| ObjectiveIn the multiple aspects of Ethology, tissue morphology and molecular biology, to observe the effect of tonifying kidney therapy on the proliferation of hippocampal in hippocampal dentate gyrus of kidney deficiency model rats and the Action mechanism in vitro. In order to the definite internal relations of the function of tonifying kidney therapy and replenishing essence and the proliferation of hippocampal neural stem cells, the control mechanism of tonifying kidney on the proliferation and differentiation of neural stem cells, to provide experimental and theories gist for the treatment of difficult and complicated clinical diseases with tonifying kidney therapy combined with stem cell technology.Methods1. The theoretical researchBy reviewing the related literature, probe into the relationship between tonifying Kidney and replenishing essence and the proliferation and differentiation of hippocampal neural stem cells and the influencing factors.2. The experimental researchUsing pure sexual intemperance (male and female ratio 5:1, rearing for 30 days), long-term of sexual intemperance (male and female ratio 2:1, rearing for 6 months), sexual intemperance combined with tired (male and female ratio 5:1 and forced swimming for 1 hour, rearing for 30 days)and gavage with adenine, product kidney deficiency model rats. In order to the ideal animal model for further research, evaluate the quality of model by comparing the results of the Morris water maze test and histomorphology index changes. To observe the effects of the tonifying kidney therapy on spatial Learning and memory abilities and histomorphology changes of relevant organs in kidney deficiency model rats, the effects of the tonifying kidney therapy on cell ultra-structure and the expression of protein related to proliferation and apoptosis in hippocampal dentate gyrus of kidney deficiency model rats, the effects of the tonifying kidney therapy on the relative expression of Wntl,β-catenin, CyclinDl and c-Myc.Results1. The theoretical researchThere is an association between tonifying Kidney and replenishing essence and the proliferation and differentiation of stem cells and a certain intervention effect of the prescription of tonifying Kidney on the proliferation and differentiation of stem cells.2. The experimental research2.1 the effects of the tonifying kidney therapy on spatial Learning and memory abilities of kidney deficiency model ratsIn the place navigation test of Morris Water Maze, compared with the normal control group, the escape latency of rats were prolonged obviously in the kidney deficiency model group (P<0.01,P<0.05), The escape latency of rats in moxibustion group was shorter than the kidney deficiency model group (P <0.05).Compared with moxibustion group, the escape latency of rats were prolonged obviously in the low dosage of Liuwei Rehumannia Pill group and the high dosage of Liuwei Rehumannia Pill group (P<0.01, P<0.05). In the first 20 seconds of the spatial probe test, compared with the normal control group, platform crossing times (times of crossing the former site of removed hidden platform) and the residence time of the former hidden platform surrounding area were reduced in the the kidney deficiency model group, but no obvious difference. Compared with kidney deficiency model group, platform crossing times was increased obviously in the high dosage of Liuwei Rehumannia Pill group (P<0.05), platform crossing times was increased in moxibustion group, but no obvious difference, the residence time of the former hidden platform surrounding area were increased obviously in the high dosage of Liuwei Rehumannia Pill group and moxibustion group (P<0.05). Compared with the low dosage of Liuwei Rehumannia Pill group, platform crossing times and the residence time of the former hidden platform surrounding area were increased obviously in the high dosage of Liuwei Rehumannia Pill group and moxibustion group (P<0.05)2.2 The effects of the tonifying kidney therapy on cell growth changes in hippocampal dentate gyrus of kidney deficiency model rats2.2.1 Results of Morris Water Maze testIn the place navigation test of Morris Water Maze, Compared with the normal control group, the escape latency of rats were prolonged obviously in the kidney deficiency model group (P<0.01). The escape latency of rats in the rest groups were shorter obviously than the kidney deficiency model group (P<0.01, P<0.05). In the first 20 seconds and 60 seconds of the spatial probe test, the residence time of the former hidden platform quadrant and the residence time of the former hidden platform surrounding area were reduced in the the kidney deficiency model group (P<0.01), compared with kidney deficiency model group, the residence time of the former hidden platform quadrant and the residence time of the former hidden platform surrounding area were prolonged in the high dosage of the Jigui shenqi pill group(P< 0.01, P < 0.05). In the first 20 seconds seconds of the spatial probe test, compared with kidney deficiency model group, the residence time of the former hidden platform surrounding area were prolonged in the high dosage of the low dosage of the Liuwei Rehumannia Pill group(P< 0.05), compared with the high dosage of Jigui shenqi pill group, the residence time of the former hidden platform surrounding area was reduced in the low dosage of Jigui shenqi pill group and moxibustion group.2.2.2 Results of the cell ultra-structure of hippocampal dentate gyrus detected by transmission electron microscopyThere were cell ultra-structure lesions of hippocampal dentate gyrus in kidney deficiency model group. The lesions showed mainly the damage of the obvious dissolution neural microfilament and microtubule structure, vacuolar degeneration of mitochondria. The cell ultra-structure lesions of hippocampal dentate gyrus were significantly alleviated in varying degrees in the intervention groups.2.2.3 Results of the expression of protein of hippocampal dentate gyrus detected by fluorescence immunocytochemistryCompared with the normal group, the expression of β-catenin, NeuN and PCNA of hippocampal dentate gyrus were obviously reduced in kidney deficiency model group(P<0.01, P<0.05), and the expression of TUNEL was increased. Compared with kidney deficiency model group, the expression of NeuN and PCNA of hippocampal dentate gyrus were obviously increased in moxibustion group (P< 0.05), the expression of β-catenin, NeuN and PCNA of hippocampal dentate gyrus were increased obviously in the rest groups (P<0.01,P<0.05) and the expression of TUNEL was obviously reduced(P<0.01, P<0.05). The curative effect of high dose group was better than that of low dose group, The curative effect of prescription group was better than that of moxibustion group.2.3 The effects of the tonifying kidney therapy on growth changes in hippocampal dentate gyrus of kidney deficiency model rats2.3.1 Results of Morris Water Maze testIn the place navigation test of Morris Water Maze, Compared with the normal control group, the escape latency of rats were prolonged obviously in the kidney deficiency model group and adenine group (P<0.01), The escape latency of rats in the Liuwei Rehumannia Pill group and Jigui shenqi pill group were shorter obviously than the kidney deficiency model group (P<0.01, P<0.05). The escape latency of rats in adenine drug intervention group was shorter obviously than adenine group (P<0.05). Compared with the Liuwei Rehumannia Pill group, The escape latency of rats in Jigui shenqi pill group was reduced. There was no obvious escape latency difference between Liuwei Rehumannia Pill group and Jigui shenqi pill group.In the spatial probe test, compared with the normal control group, platform crossing times and the residence time and distance of the former hidden platform surrounding area were reduced obviously in the the kidney deficiency model group and adenine group (P<0.01). compared with kidney deficiency model group, platform crossing times and the residence time and distance of the former hidden platform surrounding area were reduced obviously increased in Jigui shenqi pill group (P<0.01, P<0.05), platform crossing times and the residence distance of the former hidden platform surrounding area were reduced obviously increased in Liuwei Rehumannia Pill group (P<0.05). Compared with adenine group, platform crossing times and the residence time and distance of the former hidden platform surrounding area were reduced obviously increased in adenine drug intervention group (P<0.05).2.3.2 Results of tissue morphology in testis and kidney detected by microscopyThere were tissue morphology lesions of testis in kidney deficiency model group and adenine group. The lesions showed mainly that the number of spermatogenic cells and sperm reduced, atrophy spermatogenic cells inlumen, even necrosis inlumen. The tissue morphology lesions of testis were significantly alleviated in varying degrees in the intervention groups.There were tissue morphology lesions of kidney in kidney deficiency model group and adenine group. The kidney lesions in kidney deficiency model group showed mainly that hyaline cast of kidney tubules, obvious vessel congestion in renal interstitium, local inflammation. The kidney lesions in adenine group showed mainly the number of nephron and expansion of kidney tubules and fester in kidney tubules with inflammatory cells and necrosis cell debris. The tissue morphology lesions of testis were significantly alleviated in varying degrees in the intervention groups.2.3.3 Results of the expression of protein of testis and kidney detected by fluorescence immunocytochemistryCompared with the normal group, the expression of β-catenin of testis was obviously reduced in kidney deficiency model group and adenine group (P <0.01), and the expression of TUNEL was increased obviously(P< 0.01). Compared with kidney deficiency model group, the expression of β-catenin and PCNA of testis were obviously increased in Liuwei Rehumannia Pill group and Jigui shenqi pill group (P<0.01), and the expression of TUNEL was reduced obviously (P<0.01). There was no obvious difference between Liuwei Rehumannia Pill group and Jigui shenqi pill group. Compared with the normal group, the expression of PCNAof kidney was obviously reduced in kidney deficiency model group and adenine group (P<0.01), and the expression of TUNEL was increased obviously(P<0.01). Compared with kidney deficiency model group, the expression of β-catenin of testis were obviously increased in Liuwei Rehumannia Pill group (P<0.01), and the expression of TUNEL was reduced obviously in Liuwei Rehumannia Pill group and Jigui shenqi pill group (P< 0.01). Compared with kidney adenine group, the expression of β-catenin and PCNA of testis were obviously increased in adenine drug intervention group (P<0.01), and the expression of TUNEL was reduced obviously (P<0.01).2.4 The effects of the tonifying kidney therapy on the relative expression associated protein of Wnt signaling pathway in hippocampal dentate gyrus of kidney deficiency model rats.2.4.1 The result of Western blotCompared with the normal control group, the relative expression of Wntl, β-catenin, c-Myc and CyclinDl of hippocampal dentate gyrus were reduced obviously in kidney deficiency model group and adenine group (P< 0.05) Compared with kidney deficiency model group, the relative expression of Wntl, β-catenin, c-Myc and CyclinDl of hippocampal dentate gyrus were increased obviously in Liuwei Rehumannia Pill and Jigui shenqi pill group (P<0.05, P <0.01). Compared with adenine group, the relative expression of Wntl, β-catenin, c-Myc and CyclinDl of hippocampal dentate gyrus were increased obviously in adenine drug intervention group (P<0.05, P<0.01)2.4.2 The results of real-time quantitative PCRCompared with the normal control group, the relative expression of β-catenin mRNA, c-Myc mRNA and CyclinDl mRNA of hippocampal dentate gyrus were reduced obviously in kidney deficiency model group and adenine group (P<0.05, P<0.01). Compared with kidney deficiency model group, the relative expression of Wntl mRNA, β-catenin mRNA, c-Myc mRNA and CyclinDl mRNA of hippocampal dentate gyrus were increased obviously in Liuwei Rehumannia Pill and Jigui shenqi pill group (P<0.05, P<0.01). Compared with adenine group, the relative expression of β-catenin mRNA, c-Myc mRNA and CyclinDl mRNA of hippocampal dentate gyrus were increased obviously in adenine drug intervention group (P<0.05, P<0.01).Conelusion1. Long-term of sexual intemperance, sexual intemperance combined with tired too much and gavage with adenine product ideal kidney deficiency rat model. According to the requirements of the experiments and research focus in the future, we could choose an appropriate method.2. The three tonifying kidney therapies could alleviate the spatial learning and memory disturbance, the tissue morphology and the cell ultra-structure lesions of hippocampal dentate gyrus in kidney deficiency rats. Effects of tonifying kidney therapies vary by rat model.3. The prescription of tonifying Kidney could promote the proliferation of hippocampal neural stem cells in kidney deficiency rats. The regulation mechanism might be relevant for raising the relative expression of Wntl, β-catenin, c-Myc and CyclinDl, activating the Wnt signal pathway.4. There are the connections between kidney essence and neural stem cells on some level. There is a correlation between tonifying Kidney and replenishing essence and the proliferation and differentiation of hippocampal neural stem cells. The prescription of tonifying Kidney could promote the proliferation of neural stem cells. The function of tonifying Kidney and replenishing essence could be en embodied in promoting the proliferation of neural stem cells. |
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