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

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*

This study aims to explore the mechanism of Buyang Huanwu Decoction(BHD) in promoting angiogenesis after oxygen-glucose deprivation/reoxygenation(OGD/R) of mouse brain microvascular endothelial cell line(brain-derived Endothelial cells.3, bEnd.3) based on the caveolin-1(Cav1)/Yes-associated protein 1(YAP1)/hypoxia-inducible factor-1α(HIF-1α) signaling pathway. Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to analyze the blood components of BHD. The cell counting kit-8(CCK-8) method was used to detect the optimal intervention concentration of drug-containing serum of BHD after OGD/R injury of bEnd.3. The lentiviral transfection method was used to construct a Cav1 silent stable strain, and Western blot and polymerase chain reaction(PCR) methods were used to verify the silencing efficiency. The control bEnd.3 cells were divided into a normal group(sh-NC control group), an OGD/R model + blank serum group(sh-NC OGD/R group), and an OGD/R model + drug-containing serum group(sh-NC BHD group). Cav1 silent cells were divided into an OGD/R model + blank serum group(sh-Cav1 OGD/R group) and an OGD/R model + drug-containing serum group(sh-Cav1 BHD group). The cell survival rate was detected by the CCK-8 method. The cell migration ability was detected by a cell migration assay. The lumen formation ability was detected by an angiogenesis assay. The apoptosis rate was detected by flow cytometry, and the expression of YAP1/HIF-1α signaling pathway-related proteins in each group was detected by Western blot. Finally, co-immunoprecipitation was used to verify the interaction between YAP1 and HIF-1α. The results showed astragaloside Ⅳ, formononetin, ferulic acid, and albiflorin in BHD can all enter the blood. The drug-containing serum of BHD at a mass fraction of 10% may be the optimal intervention concentration for OGD/R-induced injury of bEnd.3 cells. Compared with the sh-NC control group, the sh-NC OGD/R group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, significantly increased cell apoptotic rate, significantly lowered phosphorylation level of YAP1 at S127 site, and significantly elevated nuclear displacement level of YAP1 and protein expression of HIF-1α, vascular endothelial growth factor(VEGF), and vascular endothelial growth factor receptor 2(VEGFR2). Compared with the same type of OGD/R group, the sh-NC BHD group and sh-Cav1 BHD group had significantly increased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly decreased cell apoptotic rate, a further decreased phosphorylation level of YAP1 at S127 site, and significantly increased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC OGD/R group, the sh-Cav1 OGD/R group exhibited significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC BHD group, the sh-Cav1 BHD group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at the S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. YAP1 protein was present in the protein complex precipitated by the HIF-1α antibody, and HIF-1α protein was also present in the protein complex precipitated by the YAP1 antibody. The results confirmed that the drug-containing serum of BHD can increase the activity of YAP1/HIF-1α pathway in bEnd.3 cells damaged by OGD/R through Cav1 and promote angiogenesis in vitro.
Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Signal Transduction/drug effects* ; Glucose/metabolism* ; Caveolin 1/genetics* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; YAP-Signaling Proteins ; Oxygen/metabolism* ; Endothelial Cells/metabolism* ; Cell Line ; Adaptor Proteins, Signal Transducing/genetics* ; Neovascularization, Physiologic/drug effects* ; Cell Hypoxia/drug effects* ; Angiogenesis

This study aims to explore the effects and action mechanisms of the active ingredients in Buyang Huanwu Decoction(BYHWD), namely tetramethylpyrazine(TMP) and hydroxy-safflor yellow A(HSYA), on oxygen-glucose deprivation/reglucose-reoxygenation(OGD/R)-induced inflammation and oxidative stress of microglia(MG). Network pharmacology was used to screen the effective monomer ingredients of BYHWD and determine the safe concentration range for each component. Inflammation and oxidative stress models were established to further screen the best ingredient combination and optimal concentration ratio with the most effective anti-inflammatory and antioxidant effects. OGD/R BV2 cell models were constructed, and BV2 cells in the logarithmic growth phase were divided into a normal group, a model group, an HSYA group, a TMP group, and an HSYA + TMP group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory cytokines such as interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6). Oxidative stress markers, including superoxide dismutase(SOD), nitric oxide(NO), and malondialdehyde(MDA), were also measured. Western blot was used to analyze the protein expression of both inflammation-related pathway [Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)] and oxidative stress-related pathway [nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)]. Immunofluorescence was used to assess the expression of proteins such as inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). The most effective ingredients for anti-inflammatory and antioxidant effects in BYHWD were TMP and HSYA. Compared to the normal group, the model group showed significantly increased levels of IL-1β, TNF-α, IL-6, NO, and MDA, along with significantly higher protein expression of NF-κB, TLR4, Nrf2, and HO-1 and significantly lower SOD levels. The differences between the two groups were statistically significant. Compared to the model group, both the HSYA group and the TMP group showed significantly reduced levels of IL-1β, TNF-α, IL-6, NO, and MDA, lower expression of NF-κB and TLR4 proteins, higher levels of SOD, and significantly increased protein expression of Nrf2 and HO-1. Additionally, the expression of the M1-type MG marker iNOS was significantly reduced, while the expression of the M2-type MG marker Arg-1 was significantly increased. The results of the HSYA group and the TMP group had statistically significant differences from those of the model group. Compared to the HSYA group and the TMP group, the HSYA + TMP group showed further significant reductions in IL-1β, TNF-α, IL-6, NO, and MDA levels, along with significant reductions in NF-κB and TLR4 protein expression, an increase in SOD levels, and elevated Nrf2 and HO-1 protein expression. Additionally, the expression of the M1-type MG marker iNOS was reduced, while the M2-type MG marker Arg-1 expression increased significantly in the HSYA + TMP group compared to the TMP or HSYA group. The differences in the results were statistically significant between the HSYA + TMP group and the TMP or HSYA group. The findings indicated that the combined use of HSYA and TMP, the active ingredients of BYHWD, can effectively inhibit OGD/R-induced inflammation and oxidative stress of MG, showing superior effects compared to the individual use of either component.
Oxidative Stress/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Glucose/metabolism* ; Cell Line ; Inflammation/genetics* ; Oxygen/metabolism* ; Pyrazines/pharmacology* ; Microglia/metabolism* ; NF-E2-Related Factor 2/immunology* ; NF-kappa B/immunology* ; Toll-Like Receptor 4/immunology* ; Anti-Inflammatory Agents/pharmacology* ; Humans

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*