| Objective: 900 MHz mobile phone frequency electromagnetic radiation has been used as the experimental conditions and rat testicular tissue has been taken as the research object.The testicular tissue morphology,ultrastructure,oxidative stress,Nrf2 protein expression and DNA damage repair protein(?H2AX)expression were chosen as the main observation indexes.Commonly used Chinese medicine Liuweidihuang Pills for tonifying kidney was selected as intervention factor.To observe the effect of 900 MHz cell phone electromagnetic radiation on the testicular tissue structure,ultrastructure,oxidative stress,the expression of Nrf2 protein and gamma H2 AX in male rats,and the effect of Liuweidihuang Pills on them.Methods: 50 healthy male SD rats were randomly divided into five groups and each group was divided into 10 rats: normal group,2h radiation group,4h radiation group,2h medication group,4h medication group.The rats in 2h radiation group,2h medication group,4h radiation group and 4h medication group were exposed to 900 MHz electromagnetic radiation(370?W/cm2)every day for 2 hours and 4 hours respectively,continuing for 30 d,while the rats in normal group without radiation.The rats in 2h medication group and the 4h medication group were treated with Liuweidihuang Pills suspension(0.72g/kg.d)after irradiation at the prescribed time,and the rats in other 3 groups were given with distilled water.At the end of the experiment,rats in each group were fasted for 12 hours but they could drink water.Intraperitoneal anesthesia with 10% chloral hydrate was performed,and then the rats were killed and the testicular tissue was taken.The structure and ultrastructure of testis were observed by optical microscope and electron microscope respectively.The changes of superoxide dismutase(SOD),glutathione(GSH),malondialdehyde(MDA),glutathione peroxidase(GSH-Px)oxidation and antioxidation indexes were detected by colorimetric method.Immunohistochemistry and Western blot were used to detect the protein expression of nuclear factor related factor-2(Nrf2)and DNA repair related protein(?H2AX).Results:1 General situation of rats in each groupThere were no significant differences in the mental state,behavior,food and water intake,excretion,shin and hair of the rats in each group.2 Comparison of testicular histomorphology in ratsThe morphological changes of rat testis were observed under the optical microscope: The seminiferous tubule structure of the rat testis in normal group is clear and complete,spermatogenic cells arranged orderly,distinctly,full of mature sperm in the lumen;Compared with the rats in normal group,testis seminiferous tubule structure in 2h radiation group's rat is clear and complete,and primary spermatocytes and secondary spermatocyte the number of tubules cells was not decreased,slightly loose structure;Compared with the rats in normal group,the seminiferous tubule structure,tubules spermatogonia and primary spermatocytes layer of rats in 4h radiation group were clear,secondary spermatocyte layers were slightly blurred,and mature sperms in the lumen were decreased;Compared with the rats in 2h radiation group,the 2h medication group's rat testis seminiferous tubule structure was clear and complete,spermatogenic cells arranged orderly,distinctly,full of mature sperms in the lumen,the organizational structure was close to normal group;Compared with the rats in 4h radiation group,the testis seminiferous tubule structure of 4h medication group was clear and complete,the spermatogenic cells in each layer were clear,and there were more mature sperms in the lumen.3 Comparison of testicular ultrastructure in ratsThe ultrastructural changes of testis were observed under transmission electron microscope: In the rat testis of normal group,the seminiferous tubules intact basement membrane was thickening,no obvious gap between cells,cellmembrane integrity,plump cytoplasm,nuclear clear,and mitochondrial structure had no obvious injury;Compared with the rat testis of normal group,in 2h radiation group,the seminiferous tubule basement membrane was swelling and thickening,Sertoli cell tight junction separated,and the gap between the cells and cells significantly increased,partial mitochondria were vacuolized and injured;Compared with the rat testis of normal group,the 4h radiation group's basal membrane of seminiferous tubule was swelling,Sertoli cell tight junction separated and the gap between the cells and cells increased obviously,spermatogonial membrane was integrity,and we observed cytoplasmic fragmentation,nuclear pyknosis,nuclear week gap deformity,mild expansion,mitochondrial vacuolization and cristae of mitochondria fusion disappeared;Compared with 2h radiation group,2h medication group's seminiferous tubule basement membrane was normal,Sertoli cell tight junction was normal,intercellular gap was narrowed and close to normal,mitochondrial vacuolization occasionally occurred;Compared with that of 4h radiation group,in the 4h medication group,the swelling seminiferous tubule basement membrane was significantly reduced,Sertoli cell tight junction was close to normal.The gap between the cells was significantly reduced,no obvious cell membrane damage was observed,the nucleus was clear,the mitochondrial structure was normal.In addition,in the 2h radiation group and the 4h radiation group,it showed that the seminiferous tubule basement membrane was swelling,Sertoli cell tight link were separated,mitochondrial and other ultrastructural damage occurred,but the testis injury of rats in 4h radiation group was more serious than that in the 2h radiation group.4 Comparison of the content of MDA and GSH,the activity of SOD and GSH-Px in rats testis of each groupCompared with normal group's rats,testicular MDA content in 2h radiation group and 4h radiation were significantly increased(P < 0.01),and GSH content,SOD activity and GSH-Px activity were decreased significantly(P < 0.01);Compared with that of 2h radiation group,MDA content in the rats in 2h medication group was decreased significantly(P < 0.01),SOD activitywas increased significantly(P < 0.05),there were no significant changes in GSH content and GSH-Px activity ly(P > 0.05);Compared with that of 4h radiation group,MDA content in 4h medication group was decreased significantly(P < 0.01),SOD activity and GSH content were increased significantly(P < 0.01),and there was no significantly difference in GSH-Px content(P > 0.05).In addition,compared with that of the 2h radiation group,the MDA content in the testis of the 4h radiation group was significantly increased(P < 0.01),SOD activity and GSH content were significantly reduced(P < 0.01),GSH-Px activity was not significantly changed(P > 0.05).5 The protein expression of Nrf2 in rats testis of each group was detected by immunohistochemistryCompared with that in normal group's rats,Nrf2 protein expression of rat testicular tissue in 2h radiation group and 4h radiation group were decreased obviously(P < 0.05),Compared with that in 2h radiation group,4h radiation group's Nrf2 protein expression was decreased significantly(P < 0.05);Compared with that in 2h radiation group,the Nrf2 protein in testicular tissue of 2h medication group was significantly increased(P < 0.05);Compared with that in 4h radiation group,the Nrf2 protein expression in testicular tissue of 4h medication group was significantly increased(P < 0.05).6 Nrf2 protein expression of rats testicular tissue in each group by Western blotCompared with that in normal group's rats,the expression of Nrf2 protein in testis tissue of rats in each group was significantly reduced(P < 0.01);Compared with that in 2h radiation group,the Nrf2 protein in testis tissue of2 h medication group was increased significantly(P < 0.01);Compared with that in 4h radiation group,the Nrf2 protein expression in testicular tissue of 4h medication group was increased significantly(P < 0.01).In addition,compared with 2h radiation group,the Nrf2 protein expression in testis tissue of 4h radiation group was significantly reduced(P < 0.01).7 The ?H2AX protein expression in rats testis of each group was detected by immunohistochemistryThe ?H2AX protein expression of normal group were expressed strongly in spermatogonia,primary spermatocyte,secondary spermatocyte and spermatid.Compared with that in normal group,?H2AX protein of 2h radiation group and 4h radiation group were mainly expressed in spermatogonia,while the ?H2AX protein expression was weakened in primary spermatocyte,secondary spermatocyte and spermatid.Compared with that in the testis of normal group rat,the ?H2AX protein expression in the testis of the 2h radiation group,4h radiation group and 4h medication group were decreased significantly(P < 0.05).Compared with that in the testis of 2h radiation group and 4h radiation group respectively,the expression of ?H2AX in the 2h medication group and the 4h medication group were higher in spermatogonia,primary spermatocyte,secondary spermatocyte and spermatid.Compared with the 2h radiation group,the expression of ?H2AX protein in the testis of the 2h medication group was significantly increased(P < 0.05).Compared with the 4h radiation group,the expression of ?H2AX protein in the testis of the 4h medication group was significantly increased(P < 0.05).8 ?H2AX protein expression of rats testicular tissue in each group by Western blotCompared with that in the testis of normal group's rats,the expression of?H2AX protein in testis tissue of rats in each group was significantly reduced(P < 0.01);Compared with that in the testis of 2h radiation group,the expression of ?H2AX protein in testicular tissue of 4h radiation group decreased significantly(P < 0.01).Compared with that in the testis of the 2h radiation group,the expression of ?H2AX protein in the testis of 2h medication group was increased significantly(P < 0.01).Compared with that in the testis of the 4h radiation group,the expression of ?H2AX protein in the testis of the 4h medication group was increased significantly(P < 0.01).Conclusions:1.There was no significant abnormal testicular tissue morphological changes of rats induced by 900 MHz mobile phone frequency electromagneticradiation,but there was significant ultrastructural injury,aggravated oxidative damage of rats testis,decreased antioxidant activity and lower protein expression of Nrf2 and ?H2AX in the testis.The injury situation of 4 hours' radiation was worse than that in 2 hours' situation,which suggesting there was a time effect relationship.2.The Liuweidihuang Pills can improve the morphology of rat testis and the damage of ultrastructure,and its mechanism may be associated with the increase of Nrf2 protein expression in testicular tissue,decrease of oxidative damage,increase of the expression of ?H2AX protein,the repair of DNA damage.The specific mechanism still remains to be further studied. |
PDF Full Text Request