Effect Of LBP On Damage To Testicular Tissue Of Male Rats Induced By Low Dosage Of Iozining Radiation And Damage Recovery

Ionizing radiation has been considered as another major pollution source following Three Wastes (i.e. waste water, waste gas and industrial residues) that affect people's health, the reproductive system is one of its sensitive target organs. Research has shown that ionizing radiation is an inducing factor for abortion, infertility and terate, which also can lead to reduced sexual functionality of males. Ionizing radiation could induce abberant genital cell, chromosome damage and various deformity of sperm. Testis is the spermatogenic organ, and spermatogenesis, if exposed to ionizing radiation, would induce infertility, genetic diseases and malignant tumor. It was recognized as "medicine and food" by Ministry of Health in 1998. As an important functional factor of fructus lycii, lycium barbarum polysaccharides (LBP) has lots of biological functions, such as immunoregulation, reducing blood sugar and lipid, anti-aging and anti-fatigue, however, there has been few reports on its anti-radiation effect, especially, the reproductive system damage induced by radiation and the recovery of such damage have not been evaluated so far. This study aims to discuss LBP's antagonestic effect against damage to testicular tissue induced by low dosage of iozining radiation and its contribution to damage recovery, thus providing a theoretical basis for radiation protection and adjuvant therapy of damage for professionals who are exposed to radiation for a long time and tumor patients who are receiving radiotherapy.Part I Effect of LBP on male rats'testicular tssue damage induced by low dosage of iozining radiation and damage recoveryObjective:To observe the effect of LBP on male rats'testicular tissue damage induced by low dosage of iozining radiation and damage recovery. Methods:Eighty four (84) Wistar male rats were randomly divided into the following seven (7) groups:normal control group, radiation control group 1, radiation control group 2, radiation control group 3, LBP + radiation group 1, LBP + radiation group 2 and LBP + radiation group 3 (n=12 for each group). For LBP+radiation group 1,2 and 3, gastric gavage with LBP (10 mg/kg) was given prior to each exposure to radiation, and the rats were exposed to local radiation using a 60Coy irradiation therapy machine from Monday to Friday each week for four (4) consecutive weeks (Total 20 days), and the rats were sacrificed on the 1st,7th and 14th day after the last exposure. The damage induced by radiation and damage recovery in three different stages were observed. Dosage of radiation used: 0.115Gy/rat/day/Total dosage:2.3 Gy/rat. For radiation+control group 1,2 and 3, the rats were given gastric gavage with same volume of normal saline and were exposed to same dosage of radiation, then the rats were sacrificed on the 1st,7th and 14th day respectively after last exposure. For the normal control group, the rats were given gastric gavage with same volume of normal saline without being exposed to radiation. The organ coefficients of testis and accessory sexual organs were assayed on 1st day,7th day and 14th day following last exposure, and the morphological changes occurred to testicular tissues and the advanced micro structure were observed. Results:Compared with their respective radiation control group, the testicular coefficient in all LBP+radiation control groups increased, especially in LBP+radiation group 3, the difference was most significant (P<0.01); in LBP+radiation group, the spermatogentic cells damage induced by low dosage of radiation reduced signifcantly, and the recovery of spermatogentic cells damage was promoted, the effect was greater, compared with their respective radiation control group. Conclusion:LBP has significant antagonestic effect against decrease of testicular coeffiicient induced by low dosage of iozining radiation, also, it can reduce the damage to spermatogentic cells induced by radiation and effectively promote the damage recovery on 1st day,7th day and 14th day following last radiation. And the effect was obviously greater, compared with natual recovery of their respective radiation control group.PartⅡEffect of LBP on male rats' sexual functionality damage induced by low dosage of iozining radiation and damage recoveryObjective:To observe the effect of LBP on male rats'sexual functionality damage induced by low dosage of iozining radiation and damage recovery. Methods:Same with PartⅠ. For sexual functionality experiment, eighty-four (84) healthy male rats fed in the same period were used to study their mating behaviour. Following low dose of iozining radiation, the eretion latency and sexual functionality of those male rats on 1st, 7th and 14th day respectively were observed, and finally, the sperm count, sperm vigor and the level of sexhormone T, LH and FSH in serum were measured. Results: Comparing groups LBP + radiation 1,2 and 3 with groups radiation control 1,2 and 3, the erection latency, mounting latency and ejeculation latency of the rats treated with LBP reduced significantly; the number of mountings and ejeculations increased significantly(P<0.01); the sperm count and vigor were promoted significantly (P<0.01), meanwhile, the level of T in serum increased significantly (P<0.01). Conclusion:LBP is able to significantly improve the sexual functionality of the male rats that were exposed to low dosage of ionizing radiation on 1st day,7th day and 14th day, and the effect was obviously greater, compared with radiation control group. This was supposed to be achieved through increasing sperm count and vigor and regulating hypothalamic-pituitary-gonadal axis disturbance.PartⅢEffect of LBP on antioxidant capacity of male rats' testicular tissue and testicular tissue DNA damage induced by low dosage of iozining radiation and damage recoveryObjective:To observe the effect of LBP on antioxidant capacity of male rats' testicular tissue and testicular tissue DNA damage induced by low dosage of iozining radiation and damage recovery. Methods:Same with PartⅠ. The activity of superoxide dismutase (SOD) in testicular tissue, the levels of malondialdehyde (MDA), the activity of GSH-PX and reduced glutathione (GSH) were assayed respectively on 1st day,7th day and 14th day following low dosage of radiation, and the DNA chain breakage was assayed using single cell gel electrophoresis (SCGE). Results:Compared with their respective radiation control groups, the group LBP + radiation group 1,2 and 3 showed noticeable increase in SOD activity, GSH level and GSH-PX activity, while MDA level decreased significantly (P<0.01); When treated with LBP prior to exposure, group radiation control 1,2 and 3 showed significant increase in tail length and tail frequency (%) of testicular cells, compared with their respective normal control group(P<0.01). Conclusion:LBP is able to effectively scavenge oxygen free radicals produced by radiation and has antagonestic effect against testicular cell DNA damage induced by oxidative stress, and it can contribute to the recovery of damage to testicular tissue DNA. The effect is obviously greater, compared with natual recovery of their respective radiation control group.Part IV Effect of LBP on male rats'testicular cell apoptosis induced by low dose of iozining radiationObjective:To observe the effect of LBP on male rats'testicular cell apoptosis induced by low dose of iozining radiation. Methods:Same with PartⅠ. The percentages of Bcl-2, Bax, Caspes-3, Fas, P53 and Hsp70 in testicular tissue were assayed using immunohistochemistry respectively on 1st day,7th day and 14th day following low dosage of iozining radiation; The level of mRNA in P53 and Hsp70 and protein expression were measured using RT-PCR and Western Blot methods; The percentage of apoptosis in spermatogenic cells was measured using flow cytometry; The membrane potential△ψm and Flou-3 fluorescent intensity of testicular tissue were measured using confocal laser scaning microscopy. Results:Compared with their respective radiation control group, the group LBP + radiation 1,2 and 3 showed significant increase in positive cell ratio of Bcl-2 and Hsp70, while the positive cell ratio of Bax, Caspes-3, Fas and P53 decreased significantly, especially in group LBP + radiation 3 (P<0.01); using RT-PCR and Western-Bolt methods, it indicated that the P53 mRNA level decreased significantly compared with group radiation control 3, while the Hsp70 mRNA level increased signifcantly (P<0.01); using Flow Cytometry, LBP+radiation group 1,2 and 3 showed a significant decrease in testicular cell apoptosis rate, compared with their respective radiation control group (P<0.01); using confocal laser scaning microscopy, it was found that the red/green fluorescence ratio in group LBP + radiation 1,2 and 3 increased significantly, compared with their respective radiation control group, and the mitochondrial membrane potential also increased significantly, while the fluorescent intensity of Flou-3 decreased significantly (P<0.01). Conclusion: LBP is able to significantly reduce the apoptosis of spermatogenic cells induced by iozining radiation, thus reducing the damage to testicular tissue induced by radiation through down-regulating the protein expression of pro-apoptotic gene Bax, Fas, Caspase-3 and P53, up-regulating the protein expression of antiapoptotic gene Bcl-2 and Hsp 70, promoting the mitochondrial membrane potential of testicular cells as well as reducing the concentration of Ca2+ in cells. The effect is obviously greater, compared with natual recovery of radiation control group.