Research On The Roles Of HIF-1α/ERK Pathway In The Injury Of Neuron Mitochondria In Hippocampus Induced By Microwave Exposure

Objective: With the wide application of microwave technology and information era, the opportunities of people exposed to microwave are increasing particularly all communication facilities and medical equipment. The biological effects and mechanisms of microwave are the subject of bioelectromagnetics forward. Microwave has a significant effect on central nervous system while hippocampus is the most sensitive target, but the injury mechanism is unknown. Mitochondrial structure and function are closely related to energy metabolism, which is one of the first involving targets of microwave exposure, while the HIF-1α/ERK signaling pathway may play an important role in the process of injury of hippocampal neurons in mitochondria induced by microwave. Therefore, the study of HIF-1α/ERK signaling pathway and its significance in the damage of the hippocampus induced by microwave radiation provides new targets for in-depth study of microwave injury mechanisms and prevention and control measures and approaches, and has important theoretical and practical significance for the normal operation of the health protection staff and modern technology war combatant's security.Materials and methods: (1) 192 male Wistar rats were exposed to microwave at 2.5, 5 and 10mW/cm~2. Exposure time was 6min/times, 5 times/week, and continuouing exposure for 1m. The abilities of learning and memory were detected by Morris water maze at 6h, 7d, 14d, 1m, 2m and 6m after exposure. The structure of rats'hippocampus and mitochondria ultrastructure were observed by HE and tigroid body staining and electron microscope. Meanwhile, amino acid neurotransmitters contents in hippocampus were detected by HPLC. The expression of hif-1αmRNA in hippocampus was detected by Real-time PCR. HIF-1α, ERK1/2, p-ERK1/2 expression were detected by Western Blot, IHC and image analysis. (2) PC12 cells which were induced by NGF were exposed to 30mW/cm~2 microwave. The structure and function of mitochondria were observed by electron microscope, luminometer, fluorospectrophotometer and fluoromicroscope. Real-time PCR, Western Blot, IHC and image analysis were used to detect hif-1αmRNA, HIF-1α, molecular of ERK pathway (ERK1/2, p-ERK1/2, Raf, p-c-Raf, p-MEK1/2), target gene of HIF-1α(VEGF) and energy metabolism related gene (COXⅠand COXⅣ) expression. The regulation of ERK pathway on HIF-1αand the effect of HIF-1αto mitochondria function of PC12 cells were invested by intervention means of U0126 and hypso-expression plasmid of HIF-1α.Results: (1) The changes of rats'learning and memory abilities and amino acids neurotransmitter: The AELs of 10mW/cm~2 group were longer than sham group (P<0.05) at 7d after exposure. AELs of 5 and 10mW/cm~2 group were longer than sham group at 14d (P<0.05 or P<0.01). AELs of exposed groups were all longer than sham group at 1m(P<0.05 or P<0.01). Four kinds of amino acid neurotransmitter contents in hippocampus were increased significantly at 6h, 14d and 2m in 2.5 and 5mW/cm~2 groups(P<0.05 or P<0.01). GABA contents were decreased at 6h after exposure (P<0.05), and Asp and Glu contents were decreased at 14d and 2m in 10mW/cm~2 group(P<0.05 or P<0.01). (2) The changes of hippocampus structure and ultrastructure of mitochondria: The hippocampus structure of exposed groups appeared tissue edema, osteoporosis, and different degrees of degeneration and necrosis of neurons, vascular congestion and hemorrhage and enlarged perivascular space within 6m after 2.5, 5 and 10mW/cm~2 exposure. At 2.5 and 5mW/cm~2 groups, the hippocampus injuries appeared at 7d, 1m at the most serious stage, and 2m on the recovery of the trend. In 2.5 mW/cm~2 group, the hippocampus structure recovered at 6m while in 5 mW/cm~2 group, the injuries still could be seen then. In 10mW/cm~2 group, the injury of hippocampus structure appeared earlier (6h after exposure), 7d and 14d at the most serious stage, and could not recovered at 6m. The numbers of tigroid body were decreased at different extend in exposure groups and were the most serious in 10 mW/cm~2 group. The change rule of it was agreed with HE staining. The mitochondria showed swell, cristae break, cavitation and malformed which were injured at 6h, 1m at the most serious stage, and 6m on the recovery of the trend during 6m in 5 mW/cm~2 group. (3) The mRNA and protein expression changes of HIF-1αin rats'hippocampus: There was no change of hif-1αmRNA in 2.5mW/cm~2 group, while hif-1αmRNA was increased significantly at 14d (P<0.01) and recovered at 1m in 5mW/cm~2 group, then hif-1αmRNA was expressed in 10mW/cm~2 group lower than sham group, which was decreased significantly at 6h (P<0.01), 1m (P<0.05) and 2m (P<0.05). The protein of HIF-1αin rats'hippocampus was increased at 6h and 1m (P<0.01 or P<0.05) in 2.5mW/cm~2 group and at 1m in 5mW/cm~2 group (P<0.01), and was decreased at 14d~2m in 10mW/cm~2 group (P<0.01 or P<0.05). (4) The expression changes of ERK1/2 and p-ERK1/2 in rats'hippocampus: There was no significant change within 2m in exposure groups. In sham group, p-ERK1/2 was expressed weakly positive in neuron cytoplasm of hippocampus, while there was no changes within 2m in 2.5mW/cm~2 group, and p-ERK1/2 was expressed positive or strong positive at 7d~1m in 5mW/cm~2 group (P<0.01 or P<0.05) and at 7d~2m in 10mW/cm~2 group (P<0.01 or P<0.05). (5) The structure and function changes of mitochondria of PC12 cells: The mitochondria of PC12 cells showed unevenness distribution, irregular sizes and shapes, swelling, cristae break and cavitation at 6h after 30mW/cm~2 microwave exposure. ATP contents were lower than sham group (P<0.01 or P<0.05) at 1h~3d. The activities of SDH and COX were decreased significantly (P<0.05) at 1h.ΔΨm was decreased significantly at 6h (P<0.05). ROS was higher than sham group at 1~24h (P<0.01). (6) The mRNA and protein expression changes of HIF-1αin PC12 cells: hif-1αmRNA was increased at 6h after 30mW/cm~2 microwave exposure (P<0.05). HIF-1αwas increased at 1h and 12h after 30mW/cm~2 microwave exposure (P<0.05) and was decreased at 6h (P<0.05). (7) The expression changes of ERK pathway signal moleculars ERK1/2, p-ERK1/2, Raf, p-c-Raf and p-MEK1/2: The expression of p-ERK1/2 was increased significantly at 1h after 30mW/cm~2 microwave exposure (P<0.05). There were no changes of ERK1/2, Raf, p-c-Raf and p-MEK1/2 expression at 1~24h after 30mW/cm~2 microwave exposure. (8) The changes of VEGF, COXⅠand COXⅣ: The expressions of VEGF, COXⅠandⅣwere on concordance with HIF-1αabove, i.e. VEGF, COXⅠandⅣwere decreased at 6h after 30mW/cm~2 microwave exposure (P<0.05), and were increased at 12h (P<0.05). (9) ERK1/2, p-ERK1/2 and HIF-1αexpression and function of mitochondria changes after U0126 intervention: PC12 cells were exposed to 30mW/cm~2 microwave 2h after 10μmol/l U0126 intervention, and there was no change of ERK1/2 expression, while the expression of p-ERK1/2 and HIF-1αwere decreased significantly at 1h after (P<0.05). With the same conditions, the ATP contents andΔΨm were decreased at 1~24h after exposure. (10) The effect of high expression plasmid of HIF-1αand U0126 intervention on mitochondria function of PC12 cells after 30mW/cm~2 microwave exposure: After G418 screening, the transfection efficiency of sham plasmid pEGFP-N1 (N) and objective plasmid pEGFP-N1-HIF-1α(H) to PC12 cells exceeded 90%. The expressions of HIF-1αin H group were higher than N group (P<0.01). After 30mW/cm~2 microwave exposure at 1~24h, theΔΨm step up significantly in H group (P<0.01), but there was no change of SDH activity (P>0.05). The ATP inhibition ratio in H group was lower than N group 2h after 10μmol/l U0126 intervention and 30mW/cm~2 microwave exposure, and there was no change ofΔΨm at 1h and 6h after exposure (P>0.05), but was increased at 12h and 24h (P<0.01).Conclusions: (1) Long-term microwave exposure at 2.5, 5 and 10mW/cm~2 may cause structural damage in rats'hippocampus especially in mitochondria, with the abilities of learning and memory declining and amino acid neurotransmitter disorders. These changes above were positively correlated with exposure dose. (2) HIF-1αexpression in rats'hippocampus was increased in 2.5 and 5mW/cm~2 groups, while was decreased in 10 mW/cm~2 groups. The p-ERK1/2 expression was increased in exposure groups, which indicated that HIF-1αand ERK pathway participated in the processes of hippocampus mitochondria damage induced by microwave. (3) The mitochondria structure of PC12 cells were injuried by microwave at 30mW/cm~2, and the contents of ATP, the activities of SDH and COX,ΔΨm were decreased, while ROS accumulated. (4) HIF-1αexpression in PC12 cells was fluctuated which showed step up first, then step down and up. The hif-1αmRNA showed increase temporary and the expression of p-ERK1/2 increased. It indicated that microwave exposure may activate HIF-1αand activate ERK pathway by p-ERK1/2. The changes of expression of VEGF which was the target gene of HIF-1α, COXⅠandⅣwhich were energy metabolism genes appeared the same rule of HIF-1α, which indicated that genes above participated in the processes of mitochondria damage induced by microwave exposure. (5) ERK pathway inhibited by U0126 can cause the down expression of p-ERK1/2 and HIF-1α, and the decreasing of ATP contents andΔΨm, which manifested that ERK pathway participated in the positive regulation of HIF-1αafter microwave exposure and ERK pathway played a protection role in the mitochondria damage induced by microwave exposure. (6) High expression of HIF-1αcan increase the ATP contents andΔΨm and ERK pathway inhibited by U0126 can decrease the effects of high expression of HIF-1αon ATP contents andΔΨm, which indicated that the activation of HIF-1αprotected mitochondria in the processes of mitochondria damages of PC12 cells induced by microwave exposure and the protection effect of ERK pathway on mitochondria function was implemented by HIF-1α.