Experimental Medicine
Study of autophagy and expression of LC3/P62 in hepatocytes of septic mice
Meijia Shen, Lichao Sun, Guoqiang Zhang
Published 2019-04-10
Cite as Chin J Emerg Med, 2019, 28(4): 467-472. DOI: 10.3760/cma.j.issn.1671-0282.2019.04.014
Abstract
ObjectiveTo investigate the occurrence of hepatocyte autophagy and the expression of autophagy associated proteins at different time points in sepsis mice model established by cecal ligation and puncture.
MethodsFifty male ICR mice aged 6-8 weeks were randomly (random number) divided into 5 groups: CLP 3 h group, CLP 6 h group, CLP 12 h group, CLP 24 h group and control group. The samples were collected at the corresponding time points after operation. Liver function was tested to obtain alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The expression of TNF-α and IL-6 was detected by ELISA, the inflammation of liver tissue by HE staining while the ultrastructure of autophagosome and autolysosome in liver tissue was observed by electron microscopy. Meanwhile, the expression of LC3 and P62 was detected by Western blot. and the location and quantity of autophagosome was observed by Immunofluorescence. SPSS 21.0 was used for statistical analysis. Two independent sample t tests were performed to compare the two groups, and one-way ANOVA was used for comparison between multiple groups.
ResultsALT and AST increased significantly after CLP, and peaked at 12 h, respectively (137.8 ±11.94) U/L and (475.3 ±57.34) U/L. The expression of IL-6 peaked at 6 h (2589.63 ±27.96) pg/mL, and TNF-α peaked at 3 h (320.21±8.9) pg/mL. HE staining showed activation of Kupffer cells and infiltration of single granulocyte. Electron microscope showed the formation of autophagosome and autolysosome. Compared with the control group, the ratio of LC3 II/I increased first and then decreased, peaked at 6 h (t=13.35, P<0.05). The content of P62 protein declined first and then went up, reaching the lowest level at 6 h (t=66.11, P<0.05), contrary to the trend of LC3 II/I. Immunofluorescence results illustrated that the fluorescence intensity of LC3 reached its high point at 6 h (t=12.52, P<0.05).
ConclusionsAutophagy occurred in the liver of sepsis mice model established by cecal ligation and puncture. The formation of autophagosome first increased and then decreased, reaching its peak at 6 h.
Key words:
Sepsis; Cecal ligation and puncture; Liver injury; Autophagy; LC3; P62; Inflammatory factor; Mice
Contributor Information
Meijia Shen
Emergency Department China-Japan Friendship Hospita, Beijing 100029 China
Lichao Sun
Guoqiang Zhang
