| Objective 1.To study the effects of aging on testicular Sertoli cell injury and autophagy in rats;2.To investigate the effects of icariin on testicular Sertoli cell injury and autophagy in aging rats;3.To investigate the regulatory effects and mechanisms of icariin on mouse testicular Sertoli cell line TM4 injury in vitro.Methods 1.SD male rats of SPF grade at 6,12,18 and 24 months old,10 rats in each group,were weighed and sacrificed after intraperitoneal injection of 20% urethane at a dose of 5 m L/kg.The following indexes were examed:(1)The morphological changes of rat testes was observed using HE staining;(2)The numbers of Sertoli cells in testes were assessed by labing Sertoli cell marker SOX9 using immunofluorescence;(3)The expression of autophagy-related proteins ATG5 and LC3 in testicular Sertoli cells of rats during aging were detected by double-labeled immunofluorescence;(4)The changes of autophagy-related ultrastructure in testicular Sertoli cells of rats during aging were observed by transmission electron microscopy.2.16-month-old male SD rats were randomly divided into three groups: aging model group,icariin low and high dose group,and 2-month-old male rats were purchased as adult control group,10 rats in each group.The low and high dose groups of icariin were fed with 2 mg/kg and 6 mg/kg medicated feed respectively,while the aging model group and the adult control group were fed with common maintenance feed.The rats in the above groups were kept in the same environment for 4 months.After fasting for 12 h,all the rats were sacrificed,and the following indexes were observed:(1)The testes and epididymides were weighed and their indexes were calculated;(2)Sperm count and viability were tested;(3)The testosterone(T)level of testicular tissue was detected by ELISA;(4)The testicular histomorphology of rats in each group was observed by HE staining;(5)The numbers of Sertoli cells in testes were assessed by labing Sertoli cell marker SOX9 using immunofluorescence;(6)The expression level of related cytokines(GDNF,SCF,BMP4 and PLZF)secreted by Sertoli cells in testes of rats in each group were detected by western blot;(7)The expression of ATG5 and LC3 in testicular Sertoli cells of rats in each group were detected by double immunofluorescence;(8)The ultrastructure of autophagy in testicular Sertoli cells of rats in each group were observed by transmission electron microscopy.3.1 TM4 cells were stimulated with chloroquine(CQ)at different concentrations(0,12.5,25 and 50 μM)for24 h,and the following indexes were detected:(1)The effects of different concentrationsof CQ on the morphology of TM4 cells were observed under light microscope;(2)The effects of CQ on the apoptosis of TM4 cells were detected by Hoechst33342 staining;(3)The expression of LC3 and P62 in TM4 cells stimulated by CQ were detected by western blot;(4)The ultrastructure changes of autophagy in TM4 cells were observed by transmission electron microscopy.3.2 TM4 cells were divided into control group,CQ group,icariin+CQ group and icariin group.After TM4 cells reached adherent growth state,icariin+CQ group and icariin group TM4 cells were pretreated with icariin(final concentration of 0.5 μM)for 24 h,then CQ group and icariin+CQ group were added with CQ(final concentration of 50 μM)for 24 h.The following indexes were detected:(1)The effects of icariin on the morphology of TM4 cells were observed under light microscope;(2)The effects of icariin on apoptosis of TM4 cells were detected by Hoechst33342staining;(3)The effects of icariin on the expression levels of LC3 and P62 protein in TM4 cells were detected by western blot;(4)The effects of icariin on autophagy related ultrastructure in TM4 cells were observed by transmission electron microscopy.Results 1.(1)HE staining showed that the testes of 6 and 12 months old rats had good morphology.With the increase of age,the testes of rats gradually appeared pathological changes.The testicular seminiferous tubules of 18 month old rats began to atrophy,and the testicular seminiferous tubules of 24 month old rats were severely atrophic,accompanied by the exfoliation of spermatogenic cells and the enlargement of intercellular space.The statistical results showed that the testicular seminiferous tubules diameter and the thickness of seminiferous epithelium were maintained at a high level at 6 month old,decreased from12 month old,and further decreased at 18 and 24 month old;(2)Immunofluorescence results showed Sertoli cell numbers in testes of 6 and 12 month old rats had no significantly change,but the number of Sertoli cells in testes of 18-month-old and24-month-old rats were significantly decreased;(3)The results of immunofluorescence showed that the expressions of ATG5 and LC3 in testicular Sertoli cells of rats were decreased with the increase of age;(4)Transmission electron microscopy results showed that more autophagic vesicles in the cytoplasm of Sertoli cells in 6-month-old rats were observed,and the organelles were rich and well-formed.The numbers of autophagic vesicles in Sertoli cells in 12-and 18-month-old rats were slightly reduced.However,increased endoplasmic reticulum swelling,and mitochondrial disorder were found in the cytoplasm.Autophagic vesicles in the cytoplasm of Sertoli cells in 24 month-old rats were rare.And there were a lot of vacuoles in the cytoplasm,and organelles such as endoplasmicreticulum and mitochondria were seriously damaged.2.(1)Icariin significantly increased the testicular weight,testicular index,epididymal weight and epididymal index of natural aging rats;(2)Icariin significantly increased the sperm count and viability of epididymides in aging rats;(3)The results of ELISA showed that icariin significantly increased the level of T in testes of aging rats;(4)HE results showed that icariin significantly improved the morphological changes of testicular tissues in naturally aging rats,increased the diameter of seminiferous tubules and the thickness of seminiferous epithelium;(5)The results of immunofluorescence showed that icariin could significantly increase the number of Sertoli cells in testes of aging rats;(6)Western blot showed that icariin significantly increased the expression of related cytokines GDNF,PLZF,SCF and BMP4 secreted by testicular Sertoli cells in naturally ageing rats;(7)The results of immunofluorescence showed that icariin increased the expression of ATG5 and LC3 in testicular Sertoli cells of ageing rats;(8)The results of transmission electron microscopy showed that icariin improved the ultrastructural changes related to autophagy of testicular Sertoli cells,increased the number of autophagic vesicles in Sertoli cells,and decreased the number of damaged mitochondria and endoplasmic reticulum.3.1(1)The results of light microscopy showed that TM4 cells in the control group were long fusiform,and the morphology of TM4 cells had no change after 12.5 μM CQ stimulation for 24 h;with the increase of CQ stimulation concentration,the morphology of TM4 cells changed significantly after CQ stimulation of 25 μM and 50μM,the numbers of round or elliptical cells with abnormal morphology were increased;(2)The results of Hoechst33342 staining showed that the nucleus of TM4 cells in the control group was round and light blue after Hoechst staining;after 12.5 μM CQ treatment,the changes of Hoechst staining were not obvious;after 25 μM and 50 μM CQ treatment,the chromatin concentration and nuclear pyknosis were observed under Hoechst staining microscope,and the number of TM4 cells with bright blue deep staining was gradually increased,indicating that the apoptosis of TM4 cells was gradually increased;(3)The results of western blot showed that 12.5 μM CQ could increase the expression levels of LC3Ⅱ and P62 protein in TM4 cells;with the increase of CQ concentration,the expression levels of LC3Ⅱ and P62 protein in TM4 cells were gradually increased with significant differences;(4)Transmission electron microscopy showed that 12.5 μM of CQ had no obvious effect on the number of autophagosomes and autolysosomes in TM4 cells;with the increase of CQ concentration,25 μM of CQ could cause the number of autophagosomes and autolysosomes in TM4 cells to increase,and the number of damagedorganelles in the cytoplasm were gradually increased;50 μM CQ could lead to a further increase in the number of autophagosomes and autophagolysosomes in TM4 cells,accumulation of materials to be digested in autophagy vesicles,and further increase of damaged organelles in the cytoplasm.3.2(1)Light microscopy results showed that compared with the control group,icariin had no obvious effect on the morphology of normal TM4 cells;compared with the CQ group,icariin could reduce the influence of CQ on the morphology of TM4 cells,and reduce the number of round or oval cells with abnormal morphology;(2)Hoechst33342 staining results showed that icariin could reduce the apoptosis of TM4 cells induced by CQ,but had no effect on normal TM4 cells;(3)Western blot results showed that compared with the control group,the expression levels of LC3Ⅱ and P62 protein in TM4 cells of CQ group were significantly increased,while the expression levels of LC3Ⅱ and P62 in the Icariin group were not significantly changed;Compared with the CQ group,the expression levels of LC3Ⅱ and P62 proteins in TM4 cells in the Icariin+CQ group were significantly reduced;(4)Transmission electron microscopy results showed that compared with the control group,TM4 cells in the CQ group had more autophagosomes in the cytoplasm,a large number of aggregated autophagolysosomes in the cytoplasm,and increased damaged organelles in the cytoplasm.Icariin did not cause autophagosomes and autolysosome accumulation in the cytoplasm of normal TM4 cells.Compared with CQ group,the accumulation of autophagosomes and autolysosomes in TM4 cells in Icariin+CQ group was reduced.Conclusion 1.Aging can cause testicular Sertoli cells injury and autophagy inhibition.2.Icariin improves the decline of testicular function with aging,and its mechanism may be related to upregulation of Sertoli cell autophagy and inhibition of Sertoli cell injury.3.Inhibition of autophagy can cause Sertoli cell injury,while icariin can reduce Sertoli cell injury by activating autophagy. |
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