Effect Of Different Component Of Morinda Officinalis Aqueous Extract On Hypothalamic-pituitary-testis Axis Impairment In Microwave-induced Reproductive Dysfunction

Objective: To establish microwave induced reproductive impairment animal model of adult sd male rat, and investigate the effect of the water layer and ethyl acetate layer of the Morinda Officinalis aqueous extract following ethyl acetate extraction on the impairment induced by microwave. Furthermore, to demonstrate the potential mechanism how the effective component of Morinda Officinalis affect the Hypothalamic-Pituitary-Testis axis(HPTA). Eventually, to provide the theoretical evidents for effective component screenin from Morinda Officinalis and the recovery of reproductive impairment.Part ⅠMethods: 12 SD male rats were randomly divided into 2 groups. Group A: Blank control group; Group B: Radiation group, the microwave radiation induced reproductive impairment rat group, was established by given microwave radiation with Microwave Signal Generator(900MHz) at 218μW/cm2 radiation density, 24h/d, for 10 days.3. The diversity in the body weight, sexual behavior test, testis-weight ratio, epididymis-weight ratio and the expression of Gn RH in hypothalamus were observerd.Results: 1. The radiation group’s weight gain was 19.83±12.32 g, which was statistically significant less than that of the control group.2. The sexual behavioral indexes included capture incubation period(CIP) and capture times(CT). Compared with other groups, CIP significantly increased and CT significantly decreased in radiation group. 3. Testis-weight ratio and epididymidis-weight ratio significantly decreased compared with control groups. Histological assessments were observed after Hematoxylin-eosin(HE) staining. In radiation group, spaces between each seminiferous tubule were found to widen. And there was almost no sperms found in seminiferous tubule as the black arrow showed. The layers of seminiferous tubule epithelium were incomplete and found to collapse. No sperms except for the fallen spermatogenic cells were found inside the epididymal ducts of rats in radiation group 4. Gn RH immunofluorence positive laballing were mainly found in arcuate nucleus. The immunofluorence density of Gn RH decreased in radiation group.Conclusions Reproductive impairment animal model of male adult SD rat was successfully established by 900 MHz microwave radiation at 218μW/cm2 radiation density for duration of 24h/d in ten days.Part ⅡMethods: 66 SD male rats were randomly divided into 11 groups: 1.Group A: Blank control group; 2. Group B: Radiation group; 3. Group C: Radiation + NS group; 4. Group D: Water layer of the Morinda Officinalis aqueous extract following ethyl acetate extraction treatment groups(divided into four doses gradient); 5.Group E: Ethyl acetate layer of the Morinda Officinalis aqueous extract following ethyl acetate extraction treatment groups(divided into four doses gradient). The ethyl acetate layer and the water layer were obtained after ethyl acetate extraction from Morinda Officinalis aqueous extract. The content of several trace elements(Zinc, Manganese, Selenium, Iron, Copper), which might relate withrestoration of HPTA, were compared between the water layer and ethyl acetate layer of the Morinda Officinalis aqueous extract following ethyl acetate extraction. Groups C, D and E were treated with NS, water layer and ethyl acetate layer respectively. The diversity in the sexual behavior test, and the indexes related with the different levels of HPTA such as the expression of Gn RH in hypothalamus, content of Gn RH, LH, FSH and T in the serum, morphological changes in testis and epididymis, testis-weight ratio and epididymis-weight ratio were observed and compared among groups.Results: 1. The concentrations of Zinc, Manganese and Iron were generally higher in the the water layer of the Morinda Officinalis aqueous extract following ethyl acetate extraction than that of the ethyl acetate layer. While no similar tendency were found about the distribution of the Selenium and Copper in these three samples. 2. The capture incubation period(CIP) significantly decreased in the D3/D4 groups, while the capture times(CT) significantly increased in the D4 groups compared with the control and NS groups. The other groups showed no difference with control group. 3.(1)Testis-weight ration of B~E group significantly decreased in group B compared with group A. Epididymis-weight ration significantly decreased in group B compared with group A. But testis-weight ration and epididymis-weight ration significantly increased in D4 group compared with groups B/C. The other groups showed no difference with control group.(2)The level of testosterone in microwave group showed no significantly difference compared with control group. The level of testosterone significantly increased in D3/D4 group compared with B/C groups. The other groups showed no difference with control group.(3)Histological assessments in microwave group were described as part I. The count of the seminiferous tubule cells and sperms increased gradually according to the increasing dose of water layer of the Morinda Officinalis aqueous extractfollowing ethyl acetate extraction in D groups. The morphological in testis and epididymis showed no significantly difference among B/C/E groups. No sperms except for some fallen spermatogenic cells were found inside the epididymal ducts of rats in group B. In group D3 and D4, great quantity of sperms were found in the epididymal ducts, although less than the amount of the group A. 4. The concentration of FSH showed no significant difference among groups. The level of LH in D3/D4 groups significantly decreased compared with B/C groups. The other groups showed no difference with control group. 5.(1)The serum Gn RH concentration decreased in microwave group compared with control group. In D3/D4 groups, the concentration decreased significantly compared with B/C groups, while the other groups showed no difference with control group.(2)Gn RH immunofluorence positive laballing were mainly found in arcuate nucleus. The expression declined slightly in group B compared with group A, while no statistical significance was found. The expression of Gn RH in D3/D4 group significantly decreased when compared with B/C groups. The other groups showed no difference with control group.(3)Real-time PCR indicated that expression of Gn RH-m RNA in D4 group significantly reduced compared with B/C groups.Conclusions: 1. The concentrations of Zinc, Manganese, Iron,were higher in the the water layer of the Morinda Officinalis aqueous extract following ethyl acetate extraction, which may relate to the recovery of the reproduction impairment. 2. The water layer of Morinda Officinalis aqueous extract following ethyl acetate extraction can improve the sexual behavior, promote spermatogenesis and sperm maturation by improve the various levels of HPTA. And the optimal dose for administration was 40g/kg.3. The ethyl acetate layer of Morinda Officinalis aqueous extract following ethyl acetate extraction had no significant effect on the improvement of spermatogenesis in male reproductive impairment animal model.