| It is undeniable that the aging of society is becoming ever more serious.Practical problems such as survival pressure,environmental pollution and food safety can harmfully affect health,so that the symptoms of aging come early,and the incidence of aging-related diseases increases accordingly.Aging is accompanied by an increase in reactive oxygen species(ROS)and a decrease in antioxidants,which can reduce oxidative damage by scavenging free radicals.Cerium oxide nanoparticles(CeO2NPs)has strong cell permeability,good biocompatibility,and the rapid reversible transformation between Ce4+/Ce3+on the crystal surface makes it have renewable SOD and CAT mimetic activities.It has been reported that CeO2NPs can slow down the aging process and alleviate oxidative damage of age-related diseases,but still needs more detailed studies.In this study,D-Gal-induced TM3/TM4 cells and mice were used as models to evaluate the protective effect of CeO2NPs on testicular aging,the results will provide experimental basis for the biological application of CeO2NPs.1.Effect of CeO2NPs on aging model of TM3/TM4 cells induced by D-galactoseTM3/TM4 cells were treated with different concentrations of D-Gal for 24h,48h and 72h respectively to screen the optimal treatment concentration and treatment time.TM3/TM4 cells were randomly divided into four groups:?Control group(Control):cells were cultured in common medium for 72h;? CeO2NPs control group(CeO2NPs):cells were cultured in medium with CeO2NPs(10?g/mL)for 72 hours;?Aging model group(D-Gal):cells were cultured in medium with D-Gal(150mM)for 72h;? CeO2NPs intervention group(D-Gal+CeO2NPs):cells were cultured in medium with D-Gal(150 mM)and CeO2NPs(10?g/mL)for 72h.At the end of experiment,cell viability of each group was detected by CCK-8;cell cycle and apoptosis rate were detected by FACS;the positive rate of senescent cells was detected by ?-galactosidase staining;the cell mitochondrial membrane potential and cell ROS level were measured by JC-1 and DCFH-DA probe method respectively;the testosterone secretion in TM3 cells were detected by ELISA;the contents of GSH,MDA and the activity of SOD were detected by colorimetric assay.In addition,the transcriptional level of proteins was estimated by qPCR,including testosterone synthesis-related proteins(StAR,P450scc),senescence-related proteins(P16,P21,P53),autophagy-related proteins(Beclin-1,P62,mTOR,LC3)and proteins secreted by TM4 cells(GDNF,BMP4,SCF).The protein level of P16,P21,P53,Beclin-1,P62,mTOR,LC3 were detected by Western blot.The main results were as follows:(1).Successfully established D-Gal-induced TM3/TM4 aging cell model.Compared with the control,when TM3 cells were treated by D-Gal with 150mM for 72h,the cell proliferation rate was significantly inhibited(P<0.01),SA-?-Gal activity of cells was significantly increased(P<0.01),the expression of senescence-related proteins was significantly up-regulated(P<0.01)while the cell cycle was arrested.Similar results were obtained for TM4 cells.(2).CeO2NPs ameliorated the aging phenotype of TM3/TM4 model cells induced by D-Gal.After treated with CeO2NPs,cell morphology returned to normal,almost the same as the control;ROS content(P<0.01),apoptosis rate(P<0.01)and SA-?-gal activity(P<0.05)were down-regulated;mitochondrial membrane potential(P<0.05),antioxidant level(P<0.05),autophagy level(P<0.05),transcriptional level of testosterone synthesis-associated proteins(P<0.05)and testosterone secretion level of TM3 cells(P<0.05)were increased respectively.At the same time,CeO2NPs reversed cell cycle arrest induced by D-Gal at G0/G1 phase in TM3 cells and at G2/M phase in TM4 cells(P<0.05).Additionally,the transcriptional level of GDNF,BMP4 and SCF secreted by TM4 cells was up-regulated by CeO2NPs compared with aging model group(P<0.05).2.Protective effects of CeO2NPs on testicular degeneration in aging mice induced by D-galactoseForty BALB/c male mice were randomly divided into four groups with 10 mice in each:?Control group(Control):mice were injected with normal saline subcutaneously every day for 56 days;? CeO2NPs control group(CeO2NPs):mice were injected with normal saline subcutaneously every day for 56 days,and from the 16th day,CeO2NPs(10?g/kg)were injected into tail vein,twice a week for two weeks;? Aging model group(D-Gal):mice were subcutaneously injected with D-Gal solution(200mg/kg/d)for 56 days in order to establish the aging animal models;?CeO2NPs intervention group(D-Gal+CeO2NPs):mice were subcutaneously injected with D-Gal solution(200 mg/kg/d)for 56 days,and from the 16th day,CeO2NPs(10?g/kg)were injected into tail vein,twice a week for two weeks.During the modeling period,the dynamic changes of biological characteristics of treated mice were observed.At the end of experiment,mice were sacrificed and weighed;epididymis was taken for sperm quality analysis,blood for serum testosterone analysis.Testicular tissue was sliced for ?-galactosidase staining and morphological examination;tissue homogenate was used to evaluate the contents of GSH,MDA and the activity of SOD.The transcriptional level of proteins was determined by qPCR,including StAR,P450scc,GDNF,BMP4 and SCF.The expressions of senescence-related proteins(P16,P21,P53)and autophagy-related proteins(Beclin-1,P62,m-TOR,LC3)in testis were measured by Western blot.The results showed that CeO2NPs significantly reduced the activity of SA-?-gal in testis(P<0.05).Compared with aging model group,the activities of SOD and GSHPx were significantly increased(P<0.05),while the content of MDA was significantly decreased(P<0.05);epididymis sperm quality was significantly improved(P<0.05).The transcriptional level of testosterone synthesis-related proteins(StAR,P450scc)and cytokines(GDNF,BMP4,SCF)were significantly increased(P<0.05);The expression of senescence-related proteins in testis of mice decreased(P<0.05)and the expression of autophagy-related proteins increased significantly(P<0.01).The results showed that CeO2NPs could protect the structure and function of testis from damage induced by D-Gal.In summary,CeO2NPs can improve the antioxidant capacity and autophagy level of testicular cells by scavenging ROS,promote the proliferation and self-renewal of testicular cells,and maintain the homeostasis of testicular cells,thus inhibiting the senescence of testicular cells and decelerating the degeneration of testicular structure and function. |
PDF Full Text Request