| Objective:To gain insight into the physiological effects of thyroid hormone (TH) and growth hormone (GH), and their cross-talking on brain in different development stages, we have investigated the effects of TH on GH, growth hormone receptor (GHR) and the key molecules in the signal transduction pathway of GH in hippocampus with the models of hypothyroidism rat in different development stage, and on cultured hippocampal neurons. We also tested the effects of GH and TH on the differentiation of neural stem cell (NSCs) in hippocampus.Methods:1. Observe the expression of GHR in hippocampal neurons, and the effects of TH on the expression of GH/GHR and GH signal transduction in cultured hippocampal neurons. The expression of GHR was observed in hippocampal neurons by immunohistochemistry, and the mRNA levels of GH/GHR/IGF-1 were tested by real time PCR in primary hippocampal neurons cultured with different concentrations of 3,5,3’-triiodo thyronine (T3). And then, STAT5 protein, a GH signaling pathway molecule, was measured by western blotting in the hippocampal neurons cultured with an appropriate concentration of GH, T3, Mab74.3 (GH antagonist) independently or joint together.2. Observe the effects of hypothyroidism on GH signal transduction in rats of different developmental stage. After the models of hypothyroidism in antenatal,4 weeks and 8 weeks old rats were established, the GH and GHR mRNA levels were tested using real time PCR, and the molecules including JAK2, P-JAK2, STAT5, P-STAT5 in GH signaling pathway were also detected in hippocampus of these rats models.3. Observe the effects of T3 and GH on the differentiation of rat hippocampal neural stem cells and the relationship between them. After the neural stem cells were isolated, purified and identified from rat’s hippocampus, the effects of T3 and GH with different concentration on the differentiation of these hippocampal neural stem cells were observed. After treatment of the two hormones simultaneously, the change of the diffentiation of rat hippocampal neural stem cells.Results:1. Effects of T3 on GH/GHR and the signal transduction molecule STAT5 protein levelsThere were abundant GHR on neurons membrane of rat hippocampus. After treatment with different concentrations of T3 (0,2.5nM,5nM,10 nM) for 96 hours, GH and GHR mRNA level in primary cultured hippocampal neurons were changed in different degrees. After 96 hours treatment of 5nM T3, GH and GHR mRNA level was significantly higher than in the control group (p<0.05). However, IGF-1 mRNA levels showed no significant change compare to the control group.Primary cultered hippocampal neurons of neonatal rat were treated with T3 (5nM), GH (25ng/ml), T3 (5nM)+GH (25ng/ml), T3 (5nM)+GH (25ng/ml)+ Mab74.3 (5ng/ml) after 96 h, western blotting showed that protein levels of STAT5 were changed with different degrees compared to the control group (PBS treated). In T3 treatment of neonatal rat hippocampal neurons, STAT5 protein levels were slightly higher than the control group, but there was no statistical significance (p>0.05), after GH treatment, STAT5 protein levels were increased compared to control group (P <0.05); when adding T3 and GH at the same time to the cultured hippocampal neurons, STAT5 protein levels were significantly higher than the control group (p<0.01). When adding T3 and GH, Mab74.3 simultaneously, STAT5 protein levels were decreased compared to the control group (p<0.05).2. Effects of hypothyroidism on GH and GHR and signaling molecule protein levels in rat hippocampus.The relative thyroid weight (thyroid weight/100g body weight) was significantly higher in 4 weeks and 8 weeks old hypothyroidism rats than the control rats(p<0.001). Serum FT3, FT4 levels of 4 weeks and 8 weeks old hypothyroidism rats were significantly decreased compared to the control group(p<0.001), TSH levels were significantly higher than control group in 4 weeks and 8 weeks old hypothyroidism rats(p<0.001). The results indicated that hypothyroidism rat models were successfully estabolished. The body weight of CHT rats were significantly decreased compared to control rats (p<0.001), the body weight of T3 treated CHT group (CHT+T3 group) were significantly higher than the CHT group (p<0.001), and there were no difference between CHT+T3 group and control rats. ELISA results showed that serum T3 and T4 levels of CHT rats were significantly lower than control group (p<0.001). After T3 treated, serum T3 levels in CHT+T3 group were significantly increased compared to CHT group (p<0.001). The results indicated that rat model of CHT were successfully prepared, and T3 treatment was successful.GH and GHR mRNA levels in the hippocampus of 2W old CHT group were significantly lower than control group (p<0.001), and after T3 treated, GH and GHR mRNA levels were significantly higher than CHT group (p<0.05). GH and GHR protein levels in the hippocampus of CHT group were decreased significantly compared to control group, and also significantly recovered after T3 treatment. Protein levels of JAK2, P-JAK2, STAT5, P-STAT5 in hippocampus of CHT group decreased significantly compared to the control group, and significantly recovered after T3 treatment, but some results showed that there was still a gap between the CHT+T3 group and the control group. HE and nissl staining showed that there was a neuron loss and severe structural damage in hippocampus of CHT rat at the 7W age, and there was a considerable degree of recovery after T3 treatment. Behavioral test results showed that learning and memory ability of rats in CHT group were decreased, and T3 treatment can recover it to some degree.There were no difference between hypothyroidism rats and control rats in GH and GHR mRNA levels in 4 weeks or 8 weeks old rats. Western blotting showed that in 4 weeks and 8 weeks old hypothyroidism rat, GH and GHR protein in the hippocampus were decreased significantly compared to control group. The important signal molecules in GH signaling pathway protein levels were significantly lower than the control group, but JAK2/P-STAT5 protein levels had no significant difference between hypothyroidism rats and control rats.3. After 30ng/ml GH treatment, percentage ofβⅢ-tubulin positive neurons was higher than control group (P<0.05), percentage of GFAP-positive astrocytes was slightly lower than control group (P<0.05), after 10ng/ml and 90ng/ml GH treated, percentages of neurons and astrocytes had no statistically significant difference compared to control group.After 40nM T3 treatment, percentage ofβⅢ-tubulin positive neurons was higher than control group (P<0.05), percentage of GFAP-positive astrocytes was slightly lower than control group (P<0.05); after 10nM T3 treatment, percentage of neurons was slightly increased compared to the control group (P<0.05), the proportion of astrocytes had no statistically significant difference compared to control group.5nM T3 treatment didn’t change the percentage of neurons and astrocytes compared to control group.After 10nM T3 and 10ng/ml GH treatment, percentage of neurons was significantly higher than the control group (P<0.01), 10nM T3 treatment and 10g/ml GH treatment didn’t change the percentage of neurons compared to control group. In 10nM T3, 10ng/ml GH and 10nM T3+10 ng/ml GH treatment group, the percentage of astrocytes had no significant difference compared to control group.Conclusions:This study confirmed that TH can upregulate the expression of GH and GHR in rat hippocampus, and promote GH signal transduction. During the stage of brain development, interestingly, there are upregulation both in the transcription and translation levels on GH and GHR, but after maturation of brain, just in translation level. It indicated that the regulation of TH on GH occurs mainly during the development stage of brain. Therefore, GH may mediate the function of TH to promot brain development. The viewpoint that cretinism is just the results of thyroid hormone deficiency will be renewed. |
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