| OBJECTIVE:The cross-sectional study was carried out to investigate the effect of microwave radiation on male reproductive function, while the animal study was carried out to investigate the effect of Bushen Shengjing Formula on the testis tissue in male rats after microwave radiation and the potential mechanism.MATERIALS AND METHODS:1. The soldiers direct contacted with the radar radiation were included with radiation group, and the ones without radar radiation contact were included with control group.A questionnaire survey was conducted to collect working period, working place, working hours daily, habits (smoking, drinking, etc.), phone usage, marital and reproductive history,radaion history, etc.Then semens collected were for semen analysis and flow cytometry for sperm motility, sperm concentration, sperm total number and sperm apoptosis. Blood samples were used to analyze the follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E2) levels of prolactin (PRL) and progesterone (P) level. Western blot assay was conducded for HSP70, bax, bcl-2, cyt-c, apaf-1, caspase-3, caspase-9 protein expression.2.48 male Wistar rats were randomly assigned into 4 groups:control (C) group, radiation (R) group, Bushen Shengjing Formula prevention (P) group and the Bushen Shengjing Formula treatment (T) group,12 in each group.Radiation group:Rats were radiated by S-band microwave at power density of 30mW/cm2 15 min daily for 5 days. Control group:Rats were placed in the irradiation place without radiation. Bushen Shengjing Formula prevention (P) group:7 days prior to irradiation, rats were fed Chinese decoction every day until 7 days after radiation. Bushen Shengjing Formula treatment (T) group:rats were fed Chinese decoction every day from begining of irradiation to 7 days after radiation. On Dl, D2, D7, D14,3 rats in each group were anesthetized, and blood serum, testis and semen were collected. Serums were separated for measuring hormone levels, testises were separated for viscera index, HE staining, TUNEL assay. The bilateral epididymal sperm suspension obtained were used for sperm motility and morphology.3.48 male Wistar rats were randomly assigned into 4 groups:control (C) group, radiation (R) group, Bushen Shengjing Formula prevention (P) group and the Bushen Shengjing Formula treatment (T) group,12 in each group. The treatment conditions of control group, radiation group, prevention group and treatment conditions were similar to the second part. On D1, D2, D7, D14,3 rats in each group were anesthetized, and testises were collected.HSP70, bax, Bcl-2, cyt-c, apaf-1, caspase-3, caspase-9 mRNA and protein expression in germ cells were determined by RT-PCR, western blotting and immunochemistry assasy.RESULTS:1.1) Smoking and radiation exposure are risk factors of sperm motility. Demographic factors, lifestyle factors, radiation exposure and working period did not affect the total number of sperm. BMI, stay up late affected sperm density, while radiation exposure and working period did not affect sperm density.2). BMI, smoking and staying up late were related to living cells rate and early apoptosis rate of sperm, while working period did not affect sperm apoptosis rate in radiation group.3) The level of E2 was related to radiation, and E2 in radiation group was significantly lower than that in control group; testosterone was negatively correlated with BMI, so the higher of the BMI, the lower of the testosterone; Progesterone was correlated with working period in radiation group; PRL was positively correlated with staying up late. LH was correlated with radiation. No factors were found effecting FSH level.4).HSP70 was correlated with radiation, sperm concentration, total sperm count, testosterone levels. Apaf-1 and Bcl-2 were not correlated with radiation, sperm motility, total sperm count, sperm density, hormone E2, T, P, PRL, LH, FSH. Cyt-c was correlated with testosterone levels, but not correlated with radiation, sperm motility, total sperm count, sperm density, and hormone E2, P, PRL, LH, FSH. Caspase-9 was correlated with LH, but not correlated with radiation, sperm motility, total sperm count, sperm density, hormone E2, T, P, PRL, FSH. caspase-3 was associated with radiation, but not associated with sperm motility, total sperm count, sperm density, hormone E2, T, P, PRL, LH, FSH.2.1) Rats in radiation group showed reduced activity, slow response, dull brown fur, fever.Chines medicine could release the damaging effects. After radiation testis index was significantly increased (D7), and no differences was found among radiation group, prevention group and treatment group.2) Microwave radiation could make sperm activity decreased, malformation rate increased, testicular morphology changed. Chinese medicine could play a protective role, so that damage could be reduced.3) On D7 and D14, microwave radiation can lead to T, E2, FSH, LH decreased significantly. On D7, prevention group had a protective effect on LH.4) On D1, prevention group and treatment group could play a protective role in radiation-induced apoptosis. On D2, the protective effect of traditional Chinese medicine was not obvious. On D7 and D14, no significant differences were found in apoptosis among each group.3.1) HSP70 mRNA expression in RT-PCR assay showed:on D1, HSP70 mRNA expression of prevention group and treatment group was significantly lower than the radiation group (p<0.05). On D2, D7, D14, HSP70 expression declined gradually, no significant differences were found among each groups (p>0.05). Results of immunohistochemistry were consistent with the results of t western blot:on D1, compared with control grouop, HSP70 of radiation group was significantly increased (p<0.05), while prevention group and treatment group were significantly lower than the radiation group (p<0.05).2) caspase-3 mRNA expression in RT-PCR assay showed:on D1, caspase-3 mRNA expression of treatment group was significantly lower than the radiation group (p<0.05). No significant difference was found between prevention group and radiation group(p>0.05).On D2, D7, D14, no significant differences of caspase-3 were found among each groups (p>0.05).Cleaved Caspase-3 was the active form of caspase-3. Western blot results showed that on D1, compared with treatment group and control group, cleaved caspase-3 of radiation group was significantly increased (p<0.05). On D2, D7, D14, no significant differences of caspase-3 were found among each groups (p>0.05. Immunohistochemistry results of caspase-3 showed that:on D1, D2, D7, D14, no significant differences were found among each groups (p>0.05).3) RT-PCR results of cyt-c show that on D7, compared with the control group, cyt-c mRNA of Chinese medicine prevention group declined significantly (p<0.05). On D1, D2, D14, no significant differences were found among each groups (p>0.05). Results of immunohistochemistry were consistent with the results of t western blot:On D1, D2, D7 and D14, no significant differences were found among each group (p>0.05).4) Immunohistochemistry and western blot results were consistent with RT-PCR in Bax, Bcl-2, and apaf-1 and caspase-9 expressinons. No significant differences of Bax, Bcl-2, and apaf-1 and caspase-9 were found among the groups on D1, D2, D7 and D14 (p>0.05).CONCLUSIONS:1. Microwave radiation could affect sperm motility, E2 level,LH level, P level. Male Reproductive system damage caused by microwave radiation may be related to HSP70, caspase-3 expression.2. Microwave canld make testis index increased, sperm motility decreased, malformation rate increased, testicular morphological changed, and Bushen Shengjing Formula could play a protective role. On D7 and D14, microwave radiation had an impact on hormones, while Chinese medicine had a protective effect on LH reduction induced by radiation. On D1 and D2, microwave radiation caused increased apoptosis, while Bushen Shengjing Formula prevention group might play a protective role on early apoptosis rate induced by radiation.3. Bushen Shengjing Formula might affect the germ cells, and the protective effects on germ cells may partly result from its ability to intervene in the HSP70-caspase-3 mediated apoptosis in male rats after microwave radiation on D1.
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