We examined in duction of autophagy after CLP in various organs. Liver, spleen, heart, Tofacitinib JAK mesenteric lymph nodes, and kidney were isolated at 1, 3, 6, 12, or 24 h after CLP and auto phagosome formation in these organs evaluated by west ern blotting. In the sham operated mice, the LC3 II LC3 I ratio slightly increased over the time course following surgery and declined by 24 h after surgery. In the liver, a significant increase in LC3 II LC3 I ratio was observed at 6 h after CLP, and the ratio returned to basal levels by 24 h. The same tendency was observed in the heart and spleen. In contrast, minimal or no change was seen in the ratio in the mesenteric lymph node and kidney over the time course following CLP.
Since the liver is one of the critical organs in sepsis and induction of autophagy was greatest in the liver in this study, based on LC3 II LC3 I ratios, we focused on the liver in subsequent Inhibitors,Modulators,Libraries analyses. To further investigate LC3 induction, we examined LC3 mRNA expression Inhibitors,Modulators,Libraries in the liver. The expression significantly increased 1 and 3 h after CLP compared to the sham group, Inhibitors,Modulators,Libraries indicating that the total amount of LC3 protein in the liver increased at a transcriptional level and was then converted to LC3 II post transcriptionally. Autophago some formation was also examined using GFP LC3 transgenic mice. In these mice, autophagosomes are vi sualized as cytoplasmic GFP LC3 dots by confocal mi croscopy. In agreement with the western blotting data, CLP induced an increase in GFP LC3 dots. the number peaked at 6 h and then decreased by 24 h in the liver.
No sig nificant increase in GFP LC3 dots was observed in the sham operated group. Completion of autophagy induction in the liver after CLP An increase in Inhibitors,Modulators,Libraries autophagosome numbers does not ne cessarily infer completion of the autophagy Inhibitors,Modulators,Libraries process. The autophagosome fuses with a lysosome to form an autolysosome. Blockade of autophagy at this step would also result in an increased number of autophagosomes. In order to distinguish these possibilities, fusion of autopha gosomes with lysosomes was examined by immunofluo rescence. Co localization of GFP LC3 dots and signals for LAMP1, a lysosomal marker, was evaluated in the liver after CLP. As shown in Figure 2A, increased co loca lization of LAMP1 and GFP LC3 was observed in the CLP group compared with the sham operated group at both 6 h and 24 h. At 6 h after CLP, 25.
4% of GFP LC3 dots were co localized with LAMP1 signals, and this percentage in creased to 58. 8% by 24 h selleck chemicals Calcitriol after CLP. To evalu ate autophagy flux, the amount of p62 protein was examined. As shown in Figure 2C, no significant difference was observed between the sham and CLP groups at either 6 or 24 h after the operation. However p62 protein signifi cantly increased at 24 h compared to that at 6 h in CLP group.