Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.

Lead (Pb) is a common metal, which can be toxic to the human body via the pollution of water or food, and can cause anemia and other diseases. However, what happens before hemolysis and anemia caused by Pb poisoning is unclear. Here, we demonstrated Pb can cause procoagulant activity of erythroid cells leading to thrombosis before hemolysis. In freshly isolated human erythroid cells, we observed that Pb resulted in hemolysis in both concentration- and time-dependent manners, but that no lysis occurred in Pb-exposed erythroid cells (≤20 μM for 1 h). Pb treatment did not cause shape changes at up to 0.5 h incubation but at 1 h incubation echinocyte and echino-spherocyte shape changes were observed, indicating that Pb can exaggerate a concentration- and time-dependent trend of shape changes in erythroid cells. After Pb treatment, ROS-independent eryptosis was shown with no increase of reactive oxygen species (ROS), but with an increase of [Ca2+]i and caspase 3 activity. With a thrombosis mouse model, we observed increased thrombus by Pb treatment (0 or 25 mg/kg). In brief, prior to hemolysis, we demonstrated Pb can cause ROS-independent but [Ca2+]i-dependent eryptosis, which might provoke thrombosis.

Nanoplastics are global pollutants that have been increasingly released into the environment following the degradation process of industrial and consumer products. These tiny particles have been reported to adversely affect various organs in the body, including the heart. Since it is probable that the less-developed hearts of newborn offspring are more vulnerable to nanoplastic insult during the infant feeding compared with mature hearts of adults, the acute effects of nanoplastics on the collective contractility of neonatal cardiomyocytes are to be elucidated. Here, we traced the aggregation of nanoplastics on the cell membrane and their internalization into the cytosol of neonatal rat ventricular myocytes (NRVMs) for 60 min in the presence of electrical pulses to synchronize the cardiac contraction in vitro. The time-coursed linkage of collective contraction forces, intracellular Ca2+ concentrations, mitochondrial membrane potentials, extracellular field potentials, and reactive oxygen species levels enabled us to build up the sequence of the cellular events associated with the detrimental effects of nanoplastics with positive surface charges on the immature cardiomyocytes. A significant decrease in intracellular Ca2+ levels and electrophysiological activities of NRVMs resulted in the reduction of contraction forces in the early phase (0-15 min). The further reduction of contraction force in the late phase (30-60 min) was attributed to remarkable decreases in mitochondrial membrane potentials and cellular metabolism. Our multifaceted assessments on the effect of positively surface charged nanoplastics on NRVM may offer better understanding of substantial risks of ever-increasing nanoplastic pollution in the hearts of human infants or adults.

BACKGROUND: Phagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood.
OBJECTIVE: To clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes.
METHODS: The phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM.
RESULTS: The phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM.
CONCLUSIONS: Taken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.

Ochratoxin A (OTA) is a frequent contaminant of feed and food worldwide. The toxicity of OTA on intestinal barrier was investigated in porcine intestinal epithelial cells (IPEC-J2). We observed that OTA induced intestinal barrier dysfunction as indicated by the reduction in transepithelial electrical resistance (TEER) and elevation in paracellular permeability to 4 kDa dextran. The barrier dysfunction was accompanied with tight junction disruption including a down-regulation in ZO-1 expression and redistribution of Occludin and ZO-1. Moreover, OTA exposure increased reactive oxygen species (ROS) generation, elevated the intracellular calcium level ([Ca2+]c) and activated myosin light chain kinase (MLCK). Simultaneously, NAC, a ROS scavenger, blocked OTA-induced ROS generation, [Ca2+]c elevation, barrier dysfunction and tight junction disruption, suggesting that OTA-induced ROS generation may act as a trigger. Next, we found that OTA-induced MLCK activation was inhibited by BAPTA-AM, the cytosolic Ca2+ chelator, demonstrating that OTA-induced MLCK activation is dependent on [Ca2+]c elevation. Furthermore, inhibition of MLCK with ML-7 or inhibition of [Ca2+]c elevation with BAPTA-AM markedly prevented OTA-induced barrier dysfunction and tight junction disruption. Taken together, our results indicated that OTA induces ROS generation, and then elevates the [Ca2+]c and MLCK activity in turn, which finally induces barrier dysfunction and disrupts tight junction in IPEC-J2 cell monolayers.

OBJECTIVE: To observe the changes of intracellular calcium ([Ca2＋]i) concentration and expression of calcium/calmodulin dependent protein kinaseⅡ (CaMKⅡ) in spinal dorsal horn neurons of spared nerve injury (SNI) rats, so as to explore its mechanisms underlying improvement of neuropathic pain.
METHODS: One hundred and ten SD rats were randomly divided into 5 groups: sham control, model, EA, AP-5 and L-NAME groups. The sham group underwent only a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right \"Weizhong\" (BL 40) and \"Huantiao\" (GB 30) for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg·kg－1·d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase [NOS], 60 mg·kg－1·d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, and the calcium fluorescence intensity (shown by Fluo-3/AM calcium fluorescence indicator) of the superficial layer of the lumbar spinal cord (L 4－L 6) was measured by immunohistochemical staining and the expression of spinal cord (L 4－L 6) CaMK Ⅱ protein was detected by Western blot (WB).
RESULTS: After modeling, the mechanical pain threshold was significantly decreased on day 10 and 16 after operation in comparison with the sham operation group and baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA, AP-5 and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05), while the effect of EA was significantly superior to that of AP-5 and L-NAME groups (P<0.05), suggesting a reduction of EA analgesia after administration of AP-5 and L-NAME. The concentration of intracellular [Ca2＋]i was significantly higher in the model group than in the sham group, and considerably lower in the EA, AP-5 and L-NAME groups than in the model group (P<0.01, P<0.05). Moreover, the expression level of CaMKⅡ shown by WB and immunohistochemical staining was significantly higher in the model group than in the sham group (P<0.05) and obviously lower in the EA group (not the AP-5 and L-NAME groups) than in the model group on day 16 after the intervention (P<0.05). It suggests an involvement of glutamate NMDA receptor and NMDAR-NOS/NO signaling in the analgesic effect and CaMKⅡ expression down-regulation of EA.
CONCLUSIONS: EA can ease pain in rats with neuropathic pain, which is closely related to its effect in reducing the calcium concentration and the expression of CaMKⅡ in the lumbar spinal cord, possibly mediated by glutamate NMDA receptor and NMDAR-NOS/NO signaling.

This study aimed to identify sperm proteomic signatures regulating sperm functions and fertility by: (i) comparing the sperm electrophoretic protein profiles and identifying the differentially abundant proteins among breeding bulls differing in fertility status and (ii) elucidating the possible role of one of the identified novel proteins, PEBP4 on sperm function and fertility. The grouping of bulls as fertile (n = 6) and low fertile (n = 6) was performed based on bull fertility index and infertile (n = 6) based on semen rejection rate (>33%). The sperm motility, fructolysis index, acrosomal reaction, intracellular calcium levels, and seminal plasma fructose and calcium levels were studied among fertility groups. The differentially expressed sperm proteins observed in single- and two-dimensional gel electrophoresis (2DE) were identified using Nano-LC-MS/MS. In the fertile bulls, the expression levels of calmodulin (CALM1), spermadhesinZ13 (SPADH2), and phosphatidylethanolamine-binding protein 4 (PEBP4) were significantly (p < 0.05) higher than in other fertility groups. In bovine, expression of PEBP4 a novel seminal protein was not observed in spermatozoa of infertile bulls. When the bulls were grouped based on the presence (n = 8) or absence (n = 10) of PEBP4 protein in spermatozoa, a positive significant (p < 0.05) association of this protein with the percentage of motile, type-A spermatozoa, and sperm fructose uptake was observed. Further, PEBP4 was localized in elongated spermatids, Leydig cells, excurrent duct system, and principal piece of spermatozoa. These findings suggest a crucial role for the PEBP4 protein in spermiogenesis, epididymal sperm maturation, and sperm motility. This first study in bovine indicates the positive association of PEBP4 in regulating sperm maturation, functions, and fertility and could be a potential marker for predicting semen quality and fertility.

Fluoride (F) is an environmental contaminant and industrial pollutant. Molecular mechanisms remain unclear in F induced pulmonary toxicity even after numerous studies. Tamarind fruits act as defluoridating agents, but no study was conducted in in vitro systems. Hence, we aimed to assess the ameliorative impact of the tamarind seed coat extract (TSCE) against F toxicity utilizing lung epithelial cells, A549. Cells were exposed to sodium fluoride (NaF-5 mM) alone and in combination with TSCE (750 ng/ml) or Vitamin C (positive control) for 24 h and analyzed for F content, intracellular calcium ([Ca(2+)]i) level, oxidative stress, mitochondrial integrity and apoptotic markers. TSCE treatment prevented the F induced alterations in [Ca(2+)]i overload, F content, oxidant (reactive oxygen species generation, lipid peroxidation, protein carbonyl content and nitric oxide) and antioxidant (superoxide dismutase, catalase, glutathione peroxidase and glutathione) parameters. Further, TSCE modulates F activated changes in mitochondrial membrane potential, permeability transition pore opening, cytochrome-C release, Bax/Bcl-2 ratio, caspase-3 and PARP-1 expressions. In conclusion, our study demonstrated that TSCE as a potential protective agent against F toxicity, which can be utilized as a neutraceutical.

OBJECTIVE: The current study aimed to explore the effects of apolipoprotein e (ApoE) on intracellular calcium ([Ca(2+)]i) and apoptosis of neurons after mechanical injury in vitro.
METHODS: A neuron mechanical injury model was established after primary neurons obtained from APOE knockout and wild-type (WT) mice, and four experimental groups were generated: Group-ApoE4, Group-ApoE3, Group-ApoE(-) and Group-WT. Recombinant ApoE4 and ApoE3 were added to Group-ApoE4 and Group-ApoE3 respectively, and Group-ApoE(-) and Group-WT were control groups. Intracellular calcium was labeled by fluo-3/AM and examined using laser scanning confocal microscope and flow cytometry, and the apoptosis of neurons was also evaluated.
RESULTS: The intracellular calcium levels and apoptosis rates of mice neurons were significantly higher in Group-ApoE4 than in Group-ApoE3 and Group-WT after mechanical injury. However, without mechanical injury on neurons, no significant differences in intracellular calcium levels and apoptosis rates were found among all four experimental groups. The effects of ApoE4 on intracellular calcium levels and apoptosis rates of injured neurons were partly decreased by EGTA treatment.
CONCLUSIONS: Compared with ApoE3-treatment and WT neurons, ApoE4 caused higher intracellular calcium levels and apoptosis rates of neurons after mechanical injury. This suggested APOE polymorphisms may affect neuron apoptosis after mechanical injury through different influences on intracellular calcium levels.

BACKGROUND: Ochratoxin A (OTA), a mycotoxin, causes extensive cell damage, affecting liver and kidney cells. OTA toxicity is fairly well characterized where oxidative stress is believed to play a role, however, the sequence of molecular events after OTA-exposure, have not been characterized in literature. Further, antidotes for alleviating the toxicity are sparsely reported. The aim of this study was to understand the sequence of some molecular mechanisms for OTA-induced toxicity and the cytoprotective effect of quercetin on OTA-induced toxicity.
METHODS: Time course studies to evaluate the time of intracellular calcium release and ROS induction were carried out. The time of activation and induction of two key redox- sensitive transcription factors, NF-κB and Nrf-2 were determined by nuclear localization and expression respectively. The time of expression of inflammatory marker COX-2 was determined. Oxidative DNA damage by comet assay and micronucleus formation was studied. The ameliorative effect of quercetin on OTA-induced toxicity was also determined on all the above-mentioned parameters.
RESULTS: OTA-induced calcium release, ROS generation and activated NF-κB nuclear translocation and expression. Pre-treatment with quercetin ameliorated ROS and calcium release as well as NF-κB induction and expression. Quercetin induced Nrf-2 nuclear translocation and expression. Quercetin\'s anti-inflammatory property was exhibited as it down regulated COX-2. Anti-genotoxic effect of quercetin was evident in prevention of DNA damage and micronucleus formation.
CONCLUSIONS: Quercetin modulated OTA-induced oxidative stress and redox-signaling in HepG2 cells.
CONCLUSIONS: The results of the study demonstrate for the first time that quercetin prevents OTA-induced toxicity in HepG2 cells.