Senescent Changes Of The Gonad Axis In Female Rat And The Experimental Study On The Mechanism In Anti-aging Of Traditional Chinese Medicine Heshouwuyin

As a normal and complicated life process, senescence was involved in a series of routine changing in various tissues and cells. The development and aging of hypothalamus- pituitary-gland axis (H-P-G-A) was important in life proceeding. Immune-neuroendocrine network played an important role in aging and H-P-G-A was an important component in this network.Many studies indicated that cellular senescence was implemented via some signaling transduction pathways which a lot of cytokines were involved in. When the key regulators in these pathways were changed, cellular senescence would be delayed. In this study, we had investigated the changes of some key regulators including p53, p19ARF, Rb, p16, IGF-Ⅰ, IGF-BP, TNF-α, EGF, EGFR and the changes of the ultrastructure of arcuate nucleus in the hypothalamus, adenohypophysis, ovary after taking traditional Chinese medicine Heshouwuyin (HSWY) by using the sub-acutely aging female model rats induced by D-galactose, so as to find the senescent mechanism of hypothalamus-pituitary-ovary axis (H-P-O-A) and the mechanism of HSWY in anti-aging.1. ObjectiveWe deteceted the level of mRNA p53, p19ARF, Rb, p16 and IGFBP3 by reverse transcription-polymerase chain reaction (RT-PCR), and examined the expression of protein Rb and p16 by Western-blotting, and evaluated the expression of p53, TNF-α, EGF and EGFR by immunohistochemistry (IH), and analysised the mRNA level of IGF-Ⅰby hybridization in situ (HIS) in order to find out the senescent mechanism of hypothalamus-pituitary-ovary axis (H-P-O-A) of the aging model rats and the effect of HSWY in anti-aging, so as to provide theoretical and experiment evidence to anti-aging in the female'sexual gland.2. Methods2.1 Choosing of the experimental animal and establishment of the aging model. Eight-week-old female SD rats (n=96) were separated into three groups at random: normal group, aging model group, preventing aging group. The rats in preventing aging group were re-divided into four groups: HSWY low dosage group, HSWY medium dosage group, HSWY high dosage group and Heshouwan(HSWW) group (n=16 in each group). The sub-acutely aging model rats were induced by D-galactose, and the rats in preventing aging group accepted intragastric administration of HSWY and HSWW while accepting D-galactose intraperioneal injection for 60 days.2.2 Experimental procedures. Hypothalamus, adenohypophysis and ovary tissues of each group were obtained after the rats undergoing experiment for 60 days. We detected the gene mRNA expression of p53, p19ARF, Rb, p16 and IGFBP3 by RT-PCR, and examined the protein expression of Rb and p16 by Western-blotting, evaluated the expression of p53, TNF-α, EGF and EGFR by IH, analysised the level of mRNA IGF-Ⅰby HIS, and observed the ultrastructure by transmission electron microscope (TEM).3. Results3.1 Key regulators—p53, p19ARF, Rb, p16 of signaling transduction pathways: p19ARF/p53/p21Cip1 and p16INK4a/Rb in H-P-O-A were changed in aging model, and the overexpression of these regulators could be retrieved by using HSWY.3.1.1 The result of PCR had shown that gene expression of p53 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p53 were significantly decreased (P<0.05). In the hypothalamus and adenohypophysis tissues, the gene expression of p53 in HSWY low dosage group were significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of p53 was significantly lower than in HSWW group (P<0.05). The results of IH had shown that immunostaining for p53 in arcuate nucleus, adenohypophysis and ovary tissues were significantly more intense in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for p53 were significantly decreased (P<0.05). In the adenohypophysis tissue, the immunostaining for p53 in HSWY low dosage group was significantly weaker than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the immunostaining for p53 was significantly weaker than in HSWW group (P<0.05).3.1.2 The result of PCR had shown that gene expression of p19ARF in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p19ARF were significantly decreased (P<0.01, P<0.05). In the hypothalamus tissue, the gene expression of p19ARF in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of p19ARF was significantly lower than in HSWW group (P<0.05).3.1.3 The result of PCR had shown that gene expression of Rb in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of Rb were significantly decreased (P<0.01, P<0.05). In the hypothalamus and the adenohypophysis tissues, the gene expression of Rb in HSWY low dosage group were significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of Rb was significantly lower than in HSWW group (P<0.05). The result of Western blotting had shown that expression of protein Rb in hypothalamus, adenohypophysis and ovary tissues were increased in aging rats (P<0.05). After pretreatment of HSWY in preventing aging group, the expression of protein Rb were significantly decreased (P<0.05). In the adenohypophysis tissue, the expression of protein Rb in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the expression of protein Rb was significantly lower than in HSWW group (P<0.05).3.1.4 The result of PCR had shown that gene expression of p16 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p16 were significantly decreased (P<0.01, P<0.05). In the adenohypophysis tissue, the gene expression of p16 in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). The result of Western blotting had shown that expression of protein p16 in hypothalamus, adenohypophysis and ovary tissues were increased in aging rats (P<0.05). After pretreatment of HSWY in preventing aging group, the expression of protein p16 were significantly decreased (P<0.05).3.2 Cytokines related to senescence in H-P-O-A were changed in aging model, and the abnormal expression could be retrieved by using HSWY.3.2.1 The results of IH had shown that immunostaining for EGF in ovary tissue was significantly weaker in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for EGF was significantly increased (P<0.01). The results of IH had shown that immunostaining for EGFR in arcuate nucleus, adenohypophysis and ovary tissues were significantly weaker in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for EGFR were significantly increased (P<0.05).3.2.2 The results of IH had shown that immunostaining for TNF-αin arcuate nucleus, adenohypophysis and ovary tissues were significantly more intense in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for TNF-αwere significantly decreased (P<0.01, P<0.05). In the arcuate nucleus and adenohypophysis tissues, the immunostaining for TNF-αin HSWY group were significantly weaker than in HSWW group (P<0.05).3.2.3 The results of HIS had shown that the expression of IGF-ⅠmRNA in ovary tissue was significantly decreased in model rats (P<0.01). After pretreatment of HSWY in preventing aging group, the expression of IGF-ⅠmRNA was significantly increased (P<0.01). The result of PCR had shown that gene expression of IGFBP3 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of IGFBP3 were significantly decreased (P<0.01, P<0.05).3.3 Morphologic structure of H-P-O-A in aging model was damaged, and the impairment could be restored by using HSWY.3.3.1 The morphological changes of the arcuate nucleus in the hypothalamus: By light microscope, it could be seen that there were two kinds of cells in arcuate nucleus: neurone and neuroglial cell. In model rats, there were a lot of reactive colloid cells except for the two kinds of cells above-mentioned. By transmission electron microscope(TEM), arcuate nucleus was constituted of dark neurons, pale neurons. In model rats′arcuate nucleus, it was found that the nucleolemma was indented or dissolved, and the number of lysosome was increased, and mitochondriales cristae were broken and even vanished, and rough endoplasmic reticulum losted its granules, and Golgi complex swelled, and pre- and postsynaptic membranes were unclear and synaptic cleft was fusional. In preventing aging rats′arcuate nucleus, part of the nucleolemma was dissolved, and lysosome could be seen. Part of the mitochondriales cristae was broken. Rough endoplasmic reticulum losted its granules lightly. Golgi complex swelled lightly. Pre- and postsynaptic membranes were clear and part of synaptic cleft was fusional.3.3.2 The morphological changes of the adenohypophysis: By light microscope, it could be seen that the gland cells were including three kinds of cells: acidophil, basophi and chromophobe cells. In model rats, it could be seen that the gland cells were arranged disorderly and the blood vessels were proliferated. The number of acidophil and basophi cells was decreased. By TEM, the gonadotroph of model rats changed obviously. The heterochromatin became more and the nucleolemma was dissolved. GER dilated obviously and Golgi complex increased and swelled. Mitochondriales cristae were broken and even vanished. The secretary granules were increased. In preventing aging rats′gonadotroph, rough endoplasmic reticulum were increased and dilated lightly. Golgi complex swelled lightly and part of the mitochondriales cristae was broken. The secretary granules were increased lightly.3.3.3 The morphological changes of the ovary: By light microscope, the ovary was characterized by follicles in every grade and corepus luteums. The number of primordial follicles, growing follicles and corepus luteums in model rats was less than that in normal rats. The granulosa cells were arranged disordered and the number of the saccular ectasia follicles was increased. Compared with model rats, the number of primordial follicles, growing follicles and corepus luteums was more in preventing aging rats. By TEM, in the model rats′granulosa cell, it was found that mitochondria were droped and swelled, cristae were broken and even vanished, and smooth endoplasmic reticulum were decreased and dilated, and Golgi complex swelled. In HSWY low dosage rats′granulosa cell, it could be seen that mitochondria were swelled obviously, and smooth endoplasmic reticulum were dilated lightly, and Golgi complex swelled, and lipid droplets were increased. In HSWY medium dosage rats′granulosa cell, it was found that mitochondria were increased and swelled lightly, and many smooth endoplasmic reticulum were dilated lightly, and lipid droplets were increased. The ultrastructure of HSWY high dosage rats′granulosa cell was similar to nomal rats′. And the ultrastructure of HSWW rats′granulosa cell was similar to HSWY medium dosage rats′.4. Conclusion4.1 The genes and proteins of key regulators—p53, p19ARF, Rb, p16 of signaling transduction pathways: p19ARF/p53/p21Cip1 and p16INK4a/Rb in H-P-O-A were overexpressing in aging model, and this could be depressed by using HSWY. 4.2 Cytokines related to senescence—EGF and its receptor EGFR, IGF-Ⅰand its binding protein IGFBP3 were decreased in H-P-O-A in aging model, and the expression of these cytokines could be increased by using HSWY.4.3 The protein of TNF-α—a cytokine related to senescence was overexpressing in H-P-O-A in aging model, and the level of TNF-αcould be reduced by using HSWY.4.4 Morphologic structure of H-P-O-A in aging model was damaged by D-galactose. The impairment could be restored by using HSWY, so as to the function of H-P-O-A could be improved and senescence could be delayed.