| Aging is the body’s normal and complicated life process,which is a degenerative changes in the tissues and organs. As humans enter the aging society, the elderly physiological and pathological characteristics of the body become hot study object. Just as the "2012China development report" showed, the people aged60and above in2011reached184.99million, accounting for13.7%of the national population, which is increased by0.47%compared to2010;while the people of65years and older reached122.88million, accounting for9.1%of the population, which is increased by0.25%compared to2010.While the proportion of elderly is gradually increasing as well as the sustained and rapid ageing, the elderly population structure is translated to aging furtherly,and aging trend is more obvious. Learning and memory is one of the important function of the brain, which is a multi-stage and dynamic neural activity. when aging is coming, the impairment of learning and memory will happen. Hippocampus is an important part of the limbic system and plays an important role in learning and memory, studies have shown that the structure and function of its change in aging occurs earlier and worse. At present, China’s aging and health research and development of the geriatric medicine, especially the nervous system diseases who cause serious damage to the physical and psychology in the elderly, was laged far behind the pace of aging. So the age-related changes in hippocampus to slow brain aging have become an urgent problem.Rejuvenation research in Traditional Chinese Medicine has a long history, while tonifying Kidney and strengthening essence method is extremely important, deficiency of kidney essence, reduction of marrow and flaccidity of bones.The senile animal model is extremely important in terms of the performance and mechanism in aging,as well as drug screening essay. D-galactose model is classic due to its obvious and stable features,while it also cannot fully duplicate the pathological, biochemical changes in senescence. This is a persuasive way that we can study the aging model with OR.Oviductus Ranae (OR) is dry fallopian tube in Rana temporaria chensinensis, Tasted sweet, salty, and flat. OR it beneficial to lung as well as to kidney,which is recorded both in Compendium of Materia Medica and Illustrated Pharmacopoeia; During the qing dynasty, OR is regarded as one of the eight Jane’s. It is reported that OR can clear free radical, repaire injured gene, raise immune system function, and slow down aging, etc. Oxidative stress, which is produced by free radicals,is negative to the body, and is extreme important to lead to aging and disease.It is reported that brain derived neurotrophic factor (BDNF) is associated with learning and memory closely. BDNF levels in aging is confirmed to fall. BDNF, a kind of basic protein which molecular weight was12.3KD, composed of119amino acid and synthesized from brain tissue, distributed in cortex, hippocampus and other brain regions, can be combined with high affinity receptor TrkB to stimulate various signal transduction pathways and play its biological function to promote and maintain the activity of neuron.Research has confirmed that Yi Fu Ning is given priority to perimenopausal syndrome. OR could increase the levels of SOD, Cu-Zn SOD, POD and T-AOC significantly in serum and liver of D-galactose senile rats,and improve the the pathologic changes in liver, uterus and ovaries. It is reported that OR could significantly extend the average and supreme life span of Drosophila elanogaster, performed a good effect to aging.It has not been reported that whether OR affect BDNF in aging hippocampus.we adopts female rats aged16to18months to study whether OR is good for the models and confirme the change of BDNF to discusse material related learning and memory in the hippocampus.Objective:to study the effects of OR on SOD、MDA、GSH-Px、H2O2ang the survival of drosophila melanogaster,and to investigate the mechanism of BDNF-TRKB and P21-P53-Rb signaling pathway in the process of aging.Methods:1.The lifespan of Drosophila melanogaster:800Drosophilas melanogaster with half males and half females were randomly allocated into four groups: the control group(Control)、the Vitamin E group(VE)、the high-and low-dose OR groups (OR H/L),200in each group.2.Twenty-four SD rats aged16-18months were divided into4equal groups randomly, namely the high-and low-dose OR groups(OR H/L), diethylstilbestrol (DT) group, and aged control group (AC). Another6rats of3-4months was served as young control group(YC). The rats were given oral OR at low-or high-doses (OR H/L) DT (DT) or vegetable oil (in YC or AC) till the sixth week. Weigh themselves once a week.(1) Hematoxylin and eosin (HE) and Nissl staining were used to learn the changes of the modality and number of hippocampal neurons.(2) After completion of drug administration, all the rats were sacrificed and the levels of MDA、SOD、 GSH-PX、H2O2in the serum and brain was detected.(3)8-ohdg and proteins associated with the BDNF-TrkB and P21-Rb signaling pathways with was detected by immunohistochemist、Western Blotting and fluorescence real-time quantification PCR. Results:1. The lifespan of Drosophila melanogaster:Compared with Control group, the Average and Longest lifespan of Drosophila melanogaster in VE组、 OR H/L were increased.2.All rats was not found anomalies such as breathing difficulties, diarrhoea,prof-use secretion, and exophthalmos in the process drug administration.Activity of YC is good with the growing obesity and the sof、dense and shiny hair,while rats of AC look sluggish with less activity, dried hair,dark yellow color.OR H/L and DT performenced better than AC.Compared with AC group,DT and OR H/L groups show no significant difference in the weight at the end of the experiment.Histopathological result showed the hippocampus from group AC exhibits unclear pyramidal cell line and notable loss of neurons.The Nissl’s body became clumped or granule-form, and dyeing was thick.lt was found that group AC exposure induced polarity disorder and poor development of neurons and hippocampus of offspring.The Nissl bodies in the neuron were observed to have dissolution and disappearance,accompanied by pyknosis of neurons in the CA3subregion.Patholo-gical findings showed that dissolutionn of the Nissl bodies and pyknosis of neurons were improved in OR H/L group.3. Rats in YC maintained a higher level of antioxidant enzyme.Compared with AC group, the levels of SOD、GSH-PX in serum and brain was increased significantly,and MDA%H2O2decreased significantly.4. RT-PCR experimental results showed that the expression of P19mRNA in AC was increased (95%CI:[0.603,0.646]).Compared with AC group rats, the expression of P19mRNA in DT、OR H/L were decreased (95%CI:[0.878,1.108]、[0.937,0.991]、[0.897,0.990]). Compared with DT group rats,there was no significant differences between group DT and OR H/L statistically(P=0.843,0.557).RT-PCR experimental results showed that the expression of P21mRNA in AC was increased (95%CI:[0.301,0.412]).Compared with AC group rats, the expression of P21mRNA in DT was increased(95%CI:[1.274,1.300]),while OR H/L were decreased (95%CI:[0.726,0.741].[0.432,0.463]). Compared with DT group rats, the expression of P21mRNA in OR H/L were decreased (P=0.000.0.000).RT-PCR experimental results showed that the expression of P53mRNA in AC was increased (95%CI:[0.805,0.855]).Compared with AC group rats, the expression ofP53mRNA in DT. OR H/L were decreased (95%CI:[0.942,0.971].[0.755,0.805].[0.895,0.945]). Compared with DT group rats, the expression of P53mRNA in OR H/L were decreased (P=0.000,0.007).RT-PCR experimental results showed that the expression of Rb mRNA in AC was increased (95%CI:[0.777,0.837]).Compared with AC group rats, the expression ofRb mRNA in ORH/L. DT were decreased (95%CI:[0.425,0.448].[0.913,0.932].[0.812,0.870]). Compared with DT group rats, the expression of Rb mRNA in OR H/L were decreased (P=0.000.0.000).RT-PCR experimental results showed that the expression of IGF mRNA in AC was increased (95%CI:[0.923,0.956]).Compared with AC group rats, the expression of IGF mRNA in DT. OR H/L were decreased (95%CI:[0.404,0.659].[0.715,0.759]、[0.472,0.548]). Compared with DT group rats,there was no significant differences between group DT and OR H/L statistically(P=0.055,0.961).RT-PCR experimental results showed that the expression of GLS mRNA in AC was decreased (95%CI:[1.537,2.255]).Compared with AC group rats, the expression of GLS mRNA in DT. OR H/L were decreased (95%CI:[0.700,0.738]、[0.755,0.782].[0.599,0.641]).Compared with DT group rats,there was a significant differences between group DT and OR H/L statistically(P=0.005,0.001).RT-PCR experimental results showed that the expression of Prdx-3mRNA in AC was increased (95%CI:[0.763,0.832]).Compared with AC group rats, the expression of Prdx-3mRNA in ORH/L、DT were decreased (95%CI:[0.766,0.885]、[0.763,1.055]、[0.616,0.732]). Compared with DT group rats,there was no significant differences between group DT and OR H statistically,while OR L has(P=0.368、0.006).RT-PCR experimental results showed that the expression of Mn-SOD mRNA in AC was decreased (95%CI:[1.861,2.522]).Compared with AC group rats, the expression of Mn-SOD mRNA in DT was decreased(95%CI:[0.535,0.580]), while OR H/L increased (95%CI:[1.313,1.463],[2.340,2.484]). Compared with DT group rats, the expression of Mn-SOD mRNA in OR H/L were decreased (P=0.001-.0.000).RT-PCR experimental results showed that the expression of GPX mRNA in AC was decreased (95%CI:[1.886,1.952]).Compared with AC group rats, the expression of GPX mRNA in DT, OR H/L were increased (95%CI:[1.440,1.489],[1.937,2.082],[1.718,1.775]). Compared with DT group rats, the expression of GPX mRNA in OR H/L were increased (P=0.000,0.000).RT-PCR experimental results showed that the expression of BDNF mRNA in AC was decreased (95%CI:[1.731,2.090]).Compared with AC group rats, the expression of BDNF mRNA in DT was decreased(95%CI:[0.727,0.949]), while OR H/L increased (95%CI:[1.337,1.364],[1.429,1.552]). Compared with DT group rats, the expression of BDNF mRNA in OR H/L were increased (P=0.007,0.001).RT-PCR experimental results showed that the expression of TrkB mRNA in AC was decreased (95%CI:[1.858,2.484]).Compared with AC group rats, the expression of TrkB mRNA in DT was decreased (95%CI:[0.854,1.004]), while OR H/L increased (95%CI:[1.474,1.861]、[1.345,1.531]). Compared with DT group rats, the expression of TrkB mRNA in OR H/L were increased (P=0.005,0.000).5. Experimental results of IHC showed that8-OHDG、Bax、P19ARF and P21,related to the marker for the DNA damage、proapoptotic function. the tumor suppressor and cell proliferation and regulatory mechanism respectively,were diffuse or focal mainly in the cytoplasm and nucleus of cells.The study indicated that the number and the integrated optical density(IOD) value of positive cells had a significant differences between group AC and YC statistically (P=0.000.0.000.0.024.0.004).Compared with AC group rats, the number and the IOD of8-OHDG in DT was increased while Bax. P19and P21decreased (P=0.000>1.000.0.353.0.006);8-OHDG, Bax,P19and P21positive cells in OR H were decreased (P=0.000、0.000、0.227、0.004), and OR L were decreased (P=0.000.0.000.0.166.0.007). Compared with DT group rats, the expression of8-OHDG. Bax in OR H/L were decreased (P=0.000.0.000.0.000.0.000); while there was no significant differences of P19(P=0.999.0.819); the expression of P21in OR H was decreased(P=0.037) and there was no significant differences in OR L(P=0.409).It also showed that Bcl-2and BDNF,related to the inhibition of apoptosis and neural growth、regeneration、reparation and synaptic plasticity respectively,were diffuse or focal mainly in the cytoplasm and nucleus of cells.The study indicated that the number and the integrated optical density(IOD) value of positive cells had a significant differences between group AC and YC statistically (P=0.000.0.000).Compared with AC group rats, the number and the integrated optical density (IOD) of Bcl-2and BDNF positive cells in DT was increased(P=0.000.1.000), while OR H significantly increased (P=0.000.0.000), and OR L increased(P=0.000.0.003). Compared with DT group rats, the expression of Bcl-2(P=0.000.0.000).BDNF in OR H/L were increased (P=0.000.0.015).6. Experimental results of western blot showed a significant differences that the rats in AC were lower expression of BDNF. TrkB and pRb (P=0.012.0.000.0.025), while P19and P53were lower than YC(P=0.013,0.004).Compared with AC group rats, there was no significant differences of BDNF、TrkB、pRb and P19in DT (P=0.537、1.000、1.000、0.684),while P53increased (P=0.003);the grayscale of BDNF、TrkB、pRb in OR H group were increased (P=0.001、0.000、0.001), while P19and P53decreased (P=0.463,0.003); the grayscale of BDNF, TrkB and pRb in OR L were increased (P=0.003、0.000、0.000), while P19and P53decreased (P=0.601、0.010). Compared with DT group rats, the expression of BDNF、TrkB、 pRb in OR H were increased (P=0.013,0.000,0.035), P53decreased(P=0.002),while there was no no significant differences of P19(P=0.187); the expression of BDNF、 TrkB、pRb in OR L were increased (P=0.049,0.000,0.026), P53decreased (P=0.006),while there was no no significant differences of P19(P=0.219)Conclusion:1.OR is able to prolong the lisespan of Drosophila melanogaster.2. OR has definite antioxidative effects and ameliorates the degenerative changes of the hippocampus in rat models of aging.3. Experimental results showed that the rats in AC were higher expression of P21、P53、Rb、Bax, while lower p-Rb、BDNF、TrkB、Bcl-2. After completion of drug administration, the expression of P21、P53、Rb、Bax were decreased, while p-Rb、BDNF、TrkB、Bcl-2increased、we conjecture that OR could slow down aging by feedback regulating the signal BDNF-TrkB and P21-Rb to promotes cell proliferation. |
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