A20 rescues hepatocytes from apoptosis through the NF-κB signaling pathway in rats with acute liver failure

Abstract

Background: Acute liver failure (ALF) is a disease of acute derangements in the hepatic synthetic function with defects involving innate immune responses, which was reported to be negatively regulated by tumor necrosis factor α-induced protein 3 (A20). Herein, the present study was conducted to investigate the effects the A20 protein on the proliferation and apoptosis of hepatocytes through the nuclear factor (NF)-κB signaling pathway in the rat models simulating ALF.Methods: Male Wistar rats were used to simulate ALF in the model rats. Next, the positive expression of A20 and Caspase-3 proteins was measured in liver tissues. Rat hepatocytes were separated and subjected to pyrrolidine dithiocarbamate (PDTC, inhibitor of NF-κB pathway) or A20 siRNA. Additionally, both mRNA and protein levels of A20, NF-κB, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and receptor-interacting protein 1 (RIP1) were determined. Finally, we detected the hepatocyte proliferation, cell cycle entry, and apoptosis.Results: ALF rats displayed a lower positive expression of A20 protein and a higher expression of Caspase-3 protein. Furthermore, A20 was down-regulated, while NF-κB, TRAF6, and RIP1 were all up-regulated in ALF rats. Notably, A20 inhibited activation of NF-κB signaling pathway. The blockade of NF-κB signaling pathway enhanced proliferation and cell cycle progression of hepatocytes, whereas inhibited apoptosis of hepatocytes. On the contrary, A20 siRNA reversed the above situation.Conclusion: A20 inhibits apoptosis of hepatocytes and promotes the proliferation through the NF-κB signaling pathway in ALF rats, potentially providing new insight into the treatment of ALF.

Keywords: A20; Acute liver failure; Hepatocyte; NF-κB signaling pathway; Proliferation; Tumor necrosis factor α-induced protein 3.

Figure 1. Survival analysis for rats within 48 h and expression of A20 at different time points

(A) Survival analysis within 48 h of normal rats (n=30) and ALF rats (n=30, induced by injection of D-GalN 800 mg/kg and LPS 10 μg/kg). (B) mRNA expression of A20 within 48 h in normal and ALF rats detected by RT-qPCR. (C) and (D) Western blot analysis for protein expression of A20 within 48 h in normal and ALF rats. The data were presented as a mean ± standard deviation, analyzed by one-way analysis of variance. *P<0.05, compared with the normal group.

Figure 2. HE staining, ultrastructure observation, and immunohistochemistry show that ALF rats display serious pathological injury, lower positive expression rate of A20 protein, and higher that of Caspase-3 protein

(A) Image of liver tissues in normal and ALF rats using HE staining (×200); (B) ultrastructure observation of hepatocytes in normal and ALF rats (×20000); (C) positive expression rate of A20 protein in normal and ALF rats using immunohistochemistry (×200); (D) positive expression rate of Caspase-3 protein in normal and ALF rats using immunohistochemistry (×200); measurement data were expressed as mean ± standard deviation; the data were assessed by t-test; n=6. ***, P<0.001; ****, P<0.0001.

Figure 3. RT-qPCR and Western blot analysis show that A20 is down-regulated, while NF-κB, TRAF6, and RIP1 are up-regulated in ALF rats

(A) mRNA expressions of A20, NF-κB, TRAF6, and RIP1 in liver tissues detected by RT-qPCR; (B) protein levels of A20, NF-κB, TRAF6, and RIP1 in liver tissues detected by Western blot analysis; (C) protein band patterns of A20, NF-κB, TRAF6, and RIP1 in liver tissues detected by Western blot analysis; *P<0.05, ***, P<0.001; ****, P<0.0001 compared with the normal group; measurement data were expressed as mean ± standard deviation; the data were assessed by t-test; n=6.

Figure 4. CCK-8 assay shows that A20 promotes the proliferation of hepatocytes in ALF rats

*P<0.05, compared with the control group; ^#P<0.05, compared with the blank and NC groups; measurement data were expressed as mean ± standard deviation; the data at different time points were assessed by repeated-measures analysis of variance. The experiment was conducted three times independently.

Figure 5. Flow cytometry shows that A20 promotes cell cycle progression and inhibits apoptosis of hepatocytes in ALF rats

(A) Cell cycle entry in each group determined by PI staining of flow cytometry; (B) cell proportion in different stages in each group; (C) apoptosis of hepatocytes in each group detected by Annexin V-FITC/PI double staining of flow cytometry; (D) apoptotic rate of rats in each group; *P< 0.05, compared with the control group; ^#P<0.05, compared with the blank and NC groups; measurement data were expressed as mean ± standard deviation; the data were assessed by one-way analysis of variance. The experiment was conducted three times independently.

Figure 6. RT-qPCR and Western blot analysis show that A20 inhibits activation of NF-κB signaling pathway in hepatocytes of ALF rats

(A) mRNA expressions of A20, NF-κB, TRAF6, and RIP1 in hepatocytes after transfection detected by RT-qPCR; (B) protein levels of A20, NF-κB, TRAF6, and RIP1 in hepatocytes after transfection detected by Western blot analysis; (C) protein band patterns of A20, NF-κB, TRAF6, and RIP1 in hepatocytes after transfection detected by Western blot analysis; *P<0.05, compared with the control group; ^#P<0.05, compared with the blank and NC groups; measurement data were expressed as mean ± standard deviation; the data were assessed by t-test. The experiment was conducted three times independently.