Caffeine Inhibits The Development Of Fetal Thymus In Mice By Inducing Cell Autophagy

Background:Epidemiological researches show that adverse factors(such as drinking,drugs,)during pregnancy can result in the dysplasia of embryonic organs,and increase the susceptibility of cardiovascular,immune and metabolic diseases in offspring.Researches pointed out that caffeine has the embryo toxicity.Prenatal caffeine exposure can inhibit the development of fetal thymus and result in a decreased output of the naive T cell in thymus,and also increase the susceptibility to the immune disease,such as asthma and Type 1 Diabetes Mellitus.Therefore,the molecular mechanisms of the effects of prenatal caffeine exposure on fetal thymocytes needs to be further studied.Caffeine can induce autophagy in hepatic stellate cells,skeletal muscle cells and other cells.And it has been reported that autophagy is closely related to the development of thymocytes.Therefore,we will explore the effects of prenatal caffeine exposure on the development of fetal thymocytes and clarify its molecular mechanisms from the point of the view of cell autophagy in this study.Objects:This study was designed to observe the effects of prenatal caffeine exposure on the impaired development of fetal thymocytes,and investigate the roles of autophagy in the development of fetal thymocytes.Further the molecular mechanisms of autophagy by caffeine treatment was elucidated.Methods:(1)The influences of prenatal caffeine exposure on fetal thymocytes was observed in vivo:Pregnant Balb/c mice were divided into a caffeine group and a control group.From gestational Day(GD9),the pregnant mice in caffeine group were subcutaneously administered 96 mg/kg/d of caffeine twice per day,and the control group were given equal volume of saline subcutaneously.The pregnant mice were sacrificed at GD18,fetal thymus were removed.The body and thymus were weighed,and the fetal thymus organ index was calculated.The autophagy of fetal thymocytes was observed through transmission electron microscope(TEM).Fetal thymocyte phenotypes were analyzed by flow cytometry.RT-PCR was performed to measure the mRNA expressions of genes regulating autophagy pathway(A2AR,PKA and A20)and autophagy pathway-related genes(Beclinl,Atg12 and Atg5).Western blot was performed to measure the expressions of p-PKA,Beclinl,LC3,P62 and Bcl10 in the cytoplasm.(2)Investigate the mechanisms of caffeine-induced autophagy in thymocytes in vitro:3 weeks old mice were sacrificed and prepared for primary thymocytes culture,which were divided into a control group and four caffeine groups at different concentrations(0,0.4,4,40 and 400 ?M).After 48 h of caffeine treatment,cell counting kit-8(CCK-8)was used for cell activity analysis.The autophagy related proteins was observed by Western blot,and the effects of caffeine on the cell phenotypes were detected by flow cytometry.According to the results of concentration-effects,4 ?M caffeine was selected to treat thymocytes 0,24,48 and 72 h.The changes of autophagy related proteins was observed by Western blot,According to the concentration-effects and time-effects of caffeine on thymocyte autophagy,4 ?M caffeine was selected as the does of caffeine and 48 h as the time of the administration to study the molecular mechanisms of autophagy.Firstly,to investigate whether caffeine affects thymocytes phenotypes by inducing cell autophagy,we used Bafilomycin A1(5 nM),which was an autophagy inhibitor,to treat cells with or without caffeine.Flow cytometry was used to detect the phenotypes of thymocytes.Western blot was used to detect the changes of both the autophagy relative proteins and Bc110 in thymocytes.The interasctions between autophagic marker P62 and Bcl10 were detected by Co-immunoprecipitation(Co-IP).RT-PCR was used to detect the mRNA expressions of A20.Secondly,to observe whether caffeine induce autophagy by A2AR,SCH 58261(25?M),a specific inhibitor of A2AR,was used to treat cells with or without caffeine.Then.Western blot was used to detect the expressions of autophagy marker proteins and p-PKA.Results:(1)In vivo:?Growth and development index:As compared with the control group,the weight of fetal body and the fetal thymus organ index of the fetus in caffeine group were significantly lower(P<0.05);?Fetal thymocyte phenotypes:As compared with the control group,the absolute cell numbers of DP and CD4SP were decreased(P<0.05).The absolute counts of DN and CD8SP had no significant changes beween the caffeine and the control group;? Autophagy of fetal thymocytes:The results of transmission electron microscopy showed that the thymus in caffeine group had more autophagosomes than the control group;RT-PCR results showed that the mRNA expressions of Beclin1 and Atg5 were significantly increased(P<0.05),compared with the controls.Western blot results showed that the expressions of LC3? and Beclinl were increased(P<0.05),and P62 were reduced in the caffeine group.?Changes of the autophagy singnaling pathway;In the caffeine group,the p-PKA in the fetal thymus was down-regulated 24%,while the mRNA expressions of Bcl10 and A20 were decreased compared with the control(P<0.05).(2)In vitro:?The results of cell viability:0.04 to 40 ?M caffeine reduced the viability of thymocytes in a dose-dependent manner(P<0.05),whereas 400 ?M caffeine had no significant effects on thymocyte viability compared to the control group;? Concentration-effects and time-effects of caffeine on autophagy of thymocyte:Flow cytometry analysis showed that 0.4-40 ?M caffeine significantly decreased CD4SP proportion(P<0.05),while there were no significant changes among DN,DP and CD8SP.Western blot results showed that 4 and 40 pM caffeine significantly increased the levels of autophagy related proteins.The results of time-effects of caffeine on autophagy showed that caffeine treatment for 24 h had few effects on autophagy of thymocytes,while with prolonged durations of caffeine treatment,the levels of autophagy was significantly increased(P<0.01)in a time-dependent manner at 48 and 72 h;?Caffeine-induced thymocytes phenotypic changes was mediated by increased cell autophagy:Compared with the control group,the percentages of CD4SP and CD8SP were significantly reduced after 4 ?M caffeine treatment for 48 h(P<0.05),and DN and DP were not significantly changed.Meanwhile,the levels of autophagy were increased(P<0.05),and the expressions of Bcl10 were decreased(P<0.05);Co-IP results showed that the complex of Bcl10 with P62 decreased by 32.64%.Compared with the caffeine group,the percentage of CD4SP increased(P<0.05)with the addition of Bafilomycin A1 and caffeine treatment simultaneously,the final process of autophagy was effectively blocked,which were reflected by the increased expressions of P62 and LC3?(P<0.05).The expressions of Bcl10 was also significantly increased as compared to the caffeine group(P<0.05),and Bcl10 binding to P62 increased by 41.47%.? Caffeine-induced thymocyte autophagy was mediated by A2ARs:Compared with the control group,Beclinl and LC3II were significantly increased(P<0.05),and the expressions of p-PKA was significantly decreased(P<0.05)with SCH 58261 treatment.The changes of Beclinl,LC3,and p-PKA in caffeine group were consistent with SCH 58261 and had significantly differences(P<0.05).Conclusion:Prenatal caffeine exposure could inhibit the A2AR/PKA signal pathway and promote the cell autophagy in the fetal thymocytes,which made Bcl10 binded with P62 was degraded by autophagy,and inhibited the transcriptions of A20.As a result,the differentiation of CD4SP was inhibited and the output of CD4SP was reduced.