Research On The Activation Of The Nrf2-Keap1 Signaling Pathway By Active Fraction Of KFL Protect Neural Cells Injury Induced By Microwave Exposure

The widespread use of microwave(communication, occupational exposure, medical exposure) had raised questions about its adverse effects on human health. The central nervous system is one of the most sensitive targets of microwave. Microwave exposure can cause detrimental effects on brain, especially the cognitive function. the mechanisms underlying the adverse effects are not yet elucidated. The real-time effects of microwave on neuron and its effects on neuron nerrite development were rarely reseached either. Kang-fu-ling(KFL) is a new formulation we developed for protecting neural cells injury induced by microwave exposure. Neuroprotective effects of KFL against high power microwave(HPM) and the mechanisms underlying the protective effects(based on Nrf2-Keap1 signaling pathway) were not reseached yet.In the present study, we explore the real-time effects of microwave on primary hippocampal neurons and PC12 cells(cytoplasm, mitochondrial, and nuclear) dynamic [Ca2+] using waveguide-based real-time microwave exposure system and time-lapse microfluorimetry. The microwave(SAR=4 W/kg) didn’t induce any response in primary hippocampal neurons and PC12 cells in intracellular free Ca2+. We found HPM(average power density=30m W/cm2) exposure could rise the intracellular ROS level of primary hippocampal neurons and PC12 cells and induce the oxidative stress. The HPM exposure impaired the nerrite development of primary hippocampal neuron. Oxidative stress maybe involved in the mechanism of adverse effects of HPM exposure.KFL is a new formulation we developed under the guidance of an original Prescription of Traditional Chinese Medicine. This study aimed to evaluate the potential protective effects of KFL on cognitive deficit induced by HPM and the underlying mechanism for this neuroprotection. KFL reversed HPM-induced memory loss(tested by Morris water maze)and the histopathological changes(HE stained and TEM) in hippocampus of rats through antioxidant action. KFL and its active fraction KFL-M(methanol fraction of Kang-fu-ling) both exhibited hydroxyl radical scavenging activity in a cell-free system measured by EPR spectroscopy and suppressed the oxidative stress induced by HPM.A major mechnism in cellular defense against oxidative stress is activation of the Nrf2-Keap1 signaling pathway, which controls the expression of genes(Glutamate-cysteine ligase, heme Oxygenase-1, NADPH quinone oxidoreductase 1 thioredoxin Reductase 1, and peroxiredoxin 1) whose protein products are involved in elimination of ROS by enhancing cellular antioxidant capacity. The Nrf2-Keap1 signaling pathway plays a central role in the protection of cells against oxidative demage. And KFL and KFL-M displayed a protective effect against HPM-induced oxidative stress by activation of the Nrf2-Keap1 signal pathway and its target genes(HO-1, NQO1, and GSTmu3). The Nrf2-Keap1 signaling pathway maybe involved in the neuroprotective effects of KFL against HPM-induced oxidative stress.To confirm the hypothesis that activation of the Nrf2-Keap1 signaling pathway could protect neural cells injury induced by microwave exposure. In the present study, we overexpressed the Nrf2 and Keap1 in PC12 cells to active and supress the Nrf2-Keap1 signaling pathway. And we found the activation of the Nrf2-Keap1 signaling pathway enhanced the cellular antioxidant capacity and displayed a protective effect against HPM-induced oxidative stress. The suppression of the Nrf2-Keap1 signaling pathway weakened the cellular antioxidant capacity and made the PC12 cells more vulnerable to HPM-induced oxidative stress. The t-BHQ(The agonist of Nrf2-Keap1 signaling pathway) showed the similar effects against HPM-induced oxidative stress in primary hippocampal neurons and PC12 cells. And t-BHQ reversed HPM-induced impairment in the nerrite development of primary hippocampal neuron.Here we assessed the real-time calcium levels change of primary hippocampal neurons and PC12 cells during pulsed microwave exposure using Fluo 4 and genetically encoded calcium indicator. And we demonstrated that KFL could protect cognitive defect and oxidative stress caused by HPM by modulation of ROS formation and antioxidant enzymes. Our data also suggested that some electrophilic compounds contained in KFL protect neuron via Nrf2-Keap1 signaling pathway. The project lays the foundation for using Nrf2-Keap1 pathway as preventive and therapeutic target for treatment of microwave-induced brain injury.