| Background and Objects:There are more than 170 million Chinese living in plateau areas with an altitude of more than 2500 m.Less oxygen in these regions will induce chronic hypoxia in our bodies,with delayed reaction time and impaired motor function.Extreme high-altitude exposure(>5000m)may cause more severe damage.The brain requires a precisely balanced supply of oxygen and may adapt to hypoxia by altering brain circulation to respond to changes in oxygen and carbon dioxide levels.Early development is an important period of neural development and neural stem cells’ proliferation and differentiation into neurons continually.Although neural circuits are formed in the late stages of human embryonic development and even after birth,diverse classes of functional cells are generated and migrate to appropriate locations earlier in development.However,there is still a lack of researches on how altitudes may affect the brain’s early development,especially the latent influences on human cognitive function under high-altitude hypoxia.Since neural generation,migration,and formation of synaptic connections all require substances and energy to synthesize a large number of proteins and amino acids,sufficient nutrients and oxygen supply are required.Mitochondrial dysfunction could be used as a marker for a variety of diseases that result in brain damage and cognitive impairments,for instance,hereditary mitochondrial diseases,neurodegenerative diseases,and ageing,etc.The central nervous system is most often susceptible to mitochondrial diseases,whenever in childhood and adulthood,with clinical manifestations including dyskinesia,cognitive decline,and even dementia.Despite the significant role that mitochondria play in the brain,it is still unclear how these organelles precisely regulate the process of upgrowth and neurogenesis.Cold-inducible RNA-binding protein(CIRBP)was initially screened out as a gene transcript that is induced by DNA damage and plays a key role in controlling cellular responses to various environmental stress like hypothermia and ultraviolet light.Preliminary results showed that CIRBP expression decreased after long-term hypoxic exposure.At the same time,CIRBP was involved in the learning and memory impairment of adult mice.It was further found that CIRBP was involved in regulating mitochondrial damage in adult mice.In the present study,the pregnant C57BL/6 mice and primary cultured neurons were used to determine how chronic hypoxic exposure during pregnancy might affect the maturation of offsprings’ neurons,particularly the advanced brain functions,and clarify the role of neural abnormal maturation and dysfunction in this process.How the changes in mitochondrial function might affect neuronal maturation was then explored and the critical molecular mechanism that CIRBP would perform was further elucidated.Our works may provide an effective target for prevention and treatment to brain damages induced by persistent low-oxygen exposure during pregnancy.Methods:1.Establish pregnant animal model under hypoxic exposure:Pregnant mice at Embryonic Day 14.5(E14.5)were assigned to control or hypoxic group(Abbreviated as Con or Hyp group).The experimental group was exposed to a compound-environment experimental cabin and the simulated altitude was set to 4000 m(with temperature of 22-24℃ and relative humidity of 40%-70%).They were exposed to hypoxia until 10 weeks after the birth of offspring.Then we detected the weight,body length,and brain weight at Postnatal Day 0,3,5,7,14,21 and 70(P0 d,P3 d,P5 d,P7 d,P14 d,P21 d and P70 d);The conditioned fear box test,novel object recognition test and the water maze test were used to detect the learning and memory of mice;Electrophysiological experiments were implemented for the detection of m EPSCs.Western blot was used to detect the protein levels of synapse-related molecules;Chemical kits were used to detect the intracellular glutamate concentration.2.Hypoxia during pregnancy can affect the maturation of hippocampal neurons in P7 d and P14 d miceWestern blot,RT-qPCR and Immunofluorescence staining were conducted to detect the expression of DCX(Doublecortin),MAP2(Microtubule-associated protein 2)and presynaptic membrane-related proteins in neurons of offsprings’ brains.3.Establishment of cell culture model under hypoxiaPrimary neurons were separated from the brain tissue of pregnant C57BL/6 mice(about E14.5)and cultured until the 4th day in vitro.We put them into the hypoxic workstation(made in DWS),with 1% oxygen concentration,exposed for 2,3,6 and 9 days;CCK8 assay was used to detect neuronal viability;TUNEL and Western blot were performed to detect neuronal apoptosis;Western blot,RT-qPCR and Immunofluorescence staining were performed to test the indicators related to neuron maturation;Calcium oscillations was conducted to detect primary neuronal function.4.Regulation on neuronal maturation by changes in mitochondrial function induced by hypoxiaThe ultrastructure of mitochondria in the hippocampi of mice and primary neurons exposed to hypoxia for 6 days were detected by transmission electron microscopy;The ATP level in the hippocampi of mice and primary neurons were tested by enhanced ATP kits;The kit detects the activities of respiratory chain complexes I,II,III,and V in the hippocampi of mice and primary neurons;RT-qPCR was used to detect the mRNA expression levels of respiratory chain complex-related molecules in the hippocampi of mice at P14 d;Western blot was utilized to detect the expression of mitochondrial fission-fusion related proteins in the hippocampi of mice;CCK8 assay was used to detect neuronal viability and screen out the appropriate ATP concentration;ATP supplementation on the morphology of neurons was tested by immunofluorescence staining;Western blot was conducted to detect the expression of DCX,MAP2,Tau and presynaptic membrane-related proteins in neurons.5.Regulatory effects of CIRBP on neuronal dysplasia under hypoxiaWestern blot and RT-qPCR were used to detect the level of CIRBP expression in the hippocampi of the offspring mice;Body weight changes of CIRBP KO mice exposed to hypoxia during pregnancy at P14 d were tested;The protein changes of DCX,MAP2,Tau and presynaptic membrane proteins Syn1 and Syn2 were detected in the hippocampi of mice CIRBP KO at P14 d by Western blot;Western blot and Immunofluorescence staining were used to detect the morphology of neurons,expression of DCX,MAP2,Tau and presynaptic membrane proteins extracted from neurons transfected with CIRBP-overexpressed lentivirus and CIRBP-overexpressed mice in a hypoxic environment.6.The regulation of CIRBP on mitochondrial dysfunction in neurons induced by hypoxiaATP kit detects the ATP level in CIRBP-overexpressed neurons and the neurons extracted from CIRBP-overexpressed pregnant mice under hypoxic environment;the kit detected the activities of mitochondrial respiratory chain complexes I,II,and V in neurons transfected with CIRBP-overexpressed virus and neurons extracted from CIRBP-overexpressed pregnant mice after 6 days of hypoxia exposure.Western blot and RT-qPCR were used to detect the proteins and mRNA expression levels of respiratory chain complex-related molecules.Transmission electron microscopy was used to detect the changes of mitochondrial ultrastructure in neurons extracted from CIRBP-overexpressed pregnant mice under hypoxia;Western blot was used to detect the mitochondrial fission proteins’ expression in neurons extracted from CIRBP-overexpressed pregnant mice under hypoxia.Results:1.Establishment of animal models under hypoxia during pregnancy1)C57BL/6 pregnant mice were exposed to a compound-environment simulated experimental cabin at an altitude of 4000 m until the offspring mice were born.The results indicating the growth and development of offsprings at P3 d,P5 d,P7 d,P14 d and P21 d showed that the body length in Hyp group was significantly lower than those in Con group at P7 d,P14 d,P21d(P < 0.0001)and weight lower at P5 d,P7 d,P14 d and P21 d.2)At the same time,the brain weight of the mice in Hyp group at P7 d and P14 d was lower than that of the control group,but there was no statistical significance in brain weight/body weight ratio(Br/Bo)between the two groups.The Br/Bo of mice at P14 d in Hyp group was statistically higher than that in control(P < 0.001).3)The conditioned fear box test testified that mice at P14 d in Hyp group exhibited less freezing time than those in Con group(P < 0.001),as well as less time of new object recognition and lower discrimination ratio in the new object recognition test(P < 0.0001).The results illustrated that chronic maternal hypoxia led to impairments of learning and memory functions in P14 d mice.The water maze test was then used to detect learning and memory functions of mice at P70 d.There were significant decreases in dwelling time of target quadrant in Hyp group(P < 0.05),indicating that hypoxia during pregnancy could also induce impaired learning and memory in postnatal adult mice.4)Western blot and RT-qPCR were used to detect the expression of DCX and MAP2 in brain neurons of P3 d,P5 d,P7 d and P14 d mice.The results indicated that with the prolongation of birth time,gradually increased MAP2 and decreased DCX expression level manifested that the mice developed normally.Comparing the P7 d and P14 d mice in two groups,we found that higher DCX(P < 0.01)and lower MAP2(P < 0.001)levels were notably expressed in hypoxic group.In the meantime,increased DCX(P < 0.001)and reduced MAP2(P < 0.0001)at mRNA levels were also apparently appeared in P7 d and P14 d mice of Hyp group.All above might elucidate that hypoxic exposure during pregnancy led to inhibited neuronal maturation in the mice’s brain.5)Further analysis of synapse-related proteins expressed in the brains of offspring in Hyp group showed that there was no significant difference between two groups in the expression of postsynaptic membrane proteins,including NR1,NR2 A,Glu R1 and P-831-Glu R1.The brain presynaptic membrane protein Syntaxin 1A(Stx-1A)in P14 d mice exposed to hypoxia was significantly lower than that in control(P < 0.01).2.Hypoxia during pregnancy can affect the maturation of hippocampal neurons in P7 d and P14 d mice1)By immunofluorescence staining,it was shown that within the dentate gyrus(DG),one of the hippocampal subfields,increased count(P < 0.01)and thickness(P < 0.01)of DCX+ cells were significantly observed in P14 d mice of Hyp group than those in control.The number of Neu N+ cells in the Hyp group was lower than that in the control group(P < 0.01),as well as thinner thickness than that in control(P < 0.01).2)From the analysis of Western blot results,higher DCX(P < 0.05)and lower Neu N(P <0.05)were expressed in hippocampi of P7 d mice in Hyp group.Analogously,higher DCX(P< 0.01)and lower Neu N(P < 0.01)expression were detected in hippocampi of P14 d mice in hypoxic group.3)RT-qPCR was used to examine mRNA levels of DCX and Neu N in the hippocampi of mice at P7 d and P14 d.Consistent with data of Western blot,results of RT-qPCR showed that higher DCX was expressed in P7 d mice of Hyp group(P < 0.05),while the expression of Neu N mRNA was significantly decreased(P < 0.05).Compared with the control group,the level of DCX mRNA in P14 d mice in Hyp group was significantly higher than that of the control group(P < 0.01),with a lower level of Neu N mRNA(P < 0.01),which is consistent with the protein results.4)The expression of presynaptic membrane-associated proteins(Munc13-1,Synaptotagmin1,Synaptotagmin2,Munc18-1 and VAMP2)in the hippocampi of mice at P7 d and P14 d were detected by Western blot.Compared with the control group,the expression of Munc13-1、Syn1 and Munc18-1 in P7 d mice was decreased.The protein expression of Munc13-1,Syn1,Syn2,Munc18-1 and VAMP2 were decreased in hypoxic group at P14 d.Based on the above experimental results,hypoxic exposure during pregnancy led to abnormal neuronal maturation in the hippocampus of mice at P14 d,manifested as increased number of immature neurons,decreased number of mature neurons,and lower expression of presynaptic membrane proteins,which indicated hindered synaptic maturation.5)The m EPSC results manifested that the frequency of m EPSC in hippocampal neurons of P14 d mice reduced significantly after hypoxic exposure(P < 0.001),while there was no significant difference in amplitude between the two groups,suggesting that exposure to hypoxia during pregnancy may lead to damaged presynaptic membrane of P14 d mice’s hippocampal neurons.6)Meanwhile,increased intracellular glutamate concentration in the hippocampus of the mice at P14 d was detected in the hypoxia group(P < 0.001).It could be inferred that the excitatory neurotransmitter glutamate accumulation in neurons might be closely related to decreased expression of presynaptic membrane-associated proteins,which might hinder the release of glutamate.3.Hypoxia affects maturation and function of murine primary neurons1)The effect of hypoxic exposure on neuronal cell viability was detected by CCK8 assay.The results showed that there was no significant difference in cell viability between the two groups after hypoxic exposure for 3 and 6 days.After hypoxic exposure for 9 days,the cell viability in hypoxic group was lower than that in the control group(P < 0.05).2)The effect of hypoxic exposure on cell apoptosis was detected by TUNEL test,and the results showed that cells did not appear apoptosis after exposure to hypoxia for 3 and 6 days;cells cultured under hypoxia for 9 days appeared obvious apoptosis.3)Western blot was used to detect the effect of hypoxic exposure on Cleaved-caspase3 and Caspase3 expression.The results showed that hypoxic exposure resulted in increased Cleaved-caspase3/Caspase3 expression in cells exposed to hypoxia for 9 days(P < 0.01).The above results indicated that the cell viability was not affected by hypoxic exposure for 6 days,and no sign of apoptosis was detected.4)Western blot was used to detect the expression of DCX,MAP2,Tau,Syn2,Munc18-1 and VAMP2 in neurons exposed to hypoxia for 2,3,6 and 9 days.Compared with the control group,DCX expression in neurons were consistently increased under hypoxia at 3,6 and 9days with exposure time prolonged.While the expressions of MAP2,Tau,Munc18-1,Syn2 and VAMP2 were consistently reduced to different degrees with the prolonged exposure time.5)RT-qPCR detected the mRNA levels of DCX and MAP2 in neurons exposed to hypoxia on Day 2,3,6 and 9.The results showed that compared with the control group,the levels of DCX mRNA in hypoxic group began to increase significantly at the 3rd day under hypoxic exposure and consistently increased as exposure time prolonged.The level of MAP2 mRNA decreased since the 2nd day under hypoxic exposure.The longer the exposure time continued,the more obvious the decline amplitude appeared,which was consistent with the protein results.6)Immunofluorescence staining was used to detect the fluorescent expressions of DCX and MAP2 in neurons.The results showed that the DCX/MAP2 ratio in Hyp group rose up after 3,6 and 9 days of hypoxic exposure,and the increase was more obvious with longer exposure time.At the same time,the morphology of neurons exposed to hypoxia for 6 days was significantly different from those in Con group,with evidently shorter neurites(P < 0.01).7)The effect of hypoxic exposure on neuronal calcium oscillations was detected by the laser confocal live cell workstation.The results showed after 6 days of exposure to hypoxia,the amplitude of calcium oscillations in Hyp group was significantly lower than that in Con group(P < 0.01),with increased frequency of calcium oscillations,which might not be sufficient for normal cellular function.However,both the amplitude and frequency of cellular calcium oscillations were lower in hypoxic group for 9 days than those in the control group(P < 0.05).The above results indicated that the neuronal maturation was restrained after 6 days of hypoxic exposure,as well as impaired functions.4.Hypoxia-induced changes in mitochondrial function can impact the neuronal maturation1)The ultrastructure of mitochondria was detected by transmission electron microscopy.The results showed in the hippocampi of mice at P14 d,the number of mitochondria in Hyp group was significantly decreased than that of Con group(P < 0.01),and the average mitochondrial area was significantly reduced(P < 0.05),suggesting that hypoxia affected the mitochondrial morphology.Swollen mitochondria,reduced and shortened mitochondrial cristae was also visible in neurons exposed to hypoxia for 6 days.The results indicated with the prolongation of hypoxic exposure time,the morphology of mitochondrial cristae in neurons showed progressive damage.2)The ATP level in the hippocampi of mice was tested by enhanced ATP kits.According to the results,the ATP level was significantly lower in hypoxic mice at P7 d and P14 d(P <0.01).Compared with the control group,the intracellular ATP level of neurons exposed to hypoxia for 3 days and 6 days had decreased(P < 0.0001).With the supplementation of 0.1m M ATP for 6 days,ATP level was significantly restored in Hyp group(P < 0.001);however,there was no significant difference between ATP supplementation group and Hyp group for 9days,suggesting 0.1 m M ATP could reverse the ATP decrease caused by 6 days of hypoxic exposure,but could not restore the ATP decrease caused by 9 days of hypoxia.3)The kit detects the activities of respiratory chain complexes I,II,III and V in the hippocampi of mice.Compared with the control group,the activities of respiratory chain complexes I,II and V in the hippocampi of the mice at P14 d were significantly down-regulated in hypoxic group(P < 0.01).However,there was no difference in respiratory chain complex III between the two groups.The activity of respiratory chain complexes in primary cultured neurons were also detected,indicating that the respiratory chain complex I in Hyp group was reduced in neurons with hypoxic exposure for 6 days(P < 0.05),as well as more significant declines in complexes II and V(P < 0.01).Consequently,it demonstrated that hypoxia-induced decrease of ATP level was closely related to decreased activities of respiratory chain complexes I,II,and V.4)Western blot was used to evaluate the protein levels of respiratory chain complex-related molecules in murine hippocampus.Data showed that protein levels of Ndufb-8(complex I)and ATP5a(complex V)in the hippocampus of P14 d mice in the hypoxic group were significantly lower than those in the control group(P < 0.01).The protein levels of respiratory chain complex-related molecules in neurons were also detected,which was consistent with the above results.5)RT-qPCR was used to detect the mRNA expression levels of respiratory chain complex-related molecules in the hippocampi of mice at P14 d.Data showed that the expressions of Ndufv-1(complex I),Ndufb-8(complex I),and SDHA(complex II)in the hippocampus of mice in the hypoxic group on P14 d were significantly decreased compared with those in the control group(P < 0.01).However,there was no significant difference in the mRNA levels of SDHB(complex II)and Uqcrh(complex III)between these two groups.Besides,mRNA level of ATP5a(complex V)in the hypoxic group was also significantly decreased compared with that in the control group(P < 0.001).The mRNA levels of respiratory chain complex-related molecules in neurons were also detected,which was consistent with the hippocampus of P14 d mice results.6)Utilizing Western blot to detect the expression of mitochondrial fission-fusion related proteins in the hippocampi of mice.The expression of the fission protein Drp1 in mice at P14 d was decreased in Hyp group(P < 0.05),as well as the fission protein Fis1 expression(P <0.01).The protein levels of mitochondrial fission genes Drp1 and Fis1 in neurons were also detected,suggesting that the expressions of Drp1 and Fis1 were decreased in hypoxic exposure for 6 days(P < 0.05).It was concluded that disturbed mitochondrial energy metabolism deeply led to changes in mitochondrial dynamics.7)According to the effects of ATP supplementation on the morphology of neurons tested by immunofluorescence staining,it was shown that with the exposure to hypoxia for 6 days,neurons in Hyp group had significantly shorter neurites(P < 0.001),compared with tightly recovered neurite length in hypoxia-supplemented ATP groups(P < 0.01).These suggest that ATP supplementation could partially alleviate neuronal morphological damage caused by hypoxia.Western blot detected the effect of ATP supplementation on the expression levels of DCX,MAP2 and Tau in Hyp group for 6 days.Higher expression of DCX in Hyp group were detected compared with those in Con group(P < 0.01)and recovered in the hypoxia-supplemented ATP group(P < 0.05).The expressions of MAP2 and Tau in Hyp group were decreased compared with the control group and restored to different degrees with supplementation of 0.1 m M ATP.To evaluate the effect that ATP supplementation have on the expressions of presynaptic membrane-related proteins in neurons exposed to hypoxia for 6days,it was found that Munc13-1、Syn1、Syn2、Munc18-1 and VAMP2 were all decreased in Hyp group and Syn1,Syn2,Munc18-1 and VAMP2 were recovered to some different extent in ATP-supplemented group compared with the hypoxic group.All above confirmed that hypoxia-induced abnormal neuronal maturation was caused by the reduction of mitochondrial ATP levels.5.Regulatory effects of CIRBP on neuronal dysplasia induced by hypoxia1)Western blot was used to evaluate the effect of hypoxic exposure during pregnancy on the expression of CIRBP protein in the hippocampus of offspring mice,it was found that CIRBP in mice at P0 d and P3 d was increased under hypoxia(P < 0.05),with no significant difference in those at P5 d and P7 d,whereas the expression level of CIRBP in mice at P14 d and P21 d was significantly lower than those in the control group(P < 0.01).Immunofluorescence staining was used to detect the changes of CIRBP,DCX and Neu N in the hippocampi of mice at P14 d.The results showed that lower expression of CIRBP and Neu N in the DG was detected in Hyp group with increased expression of DCX,suggesting that the decreased CIRBP expression might be involved in the process of neuronal maturation.The expression of CIRBP in neurons was detected by Western blot on 2,3,6 and 9 days during hypoxic exposure.It was found that CIRBP was significantly increased at the 2nd day under hypoxic exposure(P < 0.01),while decreased on the 3rd day(P < 0.05)and consistently lower-expressed after 6 days of hypoxic exposure(P < 0.01).RT-qPCR detected the changes of CIRBP at mRNA level in neurons exposed to hypoxia on 2,3,6 and 9 days.The results showed that CIRBP increased since the 2nd day of hypoxic exposure(P < 0.01),decreased at the 3rd day(P < 0.01),and decreased further after 6 days of exposure(P <0.001),which was consistent with the protein-level changes.2)The body weight changes of CIRBP KO mice exposed to hypoxia during pregnancy at P14 d showed that the weight of CIRBP KO mice was significantly lower than that of WT mice(P< 0.001).Compared with WT hypoxic mice,CIRBP KO mice exposed to hypoxia had a further reduction in body weight(P < 0.01).The results suggested that knockout of the CIRBP gene would affect the weight gain of mice and weight loss appears more obvious when exposed to hypoxic environment.3)The protein changes of DCX,MAP2,Tau and presynaptic membrane proteins Syn1 and Syn2 in neurons were detected in CIRBP KO mice at P14 d.The Western blot results showed that compared with WT mice,expression of DCX was increased in CIRBP KO mice at P14 d,with decreased MAP2,Tau and presynaptic membrane proteins Syn1 and Syn2,indicating that knocking out CIRBP gene hampered neuronal maturation in hippocampus of mice.Compared with WT hypoxic mice,the expression of DCX was highly increased,while the MAP2,Tau,Syn1 and Syn2 were decreased in CIRBP KO mice,suggesting that the growth of CIRBP KO mice offspring were more restricted under hypoxic exposure,indirectly pointing out that CIRBP was involved in the process that hypoxia inhibits neuron maturation.4)Immunofluorescence staining was used to detect the expression of DCX and MAP2 in CIRBP-overexpressed neurons under hypoxia.Compared with the NC + Hyp group,DCX/MAP2 decreased in CIRBP + Hyp group(P < 0.001)and the length of neuronal neurites also recovered,indicating that CIRBP might retard neuronal abnormal maturity induced by hypoxia.The fluorescent changes of the presynaptic membrane molecule Syn1 under hypoxia showed that the intensity of Syn1 decreased significantly in the NC + Hyp group after 6 days of hypoxic exposure,while the fluorescence intensity increased in CIRBP + Hyp group compared with NC + Hyp group,indicating that CIRBP might correct the neuronal presynaptic membrane damage caused by hypoxia.5)The expression of DCX,MAP2 and Tau in CIRBP-overexpressed neurons under hypoxia showed that DCX was increased in NC + Hyp group after hypoxic exposure for 6 days,while decreased in CIRBP + Hyp group compared with those in NC + Hyp group(P < 0.05).The expression of MAP2 and Tau protein decreased after hypoxic exposure,while up-regulated in CIRBP + Hyp group compared with those in NC + Hyp group(P < 0.05),further indicating that CIRBP could partially restore the abnormal neuron maturation caused by hypoxia.At the same time,the expression of presynaptic membrane-related proteins in neurons was detected.The results showed that compared with the control group,the expressions of Munc13-1,Syn1,Syn2,Munc18-1 and VAMP2 decreased after hypoxic exposure for 6 days.With the overexpression of CIRBP,the protein levels of Syn1,Syn2 and Munc18-1 were recovered to varying degrees in CIRBP + Hyp group compared with those in NC + Hyp group,but there was no statistical difference in Munc13-1 and VAMP2 level between the two groups,indicating that CIRBP could partially restore the decreased expression of presynaptic membrane proteins caused by hypoxia.6)Immunofluorescence staining was used to detect the morphology of neurons extracted from CIRBP-overexpressed pregnant mice in hypoxic environment.The results showed that the length of neuronal neurites was shorter after 6 days of hypoxic exposure(P < 0.0001)in WT Hyp group compared with WT Con group,while the neurites length of Cre-CIRBP Hyp group was significantly recovered compared with WT Hyp group(P < 0.001).7)Western blot detected the protein expression of DCX,MAP2 and Tau in neurons extracted from CIRBP-overexpressed pregnant mice under hypoxia.DCX expression increased,while MAP2 and Tau expression decreased in WT Hyp group compared with those in WT Con group(P < 0.01).DCX expression in Cre-CIRBP Hyp group was significantly decreased,while MAP2 and Tau expression increased compared with WT Hyp group(P < 0.01).The changes of neuronal presynaptic membrane-related proteins after 6 days of hypoxic exposure showed that compared with the WT Con group,the protein levels of Munc13-1,Munc18-1,Syn1,Syn2 and VAMP2 in the WT Hyp group were significantly decreased.The expressions of Munc18-1,Syn1 and Syn2 molecules in the Cre-CIRBP Hyp group were recovered to different degrees,which was consistent with the changes in CIRBP-overexpressed neurons.All above may testify that CIRBP could restore the hypoxia-induced abnormal neuronal maturation.8)The kit was used to detect intracellular glutamate concentration of neurons extracted from pregnant mice overexpressing the CIRBP gene under hypoxic conditions.The results showed that the intracellular glutamate concentration in the WT Hyp group was significantly increased compared with the WT Con group after 6 days of hypoxic exposure(P < 0.01).And the glutamate concentration of mice in the Cre-CIRBP Hyp group was significantly reversed compared with the WT Hyp group(P < 0.01).These results indicated that overexpression of CIRBP could restore the disturbance of excitatory neurotransmitter release induced by hypoxia.6.The regulation of CIRBP on mitochondrial function in neurons1)ATP kits were used to detect the expression of ATP in CIRBP-overexpressed neurons under hypoxia.It was shown that after exposure to hypoxia for 6 days,the ATP level in NC + Hyp group was significantly lower than that in NC + Con group(P < 0.0001)and the intracellular ATP level in CIRBP + Hyp group was significantly reversed compared with that in NC + Hyp group(P < 0.01).The expression of ATP levels in neurons extracted from CIRBP-overexpressed pregnant mice after 6 days of hypoxic exposure was consistent with the changes in neurons transfected with CIRBP-overexpressed virus.2)The kit was used to examine the activity changes of mitochondrial respiratory chain complexes I,II,and V in neurons transfected with CIRBP-overexpressed virus after 6 days of hypoxic exposure.The activity of respiratory chain complexes I,II,and V was decreased.After transfected with CIRBP-overexpressed virus,the activity of complexes I and V was significantly increased in CIRBP + Hyp group compared with NC + Hyp group.The activity changes of mitochondrial respiratory chain complex in neurons extracted from CIRBP-overexpressed pregnant mice were consistent with those in neurons transfected with CIRBP-overexpressed virus.3)Western blot was used to detect the changes of mitochondrial respiratory chain complex protein levels after overexpressing CIRBP under 6 days of hypoxic exposure.Data showed that protein levels of Ndufb-8(complex I)and ATP5a(complex V)were significantly decreased in NC + Hyp group compared with NC + Con group(P < 0.01).Compared with the NC + Hyp group,protein levels of Ndufb-8(complex I)and ATP5a(complex V)were significantly increased in the CIRBP + Hyp group(P < 0.05).4)RT-qPCR was used to detect the changes in mitochondrial respiratory chain complex mRNA levels after overexpressing CIRBP under 6 days of hypoxic exposure.Data showed that mRNA levels of Ndufb-8(complex I)and ATP5a(complex V)were significantly reduced in NC + Hyp group compared with NC + Con group(P < 0.01,P < 0.001).Compared with the NC + Hyp group,mRNA levels of Ndufb-8(complex I)and ATP5a(complex V)were significantly increased in the CIRBP + Hyp group(P < 0.001,P < 0.01).5)Transmission electron microscopy detected the changes of mitochondrial ultrastructure in neurons extracted from CIRBP-overexpressed pregnant mice under hypoxia.The results showed that when exposed to hypoxia for 6 days,swollen mitochondria,less and shorter mitochondrial cristae were observed in WT Hyp group.Compared with the WT Hyp group,there were a significant recovery of mitochondrial cristae morphology in Cre-CIRBP Hyp group.6)Western blot was used to detect the mitochondrial fission proteins expressions in neurons extracted from CIRBP-overexpressed pregnant mice under hypoxia.The results indicated that both of fission proteins Drp1 and Fis1 decreased in WT Hyp group when exposed to hypoxia for 6 days.Compared with WT Hyp groups,the Drp1 and Fis1 in Cre-CIRBP Hyp group were increased to different levels.Conclusions:1.Hypoxic exposure during pregnancy leads to murine retarded brain development,abnormal neuron maturation in the hippocampus of offspring P14 d mice,as well as impairment of spatial learning and memory.2.Hypoxic exposure leads to abnormal morphology and dysfunction of mitochondria in the hippocampus of P14 d mice.3.Overexpression of CIRBP restores the activity of respiratory chain complexes I and V by regulating Ndufb-8(complex I)and ATP5a(complex V),thus alleviating the energy metabolism disorder induced by hypoxia,further rectifying the abnormal neuron development caused by hypoxia exposure. |
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