Protective Mechanism of Crocin Against Zearalenone-Induced Oxidative Damage to Swine Testicle Cells.

Mycotoxin contamination poses a major challenge to public health and has garnered increasing attention across the world in recent decades. Zearalenone (ZEA), as one of the most prevalent contaminants, induces reproductive toxicity and then poses potential threats to animal health. Autophagy/beclin-1 regulator 1 (AMBRA1) is a protein critical for autophagy induction, and can enhance mitophagy by co-localizing with LC3. However, the potential health risk caused by ZEA in male germ cells of animals is unclear. This study aimed to investigate the underlying mechanisms of ZEA-induced swine testicular (ST) cell injury and to clarify the role of AMBRA1 in this process. We established ST cell models to explore the effects of AMBRA1 on ferroptosisinduced by ZEA. Multiple experimental approaches were applied to assess cell viability, mitochondrial dysfunction, oxidative stress, iron accumulation, and mitophagy. Mechanistic insights were further validated using AMBRA1 overexpression, RNA-seq, molecular docking, western blotting, immunofluorescence, and qRT-PCR analyses. In this study, ZEA induced mitochondrial structural damage and impaired mitochondrial function, leading to excessive ROS generation and loss of mitochondrial membrane potential. We also found that ZEA disrupted the iron homeostasis and thus led to the accumulation of ferrous iron, which further induce ferroptosis. In addition, ZEA reduced autophagy activity and autophagic flux, ultimately suppressing mitophagy. Of note, AMBRA1 overexpression effectively relieved ZEA-induced ferroptosis through restoration of mitophagy in ST cells. In conclusion, our study demonstrated that ZEA targeted the AMBRA1, leading to down-regulation of AMBRA1 expression, which in turn inhibited mitophagy and thus resulted in ferroptosis in ST cells. Given the potential role of AMBRA1 in ST cells, our results uncover a previously unrecognized mechanism in which AMBRA1-mediated mitophagy functions as a crucial defense target against ferroptosis in testicular cells. Importantly, our results propose a unique insight which AMBRA1 as a promising therapeutic target for counteracting mycotoxin-induced testicular injury in animals.

The effect of classical swine fever virus NS5A and NS5A mutants on oxidative stress and inflammatory response in swine testicular cells

Porcine deltacoronavirus (PDCoV) has emerged as a novel, contagious swine enteric coronavirus that causes watery diarrhea and/or vomiting and intestinal villous atrophy in nursing piglets. PDCoV-related diarrhea first occurred in the USA in 2014 and was subsequently reported in South Korea, China, Thailand, Vietnam, and Lao People's Democratic Republic, leading to massive economic losses and posing a threat to the swine industry worldwide. Currently, no treatments or vaccines for PDCoV are available. The critical step in the development of potential vaccines against PDCoV infection is the isolation and propagation of PDCoV in cell culture. This chapter provides a detailed protocol for isolation and propagation of PDCoV in swine testicular (ST) and LLC porcine kidney (LLC-PK) cell cultures supplemented with pancreatin and trypsin, respectively. Filtered clinical samples (swine intestinal contents or feces) applied to ST or LLC-PK cells produce cytopathic effects characterized by rounding, clumping, and detachment of cells. PDCoV replication in cells can be quantifiably monitored by qRT-PCR, immunofluorescence assays, and immune-electron microscopy. Infectious viral titers can be evaluated by using plaque assays or 50% tissue culture infectious dose (TCID50) assays. The ST or LLC-PK cells efficiently supported serial passage and propagation of PDCoV. After serial passage of PDCoV in either ST or LLC-PK cells, the virus can be purified further in ST cells by plaque assays.

The role of oxidative stress in zearalenone-mediated toxicity in Hep G2 cells: Oxidative DNA damage, gluthatione depletion and stress proteins induction

Porcine deltacoronavirus induces apoptosis in swine testicular and LLC porcine kidney cell lines in vitro but not in infected intestinal enterocytes in vivo

Five continuous cell lines, swine testicular (ST), human rectal tumor (HRT 18), fetal rhesus monkey kidney (MA104), bovine turbinate (BT), and quail tracheal (QT35), were evaluated and compared with chicken embryo fibroblasts (CEFs) for their ability to propagate B1 or Texas GB strains of Newcastle disease virus (NDV). The NDV Texas GB strain replicated in all the continuous cell lines used in this study. Only the ST and QT35 cells produced a cytopathic effect (CPE) similar to that produced in CEFs. However, the ST cell line remained attached while displaying CPE, whereas infected QT35 cells detached, as did the CEFs. The B1 strain of NDV replicated in ST cells, MA104 cells, and CEFs but with less CPE as compared with the Texas GB strain. Pretreatment with trypsin did not enhance CPE with either NDV strain at the level tested. Sera evaluated for neutralizing antibody titers to NDV were significantly higher in titer when the ST cell line was used and compared with CEFs. A high correlation was found between the microscopic examination and the tetrazolium dye (MTT) microassay methods for determining the viral neutralization endpoint, thus suggesting the ST cell line and MTT microassay could be used as an alternative to CEFs and microscopic examination for evaluating neutralizing antibodies titers to NDV.

As a dominant mycotoxin, zearalenone (ZEA) has attracted extensive attention due to its estrogen-like effect and oxidative stress damage in cells. In order to find a way to relieve cell oxidative stress damage caused by ZEA, we treated goat granulosa cells (GCs) with ZEA and did a whole transcriptome sequencing. The results showed that the expression level of Sesterin2 (SESN2) was promoted extremely significantly in the ZEA group (p < .01). In addition, our research demonstrated that SESN2 could regulate oxidative stress level in GCs through Recombinant Kelch Like ECH Associated Protein 1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. The overexpression of SESN2 could reduce the oxidative damage, whereas knockdown of SESN2 would aggravate the oxidative damage caused by ZEA. What's more, microRNA (miRNA) chi-miR-130b-3p can bind to SESN2 3'-untranslated region (3'UTR) to regulate the expression of SESN2. The mimics/inhibition of chi-miR-130b-3p would have an effect on oxidative damage triggered by ZEA in GCs as well. In summary, these results elucidate a new pathway by which chi-miR-130b-3p affects the KEAP1/NRF2 pathway in GCs by modulating SESN2 expression in response to ZEA-induced oxidative stress damage.

Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions.

Graphical abstractThe sperm freezing–thawing procedure is the most commonly used technique in clinics to preserve male fertility before any pathological destruction of the testis. Therefore, most studies are currently focused on optimizing this method to achieve high-quality semen after thawing. During cryopreservation, oxidative stress-induced damage affects sperm structures and decreases their fertility potential. The use of antioxidants in freezing media can protect sperm against oxidative damage. We designed this study to evaluate whether incubation of semen with human follicular fluid, which contains a wide variety of enzymatic and nonenzymatic antioxidants, can prevent the negative effects of freezing–thawing on human spermatozoa. Human semen was divided into three groups i) the 0-hour group (before freezing), ii) the control group (after freezing–thawing), and iii) the FF group (after freezing with 50% follicular fluid). The sperm motility, viability, integrity of the plasma membrane and DNA, mitochondrial membrane potential, malondialdehyde level, total antioxidant capacity, and catalase activity were assessed in these three groups. The findings showed a significant decrease in sperm motility, viability, plasma membrane and DNA integrity, mitochondrial membrane potential, total antioxidant capacity, and catalase activity and a significant increase in malondialdehyde level in the control group compared with the 0-hour group. The FF group displayed a considerable increase in sperm parameters, total antioxidant capacity, and catalase activity and a significant decrease in malondialdehyde level compared with the control group. Follicular fluid can be considered an effective supplement to improve antioxidant indices and sperm parameters during freezing–thawing.Lay summarySperm freezing is a useful method in clinics to preserve fertility in people who are affected by some problems such as diseases or chemotherapy which decrease their fertility. Although various studies are focused on optimizing this method, some challenges decrease the efficiency of this method. Oxidative stress has been reported as one of the mechanisms inducing negative effects on sperm during freezing–thawing. Therefore, the use of cryoprotectants and also some antioxidants has been suggested to increase sperm quality during freezing–thawing. In this study, we used human follicular fluid before freezing to assess sperm parameters. Our results showed that follicular fluid with antioxidant properties and other proper factors can have positive effects on human sperm during freezing–thawing and could be proposed to be added to the sperm freezing medium to improve sperm parameters, although this suggestion needs to be confirmed by further experiments.

To investigate the potential effects and mechanism of nicotinamide riboside (NR) on the oxidative stress and fibrosis model of human trabecular meshwork (HTM) cell line cells. HTM cells were pretreated with NR, followed by the induction of oxidative injury and fibrosis by hydrogen peroxide (H2O2) and TGF-β2, respectively. Cell viability was tested using Hoechst staining and MTT assays, cell proliferation was assessed by EdU assay, and cell apoptosis was detected by flow cytometry and western blotting. DCFH-DA and DHE probes were used to measure the level of reactive oxygen species (ROS), and MitoTracker staining was used to measure the mitochondrial membrane potential (MMP). Fibrotic responses, including cell migration and deposition of extracellular matrix (ECM) proteins, were detected via Transwell assays, qRT-PCR, and immunoblotting. NR pretreatment improved the viability, proliferation, and MMP of H2O2-treated HTM cells. Compared to cells treated solely with H2O2, HTM cells treated with both NR and H2O2, exhibited a reduced rate of apoptosis and generation of ROS. Compared with H2O2 pretreatment, NR pretreatment upregulated expression of the JAK2/Stat3 pathway but inhibited mitogen-activated protein kinase (MAPK) pathway expression. Moreover, 10-ng/mL TGF-β2 promoted cell proliferation and migration, which were inhibited by NR pretreatment. Both qRT-PCR and immunoblotting showed that NR inhibited the expression of fibronectin in a TGF-β2-induced fibrosis model. NR has a protective effect on oxidative stress and fibrosis in HTM cells, which may be related to the JAK2/Stat3 pathway and MAPK pathway. Our research provides the ongoing data for potential therapy of NAD+ precursors in glaucoma.

Molecular characterization and isolation of contemporary porcine sapoviruses from swine farms in the United States.

Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus that causes intestinal diseases in neonatal piglets with diarrhea, vomiting, dehydration, and post-infection mortality of 50–100%. Currently, there are no effective treatments or vaccines available to control PDCoV. To study the potential of RNA interference (RNAi) as a strategy against PDCoV infection, two short hairpin RNA (shRNA)-expressing plasmids (pGenesil-M and pGenesil-N) that targeted the M and N genes of PDCoV were constructed and transfected separately into swine testicular (ST) cells, which were then infected with PDCoV strain HB-BD. The potential of the plasmids to inhibit PDCoV replication was evaluated by cytopathic effect, virus titers, and real-time quantitative RT-PCR assay. The cytopathogenicity assays demonstrated that pGenesil-M and pGenesil-N protected ST cells against pathological changes with high specificity and efficacy. The 50% tissue culture infective dose showed that the PDCoV titers in ST cells treated with pGenesil-M and pGenesil-N were reduced 13.2- and 32.4-fold, respectively. Real-time quantitative RT-PCR also confirmed that the amount of viral RNA in cell cultures pre-transfected with pGenesil-M and pGenesil-N was reduced by 45.8 and 56.1%, respectively. This is believed to be the first report to show that shRNAs targeting the M and N genes of PDCoV exert antiviral effects in vitro, which suggests that RNAi is a promising new strategy against PDCoV infection.

Zinc (Zn) plays an important role in spermatogenesis, and carbon tetrachloride (CCl4) induces testicular oxidative damage and cell death. The objective of the present study was to define the effects of Zn deficiency in combination with CCl4 treatment on testicular apoptosis and the associated mechanisms. Mice were fed the following diets with three different Zn levels for 6weeks: normal zinc (ZN) diet (30mg Zn/kg), zinc-deficient (ZD) diet (2mg Zn/kg), and adequate zinc (ZA) diet (100mg Zn/kg). Beginning in the third week, CCl4 was intraperitoneally injected into half of the mice in each diet group six times over 3weeks. We found that Zn was distributed in various tissues and organs in normal mice and that the zinc content in the testis of normal mice was high. The Zn-deficient diet reduced the zinc concentration in the testis tissue, and the testicular/body weight ratio significantly decreased. Moreover, the TUNEL results proved that CCl4 stimulation of mice fed with a zinc-deficient diet caused marked apoptosis of testicular cells. Furthermore, the ROS levels in the testes obviously increased after Zn-deficient mice were stimulated with CCl4, whereas reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) showed reduced activities. In addition, proteins associated with the apoptosis signaling pathway were detected with ELISA kits. P-p53, cleaved caspase-3, cleaved PRAP, p-Bad, p-JNK, p-ERK, and p-NF-κB p65 increased by varying degrees under zinc deficiency or CCl4 stimulation. All the data indicated that Zn deficiency significantly enhanced the harm to the testis induced by oxidative stress and damage, while CCl4 stimulation exacerbated the oxidative damage in testicular cells, leading to apoptosis through the activation of p53, MAPK, and NF-κB.

Cardiac Metallothionein Synthesis in Streptozotocin-Induced Diabetic Mice, and Its Protection against Diabetes-Induced Cardiac Injury

Background: Under oxidative stress conditions, the increased levels of reactive oxygen species (ROS) within cells disrupt the intracellular homeostasis. Tartary buckwheat peptides exert their effects by scavenging oxidative free radicals, such as superoxide anion and hydrogen peroxide, thereby reducing oxidative damage within cells. Meanwhile, these peptides safeguard mitochondria by maintaining the mitochondrial membrane potential, decreasing the production of mitochondrial oxygen free radicals, and regulating mitochondrial biogenesis and autophagy to preserve mitochondrial homeostasis. Through these mechanisms, Tartary buckwheat peptides restore the intracellular redox balance, sustain cellular energy metabolism and biosynthesis, and ensure normal cellular physiological functions, which is of great significance for cell survival and adaptation under oxidative stress conditions. Objectives: In this experiment, a classical cellular oxidative stress model was established. Indicators related to antioxidant capacity and mitochondrial membrane potential changes, as well as pathways associated with oxidative stress, were selected for detection. The aim was to elucidate the effects of Tartary buckwheat oligopeptides on the metabolism of cells in response to oxidative stress. Methods: In this study, we established an oxidative damage model of mouse skeletal muscle myoblast (SOL8) cells using hydrogen peroxide (H2O2), investigated the pre-protective effects of Tartary buckwheat oligopeptides on H2O2-induced oxidative stress damage in SOL8 cells at the cellular level, and explored the possible mechanisms. The CCK-8 method is a colorimetric assay based on WST-8-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodiumsalt], which is used to detect cell proliferation and cytotoxicity. Results: The value of CCK-8 showed that, when the cells were exposed to 0.01 mmol/L H2O2 for 1 h and 10 mg/mL Tartary buckwheat oligopeptides intervention for 48 h, these were the optimal conditions. Compared with the H2O2 group, the intervention group (KB/H2O2 group) showed that the production of ROS was significantly reduced (p < 0.001), the malondialdehyde (MDA) content was significantly decreased (p < 0.05), and the activity of catalase (CAT) was significantly increased (p < 0.01); the mitochondrial membrane potential in the KB/H2O2 group tended to return to the level of the control group, and they all showed dose-dependent effects. Compared with the H2O2 group, the mRNA expression of KEAP1 in the KB/H2O2 group decreased, while the mRNA expression of NRF2α, HO-1, nrf1, PGC-1, P62, and PINK increased. Conclusions: Therefore, Tartary buckwheat oligopeptides have a significant pre-protective effect on H2O2-induced SOL8 cells, possibly by enhancing the activity of superoxide dismutase, reducing ROS attack, balancing mitochondrial membrane potential, and maintaining intracellular homeostasis.