Experimental Study On Senescent Mechanism Of Hypothalamus-Pituitary-Ovary Axises And Anti-Aging Effects Of Hswy

Along with the aging problem of global population increasingly outstanding, the study about senecence mechanism and antiaging agent has been research subject which is great international medical project today. The modern medicine has found that immune-nerve-endocrine network played an important regulating role in aging. Hypothalamus-pituitary-gonad axis (HPG axis) is a very important ingredient in the network. There are clinical syndromes about menstrual disorder, menostasis and autonomic nerve functional disorder; because ovary in peri-menopausal period happens retrogressive changes which lead unbalance restriction of HPOA. The research on aging mechanisms of gonad axises and anti-aging medicines has been one of research focuses. For the the past few years , People mainly studied male gonad axises by D-gal aging animal model, but the reports about female gonad axises were found very less up to now.The traditional Chinese medicine thought the renal deficiency was the senile basic reason. According to the renal deficiency senile theory, we establish therapeutic principle to invigorate kidney essence, and we institute kidney-nourishing recipe-Heshouwuyin (Hswy). Sub-acutely aging rats were induced by D-galactose. We detect Anti-oxidative ability, blood lipid, neurotransmitter, gonadal hormones and receptors, apoptosis of ovary cells and its related genes, proteins-Bax, Bcl-2, fas, fasl, Caspase-3 by using these methods including biochemistry, HPLC, ELISA, RT-PCR, Western Bloting, Tdt-mediated dUTP nick end labelling (TUNEL), immunohistochemistry, so that we can investigate aging mechanism of female gonad axises and the delaying aging effects of Hswy from biochemistry, structure, cell, gene and protein aspects. Our study will provide experimental bases for elucidating female senecence mechanism and choosing clinical antiaging agent. PartⅠStudy on the anti-oxidative ability and blood lipid changes of D-gal aging rats and the effects of Hswy on themObjectiveTo study on the anti-oxidative ability and blood lipid changes of D-gal aging rats and the effects of Hswy on anti-oxidative ability and blood lipid.Methods1. Establishment of the animal aging model and groups(1) Establishment of the aging model:105 eight-week male SD rats (SPF degree, weight 180~200g) were used in this study. D-gal (dissolved by physiological saline into 6% solution) was injected into the rats'abdominal cavity continually by 300mg/kg/d for 60 days.(2) Groups: The rats were divided into 7 groups at random, 1) normal control group(N), 15 rats; 2) model group(M), 15 rats; 3)Heshouwiyin anti-aging group, including low(Tl), middle(Tm) and high(Th) doses group, 15 rats in each; 5) self-restoring group(S-R), 15 rats; 6) VE positive control group(VE), 15 rats.2. 2ml blood was taken from the medial angle of rat eye in each group, the serum was obtained after centrifuging, and then the following gindexes were detected respectively. The parameters of serum levels of①SOD, MDA, GSH-Px and T-AOC②total cholesterol (TC), triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) were detected by using biochemistry.Results1. Change of anti-oxidative ability in serum: T-AOC, SOD and GSH-Px activities of model rats decreased significantly, MDA content increased remarkably compared with normal rats(P<0.05). There were no significant differences between self-restoring group and model group (P>0.05). T-AOC, SOD and GSH-Px increased remarkably, MDA decreased significantly after taking Hswy. The effect was most distinct in high doses Hswy group.2. Change of blood lipid in serum: The TC, TG content in serum of model rats was significantly higher than that of normal rats (P<0.05); while the HDL-C content of model rats was significantly lower than the normal rats (P<0.05). The MDA, TC, TG content in serum after taking HSWY was significantly lower than that of self-restoring rats; while the HDL-C content of the taking HSWY rats was significantly higher than that of self-restoring rats. (P<0.05)Conclusions:1. The aging model induced by D-gal was successfully established in our study.2. Hswy could obviously enhance the activity of SOD and GSH-Px of aging rats induced by D-gal, then enhances the anti-oxidative ability, and decreases the accumulation of MDA in serum.3. Hswy could obviously decrease the TC, TG level and enhance the HDL-C level in serum of sub-acutely aging rats, then improve the lipid metabolism disorder state of aging rats.PartⅡStudy on changes of hypothalamus brain neurotransmitter of aging rats and influence of Hswy on themObjectiveTo analyze the changes of hypothalamus brain neurotransmitter of aging rats and mechanisms of Hswy on anti hypothalamus aging by detecting the contents of GnRH, SP ,β-EP and monoamine neurotransmitter such as NA, DA, 5-HT in rats hypothalamus .Methods1. The rats were killed by being amputated by head and the hypothalamus was taken. Use the method of ELISA to detect SP,β-EP and GnRH. 2. The concentration of monamine transmitters including noradrenaline (NA), dopamine (DA) and 5-hydroxypatmien(5-HT) in hypothalamus were detected by high-performance liquid chromatography (HPLC), with electrochemical detection.Results1. Changes of GnRH, SP andβ-EP in hypothalamus: GnRH and SP of rats in the model group increased significantly,β-EP decreased remarkably compared with the normal group (P<0.05).β-EP increased remarkably, GnRH and SP decreased significantly after Hswy being taken. The results of high doses treating group was better than other anti-aging groups and VE positive control group (P<0.05).2. The monamine transmitters concentration in hypothalamus: The NA, DA, 5-HT concentration in hypothalamus of model rats significantly decreased compared with normal rats (P<0.05). Compared with model rats, Hswy treating groups could raise the NA, DA, 5-HT concentration significantly. The effect was most distinct in high doses treating group.Conclusions1. The study verificated synthesis of NA, DA, 5-HT,β-EP decreasing and GnRH, SP increasing, which confirmed the normal function of hypothalamus was destroyed.2. Hswy could elevate the content ofβ-EP, and reduce the determination of GnRH and SP in hypothalamus. It could raise the concentration of NA, DA, 5-HT. These results showed that Hswy had the effects on preventing and delaying senescence of hypothalamus by regulating the disorder of hypothalamus neurotransmitter.PartⅢTo Study changes of gonadal hormone and receptors of female sub-acutely aging rats and the effects of Hswy on themObjectiveTo explore changes of gonadal hormone and receptors of female sub-acutely aging rats and the effects of Hswy on delaying senescence of ovary by using Access immunoassary systerm analyzer, in situ hybridization and western bloting.Methods1. Organ index to weigh before death. Humid weights of pituitary and ovary were measured by electronic analytical balance. Pituitary index=humid weight of pituitary/body weight X100%, and ovary index= humid weight of ovary/body weight X100%.2. To detect LH, E2, FSH by Access immunoassary systerm analyzer. 3. The ovarys were taken after the rats being infused with 4% polyformaldehyde +1‰DEPC, embedded in paraffin and sectioned as usual; hybridization in situ was used to observe the FSHRmRNA and LHRmRNA expressions of ovary cells.4. The rats were killed by being amputated and the ovary was taken. ER protein was detected by western bloting.Results1. Gonadal hormone in serum: The contents of LH and FSH in model group increased significantly, and the content of E2 decreased remarkably compared with normal group (P<0.05), and there were no statistical differences compared with self-restoring group. Th and Tm could significantly enhance the content of E2 and degrade contents of LH and FSH more than VE positive control group.2. Pituitary and ovary index: Compared with normal control group, pituitary and ovary index decreased obviously in model group (P<0.05). Compared with model group, positive control group and Hswy treating groups could increase pituitary and ovary index (P<0.05), which showed dose-effect relationship.3. Change of hormone receptor in ovary: The expression of ER, FSHR, LHR in model group decreased significantly compared with normal group (P<0.05), and there were no statistical differences compared with self-restoring (P>0.05). The ER, FSHR, LHR expression of ovary cells increased remarkably. With the increase of doses, the effect was better.Conclusions1. Pituitary and ovary index of aging rats decreased. Hswy could increase Pituitary and ovary index of sub-acutely aging rats and improve E2 in serum, which showed Hswy had Estrogen-like effect.2. Hswy could regulate gonadal hormone level of sub-acutely aging rats, effect of different degrees on multi tache of HPOA. Hswy could increase E2 level in serum, decrease FSH and LH, which helped the restoring of concordant in HPOA endocrine. 3. The hormone receptor level of aging rats decreased. Hswy could up-regulate hormone receptor level, help correlated hormone play biological effect, make functional recovery of HPOA, which greatly improve the function of ovary.PartⅣThe study on Apoptosis in ovary of aging rats and the effects of Hswy on themObjectiveInvestigation on Apoptosis and the effects of Hswy on apoptosis were observed in ovary of aging rats by detecting ovary apoptosis and its related genes, proteins.Methods1. It was treated by TUNEL to detect ovary apoptosis.2. fas and fasl genes were detected by RT-PCR.3. Apoptosis-associated protein Bax, Bcl-2 and Caspase-3 were detected by histochemical method and Western Bloting.Results1. Apoptosis: The counts of apoptotic cells of model group rats were higher than normal group rats (P<0.05), and there were no statistical differences between model group and self-restoring group. Those of Hswy groups were lower than model group (P<0.05), and showed dose-effect relationship. Th has the best effect.2. The expressions of apoptosis related genes and proteins: fas and fasl gene expression of ovary increased in model group compared with normal group; the expression of Bax and Caspase-3 increased and the expression of Bcl-2 attenuated in model group. Hswy could weaken the expression of fas/fasl gene, Bax, Caspase-3 and improve the expression of Bcl-2 in ovary.Conclusions1. Apoptosis ratio of ovary aging rats increased which posed atresia folliculi intensifying because of expression of apoptosis trigger genes-Bax, fas, fasl, Caspase-3 increasing and repressed gene Bcl-2 decreasing.2. Hswy could decrease apoptosis in ovary of aging rats through controlling apoptosis related genes and protein. These might be the methods of Hswy to delay the aging of rat ovary.To sum up, our study found that HPOA and relevant biochemical indexes of D-gal aging model take place a series of senescent changes;we also discovered Hswy had anti hypothalamus-pituitary-ovary axis aging effects through improving the anti-oxidative ability and regulating blood lipid contents ability, contents of hypothalamus neurotransmitter GnRH, SP,β-EP, NA, DA, 5-HT, secretion of gonadal hormones and expression of their receptors, degrading apoptosis in ovary and adjusting apoptosis-related genes.