This study investigated the mechanism of autophagy in the differentiation processes of MC3T3-E1 cells under osteogenic induction(physiological) and alcohol(AL) intervention(pathological), as well as the mechanism by which icariin(ICA) affected osteogenic differentiation of MC3T3-E1 cells under the pathological condition of AL intervention. Osteogenic mineralized nodule staining confirmed that the cells could differentiate into osteoblasts. After determining the appropriate concentrations of AL and ICA using the CCK-8 assay, seven groups were set up in this study: complete medium(CM) group, osteogenic induction medium(OIM) group, OIM+0.25 mol·L~(-1) AL group, OIM+0.25 mol·L~(-1) AL+1×10~(-8) mol·L~(-1) ICA group, OIM+0.25 mol·L~(-1) AL+1×10~(-7) mol·L~(-1) ICA group, OIM+0.25 mol·L~(-1) AL+1×10~(-6) mol·L~(-1) ICA group, and OIM+0.25 mol·L~(-1) AL+1×10~(-5) mol·L~(-1) ICA group, with a culture period of 7 days. Alkaline phosphatase(ALP) staining was used to detect the relative ALP area. Western blot and RT-qPCR were employed to analyze the expression of osteogenesis-and autophagy-related proteins and mRNAs. Reactive oxygen species(ROS) staining was used to detect ROS levels, and apoptosis was assessed through mitochondrial membrane potential assays. The results showed that ICA increased the relative ALP area that had been reduced by AL intervention. AL down-regulated the expression levels of Wnt family member 1(Wnt1), along with the osteogenesis-related mRNAs Wnt1, β-catenin, Runt-related transcription factor 2(Runx2), osteoprotegerin(OPG), and ALP, thereby inhibiting osteogenic differentiation. ICA up-regulated the expression levels of the osteogenesis-related proteins and mRNAs that had been inhibited by AL, promoting osteogenic differentiation. AL inhibited typical autophagy, while ICA regulated Rubicon to suppress LC3-associated phagocytosis(LAP) and promote typical autophagy. ICA also reduced the ROS levels that were elevated by AL and decreased the apoptosis of osteoblasts induced by AL intervention. In conclusion, ICA can regulate Rubicon to inhibit LAP, promote typical autophagy, eliminate ROS, reduce apoptosis, and ultimately enhance the osteogenic differentiation of MC3T3-E1 cells under the pathological condition of AL intervention by modulating the Wnt/β-catenin signaling pathway.
Autophagy/drug effects* ; Animals ; Osteogenesis/drug effects* ; Mice ; Cell Differentiation/drug effects* ; Osteoblasts/metabolism* ; Ethanol/pharmacology* ; Flavonoids/pharmacology* ; Cell Line ; Reactive Oxygen Species/metabolism* ; Drugs, Chinese Herbal/pharmacology*

Metal ion-promoted chemodynamic therapy(CDT) combined with photodynamic therapy(PDT) offers broad application prospects for enhancing anti-tumor effects. In this study, glycyrrhizic acid(GA), copper ions(Cu~(2+)), and norcantharidin(NCTD) were co-assembled to successfully prepare a natural small-molecule, carrier-free hydrogel(NCTD Gel) with excellent material properties. Under 808 nm laser irradiation, NCTD Gel responded to the tumor microenvironment(TME) and acted as an efficient Fenton reagent and photosensitizer, catalyzing the conversion of endogenous hydrogen peroxide(H_2O_2) within the tumor into oxygen(O_2), and hydroxyl radicals(·OH, type Ⅰ reactive oxygen species) and singlet oxygen(~1O_2, type Ⅱ reactive oxygen species), while depleting glutathione(GSH) to stabilize reactive oxygen species and alleviate tumor hypoxia. In vitro and in vivo experiments demonstrated that NCTD Gel exhibited significant CDT/PDT synergistic therapeutic effects. Further safety evaluation and metabolic testing confirmed its good biocompatibility and safety. This novel hydrogel is not only simple to prepare, safe, and cost-effective but also holds great potential for clinical transformation, providing insights and references for the research and development of metal ion-mediated hydrogel-based anti-tumor therapies.
Hydrogels/chemistry* ; Animals ; Photochemotherapy ; Humans ; Mice ; Antineoplastic Agents/administration & dosage* ; Photosensitizing Agents/chemistry* ; Neoplasms/metabolism* ; Female ; Copper/chemistry* ; Reactive Oxygen Species/metabolism* ; Tumor Microenvironment/drug effects* ; Cell Line, Tumor ; Male

This study aimed to explore the therapeutic mechanisms of Colquhounia Root Tablets(CRT) in treating diabetic kidney disease(DKD) by integrating biomolecular network mining with animal model verification. By analyzing clinical transcriptomics data, an interaction network was constructed between candidate targets of CRT and DKD-related genes. Based on the topological eigenvalues of network nodes, 101 core network targets of CRT against DKD were identified. These targets were found to be closely related to multiple pathways associated with type 2 diabetes, immune response, and metabolic reprogramming. Given that immune-inflammatory imbalance driven by metabolic reprogramming is one of the key pathogenic mechanisms of DKD, and that many core network targets of CRT are involved in this pathological process, receptor for advanced glycation end products(RAGE)-reactive oxygen species(ROS)-phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT)-nuclear factor-κB(NF-κB)-NOD-like receptor family pyrin domain containing 3(NLRP3) signaling axis was selected as a candidate target for in-depth research. Further, a rat model of DKD induced by a high-sugar, high-fat diet and streptozotocin was established to evaluate the pharmacological effects of CRT and verify the expression of related targets. The experimental results showed that CRT could effectively correct metabolic disturbances in DKD, restore immune-inflammatory balance, and improve renal function and its pathological changes by inhibiting the activation of the RAGE-ROS-PI3K-AKT-NF-κB-NLRP3 signaling axis. In conclusion, this study reveals that CRT alleviates the progression of DKD through dual regulation of metabolic reprogramming and immune-inflammatory responses, providing strong experimental evidence for its clinical application in DKD.
Animals ; Diabetic Nephropathies/metabolism* ; Receptor for Advanced Glycation End Products/genetics* ; NF-kappa B/genetics* ; Signal Transduction/drug effects* ; Rats ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Phosphatidylinositol 3-Kinases/genetics* ; Reactive Oxygen Species/metabolism* ; Humans ; Plant Roots/chemistry* ; Rats, Sprague-Dawley ; Tablets/administration & dosage*

This study aims to elucidate the role and mechanism of clematichinenoside AR(CAR) in protecting bone marrow mesenchymal stem cells(BMSCs) from hypoxia-induced apoptosis. BMSCs were isolated by the bone fragment method and identified by flow cytometry. Cells were cultured under normal conditions(37℃, 5% CO_2) and hypoxic conditions(37℃, 90% N_2, 5% CO_2) and treated with CAR. The BMSCs were classified into eight groups: control(normal conditions), CAR(normal conditions + CAR), hypoxia 24 h, hypoxia 24 h + CAR, hypoxia 48 h, hypoxia 48 h + CAR, hypoxia 72 h, and hypoxia 72 h + CAR. The cell counting kit-8(CCK-8) assay and terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) were employed to measure cell proliferation and apoptosis, respectively. The number of mitochondria and mitochondrial membrane potential were measured by MitoTracker®Red CM-H2XRo staining and JC-1 staining, respectively. The level of reactive oxygen species(ROS) was measured with the DCFH-DA fluorescence probe. The protein levels of B-cell lymphoma-2 associated X protein(BAX), caspase-3, and optic atrophy 1(OPA1) were determined by Western blot. The results demonstrated that CAR significantly increased cell proliferation. Compared with the control group, the hypoxia groups showed increased apoptosis rates, reduced mitochondria, elevated ROS levels, decreased mitochondrial membrane potential, upregulated expression of BAX and caspase-3, and downregulated expression of OPA1. In comparison to the corresponding hypoxia groups, CAR intervention significantly decreased the apoptosis rate, increased mitochondria, reduced ROS levels, elevated mitochondrial membrane potential, downregulated the expression of BAX and caspase-3, and upregulated the expression of OPA1. Therefore, it can be concluded that CAR may exert an anti-apoptotic effect on BMSCs under hypoxic conditions by regulating OPA1 to maintain mitochondrial homeostasis.
Mesenchymal Stem Cells/metabolism* ; Apoptosis/drug effects* ; Mitochondria/metabolism* ; Animals ; Rats ; Cell Hypoxia/drug effects* ; Homeostasis/drug effects* ; Reactive Oxygen Species/metabolism* ; Rats, Sprague-Dawley ; Membrane Potential, Mitochondrial/drug effects* ; Saponins/pharmacology* ; Caspase 3/genetics* ; Male ; bcl-2-Associated X Protein/genetics* ; Bone Marrow Cells/metabolism* ; Cell Proliferation/drug effects* ; Protective Agents/pharmacology* ; Cells, Cultured

This paper explored the specific mechanism of ginsenoside Rg_1 in regulating mitochondrial fusion through the neurogenic gene Notch homologous protein 1(Notch1) pathway to alleviate hypoxia/reoxygenation(H/R) injury in HL-1 cells. The relative viability of HL-1 cells after six hours of hypoxia and two hours of reoxygenation was detected by cell counting kit-8(CCK-8). The lactate dehydrogenase(LDH) activity in the cell supernatant was detected by the lactate substrate method. The content of adenosine triphosphate(ATP) was detected by the luciferin method. Fluorescence probes were used to detect intracellular reactive oxygen species(Cyto-ROS) levels and mitochondrial membrane potential(ΔΨ_m). Mito-Tracker and Actin were co-imaged to detect the number of mitochondria in cells. Fluorescence quantitative polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels of Notch1, mitochondrial fusion protein 2(Mfn2), and mitochondrial fusion protein 1(Mfn1). The results showed that compared with that of the control group, the cell activity of the model group decreased, and the LDH released into the cell culture supernatant increased. The level of Cyto-ROS increased, and the content of ATP decreased. Compared with that of the model group, the cell activity of the ginsenoside Rg_1 group increased, and the LDH released into the cell culture supernatant decreased. The level of Cyto-ROS decreased, and the ATP content increased. Ginsenoside Rg_1 elevated ΔΨ_m and increased mitochondrial quantity in HL-1 cells with H/R injury and had good protection for mitochondria. After H/R injury, the mRNA and protein expression levels of Notch1 and Mfn1 decreased, while the mRNA and protein expression levels of Mfn2 increased. Ginsenoside Rg_1 increased the mRNA and protein levels of Notch1 and Mfn1, and decreased the mRNA and protein levels of Mfn2. Silencing Notch1 inhibited the action of ginsenoside Rg_1, decreased the mRNA and protein levels of Notch1 and Mfn1, and increased the mRNA and protein levels of Mfn2. In summary, ginsenoside Rg_1 regulated mitochondrial fusion through the Notch1 pathway to alleviate H/R injury in HL-1 cells.
Ginsenosides/pharmacology* ; Receptor, Notch1/genetics* ; Signal Transduction/drug effects* ; Mice ; Animals ; Mitochondrial Dynamics/drug effects* ; Mitochondria/metabolism* ; Cell Line ; Reactive Oxygen Species/metabolism* ; Oxygen/metabolism* ; Cell Hypoxia/drug effects* ; Cell Survival/drug effects* ; Membrane Potential, Mitochondrial/drug effects* ; Humans

To study the therapeutic effect and mechanisms of ethyl syringate(MD) on ulcerative colitis(UC), the MTT assay was used to detect the proliferation inhibition of RAW264.7 cells and HT-29 cells by different concentrations of MD(50, 100, 200, 400 μmol·L~(-1)). UC cell models were constructed by inducing RAW264.7 cells and HT-29 cells with lipopolysaccharide(LPS) and tumor necrosis factor-α(TNF-α). An animal model was established by inducing mice with 2.5% dextran sulfate sodium(DSS) to verify the therapeutic effect of MD on UC. A control group, a model group(LPS or TNF-α), and groups treated with different concentrations of MD(50, 100, 200, 400 μmol·L~(-1)) were set up in this study. Nitric oxide(NO) levels were measured using a NO detection kit. Intracellular reactive oxygen species(ROS) levels were assessed using a laser confocal microscope and ROS kit. Enzyme-linked immunosorbent assay(ELISA) was used to detect changes in the levels of interleukin-6(IL-6), TNF-α, interferon-γ(INF-γ), interleukin-10(IL-10), and myeloperoxidase(MPO) in cells and animal tissues. Western blot was used to detect the expression levels of phosphorylated Janus kinase 2(p-JAK2), Janus kinase 2(JAK2), phosphorylated signal transducer and activator of transcription 3(p-STAT3), signal transducer and activator of transcription 3(STAT3), zonula occludens-1(ZO-1), occludin, and claudin-1 in cells and animal tissues. The results showed that MD can improve the inflammatory response by inhibiting the production of NO and ROS and regulating the expression of inflammatory factors. It significantly reduced the disease activity index(DAI) in mice, improved the shortening of the colon, and repaired intestinal epithelial damage by inhibiting the activation of the JAK2/STAT3 pathway, thereby exerting anti-UC activity.
Animals ; Colitis, Ulcerative/chemically induced* ; Janus Kinase 2/genetics* ; STAT3 Transcription Factor/genetics* ; Mice ; Humans ; Signal Transduction/drug effects* ; Male ; RAW 264.7 Cells ; Reactive Oxygen Species/metabolism* ; Nitric Oxide/metabolism* ; HT29 Cells ; Salicylates/administration & dosage* ; Protective Agents/administration & dosage*

This article investigated the effect and mechanism of salidroside(SAL) on the expression of adhesion molecules in oxidized low-density lipoprotein(oxLDL)-induced mouse aortic endothelial cell(MAEC). The oxLDL-induced endothelial cell injury model was constructed, and the safe concentration and action time of SAL were screened. The cells were divided into control group, oxLDL group, low and high concentration groups of SAL, and ferrostatin-1(Fer-1) group. The cell viability was detected by CCK-8 assay; lactate dehydrogenase(LDH) leakage was measured by colorimetry; the expression of intercellular adhesion molecule 1(ICAM-1) and recombinant vascular cell adhesion molecule 1(VCAM-1) were detected by immunofluorescence; Fe~(2+),glutathione(GSH),malondialdehyde(MDA),and 4-hydroxynonenal(4-HNE) levels were detected by kit method; reactive oxygen species(ROS) was detected by DCFH-DA probe; the levels of glutathione peroxidase 4(GPX4),silent mating type information regulation 2 homolog 1(SIRT1), and nuclear factor erythroid 2-related factor 2(Nrf2) were determined by using Western blot. The inhibitors of Nrf2 and SIRT1 were used, and endothelial cell were divided into control group, oxLDL group, SAL group, ML385 group(Nrf2 inhibitor), and EX527 group(SIRT1 inhibitor). The ultrastructure of mitochondria was observed by electron microscope; mitochondrial membrane potential(MMP) was detected by flowcytometry; the expressions of SIRT1,Nrf2,solute carrier family 7 member 11(SLC7A11),GPX4,ferroportin 1(FPN1),ferritin heavy chain 1(FTH1),ICAM-1, and VCAM-1 were detected by Western blot. The results showed that similar to Fer-1,low and high concentrations of SAL could improve cell viability, inhibit LDH release and the expression of ICAM-1 and VCAM-1 in oxLDL-induced endothelial cells(P<0.05 or P<0.01). It was related to increase in GSH level, decrease in Fe~(2+),ROS,MDA, and 4-HNE level, and up-regulation of SIRT1,Nrf2, and GPX4 expression to inhibit ferroptosis(P<0.05 or P<0.01). The intervention effect of high concentration SAL was the most significant. ML385 and EX527 could partially offset the protection of SAL on mitochondrial structure and MMP and reverse the ability of SAL to up-regulate the expression of SIRT1,Nrf2,SLC7A11,GPX4,FPN1, and FTH1 and down-regulate the expression of ICAM-1 and VCAM-1(P<0.05 or P<0.01).To sum up, SAL could reduce the expression of ICAM-1 and VCAM-1 in oxLDL-induced endothelial cell, which may relate to activation of SLC7A11/GPX4 antioxidant signaling pathway mediated by SITR1/Nrf2, up-regulation of FPN1 and FTH1 expression, and inhibition of ferroptosis.
Sirtuin 1/genetics* ; Animals ; Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; NF-E2-Related Factor 2/genetics* ; Mice ; Endothelial Cells/cytology* ; Glucosides/pharmacology* ; Phenols/pharmacology* ; Cell Adhesion Molecules/genetics* ; Reactive Oxygen Species/metabolism* ; Intercellular Adhesion Molecule-1/genetics* ; Vascular Cell Adhesion Molecule-1/genetics* ; Cell Survival/drug effects*

OBJECTIVE: To investigate the effects of sodium valproate (VPA) in inhibiting Erastin-induced ferroptosis in bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanisms. METHODS: BMSCs were isolated from bone marrow of 8-week-old Spragur Dawley rats and identified [cell surface antigens CD90, CD44, and CD45 were analyzed by flow cytometry, and osteogenic and adipogenic differentiation abilities were assessed by alizarin red S (ARS) and oil red O staining, respectively]. Cells of passage 3 were used for the Erastin-induced ferroptosis model, with different concentrations of VPA for intervention. The optimal drug concentration was determined using the cell counting kit 8 assay. The experiment was divided into 4 groups: group A, cells were cultured in osteogenic induction medium for 24 hours; group B, cells were cultured in osteogenic induction medium containing optimal concentration Erastin for 24 hours; group C, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA for 24 hours; group D, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA, and 8 μmol/L EX527 for 24 hours. The mitochondrial state of the cells was evaluated, including the levels of malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS). Osteogenic capacity was assessed by alkaline phosphatase (ALP) activity and ARS staining. Western blot analysis was performed to detect the expressions of osteogenic-related proteins [Runt-related transcription factor 2 (RUNX2) and osteopontin (OPN)], ferroptosis-related proteins [glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11)], and pathway-related proteins [adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1)]. RESULTS: The cultured cells were identified as BMSCs. VPA inhibited Erastin-induced ferroptosis and the decline of osteogenic ability in BMSCs, acting through the activation of the AMPK/SIRT1 pathway. VPA significantly reduced the levels of ROS and MDA in Erastin-treated BMSCs and significantly increased GSH levels. Additionally, the expression levels of ferroptosis-related proteins (GPX4, FTH1, and SLC7A11) significantly decreased. VPA also upregulated the expressions of osteogenic-related proteins (RUNX2 and OPN), enhanced mineralization and osteogenic differentiation, and increased the expressions of pathway-related proteins (AMPK and SIRT1). These effects could be reversed by the SIRT1 inhibitor EX527. CONCLUSION VPA inhibits ferroptosis in BMSCs through the AMPK/SIRT1 axis and promotes osteogenesis.
Mesenchymal Stem Cells/metabolism* ; Ferroptosis/drug effects* ; Animals ; Valproic Acid/pharmacology* ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Cell Differentiation/drug effects* ; Cells, Cultured ; AMP-Activated Protein Kinases/metabolism* ; Osteogenesis/drug effects* ; Piperazines/pharmacology* ; Bone Marrow Cells/cytology* ; Reactive Oxygen Species/metabolism* ; Signal Transduction/drug effects*

Male infertility has become a global concern, accounting for 20-70% of infertility. Dysfunctional spermatogenesis is the most common cause of male infertility; thus, treating abnormal spermatogenesis may improve male infertility and has attracted the attention of the medical community. Mitochondria are essential organelles that maintain cell homeostasis and normal physiological functions in various ways, such as mitochondrial oxidative phosphorylation (OXPHOS). Mitochondrial OXPHOS transmits electrons through the respiratory chain, synthesizes adenosine triphosphate (ATP), and produces reactive oxygen species (ROS). These mechanisms are vital for spermatogenesis, especially to maintain the normal function of testicular Sertoli cells and germ cells. The disruption of mitochondrial OXPHOS caused by external factors can result in inadequate cellular energy supply, oxidative stress, apoptosis, or ferroptosis, all inhibiting spermatogenesis and damaging the male reproductive system, leading to male infertility. This article summarizes the latest pathological mechanism of mitochondrial OXPHOS disorder in testicular Sertoli cells and germ cells, which disrupts spermatogenesis and results in male infertility. In addition, we also briefly outline the current treatment of spermatogenic malfunction caused by mitochondrial OXPHOS disorders. However, relevant treatments have not been fully elucidated. Therefore, targeting mitochondrial OXPHOS disorders in Sertoli cells and germ cells is a research direction worthy of attention. We believe this review will provide new and more accurate ideas for treating male infertility.
Male ; Humans ; Infertility, Male/metabolism* ; Oxidative Phosphorylation ; Mitochondria/metabolism* ; Spermatogenesis/physiology* ; Sertoli Cells/metabolism* ; Oxidative Stress/physiology* ; Animals ; Reactive Oxygen Species/metabolism*

Ferroptosis, an iron-dependent programmed cell death process driven by reactive oxygen species-mediated lipid peroxidation, is regulated by several metabolic processes, including iron metabolism, lipid metabolism, and redox system. Macrophages are a group of innate immune cells that are widely distributed throughout the body, and play pivotal roles in maintaining metabolic balance by its phagocytic and efferocytotic effects. There is a profound association between the biological functions of macrophage and ferroptosis. Therefore, this review aims to elucidate three key aspects of the unique relationship between macrophages and ferroptosis, including macrophage metabolism and their regulation of cellular ferroptosis; ferroptotic stress that modulates functions of macrophage and promotion of inflammation; and the effects of macrophage ferroptosis and its role in diseases. Finally, we also summarize the possible mechanisms of macrophages in regulating the ferroptosis process at the global and local levels, as well as the role of ferroptosis in the macrophage-mediated inflammatory process, to provide new therapeutic insights for a variety of diseases.
Ferroptosis/physiology* ; Macrophages/metabolism* ; Humans ; Animals ; Iron/metabolism* ; Reactive Oxygen Species/metabolism* ; Lipid Peroxidation/physiology* ; Inflammation/metabolism*