| Aging refers to the body, after a gradual decline in physiological function, degenerative changes in tissues and organs and, ultimately, the inevitable dying process. With the development of medical science, anti-aging become a research hotspot, aging is closely related to depression of the hypothalamus-pituitary-gonadal axis function, which has important roles in gonadal. The basic charcaterof aging is cell aging. Cell aging process is achieved by means of signal transduction pathways, and many cytokines are involved in these signaling pathways. When key regulatory factors of these pathways are involved in change, the cell would be anti-aging, or to bypass the aging process to continue proliferation. In recent years, changes in senescence-associated genes and its regulation has become the main trend of aging Mechanism. Which p16/Rb and p19/p53/p21 signal transduction pathway is essential. Traditional medicine think the kidney is a fundamental internal causes of aging and Rise and Fall of kidney essence and qi determines the pace of aging. Kidney Prescription Heshouwuyin (also known as Tiannianyin) has the added liver and kidney, essence and blood, and longevity of the effect. In this study, D-galactose-induced subacute aging male rat model, using modern biological technology from biological and chemical - Organization - proteins - genetic level to explore effects of Heshouwuyin on the aging male rat reproductive function, and its anti-aging mechanism for the anti-aging and provide a experimental evidence for the research and development, accumulation of clinical information on anti-aging medicine.Part one Effects of Heshouwuyin on testis spermatogenic function of D-gal aging male ratsObjectiveTo study the effects of Heshouwuyin on cauda epididymidis sperm count, vitality and survival rate, serum LH,FSH,testosterone and hypothalamus GnRH concentrations, HE staining testes and the expression of IGF-1 mRNA in testes tissue of aging male rats.Methods1 Establishment of the animal aging model and groups105 eight-week male SD rats (SPF degree, weight 180~200g) were used in this study. Randomly take 15 rats as normal group(abdominal cavity continually injecte equal doses physiological saline), the rest rats were injected D-gal (dissolved by physiological saline into 6% solution) into the rats'abdominal cavity continually by 300mg/kg/d for 60 days.Then, carry out Morris water maze ethology test; and in the 60th days, take blood about 1.5 mL from rats endocanthion vena orbitalis posterior, centrifuged supernatant obtained, detect serum superoxide dismutase and malondialdehyde concent, to determine the success of modeling. Judgement standard: The latency obviously prolong: normal group(8.91±7.59)s,model group(30.05±3.39)s, the number of times to pass through the platform is less obviously: normal group(7.11±2.36)times/min, model group(2.36±1.08)times/min; the content of serum SOD descend obviously and MDA gose up obvilously, which are sucessed aging model rats.The aging model rats were randomly divided into model group, Heshouwuyin low,medium,high dose treat group, Heshouwu pill treat group, spontaneous recovery group, each group 15 rats. The rats in aging model group were not given any treatment; the rats in Heshouwuyin low,medium,high dose group were respectively lavaged with Heshouwuyin low,medium,high dose (9.6,19.2,38.4 g/kg/d), Heshouwu pill group was lavaged with Heshouwu pill(8.24 g/kg/d) and spontaneous recovery group was lavaged with equal doses physiological saline for 60 days.2 Methods(1) Determine the success of modeling:After 60 days, each rat does Morris water maze test: Place navigation test and Spatial probe test. Rats with 10% chloral hydrate anesthesia, through the inner canthal orbital vein blood taken at about 1.5 ml, centrifuged supernatant obtained. By xanthine enzymic method radioimmunoassay and TAB method detect serum SOD vigor and MDA content.(2)At ambient temperature, rats with 10% chloral hydrate anesthesia, taking one side of caput epididymidis of rat(the weight of each drawn line), placed in 5 mL normal saline containing 37℃water bath in a small dish of warm, shredded into a sperm suspension. To observe sperm count, activity and survival.(3) Rats with 10% chloral hydrate anesthesia, through the inner canthal orbital vein blood taken at about 1.5 mL, centrifuged supernatant obtained. By radioimmunoassay detect serum LH, FSH, testosterone concentration. The concentration of hypothalamic GnRH were measured by tissue homogenate and radioimmunoassay.(4)The testicles were taken after the rats being infused with 4% polyoxymethylene, embedded in paraffin and sectioned as usual, HE staining.(5q)The testicles were taken after the rats being infused with 4% polyoxymethylene +1‰diethyl pyrocarbonate, embedded in paraffin and sectioned as usual, hybridization in situ was used to observe the IGF-1 mRNA expressions of testicles cells.Result1 Place navigation test indicate: the first two days, the result of each group didn't show significant difference(P>0.05), and each rat latency all shortenned gradually with study time extension, the latency result of model rats obviously prolong than normal rats(P<0.05). Spatial probe test indicate, The times of through platform of model rats,which is (2.36±1.08)次/min, is obviously low than normal rats,which is (7.11±2.36)次/min.2 Detect serum SOD vigor and MDA content The vigor of SOD of model group, obviously descents, the MDA content obviously goes up, and shows significant difference (P<0.01).3 Effects of Heshouwuyin on aging rat sperm: model group, sperm activity, survival and the number of sperm than those in normal control group were significantly reduced(P<0.05); model group and the spontaneous recovery group, no significant difference(P>0.05). The sperm count of Heshouwuyin and Heshouwu pill group were higher than model group and spontaneous recovery group(P<0.05), but each treatment group no significant difference(P>0.05); the treatment group can enhance the sperm activity and survival in different levels(P<0.01), and Heshouwuyin high dose group is better than other groups(P<0.05).4 Serum LH, FSH, testosterone and hypothalamic GnRH concentration changes: model group, serum LH, FSH, and hypothalamic GnRH concentration were significantly higher and testosterone levels were significantly decreased compared with the normal group, the difference was significant(P<0.05), and the spontaneous recovery group, no significant difference(P>0.05), Heshouwuyin and Heshouwu pill group can reduce serum LH, FSH, and hypothalamic GnRH concentration in different levels, increase testosterone concentration(P<0.05); Heshouwu pill group was better than Heshouwuyin low and medium dose group(P<0.05), while Heshouwuyin high dose group was the best(P<0.05).5 HE staining showed the normal rat testis seminiferous tubules full, pipe wall thickness, spermatogenic cells rich in cavity a large number of sperm, suggesting that spermatogenic activity; the model rat testis seminiferous tubule wall epithelial was thin, levels of spermatogenic cells reduced, arranged in disorder, and degradation of cavity shedding cells appear, suggesting that spermatogenic dysfunction; treatment group seminiferous tubule wall thickening can be seen at all levels of spermatogenic cells and sperm-rich cavity can be seen.6 IGF-1 mRNA in the Leydig cells, spermatocytes, sperm cells were expressed immunoreactive products distributed in the cytoplasm, showing brown granular dispersion. The model group significantly decreased compared with the normal group(P<0.05); the model group and the spontaneous recovery group, no difference(P>0.05); treatment group can raise testicular IGF-1 mRNA expression(P<0.01). Heshouwuyin high dose group was significantly better than the other groups(P<0.05).Conclusion1 D-galactose-induced aging model is succeed.2 Heshouwuyin can significantly improve the D-galactose-induced subacute aging rat testis form of structure, improving sperm quantity, activity and survival rate, thereby improving sperm count and sperm function.3 Heshouwuyin can increase the D-galactose-induced subacute aging serum of male hormone(testosterone) levels and lower gonadotropin and gonadotropin-releasing hormone levels.4 Heshouwuyin can increase the D-galactose-induced aging rat testis cells in subacute local regulator of IGF-1 mRNA expression levels.Part two Effects of Heshouwuyin on the Rb/p53 cell signal transduction pathway of aging male rat testisObjectiveBy detecting the expression of Rb, p16, p53, p19, p21 in rat testis, explore Heshouwuyin anti-aging effects on the testis.Method1 Detected the expression of pRb, p16, p53, p19, p21 in rat testis by immunohistochemistry.2 Western Blot to detect rat testis pRb, p16, p19, p21 protein expression.3 RT-PCR to detect the rat testis p53, Rb mRNA expression.Result1 Immunohistochemical staining: slices of each group were seen pRb, p16, p53, p19, p21 staining, positive cells are mainly spermatogonia, occasionally interstitial cells and seminiferous tubule basement membrane of muscle cells in the original color. Immunoreactive product in the nucleus, usually brownish yellow granules, small part of the dispersion was like.2 Western Blot Detection of testicular tissue pRb, p16, p19, p21 protein expression: the model group rat testis p16, p19, p21 protein expression levels were significantly elevated, pRb protein expression was significantly decreased compared with the normal group there was significant difference (P<0.05); and the spontaneous recovery group, no difference (P>0.05); treatment group testis p16, p19, p21 protein expression level was significantly lower than the model group, pRb protein expression was significantly higher than model group (P<0.05). Which Heshouwuyin high dose group were significantly better than the other groups (P<0.05).3 Testicular tissue RT-PCR detection of p53, Rb mRNA expression: the model group testicular tissue p53, Rb mRNA expression level was significantly higher than the normal control group (P<0.05), and the spontaneous recovery group, no significant difference (P>0.05); treatment group in rat testis p53, Rb mRNA expression levels were significantly lower than model group (P<0.05), which Heshouwuyin high dose group were significantly better than the other groups (P<0.05).Conclusion1 pRb, p16, p53, p19, p21 mainly in the nuclei of spermatogonia testis expression.2 Aging male rat testis p16/Rb and p19/p53/p21 aging pathway key regulator p53, Rb mRNA genes and p16, p19, p21 protein expression, with Heshouwuyin treatment can play an inhibitory; pRb protein expression down, Heshouwuyin treatment can play a catalytic role.In summary, our study found that Heshouwuyin(1) to improve the aging rat spermatogenesis; (2) regulating the aging male rat testis Rb/p53 cell signal transduction pathways to achieve the improvement of testicular reproductive function in rats and delaying testicular aging. |
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