Intestinal microbiota via NLRP3 inflammasome dependent neuronal pyroptosis mediates anxiety-like behaviour in mice exposed to 3.5 GHz radiofrequency radiation.

The rapid development of 5G communication technology has increased public concern about the potential adverse effects on human health. Till now, the impacts of radiofrequency radiation (RFR) from 5G communication on the central nervous system and gut-brain axis are still unclear. Therefore, we investigated the effects of 3.5 GHz (a frequency commonly used in 5G communication) RFR on neurobehavior, gut microbiota, and gut-brain axis metabolites in mice. The results showed that exposure to 3.5 GHz RFR at 50 W/m2 for 1 h over 35 d induced anxiety-like behaviour in mice, accompanied by NLRP3-dependent neuronal pyroptosis in CA3 region of the dorsal hippocampus. In addition, the microbial composition was widely divergent between the sham and RFR groups. 3.5 GHz RFR also caused changes in metabolites of feces, serum, and brain. The differential metabolites were mainly enriched in glycerophospholipid metabolism, tryptophan metabolism, and arginine biosynthesis. Further correlation analysis showed that gut microbiota dysbiosis was associated with differential metabolites. Based on the above results, we speculate that dysfunctional intestinal flora and metabolites may be involved in RFR-induced anxiety-like behaviour in mice through neuronal pyroptosis in the brain. The findings provide novel insights into the mechanism of 5G RFR-induced neurotoxicity.

Effects of radiofrequency field from 5G communications on the spatial memory and emotionality in mice.

The rapid development of 5G network technology has gained much popularity as well as concerns about its adverse effects. In this study, we investigated the effects of 4.9 GHz (one of working frequencies of 5G communication) radiofrequency (RF) field on emotional behaviours and spatial memory in adult male mice. Open field test (OFT), tail suspension test (TST) and Y maze were used to evaluate anxiety, depression-like behaviour and spatial memory ability, respectively. It was found that the anxiety-like behaviour and spatial memory ability of mice did not change, but the depression-like behaviour was induced in mice after 4.9 GHz RF exposure. In addition, the number of neurons significantly reduced and the level of pyroptosis obviously increased in amygdala rather than hippocampus. These results suggested that 4.9 GHz RF exposure could induce depression-like behaviour, which might be associated with the neuronal pyroptosis in amygdala.

Abscopal effects of thoracic X-ray radiation on spermatogenesis in mice.

The study aimed to elucidate abscopal effects of thoracic X-ray irradiation on spermatogenesis in mice. Male C57BL/6 mice were randomly divided into sham group and radiation group, and subjected to thorax fractionated X-ray irradiation or sham irradiation with the total dose of 5 Gy/day for each animal for four consecutive days. After irradiation, sperm morphology was observed, and sperm number was counted under microscope, and sperm apoptosis was detected by flow cytometry. Meanwhile, testis index was calculated, testicular morphology was observed using haematoxylin-eosin (HE) staining, and testicular ultrastructure was observed under transmission electron microscopy. The permeability of blood-testis barrier (BTB) was detected by Evans Blue fluorescence colorimetry. The protein levels of Bcl-2 associated X protein (Bax), B-cell leukemia-lymphoma-2 (Bcl-2) and Cleaved caspase 3, promyelocytic leukaemia zinc finger (PLZF) and c-kit proto-oncogene (c-kit) in testes were determined by western blotting (WB). The location of apoptotic cells was confirmed by terminal deoxynucleotidyl transferase (TdT) enzymaticated dUTP nick end labelling (TUNEL) assay. The levels of tumor necrosis factor alpha (TNF-α), transforming growth factor-ß1 (TGF-ß1), interleukin 10 (IL-10) were measured by enzyme-linked immunosorbent assay (ELISA). The levels of Total superoxide dismutase (T-SOD) and malondialdehyde (MDA) were measured by the biochemical assay kit. Compared with sham group, the sperm quality of mice in radiation group showed decreased number and survival rate, along with increased abnormality and total apoptosis rate. The testis index of irradiated mice was lower, the testicular apoptosis was increased, and their testicular histology and ultrastructure was severely damaged. The permeability of BTB was increased, the level of PLZF in testis was decreased, and the level of c-kit was increased by irradiation. After irradiation, the levels of TNF-α, TGF-ß1, IL-10, T-SOD and MDA in testes were significantly changed. Taken together, abscopal effects of thoracic X-ray irradiation on spermatogenesis were obvious, which could decrease sperm quality and damage testicular morphology and increase the permeability of BTB, and a series of inflammation and oxidative stress factors were involved in the process. These findings provide novel insights into prevention and treatment for male reproductive damage induced by clinical thoracic irradiation.

The preventive and therapeutic effect of repetitive transcranial magnetic stimulation on radiation-induced brain injury in mice.

PURPOSE: To clarify the preventive and therapeutic effects of repetitive transcranial magnetic stimulation (rTMS) on brain injury induced by X-ray cranial irradiation, preliminarily identify the mechanism and provide a novel clinical approach for the prevention and treatment of radiation-induced brain injury (RBI). MATERIALS AND METHODS: Male C57BL/6 mice were randomly divided into the sham group, large fractionated dose (5 Gy × 4 d) group, large fractionated dose + rTMS (5 Gy × 4 d + rTMS) group, conventional fractionated dose (2 Gy × 10 d) group and conventional fractionated dose + rTMS (2 Gy × 10 d + rTMS) group. After cranial irradiation and rTMS, behavioral experiments, morphological staining and molecular biology experiments were performed. We further determined the mechanism of rTMS on the prevention and treatment of RBI, including changes in hippocampal neuronal apoptosis, neural stem cell (NSC) proliferation and differentiation, and neuronal synaptic plasticity. RESULTS: rTMS alleviated the negative effects of cranial radiation on the general health of mice and promoted their recovery. rTMS ameliorated the impairment of spatial learning and memory induced by cranial radiation, and this beneficial effect was more robust in the conventional fractionated dose group than the large fractionated dose group. Moreover, rTMS alleviated the alterations in hippocampal structure and neuronal death and had preventive and therapeutic effects against RBI. In addition, rTMS reduced hippocampal cell apoptosis, promoted NSC proliferation and differentiation in the hippocampus after cranial irradiation, and enhanced neuronal synaptic plasticity in the hippocampus. Subsequent studies showed that rTMS upregulated the expression of learning- and memory-related proteins. CONCLUSION: rTMS could alleviate learning and memory impairment caused by RBI, and the preventive and therapeutic effects of rTMS were better for the conventional fraction radiation paradigms.

Effects of 5.8 GHz microwave on hippocampal synaptic plasticity of rats.

OBJECTIVE: 5.8 GHz spectrum is gaining more attention in wireless technology. To explore the potential hazards, we investigated the effect of exposure to 5.8 GHz microwave on learning and memory ability of rats. Methods: Morris Water maze (MWM), Novel object recognition (NOR) and Fear conditioning test (FCT) were used to evaluate the ability of spatial and non-spatial memory of rats. The hippocampal morphology, the level of brain injury factors in serum and the mitochondrial membrane potential of hippocampal neurons was examined to evaluate the damage of hippocampal neurons. The density of dendritic spines, the ultrastructure of synapses and the level of PSD95, Synaptophysin, p-CREB and CREB were detected to evaluate the hippocampal synaptic plasticity. RESULTS: Compared with Sham group, there was no significant difference in the performance of ethology (in MWM, NOR, FCT) in Microwave 2 h group or Microwave 4 h group. The hippocampal morphology, the serum level of brain injury factors and the content of mitochondrial JC-1 monomer in Microwave 2 h group or Microwave 4 h group did not change obviously, compared with Sham group. The density of dendritic spines, the ultrastructure of synapse and the level of PSD95, Synaptophysin, p-CREB and CREB in hippocampus in Microwave 2 h group or Microwave 4 h group did not significantly change, compared with Sham group. CONCLUSION: Under this experimental condition, exposure to 5.8 GHz microwave could not affect the hippocampal synaptic plasticity of rats.

The Abscopal Effects of Cranial Irradiation Induce Testicular Damage in Mice.

To investigate whether the abscopal effects of cranial irradiation (C-irradiation) cause testicular damage in mice, male C57BL/6 mice (9weeks of age) were randomly divided into a sham irradiation group, a shielded group and a C-irradiation group and administered sham/shielded irradiation or C-irradiation at a dose rate of 2.33Gy/min (5Gy/d for 4 d consecutively). All mice were sacrificed at 4weeks after C-irradiation. We calculated the testis index, observed testicular histology by haematoxylin-eosin (HE) staining and observed testicular ultrastructure by transmission electron microscopy. Western blotting was used to determine the protein levels of Bax, Bcl-2, Cleaved caspase 3, glial cell line-derived neurotrophic factor (GDNF) and stem cell factor (SCF) in the testes of mice. Immunofluorescence staining was performed to detect the expression of Cleaved caspase 3 and 3ß hydroxysteroid dehydrogenase (3ßHSD), and a TUNEL assay was used to confirm the location of apoptotic cells. The levels of testosterone (T), GDNF and SCF were measured by ELISA. We also evaluated the sperm quality in the cauda epididymides by measuring the sperm count, abnormality, survival rate and apoptosis rate. The results showed that there was no significant difference in testicular histology, ultrastructure or sperm quality between the shielded group and sham group. Compared with the sham/shielded group, the C-irradiation group exhibited a lower testis index and severely damaged testicular histology and ultrastructure at 4weeks after C-irradiation. The levels of apoptosis in the testes increased markedly in the C-irradiation group, especially in spermatogonial stem cells. The levels of serum T and testicular 3ßHSD did not obviously differ between the sham group and the C-irradiation group, but the levels of GDNF and SCF in the testes increased in the C-irradiation group, compared with the sham group. In addition, the sperm count and survival rate decreased in the C-irradiation group, while the abnormality and apoptosis rate increased. Under these experimental conditions, the abscopal effects of C-irradiation induced testicular damage with regard to both structure and function and ultimately decreased sperm quality in mice. These findings provide novel insights into prevention and treatment targets for male reproductive damage induced by C-irradiation.

Cathodal tDCS exerts neuroprotective effect in rat brain after acute ischemic stroke.

BACKGROUND: Transcranial direct current stimulation (tDCS) is a non-invasive brain modulation technique that has been proved to exert beneficial effects in the acute phase of stroke. To explore the underlying mechanism, we investigated the neuroprotective effects of cathodal tDCS on brain injury caused by middle cerebral artery occlusion (MCAO). RESULTS: We established the MCAO model and sham MCAO model with an epicranial electrode implanted adult male Sprague-Dawley rats, and then they were randomly divided into four groups (MCAO + tDCS, MCAO + sham tDCS (Sham), Control + tDCS and Control + Sham group). In this study, the severity degree of neurological deficit, the morphology of brain damage, the apoptosis, the level of neuron-specific enolase and inflammatory factors, the activation of glial cells was detected. The results showed that cathodal tDCS significantly improved the level of neurological deficit and the brain morphology, reduced the brain damage area and apoptotic index, and increased the number of Nissl body in MCAO rats, compared with MCAO + Sham group. Meanwhile, the high level of NSE, inflammatory factors, Caspase 3 and Bax/Bcl2 ratio in MCAO rats was reduced by cathodal tDCS. Additionally, cathodal tDCS inhibited the activation of astrocyte and microglia induced by MCAO. No difference was found in two Control groups. CONCLUSION: Our results suggested that cathodal tDCS could accelerate the recovery of neurologic deficit and brain damage caused by MCAO. The inhibition of neuroinflammation and apoptosis resulted from cathodal tDCS may be involved in the neuroprotective process.

Effects of 220 MHz Pulsed Modulated Radiofrequency Field on the Sperm Quality in Rats.

Under some occupational conditions, workers are inevitably exposed to high-intensity radiofrequency (RF) fields. In this study, we investigated the effects of one-month exposure to a 220 MHz pulsed modulated RF field at the power density of 50 W/m² on the sperm quality in male adult rats. The sperm quality was evaluated by measuring the number, abnormality and survival rate of sperm cells. The morphology of testis was examined by hematoxylin-eosin (HE) staining. The levels of secreting factors by Sertoli cells (SCs) and Leydig cells (LCs) were determined by enzyme linked immunosorbent assay (ELISA). The level of cleaved caspase 3 in the testis was detected by immunofluorescence staining. Finally, the expression levels of the apoptosis-related protein (caspase 3, BAX and BCL2) in the testis were assessed by Western blotting. Compared with the sham group, the sperm quality in the RF group decreased significantly. The levels of secreting factors of SCs and the morphology of the testis showed an obvious change after RF exposure. The level of the secreting factor of LCs decreased significantly after RF exposure. The levels of cleaved caspase 3, caspase 3, and the BAX/BCL2 ratio in the testis increased markedly after RF exposure. These data collectively suggested that under the present experimental conditions, 220 MHz pulsed modulated RF exposure could impair sperm quality in rats, and the disruption of the secreting function of LCs and increased apoptosis of testis cells induced by the RF field might be accounted for by this damaging effect.

Effects of 1.8 GHz radiofrequency field on microstructure and bone metabolism of femur in mice.

To investigate the effects of 1.8 GHz radiofrequency (RF) field on bone microstructure and metabolism of femur in mice, C57BL/6 mice (male, age 4 weeks) were whole-body exposed or sham exposed to 1.8 GHz RF field. Specific absorption rates of whole body and bone were approximately 2.70 and 1.14 W/kg (6 h/day for 28 days). After exposure, microstructure and morphology of femur were observed by microcomputed tomography (micro-CT), Hematoxylin and Eosin (HE) and Masson staining. Subsequently, bone parameters were calculated directly from the reconstructed images, including structure model index, bone mineral density, trabecular bone volume/total volume, connectivity density, trabecular number, trabecular thickness, and trabecular separation. Biomarkers that reflect bone metabolism, such as serum total alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP), and tartrate-resistant acid phosphatase 5b (TRACP-5b), were determined by biochemical assay methods. Micro-CT and histology results showed that there was no significant change in bone microstructure and the above parameters in RF group, compared with sham group. The activity of serum ALP and BALP increased 29.47% and 16.82%, respectively, in RF group, compared with sham group (P < 0.05). In addition, there were no significant differences in the activity of serum TRACP-5b between RF group and sham group. In brief, under present experimental conditions, we did not find support for an effect of 1.8 GHz RF field on bone microstructure; however, it might promote metabolic function of osteoblasts in mice. Bioelectromagnetics. 39:386-393, 2018. © 2018 Wiley Periodicals, Inc.

Effects of 1.8 GHz Radiofrequency Fields on the Emotional Behavior and Spatial Memory of Adolescent Mice.

The increasing use of mobile phones by teenagers has raised concern about the cognitive effects of radiofrequency (RF) fields. In this study, we investigated the effects of 4-week exposure to a 1.8 GHz RF field on the emotional behavior and spatial memory of adolescent male mice. Anxiety-like behavior was evaluated by open field test (OFT) and elevated plus maze (EPM) test, while depression-like behavior was evaluated by sucrose preference test (SPT), tail suspension test (TST) and forced swim test (FST). The spatial learning and memory ability were evaluated by Morris water maze (MWM) experiments. The levels of amino acid neurotransmitters were determined by liquid chromatography-mass spectrometry (LC-MS). The histology of the brain was examined by hematoxylin-eosin (HE) staining. It was found that the depression-like behavior, spatial memory ability and histology of the brain did not change obviously after RF exposure. However, the anxiety-like behavior increased in mice, while, the levels of γ-aminobutyric acid (GABA) and aspartic acid (Asp) in cortex and hippocampus significantly decreased after RF exposure. These data suggested that RF exposure under these conditions do not affect the depression-like behavior, spatial memory and brain histology in adolescent male mice, but it may however increase the level of anxiety, and GABA and Asp were probably involved in this effect.

Enhancement of X-ray Induced Apoptosis by Mobile Phone-Like Radio-Frequency Electromagnetic Fields in Mouse Spermatocyte-Derived Cells.

To explore the combined effects of environmental radio-frequency (RF) field and X-ray, mouse spermatocyte-derived (GC-1) cells were exposed to 1950 MHz RF field at specific absorption rate (SAR) of 3 W/kg for 24 h combined with or without X-ray irradiation at 6 Gy. After treatment, the cell proliferation level was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) Assay and 5-Bromo-2-deoxy Uridine (BrdU) enzyme linked immunosorbent (ELISA) Assay. The apoptosis level was detected by annexin V flow cytometry assay, transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) Assay and Caspase-3 Activity Assay. It was found that the proliferation and apoptosis level did not change in GC-1 cells after RF exposure alone. However, compared with the X-ray group, the proliferation level significantly decreased and the apoptotic rate significantly increased in the RF+X-ray group. Moreover, a significant decrease in Bcl-2 protein expression and increase in Bax protein expression were observed. The findings suggested that RF exposure at SAR of 3 W/kg did not affect apoptosis and proliferation in GC-1 cells by itself, but that it did enhance the effects of X-ray induced proliferation inhibition and apoptosis, in which B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) might be involved.

Effects of electromagnetic pulse exposure on gelatinase of blood-brain barrier in vitro.

The biological effects of electromagnetic pulse (EMP) on the brain have been focused on for years. It was reported that gelatinase played an important role in maintaining brain function through regulating permeability in the blood-brain barrier (BBB). To investigate the effects of EMP on gelatinase of BBB, an in vitro BBB model was established using primary cultured rat brain microvascular endothelial cells (BMVEC), astrocytes and half-contact culture of these cells in a transwell chamber. Cultured supernatant and cells were collected at different time points after exposure to EMP (peak intensity 400 kV/m, rise time 10 ns, pulse width 350 ns, 0.5 pps and 200 pulses). Protein levels of cellular gelatinase MMP-2 and MMP-9, and endogenous inhibitor TIMP-1 and TIMP-2 were detected by Western blot. The activity of gelatinase in culture supernatant was detected by gelatin zymography. It was found that compared with the sham-exposed group, the protein level of MMP-2 was significantly increased at 6 h (p < 0.05), and the protein level of its endogenous inhibitor TIMP-2 did not change after EMP exposure. In addition, the protein levels of MMP-9 and its endogenous inhibitor TIMP-1 did not change after EMP exposure. Gelatin zymography results showed that the activity of MMP-2 in the inner pool and the outer pool of the transwell chamber was significantly increased at 6 h after EMP exposure compared with that of the sham group. These results suggested that EMP exposure could affect the expression and activity of MMP-2 in the BBB model.

Electromagnetic pulse activated brain microglia via the p38 MAPK pathway.

Previously, we found that electromagnetic pulses (EMP) induced an increase in blood brain barrier permeability and the leakage of albumin from blood into brain tissue. Albumin is known to activate microglia cells. Thus, we hypothesised that microglia activation could occur in the brain after EMP exposure. To test this hypothesis, the morphology and secretory function of microglia cells, including the expression of OX-42 (a marker of microglia activation), and levels of TNF-α, IL-10, IL-1ß, and NO were determined in the rat cerebral cortex after EMP exposure. In addition, to examine the signalling pathway of EMP-induced microglia activation, protein and phosphorylated protein levels of p38, JNK and ERK were determined. It was found that the expression of OX-42increased significantly at 1, 6 and 12h (p<0.05) and recovered to the sham group level at 24h after EMP exposure. Levels of NO, TNF-α and IL-10 also changed significantly in vivo and in vitro after EMP exposure. The protein level of p38 and phosphorylated p38 increased significantly after EMP exposure (p<0.05) and recovered to sham levels at 12 and 24h, respectively. Protein and phosphorylated protein levels of ERK and JNK did not change. SB203580 (p38 inhibitor) partly prevented the change in NO, IL-10, IL-1ß, TNF-α levels induced by EMP exposure. Taken together, these results suggested that EMP exposure (200kV/m, 200 pulses) could activate microglia in rat brain and affect its secretory function both in vivo and in vitro, and the p38 pathway is involved in this process.

Effects of long-term 50Hz power-line frequency electromagnetic field on cell behavior in Balb/c 3T3 cells.

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn't change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein.

[Effect of long-term power frequency electromagnetic field exposure on proliferation and apoptosis of SRA01/04 cells].

OBJECTIVE: To investigate the effect of long-term power frequency electromagnetic field (50 Hz) exposure on the proliferation and apoptosis of human lens epithelial cells (SRA01/04 cells). METHODS: SRA01/04 cells in the exponential growth phase were exposed or sham-exposed to power frequency electromagnetic field (50 Hz, 2.3 mT) for 2 hours per day, 5 days every week. After 11 weeks of exposure, the cells were collected; the cell morphology was observed under a microscope, the cell viability was measured by MTT assay, the cell cycle and apoptosis were examined by flow cytometry, and the protein expression levels of cyclin D and proliferating cell nuclear antigen (PCNA) were determined by western blot. RESULTS: Compared with the sham-exposed SRA01/04 cells, most exposed cells became rounded and more stereoscopic, and heterochromatin gathered near the nuclear membrane in some exposed cells. The MTT assay showed that the viability of exposed cells was significantly increased compared with that of the sham-exposed cells (P < 0.05). Long-term power frequency electromagnetic field exposure led to significantly increased number of cells in S phase (P < 0.05), and the proliferation index was significantly higher in the exposed cells than in the sham-exposed cells (P < 0.05). There was no significant difference in apoptotic rate between the exposed cells and sham-exposed cells (P > 0.05). The exposed cells had significantly higher protein expression levels of cyclin D and PCNA than the sham-exposed cells (P < 0.05). CONCLUSION: Long-term power frequency electromagnetic field exposure can promote cellular proliferation and change cell cycle in SRA01/04 cells, but it has no marked effect on the apoptosis of SRA01/04 cells.

Detrimental effect of electromagnetic pulse exposure on permeability of in vitro blood-brain-barrier model.

OBJECTIVE: To study the effect of electromagnetic pulse (EMP) exposure on permeability of in vitro blood-brain-barrier (BBB) model. METHODS: An in vitro BBB model, established by co-culturing brain microvascular endothelial cells (BMVEC) and astroglial cells (AC) isolated from rat brain, was exposed to EMP at 100 kV/m and 400 kV/m, respectively. Permeability of the model was assayed by measuring the transendothelial electrical resistance (TEER) and the horseradish peroxidase (HRP) transmission at different time points. Levels of BBB tight junction-related proteins were measured at 0, 1, 2, 4, 8, 12, 16, 20, 24 h after EMP exposure by Western blotting. RESULTS: The TEER level was lower in BBB model group than in control group at 12 h after EMP, exposure which returned to its normal level at 24 h. The 24 h recovery process was triphasic and biphasic respectively after EMP exposure at 100 kV/m and 400 kV/m. Following exposure to 400 kV/m EMP, the HRP permeability increased at 1-12 h and returned to its normal level at 24 h. Western blotting showed that the claudin-5 and ZO-1 protein levels were changed after EMP exposure. CONCLUSION: EMP exposure at 100 kV/m and 400 kV/m can increase the permeability of in vitro BBB model and BBB tight junction-related proteins such as ZO-1 and claudin-5 may change EMP-induced BBB permeability.

Altered expression of matrix metalloproteinases and tight junction proteins in rats following PEMF-induced BBB permeability change.

OBJECTIVE: To investigate the expression of occludin, ZO-1, MMP-2, and MMP-9 in cerebral microvasculature following Pulse Electromagnetic Field (PEMF) induced BBB permeability change. METHODS: Sprague-Dawley rats were randomized into PEMF and sham exposed groups (n = 8). After exposure to PEMF at 0.5, 1, 3, 6, and 12 h, BBB permeability was measured by Evans-Blue extravasation. The expression of occludin, ZO-1, MMP-2, and MMP-9 were detected by real-time quantitative reverse transcriptase PCR and western blotting. MMP-2 and MMP-9 activity were detected by EnzChek gelatinase assay. RESULTS: Compared with the sham group, PEMF exposure led to increased permeability of the BBB to EB, which was prolonged after exposure. BBB permeability became progressively more severe, and recovered at 6 h. The gene and protein expression of occludin and ZO-1 were significantly decreased, while MMP-2 and MMP-9 expression were significantly increased after exposure to PEMF. All levels of expression recovered 12 h following PEMF. CONCLUSION: Changes to BBB permeability were related to the alteration expression of tight junction proteins and matrix metalloproteinase after exposure to PEMF.

[Effects of electromagnetic pulse exposure on the morphological change and excretion function of BV-2 cells and possible mechanism].

OBJECTIVE: To study the effects of electromagnetic pulse (EMP) exposure on the morphological change and excretion functions of mouse microglia (BV-2) cells and possible mechanism. METHODS: BV-2 cells were divided into two groups: the group exposed to EMP at 200 kV/m for 200 pulses and sham exposure group. At 1, 6, 12 and 24 hour after exposure the cells and culture supernatant were collected. Cellular morphological change was observed under invert microscope, the levels of TNF-α, IL-1ß and IL-10 in culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA), nitric oxide (NO) and reactive oxygen species (ROS) were detected by nitrate reductase method and DCFH-DA probe, respectively. The protein and phosphorylation levels of ERK, JNK and p38 were measured by Western Blot method. After the cells pre-treated with the inhibitor of p38 (SB203580) were exposed to EMP, the levels of NO and ROS in culture supernatant were detected. RESULTS: It was found that the large ameboid shape appeared in some microglia cells exposed to EMP for 1, 6 and 12 h. Moreover, the number of microglia cells with ameboid shape increased significantly at 1 h, 6 h and 12 h after EMP exposure compared with sham group (P < 0.05). The levels of cytokines, such as TNF-α, IL-1ß and IL-10, in culture supernatant did not change obviously after EMP exposure. The levels of NO and ROS increased significantly at 1h after EMP exposure, reached the peak at 6 h, began to recover at 12 h and recovered to sham group level at 24 h (P < 0.05). Western blot results showed that the protein and protein phosphorylation levels of ERK and JNK did not change significantly after EMP exposure, however, the protein and protein phosphorylation levels of p38 increased obviously at 1 h and 6 h after EMP exposure, compared with sham group (P < 0.05). In addition, the pretreatment of p38 inhibitor (SB203580) significantly decreased NO and ROS production induced by EMP. CONCLUSION: EMP exposure may activate microglia cells and promote the production of NO and ROS in mouse microglia cells, and p38 pathway is involved in this process.

Ionizing radiation promotes migration and invasion of cancer cells through transforming growth factor-beta-mediated epithelial-mesenchymal transition.

PURPOSE: To examine whether ionizing radiation enhances the migratory and invasive abilities of cancer cells through transforming growth factor (TGF-ß)-mediated epithelial-mesenchymal transition (EMT). METHODS AND MATERIALS: Six cancer cell lines originating from different human organs were irradiated by 60Co γ-ray at a total dose of 2 Gy, and the changes associated with EMT, including morphology, EMT markers, migration and invasion, were observed by microscope, Western blot, immunofluorescence, scratch assay, and transwell chamber assay, respectively. Then the protein levels of TGF-ß in these cancer cells were detected by enzyme-linked immunosorbent assay, and the role of TGF-ß signaling pathway in the effect of ionizing radiation on EMT was investigate by using the specific inhibitor SB431542. RESULTS: After irradiation with γ-ray at a total dose of 2 Gy, cancer cells presented the mesenchymal phenotype, and compared with the sham-irradiation group the expression of epithelial markers was decreased and of mesenchymal markers was increased, the migratory and invasive capabilities were strengthened, and the protein levels of TGF-ß were enhanced. Furthermore, events associated with EMT induced by IR in A549 could be reversed through inhibition of TGF-ß signaling. CONCLUSIONS: These results suggest that EMT mediated by TGF-ß plays a critical role in IR-induced enhancing of migratory and invasive capabilities in cancer cells.

Synthetic gelatinases inhibitor attenuates electromagnetic pulse-induced blood-brain barrier disruption by inhibiting gelatinases-mediated ZO-1 degradation in rats.

Previously we found that exposure to electromagnetic pulse (EMP) induced an increase in blood-brain-barrier (BBB) permeability and the degradation of tight junction protein ZO-1 in rats. Matrix metalloproteinases (MMPs), in particular gelatinases (MMP-2 and MMP-9), play a key role in degradation of tight junction proteins, are known mediators of BBB compromise. We hypothesized that the degradation of ZO-1 by gelatinases contributed to EMP-induced BBB opening. To test this hypothesis, the mRNA level of ZO-1, protein levels of MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) were detected in rat cerebral cortex after exposing rats to EMP at 200 kV/m for 200 pulses. It was found that the mRNA level of ZO-1 was unaltered at different time points after EMP exposure. The protein levels of MMP-2 and MMP-9 significantly increased at 3 h and 0.5 h, respectively. However, TIMP-1 (inhibitor of MMP-9) and TIMP-2 (inhibitor of MMP-2) only moderately increased after EMP exposure. In addition, in situ zymography results showed that the gelatinase activity increased in cerebral microvessels at 3 h after EMP exposure. When rats were treated with gelatinases inhibitor (SB-3CT) before EMP exposure, the EMP-induced BBB opening was attenuated and the ZO-1 degradation was reversed. Our results suggested that EMP-induced BBB opening was related to gelatinase mediated ZO-1 degradation.