3, and their downstream molecules. Further analysis revealed that the activation of TLR and NLR pathways was associated with up‐regulation of damage‐associated molecular patterns (DAMPs) and RIPK3‐mediated necroptosis was involved in the generation of DAMPs in MCT‐induced PAH. Collectively, we identify RIPK3‐mediated necroptosis and its triggered TLR and NLR pathways in the progression of pulmonary vascular remodelling, thus providing novel insights into the mechanisms underlying inflammation and immunity in the pathogenesis of PAH.