Exposure to Methylmercury at Juvenile Stage Worsens Autism-like Symptoms in Adult BTBR T+tf/J Mice Due to Lack of Nuclear Factor Erythroid 2-Related Factor 2 Signaling Upregulation in Periphery and Brain.

Autism spectrum disorder (ASD) is a multifaceted developmental condition that first appears in infancy. The condition is characterized by recurrent patterns in behavior and impairments in social and vocalization abilities. Methylmercury is a toxic environmental pollutant, and its derivatives are the major source of organic mercury to human beings. Inorganic mercury, which is released from a variety of pollutants into oceans, rivers, and streams, is transformed into methylmercury by bacteria and plankton in the water, which later builds up in fish and shellfish, and then enters humans through the consumption of fish and shellfish and increases the risk of developing ASD by disturbing the oxidant-antioxidant balance. However, there has been no prior research to determine the effect of juvenile exposure of methylmercury chloride on adult BTBR mice. Therefore, the current study evaluated the effect of methylmercury chloride administered during the juvenile stage on autism-like behavior (three-chambered sociability, marble burying, self-grooming tests) and oxidant-antioxidant balance (specifically Nrf2, HO-1, SOD-1, NF-kB, iNOS, MPO, and 3-nitrotyrosine) in the peripheral neutrophils and cortex of adult BTBR and C57BL/6 (B6) mice. Our results show that exposure to methylmercury chloride at a juvenile stage results in autism-like symptoms in adult BTBR mice which are related to a lack of upregulation of the Nrf2 signaling pathway as demonstrated by no significant changes in the expression of Nrf2, HO-1, and SOD-1 in the periphery and cortex. On the other hand, methylmercury chloride administration at a juvenile stage increased oxidative inflammation as depicted by a significant increase in the levels of NF-kB, iNOS, MPO, and 3-nitrotyrosine in the periphery and cortex of adult BTBR mice. This study suggests that juvenile exposure to methylmercury chloride contributes to the worsening of autism-like behavior in adult BTBR mice through the disruption of the oxidant-antioxidant balance in the peripheral compartment and CNS. Strategies that elevate Nrf2 signaling may be useful to counteract toxicant-mediated worsening of ASD and may improve quality of life.

Genes Involved in DNA Repair and Mitophagy Protect Embryoid Bodies from the Toxic Effect of Methylmercury Chloride under Physioxia Conditions.

The formation of embryoid bodies (EBs) from human pluripotent stem cells resembles the early stages of human embryo development, mimicking the organization of three germ layers. In our study, EBs were tested for their vulnerability to chronic exposure to low doses of MeHgCl (1 nM) under atmospheric (21%O2) and physioxia (5%O2) conditions. Significant differences were observed in the relative expression of genes associated with DNA repair and mitophagy between the tested oxygen conditions in nontreated EBs. When compared to physioxia conditions, the significant differences recorded in EBs cultured at 21% O2 included: (1) lower expression of genes associated with DNA repair (ATM, OGG1, PARP1, POLG1) and mitophagy (PARK2); (2) higher level of mtDNA copy number; and (3) higher expression of the neuroectodermal gene (NES). Chronic exposure to a low dose of MeHgCl (1 nM) disrupted the development of EBs under both oxygen conditions. However, only EBs exposed to MeHgCl at 21% O2 revealed downregulation of mtDNA copy number, increased oxidative DNA damage and DNA fragmentation, as well as disturbances in SOX17 (endoderm) and TBXT (mesoderm) genes expression. Our data revealed that physioxia conditions protected EBs genome integrity and their further differentiation.

Oxygen as an important factor modulating in vitro MeHgCl toxicity associated with mitochondrial genes in hiPSCs.

Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.

Assessment of the effects of organic vs. inorganic arsenic and mercury in Caenorhabditis elegans.

Exposures to mercury and arsenic are known to pose significant threats to human health. Effects specific to organic vs. inorganic forms of these toxic elements are less understood however, especially for organic dimethylarsinic acid (DMA), which has recently been detected in pups of rodent dams orally exposed to inorganic sodium (meta)arsenite (NaAsO2). Caenorhabditis elegans is a small animal alternative toxicity model. To fill data gaps on the effects of DMA relative to NaAsO2, C. elegans were exposed to these two compounds alongside more thoroughly researched inorganic mercury chloride (HgCl2) and organic methylmercury chloride (meHgCl). For timing of developmental milestone acquisition in C. elegans, meHgCl was 2 to 4-fold more toxic than HgCl2, and NaAsO2 was 20-fold more toxic than DMA, ranking the four compounds meHgCl > HgCl2 > NaAsO2 ≫ DMA for developmental toxicity. Methylmercury induced significant decreases in population locomotor activity levels in developing C. elegans. DMA was also associated with developmental hypoactivity, but at >100-fold higher concentrations than meHgCl. Transcriptional alterations in native genes were observed in wild type C. elegans adults exposed to concentrations equitoxic for developmental delay in juveniles. Both forms of arsenic induced genes involved in immune defense and oxidative stress response, while the two mercury species induced proportionally more genes involved in transcriptional regulation. A transgenic bioreporter for activation of conserved proteosome specific unfolded protein response was strongly activated by NaAsO2, but not DMA at tested concentrations. HgCl2 and meHgCl had opposite effects on a bioreporter for unfolded protein response in the endoplasmic reticulum. Presented experiments indicating low toxicity for DMA in C. elegans are consistent with human epidemiologic data correlating higher arsenic methylation capacity with resistance to arsenic toxicity. This work contributes to the understanding of the accuracy and fit-for-use categories for C. elegans toxicity screening and its usefulness to prioritize compounds of concern for further testing.

Methylmercury chloride exposure exacerbates existing neurobehavioral and immune dysfunctions in the BTBR T+ Itpr3tf/J mouse model of autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disease characterized by impaired communication, impaired reciprocal social interaction, restricted sociability deficits, and the presence of stereotyped patterns of behaviors. Immune dysregulation has been suggested to play a possible etiological role in ASD. Recent studies have demonstrated that exposure to methylmercury chloride (MeHgCl) leads to abnormal gait, motor deficits, impaired hearing, and memory deficits; however, its effects on behavioral and immunological responses have not been adequately investigated in ASD. In this study, we investigated the effects of MeHgCl exposure on marble burying, self-grooming behaviors, sociability tests, and locomotor activities in BTBR T+ Itpr3tf/J (BTBR) mice. We also explored the possible molecular mechanism underlying the effects of MeHgCl administration on IFN-γ-, T-bet-, IL-9-, and IL-17A-producing CD4+, CXCR5+, CXCR6+, and CCR9+ cells isolated from spleens. Furthermore, the effects of MeHgCl exposure on the mRNA expression and levels of pro-inflammatory cytokines in the brain tissue and serum samples were also assessed. Our results demonstrated that MeHgCl exposure caused a significant increase in marble burying, self-grooming behaviors and a decrease in social interactions and adverse effects on locomotor activity in BTBR mice. MeHgCl exposure also significantly increased the production of CD4+IFN-γ+, CD4+T-bet+, CCR9+T-bet+, CXCR5+IL-9+, CD4+IL-9+, CXCR6+IL-17A+, and CD4+IL-17A+ cells in the spleen. Furthermore, MeHgCl exposure increased mRNA and protein levels of pro-inflammatory cytokines in the brain and serum respectively in BTBR mice. In conclusion, MeHgCl administration aggravated existing behavioral and immune abnormalities in BTBR mice.

Methylmercury chloride exposure aggravates proinflammatory mediators and Notch-1 signaling in CD14+ and CD40+ cells and is associated with imbalance of neuroimmune function in BTBR T+ Itpr3tf/J mice.

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder characterized by deficits in social interaction, communication, and repetitive behaviors. A key role for immune dysfunction has been suggested in ASD. Recent studies have indicated that inflammatory mediators and Notch-1 signaling may contribute to the development of ASD. Methylmercury chloride (MeHgCl) is an environmental pollutant that primarily affects the central nervous system, causing neurological alterations. Its effects on immunological responses have not been fully investigated in ASD. In this study, we examined the influence of MeHgCl exposure on inflammatory mediators and Notch-1 signaling in BTBR T+ Itpr3tf/J (BTBR) mice, a model of ASD. We examined the effects of MeHgCl on the IL-6-, GM-CSF-, NF-κB p65-, Notch-1-, and IL-27-producing CD14+ and CD40+ cells in the spleen. We assessed the effect of MeHgCl on IL-6, GM-CSF, NF-κB p65, Notch-1, and IL-27 mRNA levels in brain tissue. We also measured IL-6, GM-CSF, and NF-κB p65 protein expression levels in brain tissue. MeHgCl exposure of BTBR mice significantly increased IL-6-, GM-CSF-, NF-κB p65-, and Notch-1-, and decreased IL-27-producing CD14+, and CD40+ cells in the spleen. MeHgCl exposure of BTBR mice upregulated IL-6, GM-CSF, NF-κB p65, and Notch-1, and decreased IL-27 mRNA expression levels in brain tissue. Moreover, MeHgCl resulted in elevated expression of the IL-6, GM-CSF, and NF-κB p65 proteins in brain tissue. Taken together, these results indicate that MeHgCl exposure aggravates proinflammatory mediators and Notch-1 signaling which are associated with imbalance of neuroimmune function in BTBR mice.

Comparative study on toxicity of methylmercury chloride and methylmercury hydroxide to the human neuroblastoma cell line SH-SY5Y.

Toxicities of methylmercury chloride (CH3HgCl) and methylmercury hydroxide (CH3HgOH) to cultured neuroblastoma cell line SH-SY5Y in vitro are evaluated. This is the comparative study between two methylmercury compounds to find out the extent of toxicity of these compounds are toxic to SH-SY5Y cell line. Both cytotoxicity and genotoxicity experiments were carried out to find out the more toxic compound. For cytotoxicity study, four staining assay methods independently with trypan blue (TB), acridine orange/ethidium bromide (AO/EB), 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT), and neutral red (NR) were used and the comet assay method was done for genotoxicity study. The obtained toxicity data were used for probit analysis. In cytotoxicity, CH3HgCl had minimum inhibitory concentration (MIC) value in each assay method as 3 mg/L invariably; LC25 values were in the range 7.41 to 10.23 mg/L, and LC50 values were 14.79 to 15.48 mg/L; while LC75 values were 20.89 to 26.91 mg/L. Moreover, LC100 value was 30 mg/L, known from comet assay experiments for CH3HgCl. Similarly for CH3HgOH, the MIC value in each assay method was invariably 3 mg/L, the LC25 values were in the range 12.58 to 16.59 mg/L, and LC50 values were 19.49 to 23.44 mg/L; LC75 values were 27.54 to 30.90 mg/L and LC100 value was 42 mg/L in each assay done for cytotoxicity and genotoxicity studies. Computed DNA fragmentation indices in comet assays were 98.6 ± 0.57 30 mg/L with CH3HgCl and 76 ± 5.29 30 mg/L with CH3HgOH. This study clearly indicated that methylmercury chloride is more toxic than methylmercury hydroxide to SH-SY5Y cell line. Toxicity of Hg had been quantified with in vitro cultured human neuroblastoma cell line; since it has neurotoxic effects, its neural evaluation has implications in environmental health issues.

Form of Dietary Methylmercury does not Affect Total Mercury Accumulation in the Tissues of Zebra Finch.

Exposure to mercury in humans, other mammals, and birds is primarily dietary, with mercury in the methylated form and bound to cysteine in the tissues of prey items. Yet dosing studies are generally carried out using methylmercury chloride. Here we tested whether the accumulation of total mercury in zebra finch blood, egg, muscle, liver, kidney or brain differed depending on whether dietary mercury was complexed with chloride or cysteine. We found no effect of form of mercury on tissue accumulation. Some previous studies have found lower accumulation of mercury in tissues of animals fed complexed mercury. Much remains to be understood about what happens to ingested mercury once it enters the intestines, but our results suggest that dietary studies using methylmercury chloride in birds will produce similar tissue accumulation levels to those using methylmercury cysteine.

Effect of Lycium bararum polysaccharides on methylmercury-induced abnormal differentiation of hippocampal stem cells.

The aim of the present study was to observe the effects of a general extract of Lycium bararum polysaccharides (LBPs) on methylmercury (MeHg)-induced damage in hippocampus neural stem cells (hNSCs). The hippocampal tissues of embryonic day 16 Sprague-Dawley rats were extracted for the isolation, purification and cloning of hNSCs. Following passage and proliferation for 10 days, the cells were allocated at random into the following groups: Control, LBPs, MeHg and MeHg + LBPs. MTT and microtubule-associated protein 2 (MAP-2)/glial fibrillary acidic protein/Hoechst immunofluorescence tests were performed to detect the differentiation and growth of hNSCs in the various groups. The differentiation rate of MeHg-treated hNSCs and the perimeter of MAP-2-positive neurons were 3.632±0.63% and 62.36±5.58 µm, respectively, significantly lower compared with the control group values of 6.500±0.81% and 166±8.16 µm (P<0.05). Furthermore, the differentiation rate and the perimeter of MAP-2-positive neurons in LBPs groups cells was 7.75±0.59% and 253.3±11.21 µm, respectively, significantly higher compared with the control group (P<0.05). The same parameters in the MeHg + LBPs group were 5.92±0.98% and 111.9±6.07 µm, respectively, significantly higher than the MeHg group (P<0.05). The astrocyte differentiation rates in the MeHg and MeHg + LBPs group were 41.19±2.14 and 34.58±1.70, respectively (P<0.05). These results suggest that LBPs may promote the generation and development of new neurons and inhibit the MeHg-induced abnormal differentiation of astrocytes. Thus, LBPs may be considered to be a potential new treatment for MeHg-induced neurotoxicity in hNSCs.

The mechanisms associated with the development of hypertension after exposure to lead, mercury species or their mixtures differs with the metal and the mixture ratio.

Hypertension is considered to be the most important risk factor for the development of cardiovascular diseases. Beside life-style risk factors, exposure to lead and mercury species are increasingly discussed as potential risk factors. Although there are a few previous studies, the underlying mechanism by which exposure to lead and mercury disturb blood pressure regulation is not currently understood. Potential mechanisms are oxidative stress production, kidney damage and activation of the renin-angiotensin system (RAS), all of which can interact to cause dysregulation of blood pressure. Male rats (Wistar) were exposed to lead, inorganic mercury, methylmercury or two mixtures of all three metals for four weeks through the drinking water. The two mixture ratios were based on ratios of known reference values or environmental exposure from the literature. To investigate the potential mechanism of actions, blood pressure was measured after four weeks and compared to plasma nitrotyrosine or reduced/oxidized glutathione levels in liver as markers for oxidative stress. Plasma renin and angiotensin II levels were used as markers for RAS activation. Finally, kidney function and injury were assessed via urinary and plasma creatinine levels, creatinine clearance and urinary kidney-injury molecule (KIM-1). While exposure to lead by itself increased oxidative stress and kidney damage along with blood pressure, inorganic mercury did not affect blood pressure or any end-point examined. Conversely, methylmercury instead increased RAS activation along with blood pressure. Surprisingly, when administered as mixtures, lead no longer increased oxidative stress or altered kidney function. Moreover, the mixture based on an environmental ratio no longer had an effect on blood pressure, while the reference value ratio still retained an increase in blood pressure. Based on our results, the prominent mechanism of action associated with the development of hypertension seems to be oxidative stress and kidney damage for lead, while increased RAS activation links methylmercury to hypertension, but these mechanisms along with hypertension disappear when metals are present in some mixtures.

Effects of Methylmercury chloride exposure on PKCδ expression in rat developmental cerebellum / 中国免疫学杂志

Objective:To investigate the effect of Methylmercury chloride chronic exposure on PKCδexpression developing cer-ebellum.Methods:Establishment of cerebellum damage model of developmental rats by chronic MMC exposure .In Situ Hybridization and Western blot analysis were performed to detect the expression of PKC isozyme .Results: PKCδ was expressed at high levels at birth, but no significant change was observed with the increase in the time of birth corresponding to brain Hg 2+level, expression of PKCδ in cerebellum of experimental groups was markedly higher than that of control group .Conclusion: Neurotoxicity of Methylmercury chloride exposure might be mediated by PKCδexpression up-regulating in developmental cerebellum .

Cardiovascular responses to lead are biphasic, while methylmercury, but not inorganic mercury, monotonically increases blood pressure in rats.

Cardiovascular diseases, such as heart attack and stroke, are the major cause of death worldwide. It is well known that a high number of environmental and physiological risk factors contribute to the development of cardiovascular diseases. Although risk factors are additive, increased blood pressure (hypertension) is the greatest risk factor. Over the last two decades, a growing number of epidemiological studies associate environmental exposure to lead or mercury species with hypertension. However, cardiovascular effects beyond blood pressure are rarely studied and thresholds for effect are not yet clear. To explore effects of lead or mercury species on the cardiovascular system, normal male Wistar rats were exposed to a range of doses of lead, inorganic mercury or methylmercury through the drinking water for four weeks. High-resolution ultrasound was used to measure heart and vascular function (carotid artery blood flow) at baseline and at the end of the exposure, while blood pressure was measured directly in the femoral artery at the end of the 4-week exposure. After 4 weeks, blood pressure responses to lead were biphasic. Low lead levels decreased blood pressure, dilated the carotid artery and increased cardiac output. At higher lead doses, rats had increased blood pressure. In contrast, methylmercury-exposed rats had increased blood pressure at all doses despite dilated carotid arteries. Inorganic mercury did not show any significant cardiovascular effects. Based on the current study, the benchmark dose level 10% (BMDL10s) for systolic blood pressure for lead, inorganic mercury and methylmercury are 1.1, 1.3 and 1.0 µg/kg-bw/d, respectively. However, similar total mercury blood levels attributed to inorganic mercury or methylmercury produced strikingly different results with inorganic mercury having no observable effect on the cardiovascular system but methylmercury increasing systolic and pulse pressures. Therefore, adverse cardiovascular effects cannot be predicted by total blood mercury level alone and the mercury species of exposure must be taken into account.

Methylmercury chloride damage to the adult rat hippocampus cannot be detected by proton magnetic resonance spectroscopy.

Previous studies have found that methylmercury can damage hippocampal neurons and accordingly cause cognitive dysfunction. However, a non-invasive, safe and accurate detection method for detecting hippocampal injury has yet to be developed. This study aimed to detect methylmercury-induced damage on hippocampal tissue using proton magnetic resonance spectroscopy. Rats were given a subcutaneous injection of 4 and 2 mg/kg methylmercury into the neck for 50 consecutive days. Water maze and pathology tests confirmed that cognitive function had been impaired and that the ultrastructure of hippocampal tissue was altered after injection. The results of proton magnetic resonance spectroscopy revealed that the nitrogen-acetyl aspartate/creatine, choline complex/creatine and myoinositol/creatine ratio in rat hippocampal tissue were unchanged. Therefore, proton magnetic resonance spectroscopy can not be used to determine structural damage in the adult rat hippocampus caused by methylmercury chloride.

The effect of methylmercury on the epithelial apoptosis of fetal mice intestine in vitro / 重庆医科大学学报

Objective:To study the effect of methylmercury chloride(MMC) on the apoptosis and the expression of apoptosis-associated proteins in the intestinal epithelium of fetal mice.Methods:The duodenum and colon were taken from E13.5d and E14.5d fetal mice respectively,and cultured with different doses of MMC(0 ?g/L,200?g/L,400?g/L,800?g/L) for 24h.The samples were sliced into the paraffin sections.Some sections were stained with HE to count the numbers of apoptotic bodies(NAB) in the fetal intestinal epithelium with stereological method.The others were stained with the immunohistochemical method to observe the expression of apoptosis-associated proteins Bcl-2,Bax,P53 and c-Fos.Results:①In experimental groups,all the NAB in the intestinal epithelium were higher than that in control groups(P

Protective effects of garlic juice against embryotoxicity of methylmercuric chloride administered to pregnant Fischer 344 rats

In order to investigate the beneficial effects of 0.5 or 1.0 g/kg Korean garlic juice against the embryotoxicity of 20 mg/kg methylmercury chloride (MMC, CH3HgCl), pregnant Fisher 344 rats were simultaneously orally administered on day 7 of gestation. On day 20 of gestation the dams were laparotomized under ether anesthesia, and the fetuses were removed and examined for toxicity of methylmercury. Garlic juice depressed the toxicity in terms of some parameters. In the case of simultaneous treatment with 0.1 g/kg garlic juice and MMC, rates of increase were 17.5% in maternal body weight, 13.2% and 41.9% in fetal and litters' weight respectively, and 37.0% in fetal survival rate. Decreasing rates were 10.0% in maternal death rate, and 6.9% and 31.3% in pre- and post-implantation loss respectively. Decreasing rates of mercury levels in dams were 67.2% in liver, 57.6% in brain, 47.2% in kidney, 42.1% in spleen and 40.9% in blood. As well, decreasing rates of mercury level in fetuses were 54.9% in all body burden, 55.9% in liver, 46.7% in kidney and 37% in brain, respectively. The number of fetal ossification centers were reduced by 23.8% to 58.0% following simultaneous treatment with 1.0 g/kg garlic juice. These findings indicated that garlic juice effectively inhibited the embryotoxicity of methylmercury in pregnant Fischer 344 rats.

A Study of Cytotoxical Mechanism of Mercurial Compounds in RAW264.7 Cell Line / 대한산업의학회지

The effects of glucose on the productions of ATP and nitrite which are inhibited by mercury compounds, were examined in a cell culture system of RAW 264.7 cells. The cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) with cytokines, IL-1 and TNF for 24 hours. The viablility of RAW 264.7 cells at the end of culture was significantly decreased by mercury chloride or methylmercury chloride added into the media in dose-dependent manner, however the viability of RAW 264.7 cells were influenced in the concentrations lese than 0.8micrometer of mercury chloride or 0.4micrometer of methylmercury chloride. The addition of 4.5 g/l glucose to normal DMEM lowered the pH of media to the range of 6.7-6.8 after 48 hours of culture, but not for the cell survivals. This supplement of glucose to the media also prevented the inhibitions of ATP and nitrite syntheses which were caused by mercurial compounds. These results suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seams to be caused by the inhibition of ATP synthesis, especially related to the citric acid cycle.

The effect of hydrocortisone on epithelial apoptosis in fetal mice intestine / 重庆医科大学学报

Objective:To study the effect of hydrocortisone (HC) on apoptosis and expression of apoptosis- associated proteins in the intestinal epithelium of fetal mice.Methods:The duodena and colons were taken from E13.5d and E14.5d fetal mice respectively,and cultured with different doses of HC (0 ?g/ml,0.5?g/ml,1.0?g/ml,2.0?g/ml).After 24h,the samples were sliced up into the paraffin sections.Some sections were stained with HE to count the numbers of apoptotic bodies (NAB)in the fetal intestinal epithelium with stereological method.Others were stained with the immunohistochemical method to observe the expression of Bcl-2,Bax,P53 and c-Fos.Results:①In each experimental group,the NAB were higher than that of control group( P