The Role Of Apoptosis And Autophagy Of Pancreatic Islet Cells In Neonatal Remodeling And Its Mechanism

Objective: 1.To observe the changes of the apoptosis of pancreatic islet cells in neonatal mice.2.To observe the changes of the autophagy of pancreatic islet cells during the neonatal period in mice.3.To observe the expression of the apoptosis and autophagy related genes and explore the related mechanism.Methods: 1.Mice islets at week 1,3 and 8 after birth were isolated by collagenase.2.The apoptotic rate was detected with Annexin V-FITC/PI double staining by flow cytometry.3.The morphological structures of apoptotic cells and autophagosomes were observed by electron microscope.4.The expression of Cleaved Caspase-3,Bcl-2 and p22 phox were detected by immunohistochemistry.5.The apoptotic rate of islet ? cells was detected by immunofluorescence with the terminal deoxynucleotidyl transferase-mediated d UTP-X 3'nick end-labeling?TUNEL?/Insulin double staining.Double immunofluorescence staining was performed for insulin with Fas,Fas L or LC3.6.The expression of apoptosis and autophagy related genes were detected by real-time quantitative PCR.7.The expression levels of the apoptosis and autophagy related proteins were detected by Western blot.8.The expression level of malondiadehyde?MDA?of islet cells and hydrogen peroxide?H₂O₂?-stimulated INS-1 cells were detected using lipid oxidation kit.9.The cell viability of INS-1 cells stimulated by hydrogen peroxide with different concentrations was detected by the cell counting kit-8?CCK8?.10.INS-1 cells were treated with the inhibitor of p53 to downregulate the expression of p53.Western blot was used to detect the expression of the targeted proteins.The rate of apoptosis of INS-1 cells stimulated by hydrogen peroxide with different concentrations was detected by flow cytometry.Results: 1.1)The apoptotic rate of islet cells increased significantly at 3 w,which was markedly higher than that at l w and 8 w?P<0.01?.Early uhrastructural changes of apoptosis appeared at 3 w.Tunel+/Insulin+ cells increased markedly at 3 w compared with that of 1 wand 8 w?P<0.01?.2)The mRNA expression of the apoptosis-related genes dynamically changed.Among them,Fas and Fas Lwere higher than that at 1 w and 8 w?P<0.01?,while Bcl-2 was lower than that at 1 w and 8 w?P<0.01?.3)Western blot showed that compared with 1 w and 8 w,the protein levels of Fas,Fas L and Cleaved Caspase-3 were significantly increased at 3 w?P<0.01?,while Bcl-2 protein was markedly decreased at 3 w?P<0.01?.Immunohistochemistry showed that the expression of Cleaved Caspase-3 was higher at 3 w than that at 1 w and 8 w,while the Bcl-2 was the opposite.The cytoplasmic fluorescence intensity of Fas or Fas L at 3 w was significantly higher compared with that at 1 w and 8 w?P<0.01?.2.1)The typical doubled-membrane autophagosome appeared at 3 w.Compared with 1 w and 8 w,the punctated staining of LC3 in ? cells at 3 w was significantly increased.2)The mRNA level of p53 was higher at 3 w than that at 1 w and 8 w?P<0.05?,while mTOR was lower than that at 1 w and 8 w?P<0.01?.The mRNA expression of the autophagy-related genes dynamically changed.Compared with 1 w and 8 w,the mRNA levels of ULK1,Becn1 and Map1lc3 b markedly increased at 3 w?P<0.05?.Compared with 1 w,the levels of pik3c3,Atg5,Map1lc3 a and Atg16l1 inceased at 3 w?P<0.05?;the mRNA level of Atg4 d was significantly increased at 3 w and 8 w?P<0.05?.At 3 w,the mRNA expression of Atg4 c was higher than that at 8 w?P<0.05?.The mRNA levels of Atg4 a,Atg4b,Atg9 a,Atg9b,Atg12 and Atg16l2 were also increased at 3 w.3)Western blot showed that compared with 1 w and 8 w,the protein levels of p53, p AMPK,Beclin1 and LC3-II were markedly increased at 3 w?P<0.01?,while mTOR protein significantly decreased at 3 w?P<0.01?.3.1)Compared with 1 w and 8 w,the expression of p22 phox and MDA at 3 w was significantly increased?P<0.01?.H₂O₂ injured INS-1 cells in a dose-and time-dependent manner.The MDA level of INS-1 cells after H₂O₂ stimulation was markedly increased in a dose dependent manner?P<0.01?.2)The protein levels of LC3-II and p53 of INS-1 cells with 2 h and 5 h of H₂O₂ stimulation with different concentrations respectively were both increased gradually from 20 u M/2 h to 80 u M/5 h after 48 h.The expression of LC3-II was significantly increased but there was no significant change in the apoptosis after 48 h treatment with 80 u M,160 u M and 320 u M H₂O₂ for 5 h?P<0.05?.The level of LC3-II increased after 72 h with 80 u M and 160 u M H₂O₂ for 5 h?P<0.05?,but significantly decreased in the 320 u M H₂O₂-treated group.Compared with the control group,the rate of apoptosis increased in the 160 u M and 320 u M H₂O₂-treated groups after 72 h?P<0.01?.3)With the inhibitor of p53 to block the signaling pathway,the protein expression of p53,p AMPK,Beclin1,LC3 were significantly decreased but the expression of mTOR increased in H₂O₂-stimulated INS-1 cells after 48 h?P<0.05?;the protein levels of Fas and Fas L were both markedly decreased?P<0.05?accompanied with the decreased in cell apoptosis?P<0.01?after 72 h.Conclusions: 1.Neonatal mice islet cell apoptosis increased significantly at week 3,which is closely related to cell remodeling.The Fas/Fas L and Bcl-2 mediated apoptosis signaling pathways may be involved in the regulation of apoptosis in neonatal mice islet cells.2.Autophagy appeared in mice islet cells at week 3,which may participant in cell remodeling.The p53/p AMPK/mTOR/Beclin1 mediated autophagy signaling pathway may be involved in the regulation of autophagy in mice islet cells during the neonatal period.3.The level of oxidative stress increased in mice islet cells at week 3 during theneonatal period.The autophagy and apoptosis of INS-1 cells were induced in turn by the oxidative stress caused by H₂O₂ stimulation,suggesting that the oxidative stress may be the upstream activation signal to modulate the apoptosis and autophagy in which p53 might be play a crucial role.