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Effects of Heshouwuyin on the Testis Reproductive Function of Aging Male Rats

Author: GuoKaiHua
Tutor: NiuSiYun；GaoFuLu
School: Chengde Medical College
Course: Human Anatomy and Embryology
Keywords: Heshouwuyin Senescence Testis p53 Rb Sperm Testosterone
CLC: R285.5
Type: Master's thesis
Year: 2010
Downloads: 65
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Abstract


Physiological functions gradually decline, aging body mature, degenerative changes in tissues and organs, and ultimately to the death of an inevitable process. Become a research hotspot with the development of medicine, delaying aging, aging and hypothalamic - pituitary - gonadal axis function decline is closely related to, which the gonadal has important regulatory role. The basic characteristics of the aging cell aging. Cellular aging process is implemented by means of a signal transduction pathway, and many cytokines are also involved in these signaling pathways. When these pathways are involved in key regulatory factors change, the cell will be anti-aging or bypass the senescence program continue to proliferate. Recent years, the aging-related genes and their regulatory changes, has become the main trend of the senescence mechanism. Which p16/Rb, and p19/p53/p21 signal transduction pathway is essential. Traditional medicine that the fundamental internal kidney is aging, and the Rise and Fall of the kidney essence determines the speed of aging. The kidney prescriptions Heshouwuyin the The (aka Tiannianyin) have liver or kidney, essence and blood, the effect of longevity. In this study, using D-galactose induced subacute aging male rat model to investigate the function of the aging male rats testicular germ Heshouwuyin modern biological technology, from different levels of the biochemical - Organization - protein - gene level, and its anti- aging mechanism to provide experimental evidence for anti-aging, accumulated data for the research and development of clinical anti-aging medicine. The first part Heshouwuyin on the impact of the aging male rat spermatogenesis Objective: To observe Heshouwuyin D-galactose induced subacute aging model rat sperm count, motility and survival, serum LH, FSH, testosterone and the concentration of hypothalamic GnRH HE staining of testicular tissue and IGF-1 mRNA expression levels. Method: the establishment of an aging model and grouping: optional 8-week-old male SD rats (Sprague-Dawley rats, SD) 105 only, weight 180 ~ 220g, provided by the Experimental Animal Center of Hebei Medical University. Randomly selected 15 consecutive intraperitoneal injection 60 days remaining rats were used as normal control group (intraperitoneal injection of normal saline), the preparation of 6% D-galactose saline solution (300mg · kg-1 · d-1) . Then all rats Morris water maze behavioral experiments; modeling and 60 d after all rat inner canthus orbital venous blood was collected about 1.5 mL centrifugal supernatant, serum superoxide dismutase (superoxidedismutase SOD), malondialdehyde (malondialdehyde, MDA) content, to determine whether the model was successful. Criteria: Morris water maze test incubation period was significantly longer: the normal group (8.91 ± 7.59) s, the number of model group (30.05 ± 3.39) s space exploration test across the platform significantly reduced: the normal group (7.11 ± 2.36) times / min, the model group (2.36 ± 1.08) times / min. Serum SOD decreased significantly, MDA significantly increased as successful modeling. Modeling rats were divided into model group, Heshouwuyin low, the high-dose group, positive control group, negative control group (natural recovery group), 15 in each group. The model group no longer any treatment low Heshouwuyin, in the high-dose group were given Heshouwuyin (9.6,19.2,38.4 g / kg / d) orally, the positive control group was given the Polygonum multiflorum pills (8.24 g / kg / d) gavage, negative control group was given normal saline 60 d. 2 methods (1) to determine whether the aging model to build a successful 60 d: modeling, all the rats in the Morris water maze test: place navigation test (Place navigation test, PNT) and space exploration test (Spatial, probe test, SPT). Rats with 10% chloral hydrate anesthesia by the inner canthus orbital vein blood of approximately 1.5 mL, centrifuged supernatant. Xanthine enzymatic and TAB assay Serum SOD activity and MDA content were used. (2) at room temperature, the rats with 10% chloral hydrate anesthesia from rat side of the epididymal head (each weight materials consistent), placed filled with 5 mL of normal saline and 37 ° C water bath preheated The sperm suspension into a small petri dish, cut into pieces. Observed sperm count, motility and survival. (3) rats with 10% chloral hydrate anesthesia, about 1.5 mL blood by the inner canthus orbital vein, centrifuged supernatant. Serum LH, FSH, testosterone concentration detected by radioimmunoassay. Using the the homogenate method and radioimmunoassay to detect the concentration of hypothalamic GnRH. (4) rat containing 4% paraformaldehyde perfusion-fixed testis, embedded in paraffin, HE staining to detect the rat testis tissue changes. (5) rat the phosphorus amide containing 1 ‰ diethyl-focus (diethyl pyrocarbonate, DEPC) 4% paraformaldehyde perfusion-fixed, take bilateral testicular, embedded in paraffin, in situ hybridization detection rat testis cell changes of IGF-1 mRNA. Results: 1 PNT showed that 2 d before the incubation period of performance of each group was no significant difference (P gt; 0.05), with the extension of learning time each group of incubation period gradually shortened the rat model of the incubation period results with normal rats was significantly longer (P lt; 0.05). SPT showed that the number of times through the platform of the model rats (2.36 ± 1.08) times / min, was significantly lower than in normal rats (7.11 ± 2.36) times / min. Aging model group decreased SOD activity, MDA content significantly increased compared with normal control group was significantly different (P lt; 0.01). 3 Heshouwuyin aging rat sperm: the model group sperm motility, viability and number of sperm than those in the normal control group was significantly lower (P lt; 0.05); model group and natural recovery group had no significant difference (P gt; 0.05). Of Heshouwuyin the Polygonum multiflorum pill group sperm count higher than model group and natural recovery group (P lt; 0.05) between the treated group was no significant difference (P gt; 0.05); treated group could improve the degree of sperm motility and survival (P lt; 0.01), which the high doses Heshouwuyin group is better than the other groups (P lt; 0.05). 4 serum LH, FSH, testosterone and hypothalamic GnRH concentration changes: model rats serum LH, FSH, and hypothalamic GnRH concentration was significantly higher testosterone concentration decreased significantly, compared with normal group differences were significant (P lt ; 0.05), with natural recovery group no significant difference (P gt; 0.05) Heshouwuyin of Polygonum multiflorum pill could lower the concentration of serum LH, FSH, and hypothalamic GnRH, testosterone concentration (P lt; 0.05); Polygonum multiflorum pill group Heshouwuyin better than low, middle dose group (P lt; 0.05), while the best effect Heshouwuyin high dose group (P lt; 0.05). 5 HE staining showed normal rats spermatogenic small tube full tube wall thickness spermatogenic cells cavity sperm, suggesting spermatogenic active epithelial thinning; model group rat testis seminiferous tubule wall spermatogenic reduce cell level, disorganized, and appeared in the cavity the cell shedding degradation prompted spermatogenic dysfunction; treatment group seminiferous tubule wall thickening, visible spermatogenic cells cavity visible rich sperm. 6 IGF-1 mRNA in Leydig cells, spermatocytes, sperm cells were expressed the immunoreactive products distributed in the cytoplasm, diffuse granular brownish yellow. Model group significantly decreased compared with the normal group (P lt; 0.05); model group and natural recovery group differences (P gt; 0.05); medication group could improve testicular IGF-1 mRNA expression (P lt; 0.01) Heshouwuyin high dose group was significantly better than the other groups (P lt; 0.05). Conclusion: D-galactose induced aging model is successful. Polygonum multiflorum drink can significantly improve the D-galactose induced subacute aging rat testis morphology, sperm count, motility and survival, thereby improving sperm count and sperm function. 3 Heshouwuyin can improve the D-galactose induced subacute aging serum levels of the male hormone (testosterone), and lower levels of gonadotropins and gonadotropin-releasing hormone. The subacute aging rat testis cells 4 Heshouwuyin can improve due to D-galactose local regulation of IGF-1 mRNA expression levels of the factor. The second part of the aging male rat testis Rb/p53 cellular signal transduction pathways Heshouwuyin purpose: by detecting rat testis Rb, p16, p53, p19, p21 expression, explore the anti-aging effects on the testis Heshouwuyin . Method: 1 immunohistochemical assay in rat testis tissue of pRb, p16, p53, p19, p21 expression. 2 Western Blot assay groups in rat testis of pRb, p16 and p19 and p21 protein expression. 3 RT-PCR to detect the rat testis p53, Rb mRNA expression. Results: Immunohistochemical staining: groups slices are visible pRb, p16, p53, p19, p21 staining, immunoreactive cells spermatogonia and occasionally in the sperm cells, stromal cells and seminiferous tubule basement membrane myoblasts . The immunoreactive products located in the nucleus, usually brownish yellow granular, was a small part of the dispersion like. 2 Western Blot detection of testicular tissue of pRb, p16 and p19, p21 protein expression: model rats testicular tissue p16, p19, p21 protein expression level was significantly higher of pRb protein expression levels were significantly decreased compared with normal group was significant difference (P lt; 0.05); natural recovery group, no difference (P gt; 0.05); the medication group testicular tissue p16, p19, and p21 protein expression levels were significantly lower than the model group of pRb protein expression levels were significantly higher than the model group ( P lt; 0.05), the high doses Heshouwuyin group was significantly better than the rest of the group (P lt; 0.05). 3 RT-PCR detection of testicular tissue p53, Rb mRNA expression: model group testicular tissue p53, Rb mRNA expression level was significantly higher than the control group (P lt; 0.05), and natural recovery group differences (P gt; 0.05) ; the medication group rat testis p53, Rb mRNA expression level was significantly lower than the model group (P lt; 0.05), which Heshouwuyin high dose group was significantly better than the rest of the group (P lt; 0.05). Conclusion: 1 pRb, p16, p53, p19, p21 expression mainly in the nucleus of spermatogonia Key the 2 aging male rat testis p16/Rb and p19/p53/p21 senescence pathway the regulating p53, Rb mRNA gene and p16, p19, p21 protein expression increases with Heshouwuyin treatment can play the role of inhibiting the expression; pRb protein downregulation, Heshouwuyin therapy can play a role in promoting the expression. In summary, our study shows that Heshouwuyin (2) adjust the aging male rat testis Rb/p53 cell signal transduction pathways, so as to achieve the improvement of testicular germ by (1) to improve the aging rat spermatogenesis; function, and delay the aging of the rat testis.

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