CD5L-induced activation of autophagy is associated with hepatoprotection in ischemic reperfusion injury via the CD36/ATG7 axis

Abstract

Hepatic ischemia/reperfusion (I/R) injury is a side effect of major liver surgery that is difficult to prevent. I/R injury induces metabolic strain on hepatocytes and limits the tolerable ischemia during liver resection, as well as preservation times during transplantation. Additionally, I/R injury induces apoptosis in hepatocytes. CD5-like (CD5L), an inducer of autophagy, is a soluble scavenger cysteine-rich protein that modulates hepatocyte apoptosis. The aim of the present study was to determine if pharmacologic CD5L was protective against hepatic ischemia-reperfusion injury. Hepatocytes were subjected to I/R culture conditions, and apoptosis and caspase family activity were measured after I/R to model hepatic injury. Treatment with recombinant CD5L significantly suppressed apoptosis and caspase activity through modulating cellular autophagy to maintain activation of the cluster of differentiation 36 (CD36)-dependent autophagy-related 7 (ATG7) signaling pathway. The regulation loop between CD5L and the autophagy signaling pathway was identified to be associated with the inhibition of oxidative stress. Treatment with CD5L significantly inhibited cellular oxidative stress, which was confirmed by silencing the CD36 receptor or the autophagy related protein ATG7 using small interfering RNA, which reversed the antiapoptotic and antioxidative effects of CD5L on hepatocytes under I/R conditions. The results of the present study suggested that CD5L-mediated attenuation of hepatic I/R injury occurs through the CD36-dependent ATG7 pathway, accompanied by the inhibition of oxidative stress, which is associated with enhanced autophagy. In conclusion, the present study identifies CD5L as a novel therapeutic agent for hepatic I/R injury.

Keywords: CD5L; apoptosis; autophagy; hepatic ischemia/reperfusion injury; oxidative stress.

Figure 1.

Recombinant CD5L inhibits I/R-induced hepatocyte apoptosis. (A) Representative images of Annexin V/PI flow cytometry analysis of apoptotic I/R injury model cells treated with CD5L. (B) Quantified flow cytometry results demonstrated that CD5L significantly reversed the apoptotic rate in I/R injury model cells. (C) Caspase 3/7 and (D) caspase 8 activity levels in hepatocyte cell lysates were measured using ELISA. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. control; ^○P<0.05 vs. I/R. I/R, ischemia/reperfusion; CD5L, CD5-like; PI, propidium iodide.

Figure 2.

Inhibition of hepatocyte apoptosis by CD5L occurs via the CD36 receptor. (A-E) Hepatocytes were transfected with siRNA targeting CD36 or with siRNA-NT as a control. (A) Transfection efficiency was determined by reverse transcription-quantitative PCR. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. siRNA-CD36. (B) Representative flow cytometric dot plots of apoptotic cells following Annexin V/propidium iodide staining and (C) quantification of apoptosis. (D) Caspase 3/7 and (E) caspase 8 activity levels in hepatocyte cell lysates were measured using ELISA. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. control; ^▲P<0.05 vs. I/R; ^○P<0.05 vs. I/R + siRNA-CD36. (F-H) Hepatocytes were transfected with siRNA against the CD36 transcript or with siRNA-NT as a control and incubated under I/R conditions. (F) Apoptosis was analyzed by flow cytometry. (G) Caspase 3/7 or (H) caspase 8 activity from hepatocyte cell lysates were measured using ELISA. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. control. I/R, ischemia/reperfusion; CD5L, CD5-like; CD36, cluster of differentiation 36; siRNA, small interfering RNA; NT, non-targeting; PI, propidium iodide.

Figure 3.

CD5L activates the cellular autophagy process. (A-D) Hepatocytes were transfected with siRNA-CD36 or siRNA-NT, incubated under I/R conditions and treated with CD5L. Cells without any treatment was used as control. (A and C) Representative western blots and quantification of the expression levels of LC3B-II and β-actin. (B and D) Representative western blots and quantification of the expression levels of ATG7 and β-actin. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. control; ^▲P<0.05 vs. I/R; ^○P<0.05 vs. I/R + CD5L + siRNA-CD36. (E-H) Hepatocytes were transfected with siRNA targeting CD36 or siRNA-NT as a control and incubated under I/R conditions. (E and G) Representative western blots and quantification of the expression levels of ATG7. (F and H) Representative western blots and quantification of the expression levels of β-actin. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. control. I/R, ischemia/reperfusion; CD5L, CD5-like; CD36, cluster of differentiation 36; siRNA, small interfering RNA; NT, non-targeting.

Figure 4.

CD5L serves a cytoprotective function by modulating autophagy. (A-E) To determine the role of autophagy in the anti-apoptotic actions of CD5L, hepatocytes were transfected with siRNA against ATG7 or siRNA-NT. (A) The siRNA-mediated transfection efficiency was demonstrated by reverse transcription-quantitative PCR. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. siRNA-ATG7. (B-E) Hepatocytes were incubated under I/R conditions and treated with CD5L. (B) Representative flow cytometric dot plots of apoptotic cells following Annexin V/propidium iodide staining and (C) quantification of apoptosis. (D) Caspase 3/7 and (E) caspase 8 activity levels in cell lysates were measured using ELISA. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. control; ^▲P<0.05 vs. I/R; ^○P<0.05 vs. I/R + CD5L + siRNA-ATG7. (F-H) Hepatocytes were transfected with siRNA targeting ATG7 or siRNA-NT as a control and incubated under I/R conditions. (F) Apoptosis was analyzed by flow cytometry. (G) Caspase 3/7 and (H) caspase 8 activity levels in hepatocyte cell lysates were measured using ELISA. Each column represents the mean ± SD of three independent experiments. ^*P<0.05 vs. control. I/R, ischemia/reperfusion; CD5L, CD5-like; ATG7, autophagy-related 7; siRNA, small interfering RNA; NT, non-targeting.

Figure 5.

CD36 and ATG7 reverse the antioxidant effect of CD5L on hepatocytes. (A-D) Hepatocytes transfected with siRNA-CD36, siRNA-ATG7 or siRNA-NT and cultured under I/R conditions with or without CD5L. Hepatocytes without any treatment were used as controls. (A) Intracellular ROS production was analyzed by fluorescence spectrophotometry. Commercial kits were used to determine the levels of (B) SOD, (C) CAT and (D) GSH-Px activities in hepatocytes. Data represent the mean ± SD from three independent experiments. ^*P<0.05 vs. control; ^▲P<0.05 vs. I/R + CD5L; ^○P<0.05 vs. I/R + CD5L + siRNA-NT. DFS, dual fluorescent staining; I/R, ischemia/reperfusion; CD5L, CD5-like; CD36, cluster of differentiation 36; ATG7, autophagy-related 7; siRNA, small interfering RNA; NT, non-targeting; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; GHS-Px, glutathione peroxidase.