| Spermatogenesis in testis and sperm maturation in epididymis.If oxidative stress occurs in testis or epididymis,it will have an adverse effect on the semen quality of male animals.VD has both the functions of improving the quality of animal semen and anti-oxidation,so we speculate that VD has the ability to improve the anti-oxidation stress of testis or epididymis.There are many kinds of cells in testis and epididymis,in order to find out which cell plays a major role though VD,the expression of VDR(vitamin D receptor,VDR)and four enzymes involved in VD metabolism in sheep testis and epididymis were detected in this study.Epididymal epithelial cells was selected as the object,then H2O2 was used to construct an oxidative stress model.The model was stimulated by VD,and then the apoptosis rate,mitochondrial apoptosis pathway and PI3K/Akt signaling pathway were studied,in order to find out whether VD can inhibit the apoptosis of model cells and its molecular mechanism;at the same time,the antioxidant capacity and Nrf2 signaling pathway were studied to explore whether VD can improve the antioxidant capacity and its molecular mechanism.Results:1.VDR and the four enzymes related to VD metabolism were found in testis and epididymis,and the locations were the same.They were mainly distributed in Leydig cells and seminiferous tubules in testis and epithelial cells in epididymis.Because the expression of VDR in epididymis was higher than that in testis,epididymal epithelial cells were selected as the object in this study.2.Epididymal epithelial cells were cultured in vitro and stimulated by H2O2,H2O2 can lead to a dose-dependent decline in cell viability,the IC50 was 400 ?M.Under the condition,a large number of cells shrunk,the nucleus became smaller and irregular;the apoptosis rate,ROS content and MDA content in cells were increased,the ratio of GSH/GSSG decreased;the LDH content in cell culture supernatant was increased.The results showed that when 400 ?M H2O2 was used to stimulate cells,oxidative stress occurred and the oxidative stress model was successfully constructed.3.In the oxidative stress model,a large number of cells were apoptosis,Caspase 3 was activated,mitochondrial membrane potential significantly was decreased(P<0.01),Cyt C was released into cytoplasmic matrix,the expression of mitochondrial apoptosis-related genes were changed,these changes indicated that H2O2 induced apoptosis depends on mitochondrial apoptosis pathway.However,if the oxidative stress model was treated with 100 nM 1?,25-(OH)2VD3,the apoptosis rate was decreased significantly(P<0.01),and the above indexes of mitochondrial apoptosis pathway were improved.When LY294002,a PI3K/Akt signal pathway inhibitor,was used to inhibit PI3K,the ability of VD to inhibit apoptosis and improve the indexes related to mitochondrial apoptosis pathway was decreased.It was suggested that the inhibition of VD on the apoptosis of epididymal epithelial cells under oxidative stress depended on the activation of PI3K/Akt signaling pathway.4.In oxidative stress model,ROS was significantly increased(P<0.01),membrane lipid peroxidation was increased,LDH was outflowed,GSH/GSSG was decreased significantly(P<0.01),the activity of antioxidant enzyme was reduced,and the expression of antioxidant enzyme genes expression was inhibited.It was suggested that H2O2 can inhibit the antioxidant capacity of epididymal epithelial cells under oxidative stress,and thus cause oxidative damage.However,if 100 nM 1?,25-(OH)2VD3 was used to treat the oxidative stress model,all the above indexes in the cells were improved.When Nrf2 gene was silenced,the ability of VD to improve the above indexes were weakened.It was suggested that VD can improve the antioxidant capacity of epididymal epithelial cells under oxidative stress and alleviate the oxidative damage of cells,which was related to the activation of Nrf2 signaling pathway.When LY294002 was used to inhibit PI3K,the phosphorylation of Nrf2 protein was inhibited,indicated that PI3K/Akt signaling pathway can activate Nrf2.The above results showed that VD could effectively alleviate oxidative stress in epididymal epithelial cells of sheep,and its molecular mechanism was to inhibit the expression of mitochondrial apoptosis related genes through PI3K/Akt signal pathway,thus inhibiting the activation of mitochondrial apoptosis pathway,and then inhibiting the apoptosis of cells through PI3K/Akt/Nrf2 signaling pathway,the expression of antioxidant enzyme genes in oxidative stress epididymal epithelial cells were increased,so as to improve the antioxidant capacity of cells and reduce oxidative damage. |
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