BackgroundAtherosclerosis (AS) is a chronic inflammatory disease and a leading cause of global morbidity and mortality. Dysregulated expression of the long non-coding RNA (lncRNA) H19 has been implicated in AS progression. However, the underlying molecular mechanisms remain unclear.MethodsTo determine the role of H19 in AS, the AS mouse model was established using ApoE-/- mice fed a high-fat diet, and the AS cell model was generated by stimulating human umbilical vein endothelial cells (HUVECs) with oxidized low-density lipoprotein (oxLDL). H19 expression levels were subsequently measured. To investigate the underlying mechanism, H19 siRNA and was transfected into cells, and adeno-associated virus (AAV) expressing short hairpin RNA targeting H19 (AAV-sh-H19) was administered to mice to evaluate the functional impact of H19 on AS.ResultH19 was markedly upregulated in the aortae of AS mice and in the AS cell model, which correlated with enhanced adhesion molecule expression and systemic cytokine release. Mechanistically, H19 functioned as a molecular sponge for microRNA let-7a (miR-let-7a), thereby relieving the repression of its target integrin subunit beta 3 (ITGB3) and amplifying VCAM-1/ICAM-1/N-cadherin-dependent leukocyte recruitment. Luciferase reporter assays confirmed that miR-let-7a is a direct target of H19. Moreover, H19 knockdown increased miR-let-7a levels, thereby inhibiting endothelial cell adhesion and inflammatory responses. Conversely, treatment of mice with AAV-sh-H19 markedly attenuated AS lesion formation.ConclusionThese findings indicate that the H19/miR-let-7a/ITGB3 axis constitutes a targetable inflammatory checkpoint that links endothelial dysfunction to plaque initiation, highlighting H19 inhibition as a potential therapeutic target.

The most lethal diseases to have affected humankind are cardiovascular disorders. For decades, researchers have sought to elucidate the aetiology of heart disease, while clinicians have worked to prevent premature mortality. In recent years, substantial progress has been made in understanding cardiovascular conditions, accompanied by the development of systematic therapeutic strategies that extend patient survival.Advances in proteomics have enabled the analysis of clinical samples with far greater depth than was possible using traditional approaches. Immunoblotting, much like line fishing, is highly specific but inherently inefficient. Proteomics, by contrast, resembles the use of a net, allowing the comprehensive capture of molecular information from limited samples without sacrificing analytical precision. With appropriate methodologies, extensive proteomic information can be obtained through the identification of protein fragments.Studies involving a wide range of sample sources, from solid tissues to biofluids, were included in this review. Tissue samples represent the most informative material for investigating pathological processes at sites of vascular occlusion. However, tissues such as the heart and blood vessels are vital for physiological function and have limited regenerative capacity, rendering these samples rare yet highly valuable.In contrast, biofluid samples are readily accessible. Although they contain more contaminants, such as salts, and yield lower amounts of protein than tissue samples, they nonetheless provide meaningful insights into the circulatory system. Proteomic analyses have revealed that cytoskeletal organisation, extracellular matrix remodelling, immune activation, inflammation, and metabolic processes are involved in most cardiomyopathies, whereas mitochondrial dysfunction, fibrosis, and additional pathways are more prominent in other cardiovascular diseases.Comparative analyses of proteomic data across different cardiovascular conditions and patient cohorts have identified shared molecular pathways, highlighting the capacity of proteomics to uncover multiple dimensions of cardiovascular pathology. Moreover, proteomic profiling facilitates the discovery of candidate biomarkers—whether diagnostic, prognostic, or indicative of disease risk—as well as potential therapeutic targets. These putative biomarkers and targets, however, require validation in larger and more diverse cohorts.

Background Atrial fibrillation (AF) is a common cardiac arrhythmia that contributes significantly to morbidity and mortality, with associated risks such as stroke and heart failure. This study aimed to identify novel biomarkers for AF progression using a multi-omics approach, combining proteomics and metabolomics, to gain a deeper understanding of AF pathophysiology and uncover potential diagnostic and therapeutic targets. Methods We enrolled 39 participants, including 23 AF patients and 16 SR controls, and subjected their serum samples to tandem mass spectrometry-based proteomics and untargeted liquid chromatography-mass spectrometry-based metabolomics. Differential protein and metabolite expression were analyzed using bioinformatics tools, and the results were verified with in vitro assays. Results A total of 19 differentially expressed proteins and 154 altered metabolites were identified in AF patients compared to controls. Among these, pyruvate kinase M2 (PKM2) stood out due to its significant correlation with glycolytic intermediates, such as lactate and phosphoenolpyruvate. Proteomic data revealed that PKM2 expression was significantly upgulated in AF patients. In vitro experiments using angiotensin II (Ang II)-treated cardiac fibroblasts confirmed that PKM2 is associated with fibrosis and cellular remodeling in AF. PKM2 knockdown significantly reduced fibroblast proliferation and α-SMA expression, attenuating fibrotic remodeling. The data also demonstrated that PKM2 regulates key metabolic and fibrotic pathways through its interaction with STAT3 signaling. Conclusions PKM2 is a key regulator of metabolic and fibrotic remodeling in AF and holds potential as both a biomarker for diagnosis and a therapeutic target.

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide, and its progression is closely linked to mitochondrial dysfunction in cardiomyocytes. Given the high energy demands of the heart, precise regulation of mitochondrial homeostasis, including oxidative phosphorylation, reactive oxygen species balance, calcium handling, and mitophagy, is essential for maintaining cardiac function. Emerging evidence has identified mitochondrial-associated long non-coding RNAs (mito-lncRNAs) as important regulators of these processes. Mito-lncRNAs comprise both nuclear-encoded transcripts that translocate to mitochondria and mitochondrial genome-encoded lncRNAs that function within the organelle. These molecules modulate mitochondrial gene expression, respiratory chain stability, metabolic flux, and stress responses, thereby influencing the pathogenesis of acute myocardial infarction, heart failure, diabetic cardiomyopathy, pulmonary hypertension, and cardiac remodeling. In this review, we categorize mito-lncRNAs based on their genomic origin and mitochondrial localization and summarize their mechanistic roles in cardiovascular physiology and disease. Moreover, the review highlights context-dependent effects of key transcripts such as LIPCAR, MALAT1, RMRP, H19, and lncND5. We further discuss the emerging value of mito-lncRNAs as circulating biomarkers and examine the major challenges that currently limit therapeutic translation, including cardiac- and mitochondrial-specific delivery, mechanistic ambiguity, species conservation, and technical limitations in detection. A deeper understanding of mito-lncRNA biology may provide new insights into mitochondrial regulation in the heart and inform the development of novel diagnostic and therapeutic strategies for CVDs.

BackgroundCalcium oxalate nephrolithiasis represents the most common form of kidney stone disease, characterized by limited therapeutic interventions and a high rate of recurrence. Grona styracifolia (Osbeck) H.Ohashi and K.Ohashi [syn.: Desmodium styracifolium (Osbeck) Merr.; Fabaceae], a traditional Chinese medicinal plant has been empirically used to treat urolithiasis; however, the mechanisms underlying its efficacy remain poorly elucidated.ObjectiveTo investigate the anti-lithogenic mechanisms of total flavonoids of G. styracifolia capsules (DS) in calcium oxalate (CaOx) crystal deposition and their renal-associated injury.MethodsSprague-Dawley rats were subjected to an ethylene glycol-induced CaOx nephrolithiasis model. The animals received DS treatment (50, 100, and 200 mg/kg) for 1 month. Thereafter, Renal histopathology was evaluated using hematoxylin and eosin staining. Furthermore, biochemical parameters such as calcium, inorganic phosphorus, creatinine, and urea nitrogen levels were assessed. Endoplasmic reticulum stress markers and autophagy-related proteins were assessed using quantitative PCR and Western blotting.ResultsThe DS treatment significantly reduced CaOx crystal deposition and the associated renal injury compared to untreated controls. In addition, treated animals demonstrated improved renal function, as evidenced by decreased levels of calcium, creatinine, inorganic phosphorus, and urea nitrogen. Histological examination revealed attenuated renal fibrosis and improved kidney morphology. Mechanistic investigations have indicated that the administration of DS is linked to the modulation of endoplasmic reticulum stress and autophagy, processes that are disrupted by the formation of kidney stones and urinary obstruction.ConclusionThe DS significantly mitigate CaOx nephrolithiasis by modulating endoplasmic reticulum stress and autophagy. This finding highlights its traditional therapeutic application and suggests its potential development as a phytotherapeutic agent for the prevention of kidney stones.

Receptor-interacting protein kinases (RIPKs) are a family of serine/threonine kinases that regulate innate immunity, inflammation, and several modalities of regulated cell death, and are increasingly implicated in cardiovascular disease. Increasing evidence suggests that dysregulated RIPK signaling contributes to cardiomyocyte injury, fibrosis, maladaptive remodeling, mitochondrial dysfunction, and inflammatory amplification in multiple cardiac pathologies. While the roles of RIPK1 and RIPK3 in necroptosis are well recognized, recent studies indicate broader and more complex role in heart disease, including broader non-canonical functions involving mitochondrial regulation, oxidative stress, inflammasome activation, and metabolic remodeling. RIPK2 is emerging as a significant but underrecognized modulator of sterile cardiac inflammation and pathological remodeling. Similarly, RIPK4 has recently been associated with oxidative stress responses and ferroptosis, although direct cardiac evidence remains limited. This review provides an updated overview of RIPK1-RIPK4 signaling within the framework of cardiovascular disease, emphasizing both canonical necroptotic and non-canonical signaling mechanisms. We discuss the involvement of RIPKs in myocardial ischemia/reperfusion injury, heart failure, cardiac hypertrophy, diabetic cardiomyopathy, myocarditis, and doxorubicin-induced cardiotoxicity. We further summarize current and emerging therapeutic strategies targeting RIPKs, including small-molecule inhibitors, natural compounds, and endogenous regulators approaches. Finally, we highlight major knowledge gaps and translational challenges, including isoform-specific functions, temporal dynamics of RIPK activation, safety considerations, and the clinical translation of preclinical findings.

The development of temporal single-cell RNA-seq (scRNA-seq) assay enabled us to systemically investigate the effects of various types of perturbations in a time course with single-cell resolution. However, the existing temporal scRNA-seq technologies have certain limitations in reliability, detection efficacy and detection diversity. In the current study, we develop scBiopsy-seq assay, which combines a synergistic electroosmosis-electrophoresis extraction method for efficient RNA extraction and digital microfluidics with contamination-isolated hydrophobic interface for high-performance sample processing. scBiopsy-seq extracts the cytoplasm at well-controlled volume to detect >10K genes per extraction with 90% successful rate. Functional enrichment analysis revealed that the genes robustly detected by scBiopsy-seq were associated to diverse biological processes, demonstrating its superb diversity of detection. scBiopsy-seq can perform the sequential extraction of the cytoplasm from the same cell multiple times, which allows us to associate the cell phenotypic responses with its transcriptional dynamics. We employed scBiopsy-seq to analyze the temporal response to a BRD4 degrader induced transcriptional suppression, which identified the key roles of fatty acid beta oxidation in this biological process. scBiopsy-seq with similar data quality as scRNA-seq will dramatically expand the application of temporal scRNA-seq towards a broader spectrum of cell biology research. ### Competing Interest Statement The authors have declared no competing interest.

Multimodal measurement of single cells provides deep insights into the intricate relationships between individual molecular layers and the regulatory mechanisms underlying intercellular variations. Here, we reported DMF-DM-seq, a highly integrated, sensitive, and automated platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics. This platform first integrates the processes of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enables complete separation of single-cell mRNA and miRNA via a magnetic field application, resulting in a higher miRNA detection ability. DMF-DM-seq revealed differential expression patterns of single cells of noncancerous breast cells and noninvasive and aggressive breast cancer cells at both mRNA and miRNA levels. The results demonstrated the anticorrelated relationship between miRNA and their mRNA targets. Further, we unravel the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, along with important miRNA-interaction networks involved in significant signaling pathways. We also deconstruct the miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool for a comprehensive study of the expression heterogeneity of single-cell mRNA and miRNA, which will be widely applied in basic and clinical research.

Single-cell microRNA (miRNA) sequencing has allowed for comprehensively studying the abundance and complex networks of miRNAs, which provides insights beyond single-cell heterogeneity into the dynamic regulation of cellular events. Current benchtop-based technologies for single-cell miRNA sequencing are low throughput, limited reaction efficiency, tedious manual operations, and high reagent costs. Here, a highly multiplexed, efficient, integrated, and automated sample preparation platform is introduced for single-cell miRNA sequencing based on digital microfluidics (DMF), named Hiper-seq. The platform integrates major steps and automates the iterative operations of miRNA sequencing library construction by digital control of addressable droplets on the DMF chip. Based on the design of hydrophilic micro-structures and the capability of handling droplets of DMF, multiple single cells can be selectively isolated and subject to sample processing in a highly parallel way, thus increasing the throughput and efficiency for single-cell miRNA measurement. The nanoliter reaction volume of this platform enables a much higher miRNA detection ability and lower reagent cost compared to benchtop methods. It is further applied Hiper-seq to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture and discovered unreported miRNAs that regulate bone repairing.

Cells experience continuous transformation under both physiological and pathological circumstances. Single-cell RNA sequencing (scRNA-seq) is competent in disclosing the disparities of cells; nevertheless, it poses challenges in linking the individual cell state at distinct time points. Although computational approaches based on scRNA-seq data have been put forward for trajectory analysis, the result is based on assumptions and fails to reflect the actual states. Consequently, it is necessary to incorporate a "time anchor" into the scRNA-seq library for the temporal documentation of the dynamic expression pattern. This review comprehensively overviews the time-resolved single-cell transcriptomic sequencing methodologies and applications. As scRNA-seq functions as the basis for profiling single-cell expression patterns, the review initially introduces various scRNA-seq approaches. Subsequently, the review focuses on the different experimental strategies for introducing a "time anchor" to scRNA-seq, highlighting their principles, strengths, weaknesses, and comparing their adaptation in various scenarios. Next, it provides a brief summary of applications in immunity response, cancer progression, and embryo development. Finally, the review concludes with a forward-looking perspective on future advancements in time-resolved single-cell transcriptomic sequencing.

Dragon’s blood (Resina Draconis) is the red resin of Dracaena spp, which has a variety of biological activities and pharmacological effects, including anti-thrombotic, anti-inflammatory, anti-bacterial, analgesic, anti-oxidant, anti-tumor, and immunosuppressive. In China, the main source of dragon’s blood is Dranaena Cochinchinensis (Lour.) S.C.Chen. A wide array of studies have speculated that the dragon’s blood derived from Dranaena Cochinchinensis (Lour.) S.C.Chen possesses cardiovascular protective effects. It has been reported that Chinese dragon’s blood can potentially alleviate and treat conditions such as coronary heart disease, myocardial infarction, and myocardial ischemia–reperfusion through its anti-inflammatory and antioxidant properties, which have not been systematically stated in previous reviews. Moreover, the precise underlying pharmacological mechanisms through which the Chinese dragon’s blood exhibits cardioprotective effects are not fully understood. Therefore, this article discusses the pharmacological action and biomolecular mechanism of dragon’s blood from Dranaena Cochinchinensis (Lour.) S.C.Chen and how it prevents and protects against cardiovascular diseases. The review article concludes with prospects for further application of dragon’s blood in respect to cardiovascular diseases.

Citrus grandis fruit is a famous traditional Chinese medicine with various bioactivities, including cardioprotective effects. Polysaccharides are one of the key active ingredients responsible for its cardioprotective effects. This study aimed to investigate the structure and cardioprotective effect of a homogeneous polysaccharide from C. grandis fruit (CGP80-1) and explore its mechanism against myocardial ischemia-reperfusion (MI/R) injury. Structure analysis showed that CGP80-1 (11,917 Da) is an arabinan with compact coil chain conformation, containing →5)-α-L-Araf-(1→, →3,5)-α-L-Araf-(1→, and →2,3,5)-α-L-Araf-(1→ as the backbone, as well as →5)-α-L-Araf-(1→ and t-α-L-Araf as side-chains substituted at the C2 and C3 positions. Pharmacological experiments showed that pre-treatment with CGP80-1 could effectively alleviate MI/R injury by improving endogenous antioxidant enzymes and cardiac enzymes, reducing reactive oxygen species levels, and regulating apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. The protective effects were correlated with the Nrf2/Keap1 and IRE1/GRP78 signaling pathways. Further analysis of structure-activity relationships revealed that the myocardial protection effects of CGP80-1 might be attributed to its appropriate molecular weight, high arabinose content, and unique compact coil chain conformation. Overall, our results provide insight into the chemical structure of CGP80-1 and its mechanism of action, suggesting that CGP80-1 could be a candidate drug for myocardial protection.

Mapping genome-wide DNA-protein interactions (DPIs) provides insights into the epigenetic landscape of complex biological systems and elucidates the mechanisms of epigenetic regulation in biological progress. However, current technologies in DPI profiling still suffer from high cell demands, low detection sensitivity, and large reagent consumption. To address these problems, we developed DMF-ChIP-seq that builds on digital microfluidic (DMF) technology to profile genome-wide DPIs in a highly efficient, cost-effective, and user-friendly way. The entire workflow including cell pretreatment, antibody recognition, pA-Tn5 tagmentation, fragment enrichment, and PCR amplification is programmatically manipulated on a single chip. Leveraging closed submicroliter reaction volumes and a superhydrophobic interface, DMF-ChIP-seq presented higher sensitivity in peak enrichment than other current methods, with high accuracy (Pearson Correlation Coefficient (PCC) > 0.86) and high repeatability (PCC > 0.92). Furthermore, DMF-ChIP-seq was capable of processing the samples with as few as 8 cells while maintaining a high signal-to-noise ratio. By applying DMF-ChIP-seq, H3K27ac histone modification of early embryonic cells during differentiation was profiled for the investigation of epigenomic landscape dynamics. With the benefits of high efficiency and sensitivity in DPI analysis, the system provides great promise in studying epigenetic regulation during various biological processes.

Anemarrhena asphodeloides polysaccharide (AAP70-1) was reported to have immunomodulatory effects in our previous report. To further improve the immunomodulatory effects of AAP70-1, an A. asphodeloides polysaccharide-zinc complex (AAP-Zn) was synthesized using a ZnCl2 modification method, and the potential mechanisms by which AAP-Zn activates macrophages were investigated. The results showed that the structural features of AAP-Zn were similar to those of AAP70-1 with a Zn content of 0.2 %, confirming that Zn mainly interacted with AAP70-1 by forming ZnO coordination bonds and Zn…OH bonds. In addition, the administration of AAP70-1 and AAP-Zn effectively improved the immunomodulatory effects by enhancing phagocytosis and upregulating the mRNA expression of cytokines (TNF-α, IL-6, IL-1β, and IL-18), as well as increasing the production levels of nitric oxide (NO) and reactive oxygen species (ROS) in zebrafish embryos. The intracellular mechanism by which AAP-Zn activates macrophages was found to involve activation of the NF-κB and MAPK signaling pathways. Our findings suggested that AAP-Zn may be a potential immunopotentiator in the field of biomedicine or functional foods.

Single-cell RNA sequencing (scRNA-seq) plays a critical role in revealing genetic expression patterns at the single-cell level for cell type identification and rare transcript detection. Although there have been great advances in scRNA-seq methodologies, existing technologies still suffer from complexity and high cost, and an integrated platform for complete library construction is still lacking. Herein we describe Cilo-seq for high-performance scRNA-seq library construction in a single device with programmed and addressable droplet handling based on digital microfluidics. The platform is simultaneously accessible for convenient single-cell isolation, efficient nucleic acid amplification, low-loss nucleic acid purification and high-quality library preparation by leveraging specific interface design, tiny reaction volume, auxiliary magnetic field control and accurate droplet control. With a closed hydrophobic interface, the platform further reduces nucleic acid loss and exogenous background interference. Cilo-seq provides excellent detection sensitivity (1.4-fold improvement over tube-based methods), accuracy (R = 0.98) and cost efficiency (10-fold decrease in cost compared to tube-based methods), and holds great promise for studies of single-cell RNA biology.

The mortality rate of cardiovascular disease ranks first in the world. Its pathogenesis involves not only internal factors such as immunity, inflammation, metabolic disorders, and self-development but also external factors such as the environment. In the last decade, the emergence of single-cell technology has greatly promoted the development of disease research. Among them, the more mature single-cell RNA sequencing can carry out high-throughput analysis of single cells while studying with single-cell resolution. This technology enables people to characterize the heterogeneity of single cells, identify rare cell types in heart and blood vessels, and construct human heart cell map. With the data analysis of bioinformatics experts, it can also reconstruct the development track of the heart, to construct a map of heart development. Single-cell sequencing plays an important role in analyzing the human physiological structure and disease progression due to its advantages of single-cell resolution. The possibility of combining other omics technologies is proposed by summarizing the existing application examples and advanced technologies like spatial transcriptome. In this review, we summarize the current single-cell sequencing technologies (plate-based and droplet-based) and describe the data analysis process. The latest findings in cardiovascular disease using single-cell RNA sequencing technology are described. Finally, we discussed the shortcomings of single-cell RNA sequencing technology. At the same time, the possibility of the combination of single-cell RNA sequencing and spatial omics technology, and how to apply it to the study of cardiovascular diseases is discussed.

Diabetic peripheral neuropathy (DPN) is one of the most common chronic complications of diabetes. Symptoms of DPN mainly include spontaneous intractable pain that is diffuse and continuous and can last from several weeks to several months. DPN is associated with a high mortality rate and poor prognosis. Its pathogenesis is not fully understood, and clinical treatment is focused on relieving its clinical symptoms, as well as improving blood sugar control and cardiovascular risk factors. DPN and its clinically effective treatments need to be studied. This study discusses the treatment methods and pathogenesis of DPN, summarizes the related research progress, and attempts to provide a reference for DPN research.

Ischemic heart disease (IHD) is a high-risk disease in the middle-aged and elderly population. The ischemic heart may be further damaged after reperfusion therapy with percutaneous coronary intervention (PCI) and other methods, namely, myocardial ischemia–reperfusion injury (MIRI), which further affects revascularization and hinders patient rehabilitation. Therefore, the investigation of new therapies against MIRI has drawn great global attention. Within the long history of the prevention and treatment of MIRI, traditional Chinese medicine (TCM) has increasingly been recognized by the scientific community for its multi-component and multi-target effects. These multi-target effects provide a conspicuous advantage to the anti-MIRI of TCM to overcome the shortcomings of single-component drugs, thereby pointing toward a novel avenue for the treatment of MIRI. However, very few reviews have summarized the currently available anti-MIRI of TCM. Therefore, a systematic data mining of TCM for protecting against MIRI will certainly accelerate the processes of drug discovery and help to identify safe candidates with synergistic formulations. The present review aims to describe TCM-based research in MIRI treatment through electronic retrieval of articles, patents, and ethnopharmacology documents. This review reported the progress of research on the active ingredients, efficacy, and underlying mechanism of anti-MIRI in TCM and TCM formulas, provided scientific support to the clinical use of TCM in the treatment of MIRI, and revealed the corresponding clinical significance and development prospects of TCM in treating MIRI.