Background Protein phosphatase 4 (PP4), a member of the PP2A family, is an evolutionarily highly conserved serine/threonine phosphatase. Its dephosphorylation is one of the mechanisms regulating cell proliferation, apoptosis, differentiation and other important functions. The regulatory role of PP4 in nonalcoholic fatty liver disease is unclear.

Objective To study the effects of PP4 on lipid metabolism, injury and apoptosis of lipid deposited hepatocytes.

Methods Mouse liver cells AML12 were divided into control group, PP4 inhibitor treatment group, oleic acid treatment group and oleic acid + PP4 inhibitor group. Triglyceride (TG) and aminotransferase (ALT, AST) contents were detected by spectrophotometry, and reactive oxygen species (ROS), cell cycle and apoptosis were detected by flow cytometry. Lipid metabolism and apoptotic gene expression were detected by qPCR. AML12 mouse liver cells with PP4 overexpression were divided into control group, PP4 overexpression group, PP4 interference group, oleic acid treatment group and PP4 overexpression + oleic acid treatment group. qPCR was used to detect the expression of key enzyme genes of lipid metabolism, and oil red O staining was used to detect lipid accumulation. Western blot assay was used to detect the expression of key enzyme protein of lipid metabolism, and to explore the effects of PP4 on lipid metabolism, injury and apoptosis of lipid-accumulating hepatocytes.

Results Compared with the control group, the expression levels of PP4, liver fibrosis gene Timp1, anti-apoptotic gene Bcl-2, apoptotic gene Bax and apoptotic gene Caspase 3 increased in hepatocytes of oleic acid treatment group (P＜0.05), and the expression levels of key lipid metabolism enzyme genes CPT1, ACC1 and FAS also increased (P＜0.05), so did the supernatant TG, transaminase ALT and AST (P＜0.05); The percentage of ROS positive cells increased (P＜0.001), while the proportion of G0/G1 phase cells decreased (P＜0.01), the proportion of S phase cells increased (P＜0.05), and the apoptosis of cells increased (P＜0.001). Compared with oleic acid treatment group, the expression levels of liver fibrosis gene Cola1, apoptotic gene Bax and apoptotic gene Caspase 3 decreased in oleic acid + PP4 inhibitor treatment group (P＜0.05); The expression levels of ACC1 and FAS, the key enzymes of lipid metabolism, and aminotransferase ALT and AST were also decreased (P＜0.05); The percentage of ROS positive cells decreased (P＜0.05), the proportion of G0/G1 phase increased (P＜0.01), while the proportion of S phase decreased (P＜0.05), and the apoptosis decreased (P＜0.001). CPT1 and FAS expression levels were decreased after PP4 interference (P＜0.05), while CPT1 and FAS expression levels were increased after PP4 overexpression (P＜0.05); After PP4 overexpression and oleic acid treatment, oil red O staining showed increased lipid deposition. After oleic acid treatment, the expressions of key enzyme proteins PP4, ACC1 and CPT1 were increased, while the expressions of CPT1 were decreased.

Conclusion PP4 is involved in lipid metabolism, injury and apoptosis of hepatocytes treated with oleic acid. Inhibition of PP4 activity can reduce lipid accumulation in hepatocytes, and increase the accumulation of PP4 overexpressed lipids.