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*

OBJECTIVE: To explore the mechanism by which ω-3 polyunsaturated fatty acids (hereinafter referred to as "ω-3") exert antioxidant stress protection and promote osteogenic differentiation in MC3T3-E1 cells, and to reveal the relationship between ω-3 and the key antioxidant stress pathway involving nuclear factor E2-related factor 2 (Nrf2) and NAD (P) H quinone oxidoreductase 1 (NQO1) in MC3T3-E1 cells. METHODS: The optimal concentration of H ₂O ₂ (used to establish the oxidative stress model of MC3T3-E1 cells in vitro) and the optimal intervention concentrations of ω-3 were screened by cell counting kit 8. MC3T3-E1 cells were divided into blank control group, oxidative stress group (H ₂O ₂), low-dose ω-3 group (H ₂O ₂+low-dose ω-3), and high-dose ω-3 group (H ₂O ₂+high-dose ω-3). After osteoblastic differentiation for 7 or 14 days, the intracellular reactive oxygen species (ROS) level was measured by fluorescence staining and flow cytometry, and the mitochondrial morphological changes were observed by biological transmission electron microscope; the expression levels of Nrf2, NQO1, heme oxygenase 1 (HO-1), Mitofusin 1 (Mfn1), and Mfn2 were detected by Western blot to evaluate the cells' antioxidant stress capacity; the expression levels of Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were detected by immunofluorescence staining and Western blot; osteogenic potential of MC3T3-E1 cells was evaluated by alkaline phosphatase (ALP) staining and alizarin red staining. RESULTS: Compared with the oxidative stress group, the content of ROS in the low and high dose ω-3 groups significantly decreased, and the protein expressions of Nrf2, NQO1, and HO-1 significantly increased ( P<0.05). At the same time, the mitochondrial morphology of MC3T3-E1 cells improved, and the expressions of mitochondrial morphology-related proteins Mfn1 and Mfn2 significantly increased ( P<0.05). ALP staining and alizarin red staining showed that the low-dose and high-dose ω-3 groups showed stronger osteogenic ability, and the expressions of osteogenesis-related proteins RUNX2 and OCN significantly increased ( P<0.05). And the above results showed a dose-dependence in the two ω-3 treatment groups ( P<0.05). CONCLUSION ω-3 can enhance the antioxidant capacity of MC3T3-E1 cells under oxidative stress conditions and upregulate their osteogenic activity, possibly through the Nrf2/NQO1 signaling pathway.
Oxidative Stress/drug effects* ; NF-E2-Related Factor 2/metabolism* ; NAD(P)H Dehydrogenase (Quinone)/metabolism* ; Animals ; Mice ; Osteogenesis/drug effects* ; Cell Differentiation/drug effects* ; Fatty Acids, Omega-3/pharmacology* ; Signal Transduction/drug effects* ; Osteoblasts/drug effects* ; Reactive Oxygen Species/metabolism* ; Cell Line ; Hydrogen Peroxide/pharmacology* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Antioxidants/pharmacology* ; Heme Oxygenase-1/metabolism*

OBJECTIVE: To analyze the influences of dihydromyricetin on the proliferation and apoptosis of chondrocytes in osteoarthritis induced by hydrogen peroxide (H₂O₂) through reactive oxygen species (ROS)/p38 mitogen activated protein kinase (p38-MAPK) pathway. METHODS: Five C57BL/6J mice were euthanized by cervical dislocation after anesthesia. Chondrocytes were extracted and cultured.After passage, the chondrocytes were divided into control group, H2O2 group (0.8 μmol·L^-1 H₂O₂), dihydromyricetin low concentration group (0.8 μmol·L^-1 H₂O₂+20 μmol·L^-1 dihydromyricetin), dihydromyricetin high concentration group (0.8 μmol·L^-1 H₂O₂+80 μmol·L^-1 dihydromyricetin), and ROS inhibitor N-acetylcysteine (NAC) group (0.8 μmol·L^-1 H₂O₂+5 mmol·L^-1 NAC). The activity of chondrocytes was measured by methyl thiazolyl tetrazolium (MTT) assay. The apoptosis rate of chondrocytes was measured by Hoechst 33342 method. The level of ROS in chondrocytes was measured by 2, 7-dichlorofluorescein diacetate (DCFH-DA) fluorescence probe.The level of Type II collagen α1 (Col2α1) mRNA was measured by qRT-PCR.And the expression of Col2α1, p-p38-MAPK/p38-MAPK, B cell lymphoma gene-2 (Bcl-2) and Bcl-2 associated X protein (Bax) proteins was detected by Western blot. RESULTS: The chondrocytes showed swirling fibrous mass, and the expression of COL2α was positive. Compared with the control group, the chondrocyte viability, apoptosis rate, ROS fluorescence intensity, p-p38-MAPK/p38-MAPK, and the expression of Bax protein in H2O22 group increased, the level of Col2α1 mRNA, and the expression of Col2α1 and Bcl-2 proteins decreased (P<0.05). Compared with H₂O₂ group, the chondrocyte viability, apoptosis rate, ROS fluorescence intensity, p-p38-MAPK/p38-MAPK, and the expression of Bax protein in dihydromyricetin low concentration group, dihydromyricetin high concentration group, and NAC group decreased, the level of Col2α1 mRNA, and the expression of Col2α1 and Bcl-2 proteins increased (P<0.05). CONCLUSION Dihydromyricetin may inhibit chondrocyte apoptosis, inflammatory reaction and oxidative stress by inhibiting ROS/p38-MAPK pathway. Dihydromyricetin may be a potential drug for treating osteoarthritis.
Animals ; Chondrocytes/metabolism* ; Apoptosis/drug effects* ; Hydrogen Peroxide/toxicity* ; Osteoarthritis/physiopathology* ; Mice, Inbred C57BL ; Reactive Oxygen Species/metabolism* ; Mice ; Flavonols/pharmacology* ; p38 Mitogen-Activated Protein Kinases/genetics* ; Cell Proliferation/drug effects* ; Male ; Signal Transduction/drug effects* ; MAP Kinase Signaling System/drug effects* ; Cells, Cultured

Objective To investigate the effects of ROCK-siRNA transfection on endothelial cell senescence and endothelial microparticles (EMPs) induced by angiotensin II (Ang II). Methods Human umbilical vein endothelial cells (HUVECs) were treated with Ang II (1.0 μmo/L) to induce cellular senescence models, followed by transfection with ROCK-siRNA. The cells were divided into four groups: control group, model group, negative transfection control group (Ang II combined with NC-siRNA), and ROCK-siRNA transfection group (Ang II combined with ROCK-siRNA). Cellular senescence was assessed by SA-β-Gal staining. EMP levels in cell supernatants and intracellular reactive oxygen species (ROS) levels were assessed using flow cytometry. The expression levels of silenced information regulator 1(SIRT1) and p53 protein in each group were analyzed by Western blotting. Results Following ROCK-siRNA transfection, the number of senescent cells induced by Ang II was significantly reduced, accompanied by decreased CD31⁺ EMP levels and suppressed intracellular ROS levels. Meanwhile, the expression levels of SIRT1 were up-regulated, while the expression levels of p53 were down-regulated. Conclusion Silencing ROCK expression suppresses EMP release, reduces ROS generation, regulates the expression of SIRT1 and p53, and ultimately attenuates Ang II-induced endothelial cell senescence.
Humans ; Angiotensin II/pharmacology* ; Cellular Senescence/genetics* ; Human Umbilical Vein Endothelial Cells/cytology* ; RNA, Small Interfering/metabolism* ; Reactive Oxygen Species/metabolism* ; Sirtuin 1/genetics* ; Transfection ; Tumor Suppressor Protein p53/genetics* ; Cell-Derived Microparticles/drug effects* ; rho-Associated Kinases/metabolism* ; Endothelial Cells/metabolism* ; Cells, Cultured

Depression currently affects about 280 million people worldwide and its prevalence has been increasing dramatically, especially among the young and people of reproductive age, which consequently leads to an increase in antidepressant consumption. Antidepressants are associated with sexual dysfunction in both men and women; however, their role in male fertility has been scarcely studied. Fluoxetine and sertraline, two serotonin reuptake inhibitors (SSRIs), are among the most prescribed antidepressants worldwide. To determine their possible effects, human sperm cells were exposed to either sertraline or fluoxetine at concentrations previously found in blood and seminal fluid of patients undergoing treatment. Spermatozoa were incubated for up to 24 h at 37°C and 5% CO 2 , and important functional parameters such as sperm motility, viability, mitochondrial membrane potential, cellular reactive oxygen species (ROS) production, chromatin/DNA integrity, acrosome status, and tyrosine phosphorylation were assessed. At low levels, fluoxetine consistently decreased progressive motility throughout time while promoting fluctuations in ROS levels and sperm capacitation. Nevertheless, it did not affect viability, mitochondrial membrane potential, acrosome reaction nor chromatin/DNA integrity. Sertraline, on the other hand, had little to nonsignificant impact at low doses, but affected almost all tested parameters at supratherapeutic concentrations. Altogether, our results suggest that both antidepressants may impair sperm function, possibly through different mechanisms of action, but fluoxetine is the only exhibiting mild negative effects at doses found in vivo .
Humans ; Male ; Spermatozoa/drug effects* ; Fluoxetine/pharmacology* ; Sperm Motility/drug effects* ; Sertraline/pharmacology* ; Reactive Oxygen Species/metabolism* ; Antidepressive Agents/pharmacology* ; Membrane Potential, Mitochondrial/drug effects* ; Sperm Capacitation/drug effects* ; Selective Serotonin Reuptake Inhibitors/pharmacology* ; Cell Survival/drug effects* ; Acrosome Reaction/drug effects*

Triclocarban (TCC) is a broad-spectrum antimicrobial widely used in various personal care products, textiles, and children's toys. TCC has potential reproductive and developmental toxicity in animals. However, little is known regarding the effect of TCC on human sperm function. In this study, an in vitro assay was used to investigate the effects of TCC on normal human spermatozoa and the possible underlying mechanisms involved. Semen from healthy male donors was collected and cultured in complete Biggers, Whitten and Whittingham (BWW) and low-sugar BWW media, followed by treatment with TCC at concentrations of 0, 0.1 µmol l -1 , 1 µmol l -1 , 10 µmol l -1 , and 100 µmol l -1 for 4 h. TCC was found to reduce the sperm total motility and progressive motility. Moreover, the sperm kinematic parameters, straight-line velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL) were affected in a dose-dependent manner. After treatment with TCC at the lowest effective concentration of 10 µmol l -1 , TCC caused a significant decrease in mitochondrial adenosine triphosphate (ATP) production and mitochondrial membrane potential (MMP) and a significant increase in reactive oxygen species (ROS), similar to the observations with the positive control carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), suggesting that TCC may decrease sperm motility by affecting the oxidative phosphorylation (OXPHOS) pathway. In a sugar-free and low-sugar BWW culture environment, TCC enhanced the damaging effect on sperm motility and ATP, MMP, and lactate decreased significantly, suggesting that TCC may also affect the glycolytic pathway that supplies energy to spermatozoa. This study demonstrates a possible mechanism of TCC toxicity in spermatozoa involving both the OXPHOS and glycolysis pathways.
Male ; Sperm Motility/drug effects* ; Humans ; Carbanilides/pharmacology* ; Oxidative Phosphorylation/drug effects* ; Glycolysis/drug effects* ; Membrane Potential, Mitochondrial/drug effects* ; Adenosine Triphosphate/metabolism* ; Spermatozoa/metabolism* ; Reactive Oxygen Species/metabolism* ; Mitochondria/metabolism*

OBJECTIVE: Photodynamic therapy has become an important method in clinical tumor treatment. This study aimed to investigate the effects of hematoporphyrin on multiple myeloma (MM) and its potential applications. METHODS: The MM cell line RPMI 8226 was treated with hematoporphyrin derivative (HPD), and CCK-8 assay was used to determine cell viability, apoptosis was detected by flow cytometry, intracellular reactive oxygen species (ROS) levels were measured using a detection kit combined with flow cytometry, and Western blot assay was used to detect apoptosis-related proteins and key signaling pathway protein levels. RESULTS: The optimal incubation time for the maximum absorption of HPD in RPMI 8226 cells was 4 hours. HPD significantly inhibited the proliferation of RPMI 8226 cells in a dose- and illumination time-dependent manner ( r =0.981; r =0.961). Additionally, HPD induced apoptosis in RPMI 8226 cells, but had no significant inhibitory effect on peripheral blood mononuclear cells derived from healthy individuals. HPD combined with illumination treatment significantly increased the intracellular ROS level, upregulated the expression of apoptosis-related proteins such as cleaved PARP, cleaved caspase-3 and Bax, and down-regulated the expression of proteins that maintain cell survival, such as NF-κB and Akt. CONCLUSION The HPD can inhibit the proliferation and induce apoptosis of multiple myeloma cells.
Humans ; Multiple Myeloma/pathology* ; Hematoporphyrins/pharmacology* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism* ; Cell Proliferation/drug effects* ; Photochemotherapy ; Cell Survival/drug effects* ; Signal Transduction

OBJECTIVE: To investigate the effects and mechanisms of hydroxysafflor yellow A (HSYA) on replicative senescence in human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS: hUC-MSCs were cultured to construct a replicative senescence model through continuous amplification in vitro. Cells at passage 2 served as the control group, while cells at passage 10 were designated as the senescence group. The senescent cells were cultured in a culture medium containing HSYA. Cell viability was detected by the CCK-8 assay, and cell confluence was analyzed using the Incucyte S3 live-cell analysis system. The optimal concentration and time point were determined and utilized for subsequent experiments. Senescent cells were pretreated with 0.01 mg/ml HSYA, and the proportion of senescence-associated β-galactosidase (SA-β-gal) positive cells was detected to assess the senescence state. The relative telomere length was detected by qPCR. Reactive oxygen species (ROS) levels were measured using the fluorescent probe DCFH-DA. Mitochondrial membrane potential was assessed by JC-1 staining. The expression of p53, p16, p21, OCT4, and SOX2 genes was detected by qPCR. The expression of p16, p53, OCT4, and SOX2 proteins was analyzed by Western blot. RESULTS: HSYA significantly decreased the SA-β-gal positive staining rate, inhibited telomere attrition, reduced the ROS accumulation, increased mitochondrial membrane potential in senescent cells. Additionally, HSYA downregulated the expression of p53 and p16, and upregulated the expression of OCT4. HSYA decreased p16 protein level and increased OCT4 and SOX2 protein levels. CONCLUSION HSYA may ameliorate replicative senescence in hUC-MSCs by modulating the p53 and p16 signaling pathways and suppressing oxidative stress.
Humans ; Mesenchymal Stem Cells/drug effects* ; Cellular Senescence/drug effects* ; Chalcone/pharmacology* ; Oxidative Stress/drug effects* ; Quinones/pharmacology* ; Umbilical Cord/cytology* ; Reactive Oxygen Species/metabolism* ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Tumor Suppressor Protein p53/metabolism* ; Membrane Potential, Mitochondrial ; Cell Proliferation

OBJECTIVE: To elucidate the role and underlying mechanism of ubiquitin-specific protease 35 (USP35) in ferroptosis of rheumatoid arthritis-fibroblast like synoviocytes (RA-FLS), thereby enhancing our comprehension of the pathogenesis of RA and identifying potential therapeutic targets for its treatment. METHODS: (1) RA-FLS were cultured in vitro and transduced with lentiviral vectors to establish stable cell lines: A USP35-knockdown line (short hairpin ribonucleic acid of USP35, shUSP35) and its control (negtive control of short hairpin ribonucleic acid, shNC), as well as a overexpression of USP35 line (USP35 OE) and its control (Vector). To investigate the role of USP35 in ferroptosis regulation, a ferroptosis model was induced in RA-FLS by treatment with 1 μmol/L Erastin. The cells were divided into six groups: shNC, shNC + Erastin, shUSP35 + Erastin, Vector, Vector + Erastin, and USP35 OE + Erastin. (2) Cell viability was detected using the cell counting kit-8 (CCK-8). (3) Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione/glutathione disulfide (GSH/GSSG) ratios, and Ferrous ion (Fe²⁺) levels were measured using specific assay kits to evaluate oxidative stress, lipid peroxidation, and glutathione redox status in the cells. (4) Protein expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were detected using Western blotting to investigate their potential involvement in USP35-mediated ferroptosis regulation. RESULTS: (1) Compared with the shNC +Erastin group, the cell viability of the shUSP35+Erastin group was significantly decreased (P < 0.001), while it was notably increased in the USP35 OE+Erastin group compared with the Vector+Erastin group (P < 0.001). These findings indicated that USP35 could alleviate the inhibitory effect of Erastin on RA-FLS cell viability. (2) In comparison to the shNC+Erastin group, the levels of ROS (P < 0.001), MDA (P < 0.05), and Fe²⁺ (P < 0.001) were significantly elevated, and the GSH/GSSG ratio was increased (P < 0.05) in the shUSP35+Erastin group. Conversely, the levels of ROS (P < 0.001), MDA (P < 0.05), and Fe²⁺ (P < 0.05) were significantly decreased, and the GSH/GSSG ratio was decreased (P < 0.05) in the USP35 OE+Erastin group compared with the Vector+Erastin group. These results suggested that USP35 could inhibit Erastin-induced oxidative stress and lipid peroxidation in RA-FLS. (3) In Erastin-induced RA-FLS, the expression of USP35 was positively correlated with the protein levels of SLC7A11 and GPX4, indicating a potential mechanism by which USP35 regulated ferroptosis in these cells. CONCLUSION USP35 inhibits ferroptosis in RA-FLS, potentially through the increased expression of SLC7A11 and GPX4.
Ferroptosis ; Humans ; Arthritis, Rheumatoid/metabolism* ; Synoviocytes/pathology* ; Reactive Oxygen Species/metabolism* ; Ubiquitin-Specific Proteases/metabolism* ; Fibroblasts/pathology* ; Cell Survival ; Piperazines/pharmacology* ; Endopeptidases/metabolism* ; Cells, Cultured ; Cell Line ; Amino Acid Transport System y+

OBJECTIVE: To investigate the inhibitory effect of Tanreqing Injection (TRQ) on the activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in macrophages infected with influenza A virus and the underlying mechanism based on mitophagy pathway. METHODS: The inflammatory model of murine macrophage J774A.1 induced by influenza A virus [strain A/Puerto Rico/8/1934 (H1N1), PR8] was constructed and treated by TRQ, while the mitochondria-targeted antioxidant Mito-TEMPO and autophagy specific inhibitor 3-methyladenine (3-MA) were used as controls to intensively study the anti-inflammatory mechanism of TRQ based on mitophagy-mitochondrial reactive oxygen species (mtROS)-NLRP3 inflammasome pathway. The levels of NLRP3, Caspase-1 p20, microtubule-associated protein 1 light chain 3 II (LC3II) and P62 proteins were measured by Western blot. The release of interleukin-1β (IL-1β) was tested by enzyme linked immunosorbent assay, the mtROS level was detected by flow cytometry, and the immunofluorescence and co-localization of LC3 and mitochondria were observed under confocal laser scanning microscopy. RESULTS: Similar to the effect of Mito-TEMPO and contrary to the results of 3-MA treatment, TRQ could significantly reduce the expressions of NLRP3, Caspase-1 p20, and autophagy adaptor P62, promote the expression of autophagy marker LC3II, enhance the mitochondrial fluorescence intensity, and inhibit the release of mtROS and IL-1β (all P<0.01). Moreover, LC3 was co-localized with mitochondria, confirming the type of mitophagy. CONCLUSION TRQ could reduce the level of mtROS by promoting mitophagy in macrophages infected with influenza A virus, thus inhibiting the activation of NLRP3 inflammasome and the release of IL-1β, and attenuating the inflammatory response.
Mitophagy/drug effects* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Animals ; Macrophages/virology* ; Inflammasomes/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Mitochondria/metabolism* ; Reactive Oxygen Species/metabolism* ; Influenza A virus/physiology* ; Interleukin-1beta/metabolism* ; Cell Line ; Injections