| Objective: To explore the effects of the continuous fluorine combined with aluminum exposure during gestation and lactation periods on spatial learning and memory abilities and the c AMP-PKA-CREB signaling pathway in the 2nd offsprings’ hippocampus. Methods: Thirty-six pregnant rats were randomly divided into 9 groups(four in each). The exposure of fluorine and aluminium in maternal rats was conducted by drinking toxic water freely, concentration of Na F and Al Cl3 in drinking water for each group was(0, 0),(100, 0),(200, 0),(0, 500),(0, 1000),(100, 500),(100, 1000),(200, 500),(200,1000)mg/L. The pregnant rats were treated from pregnant day 0 to postnatal day 21(PND21) of the first generation offspring rats. When the first generation rats were at PND90, 6 of the 1st offspring rats were selected randomly in each group and female-male ratio was 2:1. 8 of the 2nd offsprings’ rats, half male and half female,were selected randomly in each group and treated doses followed the same groups as above mentioned from PND22 to PND60.The 2nd offsprings’ rats were weighed once 2 week. Before killed, physiological development and the neurobehavioral reflection indexes of the 2nd offsprings were observed, learning and memory abilities were tested by the Morris water maze, 12 hour urine was collected. After killed, the 2nd offsprings’ brains were kept, fluorine in urine and brain tested by ion selected electrode method, and aluminium in urine and brain tested by inductively coupled plasma atomic emission spectrometry; the protein content of c AMP and m RNA expression levels of PKA and CREB and protein expression levels of PKA and phosphorylated CREB(p-CREB) were detected by enzyme-linked immuno sorbent assay(Elisa), real-time polymerase chain reaction( Real-time PCR)and immuohistochemistry, respectively. Results: 1.Concentration of fluorine and aluminum in urine and brain: Compared with the control group, except the groups only exposed to aluminum, the concentration of fluorine in urine and brain of the 2nd offspring rats from the other tested groups was increased(P<0.05); except the groups only exposed to fluorine, aluminum in urine and brain of the 2nd offspring rats from the other test groups was elevated significantly(P<0.05); and the concentration of fluorine and aluminum in urine and brain of the 2nd offspring rats from the high fluorine + low aluminum group was higher than those from the other test groups. 2. The growth and neurobehavior reflection of 2nd offspring’ rats: Compared with the control group, except the low fluorine group, the high aluminum group and the low fluorine + low aluminum group, weight of the 2nd offspring rats from the other tested groups was lower(0 week); the weight of the 2nd offspring rats from the high aluminium group was higher, but that was lower from the low fluorine+high aluminum group and the high fluorine+low aluminum group(the 2nd week and the 4th week), the difference was remarkable(P<0.05); except that from the fluorine groups and the high aluminum group, the weight of the 2nd offspring rats from the other test groups was lower(the 6th week); except the weight of the 2nd offspring rats from the low fluorine group was higher, that from the other test groups were lower, the differences were significant(P<0.05)(the 8th week). Brain weight coefficients, teething and eye opening time of the 2nd offspring rats from every test group had no significant difference. Compared with the control group, time of surface righting reflex was significantly prolonged in the high fluorine group, the low fluorine+low aluminum group and the high fluorine+low aluminum group(P<0.05); except the low fluorine group, the low aluminum group and low fluorine+low aluminum group, time of cliff avoidance reflex were significantly prolonged in the other groups(P<0.05); 3. Spatial learning and memory ability: Compared with the control group, except the low fluorine group, the low aluminum group and the low fluorine + low aluminum group, the escape latency in the 2nd day of the 2nd offspring rats from the other groups was significantly prolonged(P<0.05); except the low fluorine+low aluminum group, the escape latency in the 3rd day and the 4th day of the 2nd offspring’rats from other group was significantly prolonged(P<0.05). Compared with the control group, except those from the low fluorine group, low aluminum group and the low fluorine+low aluminum group, the time of first reaching platform and the times of across platform of the offspring’ rats were markedly prolonged(P<0.05),and reduced in the other groups(P<0.05),respectively. 4. Expression of proteins and genes correlative to c AMP-PKA-CREB signaling pathway in the hippocampus: Compared with the control group, the c AMP contents in the hippocampuses from the fluorine groups, the low aluminum group and the low fluorine + high aluminum group decreased,(P<0.05); the protein expression levels of PKA in the hippocampuses from the fluorine groups and all the combined fluorine with aluminium groups decreased, but the protein expression level of PKA in the hippocampuses from the aluminium group increased; the protein expression levels of p-CREB in the hippocampuses from all the experimental groups decreased; the PKA m RNA expression levels in the hippocampuses from the the low aluminum group and low fluorine+high aluminum group elevated(P<0.05); the expression levels of CREB m RNA in the hippocampuses from the low aluminium group,the low fluorine + high aluminum group and the high fluorine + high/low aluminium groups elevated(P<0.05). 5. Effects of the combined fluorine and aluminum on every indexes: There were antagonism effects of the combined fluorine and aluminum exposure on the spatial learning and memory abilities, m RNA expression levels of CREB and PKA and the protein contents of c AMP in the hippocampuses and the protein contents of PKA and p-CREB in the brain tissues, however, there were synergism effects of the combined fluorine and aluminum exposure on the weight in the latter stages; Conclusions: Under the experimental condition, 1. Exposure of continuous fluorine combined with aluminum inhibited growth and development of the 2nd offspring rats; 2. Exposure of continuous fluorine combined with aluminum did harm to the spatial learning and memory abilities of the 2nd rat offspring rats; 3. Exposure of continuous fluorine combined with aluminum maybe damage the learning and memory abilities of the 2nd rat offspring rats through inhibiting c AMP-PKA-CREB signaling pathway in the hippocampus. 4. Combined effects of fluorine and aluminum mainly were antagonism interaction. |
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