| Chapter one: the effects of sodium nitrite exposure on blood,maternal-fetal interface and offspring nervous system of SD pregnant ratsObjective To investigate effects of sodium nitrite exposure on blood,maternal-fetal interface and offspring nervous system of SD pregnant rats.Methods Pregnant SD rats were randomly divided into four groups:control group(drinking water during pregnancy),low dose group(drinking water containing 0.05% sodium nitrite),medium dose group(drinking water containing 0.15% sodium nitrite)and high dose group(drinking water containing 0.25% sodium nitrite).On the 19 th day of gestation,the number,sex,body weight,brain weight and other indicators of fetal rats in each group were observed.Blood,placenta and brain tissue of pregnant rats were collected for follow-up detection.The expression levels of peroxynitrite,nitric oxide,malondialdehyde and superoxide dismutase in blood,IL-1β and TNF-α in blood and placental tissues of pregnant rats were determined by enzyme-linked immunosorbent assay.Flow cytometry was used to detect ROS levels in fetal cerebral cortex.Results The statistical results showed that there were no significant differences in the number,sex and survival rate of fetal rats in each group,but the body weight and brain weight of fetal rats in medium and high dose groups were significantly reduced compared with the control group(P<0.05).The contents of peroxynitrite and nitric oxide in blood of pregnant rats increased gradually with the increase of sodium nitrite exposure concentration during pregnancy,and both had statistical significance(P < 0.05).The levels of malondialdehyde(MDA)and superoxide dismutase(SOD)in blood were significantly increased in medium and high dose groups.The levels of IL-1β and TNF-α in blood were significantly increased in low,medium and high dose groups.The levels of IL-1β in placenta tissue were significantly increased in high dose group while TNF-α were significantly increased in medium and high dose groups.ROS levels in fetal cerebral cortex increased significantly with the increase of sodium nitrite exposure.Conclusion Sodium nitre intake during pregnancy causes stress response and inflammatory environment in peripheral blood of pregnant SD rats,accumulation of inflammatory factors in placenta,and increase the level of ROS in fetal brain tissue.Chapter two: the effects of sodium nitrite on energy,inflammation and apoptosis of rats primary nerve cellsObjective To further verify the effects of sodium nitrite on energy,inflammation and apoptosis of rats primary nerve cells.Methods The model of primary nerve cells cultured in SD rat brain tissue was established.The model was randomly divided into control,low,medium and high dose groups.The control group was treated with neuronal culture medium,and the other three groups were treated with low(40μmol/L),medium(60μmol/L)and high(80μmol/L)peroxide nitrite on the basis of neuronal culture medium,respectively.CCK8 was used to detect cell viability,NSE and TUBB3 were used to identify cell types and compare the difference between groups.TUNEL staining was used to detect cell apoptosis.Phosphomolybdic colorimetric method was used to compare the ATP level of primary nerve cells in each group and the activity of mitochondrial respiratory chain complex ⅱ and complexⅳ.PCR and WB were used to detect the transcription and protein expression of IL-1β and TNF-α,and the expression of Bax,Bcl-2 and Caspase 3 apoptosis proteins in each group were compared.Results The primary nerve cell culture model was successfully established and treated with peroxynitrite.Compared with the control group,the cell viability in low,medium and high dose groups were significantly decreased,the expression of NSE and TUBB3,intracellular ATP content and the activities of mitochondrial complex ⅱ and complexⅳ were significantly decreased,and the occurrence of apoptosis was gradually increased.The expression of IL-1β protein and transcription level were significantly increased,but the difference of TNF-α was only statistically significant in medium-dose and high-dose groups.The apoptotic protein Bax and activated Caspase 3 increased gradually,while the Bcl-2 decreased gradually.The expression of apoptotic protein between groups was consistent with TUNEL staining resultsConclusion Peroxynitrite can affect the activity of mitochondrial complex of primary nerve cells and cause energy metabolism disorder,increase the expression of inflammatory factors,thus slowing down cell proliferation and inducing cell apoptosis.Chapter three: The rescue effect and mechanism of PUFA ω-3 on SD pregnant rats and their offspring nervous system injury caused by sodium nitriteObjective To confirm that PUFA ω-3 can alleviate the injury of SD pregnant rats and their offspring nervous system caused by sodium nitrite exposure,and to explore the mechanismMethods Pregnant rats were randomly divided into three groups:control group(normal drinking water),model group(drinking water containing 0.25% sodium nitrite)and PUFA ω-3 group(drinking water containing 0.25% sodium nitrite,supplemented with an additional 4%PUFA ω-3).The number,sex,body weight and brain weight of fetal rats were recorded on the 19th day of gestation,and blood,placenta and brain tissue of pregnant rats were collected for subsequent detection.The contents of malondialdehyde(MDA)and superoxide dismutase(SOD)in blood and the expressions of IL-1β and TNF-α in placental tissue were detected by ELISA.The expressions of OCLN,TJP1 and SOX2,NSE and TUBB3 proteins in fetal cerebral cortex were detected by immunohistochemistry and immunofluorescence methods,respectively.The levels of ROS and mitochondrial membrane potential were analyzed by flow cytometry.The transcription and protein levels of IL-1β and TNF-α were detected in the cerebral cortex of fetal rats,and the content of ATP and the activity of mitochondrial complex ⅱ and ⅳwere determined.Transmission electron microscopy was used to further observe the structural changes of cortical organelles,and to investigate whether Nrf-1/Hmox1 and PI3K/AKT pathways are involved in the protective effect of PUFA ω-3.Then the primary neuron model was cultured and randomly divided into three groups: control group(normal cell culture),model group(treated with 80μmol/L pernitrite)and PUFA ω-3 group(treated with20μmol/L PUFA ω-3,others are the same as the model group).CCK8 was used to evaluate the cell viability of each group and flow cytometry was used to detect the apoptosis rate of each group,so as to observe the protective effect of PUFA ω-3 on nerve injury in vitro.Considering that the PI3K/AKT pathway has been confirmed in vivo to be involved in the protection of PUFA ω-3 against nerve injury,we brought inhibitors of PI3K and AKT for further verification in vitro.After treating 10μmol/L PI3K or 3μmol/L AKT inhibitors respectively in the model group or PUFA ω-3 group,CCK8 and flow cytometry were used to evaluate the cell activity and apoptosis rate of each group,and the expression of various indicators in PI3K/AKT pathway was detected by WB.Results PUFA ω-3 significantly improved the weight and brain weight loss of SD fetal rats induced by sodium nitrite,and PUFA ω-3significantly reversed the increase of malondialdehyde content and decrease of superoxide dismutase content in blood and the increase of IL-1β and TNF-α in placenta induced by sodium nitrite in pregnant rats.Compared with model group,the expressions of OCLN,TJP1,SOX2,NSE and TUBB3 in PUFA ω-3 group were significantly increased,while the expressions of IL-1β,TNF-α and ROS were significantly decreased.PUFA ω-3 increased the mitochondrial membrane potential and the activity of mitochondrial complex ⅱ and ⅳ,and corrected the mitochondrial structural disorder in cortical nerve cells of fetal rats.In addition,PUFA ω-3 reversed the transcription and protein levels of Nrf-1and Hmox1 in the model group,and increased the expression levels of p-PI3K and p-AKT.In vitro primary nerve cell model,PUFA ω-3 significantly increased cell survival rate and decreased cell apoptosis.After the addition of PI3K and AKT inhibitors,the enhancement of cell activity and the inhibition of apoptosis were significantly reduced.The influence of PI3K/AKT pathway related indicators caused by PUFA ω-3 were significantly changed.Conclusion PUFA ω-3 can reduce blood oxidative stress and placental inflammation in pregnant rats,and ameliorate the effects of sodium nitrite on fetal rats.PUFA ω-3 can reduce the oxidative stress level of fetal brain,improve the energy metabolism of fetal brain and reduce the apoptosis of nerve cells,and save fetal brain tissue damage caused by exposure to sodium nitrite during pregnancy.In addition,we confirmed that PUFA ω-3 protects the progeny nervous system through the PI3K/AKT pathway. |
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