Preventive Effect And Mechanisms Of LSPCs On Immune Damage By Extremely Low Frequency Electromagnetic Field Exposure And Calcium Signal Regulation In Hippocampal Tissue

Extremely low frequency electromagnetic field（ELF-EMF） refers to the electromagnetic field with the frequency of 0³00 Hz generated by power transmission line of household electric appliances, electric power facilities. In recent years, a large number of studies have shown that, the ELF-EMF has adverse effects on body’s immune system, nervous system and reproductive system. Among them, the immune system performs many important functions in the body, ELF-EMF will pose a huge threat to the immune system. At present, most of researches involving ELF-EMF are still focused on the risk assessment stage, lack of related protective measures.Based on these, in this thesis, we take lotus seedpot procyanidins（LSPCs） as experimental material, male ICR mice as animal model, research the effects of ELF-EMF on immune function and explore the preventive effects of LSPCs and preventive approach. In addition, the effects of ELF-EMF and LSPCs on cognitive impairment associated with Ca²⁺ signaling in mice hippocampus were also studied. We expect these works can provide theoretical basis for the development of daily diet against ELF-EMF exposure. The main research contents are as follows: 1. Protective effect of LSPCs on immune function induced by ELF-EMFThe mice were divided into normal group, ELF-EMF group and LSPCs prevention groups ELF-EMF+LSPCs（30, 60, and 90 mg/kg·bw·day）. Based on mice injury models, we set 50 Hz, 8mT, 28 days, 4h per day as the experimental conditions. Among them, LSPCs prevention groups had been gavaged LSPCs for 15 days before irradiation, and continuously treated with same dose of LSPCs till the end of the experiment.（1） Effect of LSPCs on immune function induced by ELF-EMFThe immunological indexes, such as organ index, peripheral blood analysis, the activity of IL-4 and INF-γ in blood and liver tissue were measured in this experiment. Our results indicated that spleen and thymus indexes were significantly decreased（p<0.05, p<0.01）, compared with the control, WBC number, RBC number and hemoglobin in blood reduced, and the concentration of IL-4 and INF-γin plasma and liver also decreased. However, pretreatment with LSPCs improved these levels. Especially in ELF-EMF+LSPCs90 group, the spleen and thymus indexes increased significantly（p<0.05, p<0.01）, the expression of IL-4 and INF-γ（p<0.05） in plasma, and the expression of IL-4（p<0.05） and INF-γ（p<0.01） in liver were significantly raised. Conclusion: LSPCs can effectively reduce the immune function injury induced by ELF-EMF and regulate immune activity.（2） Protective effect of LSPCs on ELF-EMF exposure induced damages in mice spleenELF-EMF and LSPCs intervention effects on immune function of spleen in mice were studied in this experiment. We measured spleen T\B lymphocyte proliferation and NK cell activity in each group of mice, the expression of SOD, CAT, GSH and cytokine expression such as INF-γ, TNF-α, IL-2, IL-6 and IL-10 in spleen. We also measured cell apoptosis and proteins expression related to apoptosis.We measured proliferation of T and B lymphocyte by MTT colorimetry, activity of NK cell by LDH release assay, related protein expression by Western blot, apoptosis by flow cytometry.Comparing with the ELF-EMF group, MTT stain and LDH release assay showed that the proliferation function and NK-cell activity of lymphocytes T and B decreased significantly in ELF-EMF group（p<0.01）. The level of SOD, CAT, GPx expression of spleen were significantly reduced. At the same time, the expression of cytokines disordered. In addition, ELF-EMF induced mice spleen cells stagnate in the G0 / G1 phase, a marked increase in the ratio of apoptotic cells, DNA damage seriously（p<0.01）. ELF EMF irradiation also decreased the expression of Bcl-xl and Bcl-2, promoted the expression of apoptosis proteins Bax, finally activated caspase 3 protein（p<0.01）, resulting in the spleen cell apoptosis.However, LSPCs had a significant improve effect on these changes. Especially in 90 mg/kg bw LSPCs group. All studies showed that LSPCs can effectively improve spleen function injury caused by ELF EMF, return the expression of antioxidant enzyme protein and cytokines to normal levels, alleviate the spleen cells apoptosis and DNA damage, thus effectively regulate the expression of key protein relate to apoptosis. These results illustrated that LSPCs had a significantly preventive effect on the spleen immune function damaged by ELF EMF.2. Extremely low frequency electromagnetic field exposure causes cognitive impairment associated with Ca²⁺ signaling in mice hippocampus: reversal by procyanidins extracted from the lotus seedpodThe experiment designs and the groups were as above, after ELF-EMF exposure in selected experimental conditions end, mice hippocampus were taken out. Fluorescently labeled cells by using Fluo-3/AM were measured the free Ca²⁺ concentration; Western blotting detected the expression of the proteins related with learning and memory and its downstream Ca²⁺ signaling pathway regulatory proteins.Results showed that, compared with control, the expression of Gα（i） in hippocampal tissues enhanced as well as the contents of IP3 and DAG, and intracellular calcium levels significantly increased in the ELF-EMF group. In addition, Western blotting found that the expression of PKA, PKC-beta II and PP2 B increased, the expression of Ca MKII decreased. p-CREB phosphorylation expression levels of hippocampal tissue of ELF-EMF group significantly decreased, further reduced the expression of the downstream molecules BDNF, and damaged learning and memory function.Pretreated with LSPCs significantly improve the expression of key proteins in the downstream of calcium signaling pathway which involves learning and memory damage induced by ELF-EMFt, there is a dose-effect relationship in the experimental concentration range, especially in 90 mg/kg bw LSPCs group close to the normal group.The above results showed that LSPCs could decrease the expression of Gα（i） in hippocampal tissues, reduce the inflow of Ca²⁺, and then to control the expression of PP2B\Ca MKII and downstream protein BDNF, improve learning and memory function in mice. In conclusion, LSPCs could improve the function of learning and memory through regulating Ca²⁺ signaling pathway.