蛋白磷酸酶4对脂质沉积肝细胞脂代谢及凋亡的影响

Effect of protein phosphatase 4 on lipid metabolism and apoptosis of hepatocytes with lipid deposition

背景蛋白磷酸酶4(protein phosphatase 4,PP4)的去磷酸化作用是调节细胞增殖、凋亡、分化和其他重要功能的机制之一,PP4在非酒精性脂肪性肝病中的调节作用尚不明确。目的研究PP4在脂质沉积肝细胞的脂代谢、损伤及凋亡中的作用。方法以小鼠肝细胞AML12为研究对象,分为对照组、PP4抑制剂处理组、油酸处理组和油酸+PP4抑制剂组,利用分光光度法检测三酰甘油(triglyceride,TG)及转氨酶[丙氨酸转氨酶(alanine transaminase,ALT)、天冬氨酸转氨酶(aspartate transaminase,AST)]含量,流式细胞术检测细胞活性氧(reactive oxygen species,ROS)、细胞周期、细胞凋亡,qPCR检测脂质代谢及凋亡基因表达情况。以AML12小鼠肝细胞PP4过表达系为研究对象,分为对照组、PP4过表达组、PP4干扰组、油酸处理组和PP4过表达+油酸处理组,qPCR检测脂质代谢关键酶基因表达情况,油红O染色法检测脂质蓄积情况,Western blot技术检测脂质代谢关键酶蛋白表达情况,探索PP4对脂质蓄积肝细胞脂质代谢、损伤及凋亡的影响。结果(1)与对照组相比,油酸处理组肝细胞PP4、肝纤维化基因Timp1、抗凋亡基因Bcl-2、凋亡基因Bax、凋亡基因Caspase 3表达水平升高(P<0.05),脂代谢关键酶基因CPT1、ACC1、FAS表达水平升高(P<0.05);TG、ALT、AST升高(P<0.05);ROS阳性细胞百分比增加(P<0.001),G0/G1期细胞占比减少(P<0.01),S期增加(P<0.05),细胞凋亡增加(P<0.001)。(2)与单纯油酸处理组相比,油酸+PP4抑制剂处理组肝纤维化基因Cola1、凋亡基因Bax、凋亡基因Caspase 3表达水平降低(P<0.05);脂代谢关键酶基因ACC1、FAS表达水平降低(P<0.05);ALT、AST降低(P<0.05);ROS阳性细胞百分比减少(P<0.05),细胞G0/G1期占比增加(P<0.01),S期减少(P<0.05),细胞凋亡降低(P<0.001)。(3)PP4干扰后脂代谢关键酶基因CPT1、FAS表达水平降低(P<0.05),PP4过表达后脂代谢关键酶基因CPT1、FAS表达水平升高(P<0.05);PP4过表达加油酸处理后与对照组加油酸处理后油红O染色显示脂质沉积增加(P<0.05);油酸处理后脂代谢关键酶蛋白PP4、ACC1表达升高,CPT1表达下降(P<0.05)。结论PP4参与油酸处理肝细胞脂质代谢、损伤及凋亡,抑制PP4活性能够减少肝细胞脂质蓄积,PP4过表达脂质蓄积增加。

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.