Biological Research最新文献

Background: Oxidative stress, a source of genotoxic damage, is often the underlying mechanism in many functional cell disorders. Extracellular vesicles (EVs) have been shown to be key regulators of cellular processes and may be involved in maintaining cellular redox balance. Herein, we aimed to develop a method to assess the effects of EVs on DNA oxidation using porcine seminal plasma extracellular vesicles (sEVs).

Results: The technique was set using a cell-free plasmid DNA to avoid the bias generated by the uptake of sEVs by sperm cells, employing increasing concentrations of hydrogen peroxide (H₂O₂) that generate DNA single-strand breaks (SSBs). Because SSBs contain a free 3'-OH end that allow the extension through quantitative PCR, such extension -and therefore the SYBR intensity- showed to be proportional to the amount of SSB. In the next stage, H₂O₂ was co-incubated with two size-differentiated subpopulations (small and large) of permeabilized and non-permeabilized sEVs to assess whether the intravesicular content (IC) or the surface of sEVs protects the DNA from oxidative damage. Results obtained showed that the surface of small sEVs reduced the incidence of DNA SSBs (P < 0.05), whereas that of large sEVs had no impact on the generation of SSBs (P > 0.05). The IC showed no protective effect against DNA oxidation (P > 0.05).

Conclusions: Our results suggest that the surface of small sEVs, including the peripheral corona layer, may exert a protective function against alterations that are originated by oxidative mechanisms. In addition, our in vitro study opens path to detect, localize and quantify the protective effects against oxidation of extracellular vesicles derived from different fluids, elucidating their function in physiopathological states.

Background: Male factor infertility (MFI) is responsible for 50% of infertility cases and in 15% of the cases sperm is absent due to germ cell aplasia. Human induced pluripotent stem cell (hiPSC)-derived spermatogonial stem cells (hSSCs) could serve as an autologous germ cell source for MFI in patients with an insufficient sperm yield for assisted reproductive technology (ART). The endocannabinoid system (ECS) has been implicated to play a role in mouse embryonic stem cells (mESCs) and the human testicular environment. However, the contribution of the ECS in hiPSCs and hiPSC-derived hSSCs is currently unknown. Here, we aimed to assess whether hiPSCs and hiPSC-derived hSSCs are regulated by components of the ECS and whether manipulation of the ECS could increase the yield of hiPSC-derived SSCs and serve as an autologous cell-based source for treatment of MFI.

Methods: We reprogrammed human dermal fibroblasts (hDFs) to hiPSCs, induced differentiation of hSSC from hiPSCs and evaluated the presence of ECS ligands (AEA, 2-AG) by LC/MS, receptors (CB1R, CB2R, TRPV1, GPR55) by qPCR, flow cytometry and immunofluorescent labeling. We then examined the efficacy of endogenous and synthetic selective ligands (ACPA, CB65, CSP, ML184) on proliferation of hiPSCs using real-time cell analysis (RTCA) and assessed the effects of on CB2R agonism on hiPSC pluripotency and differentiation to hSSCs.

Results: hiPSCs from hDFs expressed the pluripotency markers OCT4, SOX2, NANOG, SSEA4 and TRA-1-60; and could be differentiated into ID4+, PLZF + hSSCs. hiPSCs and hiPSC-derived hSSCs secreted AEA and 2-AG at 10^- 10 - 10^- 9 M levels. Broad expression of all ECS receptors was observed in both hiPSCs and hiPSC-derived hSSCs, with a higher CB2R expression in hSSCs in comparison to hiPSCs. CB2R agonist CB65 promoted proliferation and differentiation of hiPSCs to hiPSC-hSSCs in comparison to AEA, 2-AG, ACPA, CSP and ML184. The EC₅₀ of CB65 was determined to be 2.092 × 10^- 8 M for support of pluripotency and preservation of stemness on hiPSCs from 78 h. CB65 stimulation at EC₅₀ also increased the yield of ID4 + hSSCs, PLZF + SSPCs and SCP3 + spermatocytes from day 10 to 12.

Conclusions: We demonstrated here for the first time that stimulation of CB2R results in an increased yield of hiPSCs and hiPSC-derived hSSCs. CB65 is a potent CB2R agonist that can be used to increase the yield of hiPSC-derived hSSCs offering an alternative source of autologous male germ cells for patients with MFI. Increasing the male germ/stem cell pool by CB65 supplementation could be part of the ART-associated protocols in MFI patients with complete germ cell aplasia.

Background: Ischemic reperfusion (IR) generates reactive oxygen species (ROS) that inevitably result in myocardial cell death and heart failure. The regenerative power of cardiac progenitor/stem pools (CSCs), especially the Sca1⁺ population, in response to IR injury remains unclear.

Methods: Our work sought to investigate whether small extracellular vesicles (sEVs) isolated from bone marrow-mesenchymal stem cells (BMMSCs) could rescue CSCs, specifically Sca-1+/CSCs, from IR by increasing their proliferative capacity and limiting their apoptosis in vitro. The Sca-1+/CSCs-IR model was induced by the oxygen-glucose deprivation/reoxygenation method (OGD/R). The effects of treatment with BMMSCs-derived sEVs on oxidative stress, cell proliferation, apoptosis, and cell cycle were assessed. To further test the mechanistic action, we assessed the PTEN/pAkt/HIF-1α pathway.

Results: Compared to hypoxic untreated CSCs, BMMSCs-derived sEVs-treated cells had shifted from their quiescent to proliferative phase (p > 0.05) and showed decreased apoptosis (p < 0.001). sEVs-treated CSCs were predominately in the S phase (11.8 ± 0.9%) (p < 0.01). We identified an abundance of miRNA-21-5P in BMMSCs. HIF-1α expression was highest in CSCs treated with sEVs (p < 0.05). Moreover, miRNA-21-5p-rich sEVs shifted the redox state, reducing oxidative stress and promoting balance (p > 0.05).

Conclusion: Conditioning Sca-1+/CSCs, an essential population in the postnatal heart, with sEVs rich in miRNA-21 robustly enhanced the proliferation, and synthesis phase of the cell cycle, and stabilized HIF-1α while alleviating oxidative stress and apoptosis. Such sEVs rich in miRNA-21-5p can be further used as a preconditioning tool to enhance endogenous Sca-1+/CSCs regeneration in response to IR injury.

Background: Serotonin (5-HT) is known to be synthesized and accumulated in the vertebrate retina through the 5-HT transporter, SERT. While manipulation of the serotonergic system has been shown to impact visual processing, the role of 5-HT and SERT as modulators of retinal synaptic function remains poorly understood.

Results: Using mouse retinal slices, we show that acute application of 5-HT produces a cell-type specific reduction in light-evoked excitatory responses (L-EPSC) in ON-OFF retinal ganglion cells (RGCs), but not in ON RGCs. Similarly, increasing 5-HT tone by acute application of citalopram, a selective 5-HT reuptake inhibitor, also reduces L-EPSC in ON-OFF RGCs while not affecting ON RGCs. Importantly, citalopram-mediated reduction of L-EPSC was absent in ON-OFF RGCs recorded from SERT null retina, highlighting the role of SERT in regulating light-evoked responses in RGCs. The effects of both exogenous and endogenous 5-HT on L-EPSC in ON-OFF RGCs are likely due to a presynaptic reduction in excitatory synaptic strength as 5-HT and citalopram reduced the frequency but not the amplitude of spontaneous excitatory currents (sEPSCs) in ON-OFF RGCs. Moreover, 5-HT and citalopram had no effect on currents elicited by the direct activation of postsynaptic receptors in RGCs by brief application of glutamate in the inner retina.

Conclusions: Altogether these findings indicate that 5-HT modulates excitatory inputs onto RGCs in a cell-type specific manner and highlight that in the adult mouse retina, 5-HT-mediated effects onto RGCs are tightly controlled by the 5-HT transporter SERT.

Background: Electroporation (EP) leverages electric pulses to permeabilize cell membranes, enabling the delivery of therapeutic agents like calcium in cancer treatment. Calcium electroporation (CaEP) induces a rapid influx of calcium ions, disrupting cellular calcium homeostasis and triggering cell death pathways. This study aims to compare the cellular responses between microsecond (µsEP) and nanosecond (nsEP) electroporation, particularly in terms of oxidative stress, immune response activation, and cancer stem cell (CSC) viability in drug-resistant (LoVo Dx) and non-resistant (LoVo) colorectal cancer cell lines.

Results: Both µsEP and nsEP, particularly when combined with Ca²⁺, significantly reduced the viability of cancer cells, with nsEP showing greater efficacy. Reactive oxygen species (ROS) levels increased 5-fold in malignant cells following nsEP, correlating with decreased ATP production and mitochondrial dysfunction. Nanosecond CaEP (nsCaEP) also induced significant expression of aspartate-β-hydroxylase (ASPH), a protein linked to calcium homeostasis and tumor progression. Moreover, nsEP led to heightened expression of heat shock proteins (HSP27/70), indicating potential immune activation. Interestingly, nsEP without calcium drastically reduced the expression of CD133, a marker for CSCs, while the addition of Ca²⁺ preserved CD133 expression. The expression of death effector domain-containing DNA binding protein (DEDD), associated with apoptosis, was significantly elevated in treated cancer cells, especially in the nucleus after nsCaEP.

Conclusions: The study confirms that nsEP is more effective than µsEP in disrupting cancer cell viability, enhancing oxidative stress, and triggering immune responses, likely through HSP overexpression and ROS generation. nsEP also appears to reduce CSC viability, offering a promising therapeutic approach. However, preserving CD133 expression in the presence of calcium suggests complex interactions that require further investigation. These findings highlight the potential of nsCaEP as an innovative strategy for targeting both cancer cells and CSCs, potentially improving treatment outcomes in colorectal cancer. Further studies are needed to explore the exact cell death mechanisms and optimize protocols for clinical applications.

Background: Cavernous nerve injury-induced erectile dysfunction (CNI-ED) is a common complication following radical prostatectomy and severely affects patients' quality of life. The mitochondrial impairment in corpus cavernosum smooth muscle cells (CCSMCs) may be an important pathological mechanism of CNI-ED. Previous studies have shown that transplantation of human adipose derived stem cells (ADSC) can alleviate CNI-ED in a rat model. However, little is known about the effect of human umbilical cord mesenchymal stem cells (hUC-MSC) on CNI-ED. It remains unclear whether hUC-MSC can ameliorate mitochondrial damage in CCSMCs. In this study, we aimed to investigate the impacts of hUC-MSC on the mitochondrial mass and function of CCSMCs, as well as elucidate its underlying molecular mechanism.

Methods: The CNI-ED rat model was established by bilaterally crushing cavernous nerves. Subsequently, hUC-MSC were transplanted into the cavernosum and ADSC were injected as a positive control group. Erectile function evaluation and histological detection were performed 4 weeks after cell transplantation. In vitro, CCSMCs underwent hypoxia and were then co-cultured with ADSC or hUC-MSC using a transwell system. The mitochondrial mass and function, as well as signaling pathways, were investigated. To explore the role of the SIRT1/PGC-1α/TFAM pathway in regulating mitochondrial biogenesis of CCSMCs, we knocked down SIRT1 by siRNA.

Results: The administration of hUC-MSC significantly improved erectile function of CNI-ED rats and reduced the ratio of collagen to smooth muscle. Specifically, hUC-MSC treatment restored mitochondrial mass and function in CCSMCs injured by CNI or hypoxia, and inhibited the apoptosis of CCSMCs. Mechanistically, the application of hUC-MSC activated SIRT1/PGC-1α/TFAM pathway both in rat penile tissues and CCSMCs. In addition, knockdown of SIRT1 in CCSMCs abolished the protective effects of hUC-MSC on mitochondrial mass and function, while leading to an increase in cellular apoptosis.

Conclusions: hUC-MSC contribute to the recovery of erectile function in CNI-ED rats by restoring mitochondrial mass and function of CCSMCs through the SIRT1/PGC-1α/TFAM pathway. Our present study offers new insights into the role and molecular mechanisms of hUC-MSC in regulating mitochondrial homeostasis, thereby facilitating the restoration of the erectile function in CNI-ED.

Parkinson's disease (PD) is a progressive age-related neurodegenerative disease whose annual incidence is increasing as populations continue to age. Although its pathogenesis has not been fully elucidated, oxidative stress has been shown to play an important role in promoting the occurrence and development of the disease. Long noncoding RNAs (lncRNAs), which are more than 200 nucleotides in length, are also involved in the pathogenesis of PD at the transcriptional level via epigenetic regulation, or at the post-transcriptional level by participating in physiological processes, including aggregation of the α-synuclein, mitochondrial dysfunction, oxidative stress, calcium stabilization, and neuroinflammation. LncRNAs and oxidative stress are correlated during neurodegenerative processes: oxidative stress affects the expression of multiple lncRNAs, while lncRNAs regulate many genes involved in oxidative stress responses. Oxidative stress and lncRNAs also affect other processes associated with neurodegeneration, including mitochondrial dysfunction and increased neuroinflammation that lead to neuronal death. Therefore, modulating the levels of specific lncRNAs may alleviate pathological oxidative damage and have neuroprotective effects. This review discusses the general mechanisms of oxidative stress, pathological mechanism underlying the role of oxidative stress in the pathogenesis of PD, and teases out the mechanisms through which lncRNAs regulate oxidative stress during PD pathogenesis, as well as identifies the possible neuroprotective mechanisms of lncRNAs. Reviewing published studies will help us further understand the mechanisms underlying the role of lncRNAs in the oxidative stress process in PD and to identify potential therapeutic strategies for PD.

Fluoride (F), as a natural element found in a wide range of sources such as water and certain foods, has been proven to be beneficial in preventing dental caries, but concerns have been raised regarding its potential deleterious effects on overall health. Sodium fluoride (NaF), another form of F, has the ability to accumulate in reproductive organs and interfere with hormonal regulation and oxidative stress pathways, contributing to reproductive toxicity. While the exact mechanisms of F-induced reproductive toxicity are not fully understood, this review aims to elucidate the mechanisms involved in testicular and ovarian injury. In males, F exposure at different doses has been associated with reduced testis weight, reduced sperm quality in terms of count, motility, and viability, as well as abnormal sperm morphology and disruption of seminiferous tubules by altering hormone levels (especially testosterone), impairing spermatogenesis, and inducing oxidative stress and zinc deficiency. Similarly, administration of F can impact female reproductive health by affecting ovarian function, hormone levels, oocyte quality, and the regularity of the estrous cycle. However, the impact of F exposure on LH, FSH, and GnRH levels is controversial between males and females. In both males and females, F exerts its adverse effects by triggering apoptosis, autophagy, inflammation, mitochondrial dysfunction, reduction in ATP synthesis, and modulation of important genes involved in steroidogenesis. Furthermore, genetic susceptibility and individual variations in F metabolism may contribute to different responses to fluoride exposure.

Bees are essential pollinators that contribute to maintaining biodiversity and increasing agricultural production. However, by foraging on agricultural crops, bees may become contaminated with compounds used for pest control. In this study, we exposed bee (Apis mellifera L.) colonies to the insecticide imidacloprid (IMD) under field conditions to assess the occurrence of oxidative stress in larvae and pupae and investigate morphological changes in the fat body and midgut of larvae and midgut of adult bees. The apiary area was divided into three groups: control, commercial formulation containing IMD (Evidence^® 700WG) (IMD_CF), and IMD active ingredient (Sigma-Aldrich) (IMD_AI). Treatment groups were fed syrup containing 1 µg L^-1 IMD, whereas the control group was fed syrup only. Compared with the control, larvae exposed to IMD_CF or IMD_AI for 42 days exhibited morphological changes in the external body, midgut, and fat body. The midgut of adult bees contaminated with IMD_CF showed only structural remnants of the peritrophic membrane and absence of regenerative cell nests. Oxidative stress analyses revealed that IMD_CF-exposed larvae had higher nitrite and carbonylated protein contents and lower catalase and superoxide dismutase activity than control individuals. In pupae, IMD_AI decreased catalase activity while increasing superoxide dismutase activity. These findings indicate that IMD has the potential to significantly impact the development of bees and their colonies by disrupting vital organs responsible for normal physiological functioning and overall activities of individuals. Oxidative stress, which was detected at different stages of bee development, may induce lipid, protein, and DNA oxidation, leading to cell death.