| Bisphenol A (BPA) is a industrial raw material which widely exist in the environment. The adverse effects of BPA on reproductive system have drawn much attention in resent years. Study shows that lycium barbarum polysaccharides (LBP) can effectively improve the antioxidant ability and regulate the reproductive endocrine system, so as to protective the body against the spermatogenic damage induced by BPA. The present study focus on the protective effects and mechanism of LBP on spermatogenic cell apoptosis induced by BPA through the expression of Caspase-3, Bcl-2/Bax, Fas/FasL proteins and DNA ploid detected by the methods of flowcytomery and immunohistochemical technique. Fifty adult male Kunming mice were randomly divided into five groups after normally bred for seven days, which were the normal control group, BPA group and LBP groups in three different doses, with 10 mice in each group. Except the normal control group which was injected with olive oil, the other four groups were administrated with 20 mg·kg-1 BPA by intraperitoneal injection consecutively for one week. Meanwhile the mice in three LBP groups were given 50, 100 and 200 mg·kg-1 LBP by intragastric administration respectively, and the normal control group and BPA group was treated with the same dosage of physiological saline. Histopathological changes of the testicualr tissue were observed, immunohistochemical assay was applied to detect the expression of Caspase-3, Bcl-2/Bax and Fas/FasL proteins. The apoptosis of spermatogenic cells was detected by analyzing DNA ploid with the methods of flowcytomery. The testicular histopathological results showed that LBP obviously improved the recover of damaged testicular tissue, and the seminiferous tubules structure also markedly improved in the 100 and 200 mg/kg LBP groups compared with BPA control group. Immunohistochemical results showed that the expression of positive cells with Caspase-3 proteins in the BPA group was significantly higher than that in the control group(P<0.01). After supplement with different doses of LBP, the expression of Caspase-3 positive cells were significantly decreased compared with BPA group (P<0.01). BPA can enhance the expression of pro-apoptotic Bax proteins (P<0.01), diminish the expression of anti-apoptotic Bcl-2 (P<0.05). After supplement with LBP, the expression of Bax were significantly decreased especially in the 200mg/kg LBP group than that in the model group (P<0.01). While the expression of Bcl-2 gradually increased with the increasing doses of LBP, the ratio of Bcl-2/Bax were increased accompanied with the increase of LBP doses. The expression of Fas and FasL proteins were both significantly higher than that in the control group (P<0.01). Compared with BPA group, the expression of Fas and FasL in 100 mg/kg and 200 mg/kg LBP group were significantly decreased after supplement with LBP (P<0.01). Flowcytomery results showed that the percentage of apoptosis in BPA group was significantly higher than in the control group (P<0.01). The apoptosis rate in all the three LBP groups were descend than BPA group after added with LBP, in which 100 mg/kg and 200 mg/kg LBP groups have significant difference compared with BPA group (P<0.01) but no obvious difference compared with the control group. The result of DNA ploid showed that the percentage of 1C in BPA group was significantly decreased than in the control group (P<0.05). The percentage of 1C and 4C were both increased after added with LBP, although no statistically significant. The percentage of 4C was significantly improved in LBP groups than in the control group (P<0.05).Conclusion: LBP can regulate the expression of apoptotic proteins through enhancing the expression of anti-apoptotic proteins (Bcl-2) and down-regulating the expression of pro-apoptotic proteins (Bax) in mitochondrial pathway. Supplement with LBP also inhibit the expression of Fas and FasL proteins which related with death receptor pathway and reduce the combination of Fas and FasL to inhibit the activation of Caspase-3, who is an apoptotic executive protein that can trigger off the cell apoptosis. LBP can also reduce the percentage of apoptotic spermatogenic cells and increase the percentage of 4C cells, so as to ensure the primary spermatocytes further develop into sperm cells. Ultimately, LBP exerts its potective effects on spermatogenic cells to ensure the generation quantity of mature sperm, ensure testicular reproductive functions, thus alleviate the reproductive damage induced by BPA in adult male mice. |
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