| Background:Although studies have confirmed cyclophosphamide (CP) can lead to gonadal damage, it is still widely used since it is the effective drugs of first choice for many kinds of childhood cancer, autoimmune diseases such as nephrotic syndrome, and other diseases. Clinical retrospective studies and animal experiments confirmed that CP do harm to male spermatogenesis[1-9]. There were some researches about underling mechanism of immature testicular damage caused by CP, but it is not elucidated till now, which accordingly bring difficulties to find out effective protection measures. Over the past few decades, mechanism study of CP reproductive toxicity mainly focused on direct damage to spermatogonial followed by apopotosis and necrosis, which result from phosphoamide caryolysine---anti-tumor metabolism ingredients of CP. Recent studies indicated that oxidatives stress injury to testis and niche of spermatogenesis may principally contribute to the CP reproductive toxicity[10, 11]CP is a kind of precursor drugs, which has no biological activity such as anti-tumor until it was metabolized in the body by cytochrome p450 metabolism [1]. CP and ACR is the main metabolic by-product of ACR in the liver, kidney, heart and other organ toxicity has been reported. ACR can lead to hemorrhagic cystitis was widely accecpted[12]. CYP3A4. and CYP3A5 the main two kinds of metabolism-dependent key enzyme of CP metabolize into ACR, are not only located in the liver and kidney, but also higher express in Sertoli cells of male gonad [13]. Moreover, ACR is a strong oxidant, so we hypothesis that ACR oxidative stress injury may be one of the main mechanisms of CP induced immature testicular toxicity.There are many Chinese herbs containing antioxidant ingredients to remove free radicals, Ginkgo biloba (EGB) is a typical representative [14]. Studies confirmed that ginkgetin, the main active ingredient of EGB, have the capacity of antioxidant and oxygen free radical scavenging. Recent studies suggest that ginkgetin can also inhibit the proliferation of tumor cells [15, 16]. Therefore ginkgetin maybe play the dual role when administrate accompany with CP, antitumor and protecting the male gonad.Objective: The purpose of this study is to investigate the role of acrolein-the representatives'toxic metabolite of CP, in CP induced oxidative stress injury of immature rat's testis. The experiment was carrying out on CP chemotherapy induced immature rat's testis injury model in vivo and primary cultured immature sertoli cells of the rat testis in vitro. And on this basis, we explore whether the antioxidants- ginkgetin can protect immature rat testis from acrolein-induced oxidative damage invitro and in vivo, which will provid an effective method to protect immature testis from CP-induced male reproductive toxicity and lay an important foundation for clinical application.Methods: 1) CP induced immature testicular injury rats'model was adopted in vivo. Immunohistochemical was used to explore the relationship between ACR and CP Induced testicular injury, and the target cells. Simultaneous Detection of testicular tissue antioxidant enzyme activity and changes of special markers of oxidative stress response, which aimed to study relationship between CP induced testicular trauma and oxidative stress; Light microscopy and electron microscopy observation of the morphological changes of testicular tissue. Preliminary study mechanism of CP induced oxidative stress in immature rats in vivo; 2) Identify the purity of primary cultured Sertoli cells in vitro. Mechanism of ACR lead Sertoli cells toxicity studies on the model in vitro. Optical microscope was adopted to observe cell morphology; ICC was used to detect ACR modified protein antigen to determine the target cell. Spectrophotometric determination of Sertoli cells and the main antioxidant enzyme markers of oxidative stress change, Semi-quantitative RT-PCR analysis of gene expression changes in antioxidant enzyme related, ACR cells in vitro to explore the possible mechanism of injury; 3) The study based on ACR induced Sertoli cells oxidative stress injury in vitro, plus standard ginkgo Biloba extract ginkgetin as a intervention, the use of light microscopy to observe changes of cell morphology, Spectrophotometric Determination changes of Sertoli cells'main antioxidant enzyme activity and markers of oxidative stress, Semi-quantitative RT-PCR analysis of gene expression changes in antioxidant enzyme related, ACR ginkgetin Sertoli cells in vitro study of the damage caused by the oxidation of emergency protective effect through intimate; 4)Experimental, immature rats ginkgetin solution for 4 1 week after CP testicular toxicity to the chemotherapy dose chemotherapy Optical microscopy observation testicular tissue morphology, Acute testis antioxidant enzyme analytical method and its related enzymes SOD and GSH content GR activity. convalescent serum sex hormone changes with sperm counts and sperm functional understanding of change Discussion of ginkgetin in vivo toxicity of CP immature rats.Results:1 Acute testis injury of CP in vivo:1) ACR antigen mainly located in the Sertoli cells'nucleus, ACR modified protein () content of was significantly higher than that of the control group (P <0.05) Correlate correlation analysis showed ACR-Pro content was positively correlated with CP dose (r=0.7 63, P <0.05. ).2) Antioxidant capacity: T-AOC, GR, SOD activity in the experimental group was significant difference compared with that of control group (P <0.05) Correlate correlation analysis showed that three indicators and CP dose was negatively correlated (r=-0.893, -0.934, -0.950; P <0.01);3) Oxidative stress Logo: MDA and OH., the experimental group than the control group there was a significant difference (P <0.05) Correlate correlation analysis showed that the two indicators with CP dose positive correlation (r = 0.888, 0.937;P<0.01).2 CP recovery after intraperitoneal injection (4w) rat testis injury results1) Testicular Index (testicular mass/ body weight): the experimental group than that of control decreased significantly (P <0.05) and it was negative correlation with CP dose (P <0.01 r=-0.779);2) Organizational changes :HE stain showed that the experimental group testicular tissue morphology were varying degrees of damage, and the injury degree is correlated with CP dose; electron microscope found that Sertoli cell mitochondria, endoplasmic reticulum injury obviously, these suggested the major toxicant target of CP is Sertoli cells.3) The sperm head count: the experimental groups were significantly lower than control, (P <0.01), Correlate correlation analysis showed the sperm count with CP dose was negatively correlated, The correlation coefficient of -0.942, P = 0.00. 4) Sex Hormones: ITT of experimental group was significantly higher than the control group (P<0.05). And ITT was positive correlation with CP dose (r = 0.893, P<0.01); Experimental groups'serum FSH and LH compared with that of the control group were either significantly higher(P<0.01) and the same period of the experimental group and control group in serum T levels, no significant difference (P> 0.05). Serum FSH and LH were also positive correlated with CP dose (r=0.865, 0.850; P <0.05).3 Primary culture of immature rat Sertoli cell morphology: Adherent growth, irregular polygon, fusion formation processes are very strong links between the cells, Visible nucleus, 2-4 nucleolus. Identification of viable cells was 92% -95%, its purity was 95% (P <0.05).4 Detection sertoli cell injury after incubated with ACR for 3 and 12 hours:1) MTT: cell activity of each experiment groups were significantly negative correlate with the control group (P <0.05) The CD50, of ACR were respectively 200 and 105μM.2) ICC: ACR-Pro of the experimental group was significantly increased than the control (P <0.05), the positive expression mainly located in the nucleus.3) Oxidative stress signs: MDA and OH. the experimental group. Content compared with the normal control group was significantly higher (P <0.05) MDA and OH. ACR and the content of the dose and time correlated (P <0.05).4) Antioxidant capacity: the experimental groups'T - AOC. GR and SOD activity decreased significantly than the control group (p <0.05) and three indicators compared with ACR dose or the time negatively correlated (P <0.05).5 Protect effect of Sertoli cells exposure to ACR by Ginkgetin in vitro1) Dynamic light microscope for observation found that the cells'morphology of add Ginkgetin and vitamin E before exposed to ACR, there was no obvious changes compared the normal control; Adding Ginkgetin and ACR together or Ginkgetin incubated with ACR before adding to cells showed no protect effect.2) MTT: Adding Ginkgetin and ACR simultaneously, ACR and Ginkgetin incubated before add to cells, cell activity were neither significantly different compared with the negative control (P>0.05). Cell activity of Pre-Ginkgetin and vitamin E group were significantly increased than the negative control group (P<0.05) However, the role of Ginkgetin significantly stronger than vitamin E (P<0.05).3) ICC detection ACR-Pro results: Adding Ginkgetin and ACR simultaneously, ACR and Ginkgetin incubated before add to cells, both were significant different compared with the negative control (P>0.05). Pre-Ginkgetin and vitamin E group than the negative control group were significantly increased (P<0.05) However, the role of Ginkgetin significantly stronger than vitamin E (P <0.05).4) Antioxidant capacity: T - AOC, SOD and GR activity of Ginkgetin add with ACR simultaneously Group, ACR incubated Ginkgetin before add to cells were not significantly different compared with the negative control (P>0.05). Pre-Ginkgetin and vitamin E group cell activity than the negative control group were significantly increased (P<0.05) However, the effect of Ginkgetin was significantly stronger than vitamin E (P <0.05).5) Oxidative stress signs: MDA and OH. content of Ginkgetin adding simultaneously with ACR Group, Ginkgetin incubated with ACR before add to cells were no significantly different compared with the negative control (P>0.05). Pre-Ginkgetin and vitamin E group cell activity than the negative control group was significantly lower (P <0.05) However, the effecting of Ginkgetin significantly stronger than vitamin E (P <0.05).6) Antioxidant enzyme gene expression: Cu-Zn SOD, GPx mRNA RT-PCR showed that Ginkgetin and the addition of ACR Group, Ginkgetin incubated with ACR before add to cells were not significantly different with the negative control (P>0.05). Pre-Ginkgetin and vitamin E group than the negative control group were significantly increased (P <0.05) However, the effect of Ginkgetin significantly stronger than vitamin E (P <0.05).6 Protect effect of test Ginkgetin from CP testicular toxicity in vivo1) Acute phase (24 hours): Ginkgetin lower MDA and OH. content of testicular tissue after CP adminstration significantly (P<0.05), significantly raise T - AOC, GR and SOD activity (P <0.05); Significantly increased Mn SOD, Cu-Zn SOD and GPx mRNA expression (P <0.05); Significantly lower ACR antibody of testicular tissue.2) Recovery phase (4 weeks): advance gavage of Ginkgetin, testis index was no significant difference compared with normal (P> 0.05), Conventional histological examination, pre-Ginkgetin gavage before CP administration, somniferous tubules were structural integrity, there were no obvious injury of Sertoli cells and spermatogonial. Ultra structural examination showed that Sertoli structural integrity and mitochondrial structure basically normal. Ginkgetin gavage rats pretreated with CP the sperm head counts when compared with control were significantly increased (P <0.05). Advance Ginkgetin gavage rat, CP chemotherapy convalescent serum FSH, LH concentration and the ITT compared with the normal control group were not significantly different (P> 0.05). Conclusion:1 CP-induced oxidative stress is one of the principal mechanisms for immature testicular toxicity. ACR is the main factor toxicity, the role of Sertoli cell is the target cells CP testicular tissue in vivo oxidation can lead to peace Value destruction reduced antioxidant capacity, the accumulation of oxygen radicals, cells pose health subtle environmental damage. lead spermatogenesis dysfunction.2 ACR can significantly lower antioxidant capacity and oxidative stress response, decrease the free radical scavenging capacity, reduce cell viability of Sertoli cells in vitro.3 Ginkgetin can raise the antioxidant capacity of the cells under ACR oxidative stress pressure, ACR antagonistic to the cell oxidative stress, the removal of oxygen free radical oxidative stress, thus breaking the vicious cycle of oxidative stress, protecting cells from damage. 4 Ginkgetin protect the immature testis under CP chemotherapy to reduce the CP-induced oxidative stress in vivo. Protect the spermatogenesis micro environment, recovery chemotherapy testicular tissue morphology and normal weight, while maintaining normal spermatogenesis.
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