Improved Cell Viability Research Articles

Chlorogenic acid alleviates IPEC-J2 pyroptosis induced by deoxynivalenol by inhibiting activation of the NF-κB/NLRP3/caspase-1 pathway

BackgroundDeoxynivalenol (DON) is a mycotoxin that severely pollutes feed ingredients, and methods for reducing DON toxicity have become a significant research direction. Chlorogenic acid (CGA) is an active polyphenol found in some plants, which has anti-inflammatory and antioxidant properties and a protective effect on animal intestinal health. The effects of CGA on DON-induced pyroptosis in the intestinal porcine epithelial cell line-J2 (IPEC-J2) and its potential mechanism were explored in this study.ResultsIPEC-J2 cells viability and membrane integrity were inversely correlated with DON concentration. Compared to those in the group treated with DON alone at 2,500 ng/mL, pretreatment with 80 μmol/L CGA for 4 h significantly improved cell viability (P < 0.01), and the alleviation of typical pyroptotic symptoms induced by DON were observed, including reduced cellular DNA fragmentation, decreased release of lactate dehydrogenase (LDH), normalized ROS levels, restoration of extracellular Ca2+ and K+ contents to normal levels (P < 0.01 ), as well as suppressed the enzyme activities of caspase-1 and caspase-4 (P < 0.01). Additionally, the mRNA expression levels of TNF, MDP, NOD2, TLR4, ASC and GSDMD were significantly improved (P < 0.01), while both mRNA and protein expression levels of NF-κB, NLRP3, caspase-1, IL-1β and IL-18 were significantly upregulated (P < 0.01) in the CGA + DON group, compare to those in the DON group.ConclusionPretreatment with 80 μmol/L CGA for 4 h effectively alleviated pyroptosis in IPEC-J2 cells induced by 2,500 ng/mL of DON through inhibiting activation of the NF-κB/ NLRP3/capase-1 pathway.

Three-Dimensional Bioprinting for Retinal Tissue Engineering

Three-dimensional bioprinting (3DP) is transforming the field of regenerative medicine by enabling the precise fabrication of complex tissues, including the retina, a highly specialized and anatomically complex tissue. This review provides an overview of 3DP’s principles, its multi-step process, and various bioprinting techniques, such as extrusion-, droplet-, and laser-based methods. Within the scope of biomimicry and biomimetics, emphasis is placed on how 3DP potentially enables the recreation of the retina’s natural cellular environment, structural complexity, and biomechanical properties. Focusing on retinal tissue engineering, we discuss the unique challenges posed by the retina’s layered structure, vascularization needs, and the complex interplay between its numerous cell types. Emphasis is placed on recent advancements in bioink formulations, designed to emulate retinal characteristics and improve cell viability, printability, and mechanical stability. In-depth analyses of bioinks, scaffold materials, and emerging technologies, such as microfluidics and organ-on-a-chip, highlight the potential of bioprinted models to replicate retinal disease states, facilitating drug development and testing. While challenges remain in achieving clinical translation—particularly in immune compatibility and long-term integration—continued innovations in bioinks and scaffolding are paving the way toward functional retinal constructs. We conclude with insights into future research directions, aiming to refine 3DP for personalized therapies and transformative applications in vision restoration.

Protective Effects of Spirulina Against Lipid Micelles and Lipopolysaccharide-Induced Intestinal Epithelium Disruption in Caco-2 Cells: In Silico Molecular Docking Analysis of Phycocyanobilin

Damage to intestinal epithelial cells is present in obesity and other diseases because of inflammatory and oxidative processes. This damage compromises the gastrointestinal barrier, killing enterocytes, altering intestinal permeability, and eliciting abnormal immune responses that promote chronic inflammation. Recent evidence shows that spirulina is a potent natural agent with antioxidant and anti-inflammatory properties. Objectives: This study was conducted to evaluate the effect of spirulina aqueous extract (SPAE) on the alterations of the intestinal epithelium induced by lipid micelles (LMs) and/or inflammation induced by lipopolysaccharides (LPSs) in the Caco-2 cell line. Methods: In the current research, we assessed the protective actions of SPAE against cytotoxicity, oxidative stress, inflammation, and epithelial barrier perturbation by using an in vitro model, the intestinal Caco-2 cells, treated with LPSs and/or LMs. We also performed an in silico molecular docking analysis with spirulina’s bioactive compound, phycocyanobilin. Results: Our results showed that SPAE has no cytotoxic effect on Caco-2 cells. On the contrary, it improved cell viability and exhibited anti-inflammatory and antioxidant actions. SPAE also protected against endoplasmic reticulum stress and tight junction proteins, thus improving the epithelial barrier. The in silico study revealed a strong binding affinity of the phycocyanobilin compound with human SOD and NADPH oxidase and a good binding affinity towards COX-2 and iNOS. Conclusions: Taken together, these findings demonstrate the beneficial actions of SPAE on Caco-2 cells, suggesting it may be useful in preserving the epithelial intestinal barrier in human conditions involving oxidative stress and inflammation such as obesity.

Rational design to enhance the catalytic activity of acetylcholinesterase and mitigate trichlorfon toxicity in vitro

Trichlorfon (TCF) is a widely used organophosphate pesticide whose inhibition of acetylcholinesterase (AChE) results in neurotoxicity and significant biosafety risks. Addressing these concerns requires effective strategies to mitigate TCF-induced toxicity and safeguard exposed organisms. In this study, we explored the potential of a catalytic activity enhanced Culex pipiens AChE mutant to mitigate TCF-induced cytotoxicity through rational design. A double-point mutant, M5 (I198M/Y249F), was developed by combining molecular dynamics (MD) simulations with structural feature analysis to reshape the active pocket, which demonstrated enhanced catalytic efficiency and maintained thermostability. Its functional activity and improved catalytic performance were further confirmed by activity staining on non-denaturing gels. The analysis of the catalytic mechanism and the reduction in Molecular Mechanics-Generalized Born Surface Area (MM/GBSA) free energy revealed an increase in substrate affinity for M5. Additionally, the application of exogenous M5 not only restored endogenous AChE activity in NIH/3T3 cells exposed to TCF but also reduced reactive oxygen species (ROS) accumulation and apoptosis, thereby improving cell viability. In silico studies indicate that the stable interaction between M5 and TCF promotes the targeted depletion of TCF, effectively neutralizing its toxic effects. These findings indicate that M5 has potential as an enzyme-based antidote for organophosphate pesticide, offering a novel strategy for protecting non-target species from pesticide-induced damage.

Vibrotactile stimulation at 40 Hz inhibits Aβ-induced changes in SH-SY5Y, BV2 cells, and pericytes

Alzheimer’s disease (AD) poses a major societal challenge, yet no definitive cure exists. Noninvasive brain stimulation methods, such as transcranial magnetic stimulation and transcranial direct current stimulation, have shown promise in alleviating cognitive symptoms associated with neurodegenerative disorders. This study investigated the effects of 40 Hz vibrotactile stimulation on AD-related cellular responses using SH-SY5Y neuroblastoma cells, primary human brain pericytes, and BV2 microglia. SH-SY5Y cells and brain pericytes treated with oligomeric beta-amyloid (Aβ) underwent 40 Hz vibrational stimulation for varying durations. Cell viability was determined via the CCK-8 assay, while intracellular calcium levels in pericytes were assessed. Protein expression was measured using western blotting, and gene expression was quantified via a real-time quantitative polymerase chain reaction. Detailed vibrational parameters were employed to ensure precise stimulation. Notably, 40 Hz vibrotactile stimulation improved cell viability in Aβ-exposed SH-SY5Y cells, reduced intracellular calcium ion (Ca2+) levels in Aβ-treated pericytes, activated autophagy, and mitigated tau hyperphosphorylation in SH-SY5Y cells. Additionally, it exhibited anti-neuroinflammatory properties in BV2 microglia. These findings highlight the potential of 40 Hz vibrotactile stimulation as a therapeutic strategy for AD.

LncRNA AP001007 protects human renal tubular epithelial HK-2 cells and kidney organoids from LPS-induced injury

The regulation of long non-coding RNAs (lncRNAs) has been implicated in the pathogenesis of sepsis-induced acute kidney injury (SI-AKI). Nevertheless, the specific roles of individual lncRNAs in this process remain unclear. This study investigated the expression of lncRNA AP001007 in lipopolysaccharide (LPS)-induced HK-2 cells and in the peripheral blood of sepsis patients. The result shows that LPS treatment downregulated the expression of AP001007 in HK-2 cells and that circulating levels of AP001007 were lower in sepsis patients. Furthermore, overexpressing AP001007 in HK-2 cells improved cell viability, mitochondrial activity, and survival when exposed to LPS. Additionally, LPS-treated HK-2 cells secreted fewer pro-inflammatory cytokines when AP001007 was overexpressed. Similar protective effects were observed in human kidney organoids (HKOs) subjected to LPS. These findings suggest that AP001007 confers protection against LPS-induced damage in HK-2 cells and HKOs, highlighting its potential as a regulator of SI-AKI.

Predication on organs-on-chips applications the systematic treatment of breast cancer

Abstract. With the continuous development of cancer therapy, traditional in vitro models have been unable to reproduce the structure of organs and tissues of various parts of the human body, fluid flow and mechanical stimulation characteristics, and the human tumor microenvironment, which is crucial to the study of the behavior and progress of cancer, can not be presented through the animal model in vivo, which shows that the traditional physiological model of cancer can no longer meet the research of human anti-cancer therapy. Benefits from the continuous infusion of cell cultures through the microchannel, the device of organ-on-a-chip improves cell viability and activity and facilitates the simulation of the tumor cell microenvironment, which is expected to further replace traditional zoological experiments. This study reviews a method to simulate the microenvironment of the vitro breast cancer metastasis combined with microfluidic technology, and the application of this device in relevant researches of breast cancer cell metastasis and screening of breast cancer therapeutics.

Protective Effects of Ambroxol on Aβ and α-Synuclein-Induced Neurotoxicity Through Glucocerebrosidase Activation in HT-22 Hippocampal Neuronal Cells.

Dementia with Lewy bodies (DLB) is a progressive neurodegenerative disorder marked by the accumulation of α-synuclein (αSyn), often co-existing with amyloid β (Aβ) pathology. Current treatments are largely symptomatic, highlighting a critical need for disease-modifying therapies. Evidence suggests that αSyn aggregates contribute to neuronal death in DLB, particularly when exacerbated by Aβ. Given the role of autophagy in clearing misfolded proteins, exploring agents that promote this pathway is essential for developing effective treatments. Ambroxol (AMBX), a mucolytic drug, has demonstrated potential in activating glucocerebrosidase (GCase), an enzyme that enhances lysosomal function and facilitates the autophagic clearance of toxic protein aggregates, including αSyn. This study aims to evaluate AMBX's neuroprotective effects in a cellular model of DLB, with the goal of identifying new therapeutic agents that target the underlying pathology of DLB. In this study, HT-22 hippocampal neuronal cells were exposed to αSyn and Aβ, followed by AMBX treatment. Our results showed that AMBX significantly improved cell viability and reduced apoptosis in cells co-treated with αSyn and Aβ. Additionally, AMBX restored GCase activity, promoted autophagy, and reduced oxidative stress, which in turn mitigated αSyn aggregation and phosphorylation. These findings suggest that by activating GCase and enhancing autophagy, AMBX may help alleviate DLB-associated neurodegeneration. This study underscores the potential of AMBX as a therapeutic agent for DLB and supports further investigation in animal models and clinical trials to validate its efficacy in neurodegenerative disease contexts.

Ginsenoside Rg1 Prevents and Treats Acute Pulmonary Injury Induced by High-Altitude Hypoxia.

This study aimed to investigate the protective effects of ginsenoside Rg1 on high-altitude hypoxia-induced acute lung injury (ALI) and elucidated its molecular targets and related pathways, specifically its association with the fluid shear stress pathway. Using a combination of bioinformatics analysis and both in vivo and in vitro experiments, we assessed the role of ginsenoside Rg1 in mitigating physiological and biochemical disturbances induced by hypoxia. In the in vivo experiments, we measured arterial blood gas parameters, levels of inflammatory cells and cytokines, erythrocyte and platelet parameters, and conducted histological analysis in rats. The in vitro experiments utilized human pulmonary microvascular endothelial cells (HPMECs) and A549 cells to examine cell viability, intracellular reactive oxygen species (ROS) and Ca2⁺ levels, and mitochondrial function. The results of the in vivo experiments demonstrate that ginsenoside Rg1 significantly increased arterial blood oxygen partial pressure and saturation, elevated arterial blood glucose levels, and stabilized respiratory and metabolic functions in rats. It also reduced inflammatory cells and cytokines, such as tumor necrosis factor-α and interleukin-6, and improved erythrocyte and platelet abnormalities, supporting its protective role through the regulation of the fluid shear stress pathway. Histological and ultrastructural analyses revealed that Rg1 significantly protected lung tissue structure and organelles. In vitro experiments further confirmed that Rg1 improved cell viability in HPMEC and A549 cells under hypoxic conditions, decreased intracellular ROS and Ca2⁺ levels, and enhanced mitochondrial function. These findings collectively demonstrate that ginsenoside Rg1 exerts significant protective effects against high-altitude hypoxia-induced ALI by enhancing oxygen delivery and utilization, reducing inflammatory responses, and maintaining cellular metabolism and vascular function. Notably, the protective effects of Rg1 are closely associated with the regulation of the fluid shear stress pathway, suggesting its potential for treating high-altitude hypoxia-related diseases.

Antiviral activity of isoliquiritigenin against SVCV in aquaculture: A dual approach of immune modulation and viral inhibition

Isoliquiritigenin, a flavonoid from Glycyrrhiza glabra, demonstrates potent antiviral properties by enhancing host defenses through antioxidative and immunomodulatory mechanisms. Our in vitro data demonstrate that isoliquiritigenin significantly inhibits spring viremia of carp virus (SVCV) at concentrations up to 100 μg/ml without inducing cytotoxicity, effectively reducing viral replication and the production of new viral particles. Treatment with isoliquiritigenin markedly reduced the cytopathic effects in EPC cells, improving cell viability by more than 50 %. Furthermore, isoliquiritigenin enhances resistance to SVCV infection by significantly reducing intracellular the viral load when cells are pretreated for up to 24 h. This inhibition primarily occurs during the viral replication and assembly phases, without affecting viral binding or entry. Additionally, isoliquiritigenin strengthens antioxidant defenses and modulates immune responses, thereby reducing oxidative damage and interferon production. In vivo experiments with juvenile carp corroborate the antiviral efficacy of isoliquiritigenin, showing significantly reduced mortality rates and the viral load in SVCV-infected fish. These findings highlight the potential of isoliquiritigenin as an antiviral agent in aquaculture, providing a sustainable alternative to traditional chemotherapeutics by concurrently inhibiting viral replication and enhancing host immune defenses.

Astaxanthin mediated repair of tBHP-Induced cellular injury in chondrocytes

ABSTRACT Objective This study investigates how astaxanthin (AST) counters tert-butyl hydroperoxide (tBHP)-induced cellular damage in C28/I2 chondrocytes, focusing on the circ-HP1BP3/miR-139-5p/SOD1 signaling pathway and its use in sustained-release microspheres for osteoarthritis treatment. Methods We employed a variety of techniques including real-time quantitative PCR, Western blot, ELISA, and dual-luciferase reporter gene assays to explore AST's molecular effects. Additionally, the efficacy of AST-loaded sustained-release microspheres was evaluated in vitro and in a mouse model of osteoarthritis. Results AST significantly enhanced SOD1 expression, reducing apoptosis and inflammation in damaged cells. The AST-loaded microspheres showed promising in vitro drug release, improved cell viability, and reduced oxidative stress. In the osteoarthritis mouse model, they effectively decreased joint inflammation and increased the expression of chondrocyte markers. Conclusion Astaxanthin effectively mitigates oxidative stress and inflammation in chondrocytes via the circ-HP1BP3/miR-139-5p/SOD1 pathway. The development of AST-loaded microspheres offers a novel and promising approach for osteoarthritis therapy, potentially extending to osteoarthritis treatment.

Rivaroxaban as a protector of Oxidative Stress-induced Vascular Endothelial Glycocalyx Damage via The IQGAP1/PAR1-2/PI3K/Akt Pathway.

The vascular endothelial glycocalyx, crucial for blood vessel integrity and homeostasis, is vulnerable to oxidative stress, leading to endothelial dysfunction, which strongly correlates with cardiovascular disease (CVD). This study investigates the protective effects of rivaroxaban, a FXa inhibitor, on the glycocalyx under oxidative stress condition. We examined the impact of rivaroxaban on human umbilical vein endothelial cells (HUVECs) exposed to acute and chronic H₂O₂-induced oxidative stress. Rivaroxaban dose-dependently suppressed syndecan-1, a key component of the glycocalyx, shedding from cell surface, and enhanced protease-activated receptor (PAR)1-PAR2/ phosphatidylinositol-3-kinase (PI3K)-dependent cell viability after acute induction of H2O2. This protective effect was linked to the translocation of IQGAP1, a scaffold protein that modulates the actin cytoskeleton, to the perinucleus from the cell membrane. Under chronic H2O2 treatments, rivaroxaban improves cell viability accompanied by an increase in hyaluronidase activities, aiding the turnover and remodeling of hyaluronic acid within the glycocalyx. We identify that rivaroxaban protects against oxidative stress-induced endothelial glycocalyx damage and cell viability through IQGAP1/PAR1-2/PI3K/Akt pathway, offering a potential to be a therapeutic target for CVD prevention.

Isolation of Lessertiosides A and B and Other Metabolites from Lessertia frutescens and Their Neuroprotection Activity.

Lessertia frutescens (synonym Sutherlandia frutescens) is an important South African medicinal plant used traditionally to treat different human pathologies and is considered an adaptogenic plant. This study sought to isolate compounds from the plant and determine their protective potentials using SH-SY5Y cells and MPP+ (1-methyl-4-phenylpyridinium) to mimic Parkinson's disease. The phytochemical analysis of a 70% aqueous methanolic extract of L. frutescens leaves resulted in the isolation and identification of 11 pure compounds (1-11), among which compounds 1 and 2 were identified as new metabolites. The new compounds were characterised using IR, UV, NMR, and HRESIMS and were given the trivial names lessertiosides A (1) and B (2). Additionally, the flavonoids 8-methoxyvestitol (7) and mucronulatol (8) were isolated for the first time from the plant. The biological actions show that the isolated compounds had negligible toxicity on SH-SY5Y cells and improved cell viability in the cells exposed to MPP+. Furthermore, as a mechanism of action, the compounds could sustain cellular ATP generation and prevent MPP+-induced apoptotic cell death. Our findings provide evidence for the neuroprotective properties of compounds isolated from L. frutescens in MPP+-induced neuronal damage for the first time and create an avenue for these compounds to be further investigated to elucidate their molecular targets.

An RGD-Conjugated Prodrug Nanoparticle with Blood-Brain-Barrier Penetrability for Neuroprotection Against Cerebral Ischemia-Reperfusion Injury.

Cerebral ischemia-reperfusion injury significantly contributes to global morbidity and mortality. Loganin is a natural product with various neuroprotective effects; however, it lacks targeted specificity for particular cells or receptors, which may result in reduced therapeutic efficacy and an increased risk of side effects. To address the limitations of loganin, we developed LA-1, a novel compound incorporating an Arg-Gly-Asp (RGD) peptide to target integrin receptor αvβ3, enhancing brain-targeting efficacy. LA-1 exhibited optimal nanoscale properties, significantly improved cell viability, reduced ROS production, and enhanced survival rates in vitro. In vivo, LA-1 decreased infarct sizes, improved neurological function, and reduced oxidative stress and neuroinflammation. Proteomic analysis showed LA-1 modulates PI3K/Akt and Nrf2/HO-1 pathways, providing targeted neuroprotection. These findings suggest LA-1's potential for clinical applications in treating cerebral ischemia-reperfusion injury.

Regulation of mitochondrial autophagy by lncRNA MALAT1 in sepsis-induced myocardial injury

BackgroundSepsis-induced myocardial injury (SIMI) is a severe complication of sepsis, contributing significantly to mortality. Mitochondrial dysfunction and dysregulated autophagy are implicated in SIMI pathogenesis. Long non-coding RNA MALAT1 has been associated with various diseases, including sepsis, but its role in SIMI remains unclear.ObjectiveThis study aimed to investigate the role of lncRNA MALAT1 in SIMI, specifically in the regulation of mitochondrial autophagy.MethodsA sepsis-induced cardiomyopathy model was established in mice, and the cardiac tissues were analyzed. The expression of lncRNA MALAT1 was modulated and its effects on mitochondrial autophagy, myocardial injury, inflammation, and apoptosis were assessed. Furthermore, the interaction between MALAT1 and miR-146a was explored, as well as the involvement of the TLR4/NF-kB/MAPK signaling pathway.ResultsActivation of mitochondrial autophagy by urolithin A (UA) alleviated SIMI, inflammation, and cardiac dysfunction. Downregulation of MALAT1 enhanced mitochondrial autophagy, stabilized the mitochondrial membrane potential, and inhibited mitochondrial reactive oxygen species (ROS) production, leading to improved cell viability and reduced myocardial injury. Furthermore, MALAT1 interacted with miR-146a, and their modulation influenced mitochondrial autophagy, myocardial injury, and inflammation. The TLR4/NF-kB/MAPK signaling pathway was implicated in these processes.ConclusionOur findings suggest that lncRNA MALAT1 plays a crucial role in SIMI by modulating miR-146a-mediated mitochondrial autophagy and the TLR4/NF-kB/MAPK signaling pathway. These results provide new insights into the pathogenesis of SIMI and potential therapeutic targets.

Daphnetin Protects Schwann Cells Against High-Glucose-Induced Oxidative Injury by Modulating the Nuclear Factor Erythroid 2-Related Factor 2/Glutamate-Cysteine Ligase Catalytic Subunit Signaling Pathway.

Diabetic peripheral neuropathy (DPN), a common complication of diabetes mellitus, is primarily characterized by damage to Schwann cells caused by oxidative stress under hyperglycemic conditions. Recently, we demonstrated the ability of coumarin-rich Ficus formosana Maxim. to alleviate DPN in ovariectomized diabetic mice. However, the underlying mechanisms remain unclear. In this study, we established an in vitro DPN model using RSC96 Schwann cells exposed to high glucose levels. Daphnetin, a natural coumarin found abundantly in Ficus formosana Maxim., was co-incubated with Schwann cells in a high-glucose medium to investigate its protective effects against DPN. The free radical scavenging capacity of daphnetin was evaluated, along with assessments of cell viability, apoptosis, H2O2 levels, and the expression of proteins by the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutamate-cysteine ligase catalytic subunit (GCLC) pathway in RSC96 Schwann cells. The results showed that daphnetin was non-toxic within the tested concentration range of 6.25 μM to 50 μM in RSC96 Schwann cells. Moreover, daphnetin significantly improved cell viability, exhibited strong antioxidant activity, reduced H2O2 levels, and regulated the Nrf2/GCLC pathway protein expressions in RSC96 cells cultured in high-glucose medium. Additionally, daphnetin influenced apoptosis-related proteins by decreasing the expression levels of Bax and Caspase 3, while increasing the Bcl-2 expression level in high-glucose-treated RSC96 cells. These findings suggest that daphnetin may alleviate oxidative stress induced by high glucose levels through activation of the Nrf2/GCLC pathway and inhibition of Schwann cell apoptosis, underscoring its potential as a therapeutic agent for DPN.

Functional Mechanism and Clinical Implications of lncRNA LINC-PINT in Delayed Fracture Healing

Background Fracture healing can be impeded or even compromised by various factors, resulting in a growing number of patients suffering. The lncRNA LINC-PINT has garnered attention for its latent role in enhancing fracture healing, but its specific functions in this process remain unclear. Objectives The primary objective of this study is to investigate the clinical relevance and underlying molecular mechanisms of LINC-PINT in delayed fracture healing (DFH), while also assessing its potential as an early diagnostic biomarker. Materials and methods The expression levels of LINC-PINT were measured in the serum of DFH patients and those with normal fracture healing using RT-qPCR. In MC3T3-E1 cells, the study investigated the influence on the expression of differentiation-related protein, cell viability, and apoptosis through the modulation of LINC-PINT and miR-324-3p. To elucidate the targeting relationship between LINC-PINT, miR-324-3p, and BMP2, a dual-luciferase reporter assay was employed. Results The findings revealed a significant downregulation of LINC-PINT expression in DFH patients. LINC-PINT showed high sensitivity and specificity as a diagnostic marker for DFH. In MC3T3-E1 cells, LINC-PINT overexpression markedly enhanced the expression levels of ALP, OCN, Runx2, and OPN, improved cell viability, and inhibited apoptosis. LINC-PINT negatively regulated miR-324-3p, and the effects of LINC-PINT were counteracted by miR-324-3p. LINC-PINT was found to regulate BMP2 by targeting miR-324-3p. Conclusion LINC-PINT could serve as an early diagnostic biomarker for DFH and slow the progression of DFH by modulating BMP2 through the targeted regulation of miR-324-3p. This research presents new molecular targets for the diagnosis and treatment of DFH.

Role of vitamin K2 and vitamin K cycle enzymes in aortic valvular interstitial cell calcification

Abstract Introduction Calcific aortic valve disease (CAVD) is the most common valve disease. Currently, there is no medical treatment available for CAVD. Vitamin K antagonists promote CAVD while population-based studies suggest that dietary intake of vitamin K reduces the incidence of CAVD in adults. However, randomized trials have so far not proven a clear benefit of vitamin K supplementation on the progression of AVS. Consequently, there is a need for a deeper understanding of the role of vitamin K in valvular calcification. This study aims to further investigate mechanisms of action of vitamin K during valvular calcification. Methods and Results Aortic valve samples were obtained from patients undergoing surgical aortic valve replacement for CAVD and valvular interstitial cells (VIC) were isolated by collagenase II digestion (Fig. 1). VIC expressed typical markers like Vimentin and α-SMA and were cultured in a control medium (CM) or further calcified in a pro-calcifying medium (PCM) containing sodium dihydrogen phosphate and ascorbic acid. PCM led to upregulation of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) expression, and ALP-activity after 7 and 21 days of culture. Moreover, alizarin red staining visualized significant calcium mineral deposition after 21 days of culture (Fig. 2). The addition of the most potent vitamin K2 derivative (Menaquinone-7, MK-7) improved cell viability under both control and calcifying conditions (Fig. 3). Moreover, MK-7 protected VIC from ferroptotic cell death (Fig. 4) and increased expression of the vitamin K-dependent Matrix-Gla-protein (MGP), a major inhibitor of ectopic calcification (Fig. 5). To fulfill its function as an activator of vitamin K-dependent proteins like MGP, vitamin K needs to be reduced to vitamin K hydroquinone by the vitamin K epoxide reductase enzymes VKORC1 and VKORC1L1. In VIC, using our calcification protocol, treatment with PCM led to the downregulation of VKORC1, but not of VKORC1L1, an enzyme with known anti-oxidative properties (Fig. 6). We next performed siRNA-mediated knockdown of VKORC1 and VKORC1L1 in VIC and observed calcification after 7 and 21 days of culture by measuring expression of ALP, RUNX2, quantifying ALP activity, and staining for calcium minerals (alizarin red). Intriguingly, VKORC1L1-knockdown sharply increased the expression of ALP. To further elucidate these findings, we will perform overexpression experiments to analyze, if VKORC1L1 can alleviate PCM-induced calcification Conclusion: Vitamin K2 inhibits in vitro calcification of valvular interstitial cells and upregulates the calcification inhibitor Matrix-Gla-Protein. Our results suggest the involvement of the vitamin K cycle enzyme VKORC1L1, which is known to exhibit anti-oxidative and cytoprotective effects. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding.

Screening of active components of melastoma dodecandrum lour. against diabetic osteoporosis using cell membrane chromatography-mass spectrometry.

Melastoma dodecandrum Lour. (MD), a traditional botanical drug known for its hypoglycemic, antioxidant, and anti-inflammatory properties, is commonly used to treat diabetes, osteoarthritis, and osteoporosis. However, its specific active components against diabetic osteoporosis remain unclear. This study aimed to identify the key active components in MD using cell membrane chromatography coupled with mass spectrometry and validate their effects in vitro. An AGEs-induced osteoblast injury model was established. MTT assays measured cell viability, and ALP activity was assessed using a biochemical kit. Western blotting was employed to detect the expression levels of osteoblast-related proteins OCN and RUNX2 and the AGE receptor protein RAGE. ELISA was used to determine the levels of SOD, MDA, CAT, and GPx. PCR quantified TNF-α expression to evaluate the protective effects and potential mechanisms of MD. The AGEs-induced osteoblast cell membrane chromatography-mass spectrometry method facilitated the rapid identification of potentially active compounds based on their affinity for the osteoblast cell membrane. Cell experiments further validated the activity of the characteristic component isovitexin. MD significantly improved cell viability in AGEs-damaged osteoblasts, enhanced ALP, SOD, CAT, and GPx activities, reduced MDA levels, increased OCN and RUNX2 protein expression, and decreased TNF-α mRNA and RAGE protein expression. Cell membrane chromatography identified 20 chemical constituents, including 13 flavonoids, 4 organic acids, 1 phenylpropanoids, 1 terpenoids, and 1 alkaloid. Cell experiments have confirmed that isovitexin has significant protective activity against osteoblasts and can inhibit the expression of specific receptor RAGE on the osteoblast membrane, consistent with the effect of MD. MD and its active component, isovitexin, provide protective effects against AGEs-induced osteoblast injury, offering a basis for subsequent drug development.

Pharmacological Preconditioning with Fenofibrate in Cardiomyocyte Cultures of Neonatal Rats Subjected to Hypoxia/Reoxygenation, High Glucose, and Their Combination.

Pharmacological preconditioning is an alternative to protect the heart against the consequences of damage from ischemia/reperfusion (I/R). It is based on the administration of specific drugs that imitate the effect of ischemic preconditioning (IPC). Peroxisomal proliferator-activated receptors (PPARs) can prevent apoptosis in pathologies such as I/R and heart failure. Therefore, our objective was to determine if the stimulation of PPARα with fenofibrate (feno) decreases the apoptotic process induced by hypoxia/reoxygenation (HR), high glucose (HG), and HR/HG. For that purpose, cardiomyocyte cultures were divided into the following groups: Group 1-control (Ctrl); Group 2-HR; Group 3-HR + 10 μM feno; Group 4-HG, (25 mM glucose); Group 5-HG + feno; Group 6-HR/HG, and Group 7-HR/HG + feno. Our results indicate that cell viability decreases in neonatal cardiomyocytes undergoing HR, HG, and their combination, while feno improved cell viability. Feno treatment decreased apoptosis compared with HG-, HR-, or HG/HR-vehicle-treated. Nuclear- and mitochondrial-apoptosis markers increased in neonatal cardiomyocytes from HR, HG, and HR/HG; while the cytotoxicity decreased in cells treated with feno. In addition, the expression of Bax, Bad, and caspase 9 decreased due to feno, while 14-3-3ɛ and Bcl2 were increased. Inner mitochondrial cytochrome C increased with feno in every condition, as well as mitochondrial activity. Feno treatment prevented injury in the ultrastructure and in the mitochondrial membranes. Thus, our results suggest that feno decreases apoptosis in neonatal cardiomyocytes, improving the ultrastructure of mitochondria in the pathological conditions studied.