利拉鲁肽对棕榈酸诱导HepG2细胞凋亡的影响及其可能机制

Effect and mechanism of liraglutide on the apoptosis of human hepatocellular carcinoma HepG2 cells induced with palmitic acid

目的观察利拉鲁肽是否通过影响丝裂原活化蛋白激酶(MAPKs)通路起到保护HepG2细胞拮抗脂毒性的作用。方法用400μmol/L棕榈酸诱导HepG2细胞,同时予终浓度为100 nmol/L利拉鲁肽处理细胞,此外分别提前加入JNK抑制剂(SP600125)和p38 MAPK抑制剂(SB203580),检测细胞凋亡率、丙二醛(MDA)含量、caspase3活性,蛋白质印迹法检测p38丝裂原活化蛋白激酶(p38 MAPK)、c-jun氨基末端激酶(JNK)、细胞色素氧化酶P450 2E1(CYP2E1)、葡萄糖调节蛋白78(GRP78)、活化的半胱氨酸天冬氨酸蛋白酶3(cleaved caspase3)、B细胞淋巴瘤相关蛋白X(Bax)、B细胞淋巴瘤2(Bcl-2)蛋白、C/EBP同源蛋白(CHOP)的表达。多个样本均数比较采用LSD检验或DunnettT3检验。结果棕榈酸能够增加HepG2细胞p38 MAPK、JNK磷酸化水平(P<0.05),升高GRP78、CHOP、CYP2E1、MDA、Bax、caspase3的表达和细胞凋亡率,抑制Bcl-2的表达(P值均<0.05)。SP600125和SB203580能够抑制棕榈酸引起的氧化应激和凋亡(包括CYP2E1、MDA、Bax、Bcl-2、caspase3、CHOP)(P值均<0.05);利拉鲁肽能够降低p38 MAPK、JNK磷酸化水平,能够调控细胞凋亡相关蛋白(Bax、Bcl-2、caspase3、CHOP)的表达(P值均<0.05);而在两种抑制剂预处理后,利拉鲁肽对细胞凋亡蛋白(Bax、Bcl-2、caspase3、CHOP)的影响无统计学意义(P值均>0.05)。结论棕榈酸对HepG2细胞具有较强的脂毒性,诱导细胞发生凋亡,胰高血糖素样肽-1类似物利拉鲁肽可能通过介导p38 MAPK和JNK途径改善棕榈酸对细胞的脂毒性损伤。

Objective To observe whether liraglutide protects HepG2 cells from lipotoxicity by affecting mitogen-activated protein kinase(MAPKs)pathway.Methods HepG2 cells were induced with 400μmol/L palmitic acid,and cells were treated with a final concentration of 100 nmol/L liraglutide.In addition,JNK inhibitor(SP600125)and p38 MAPK inhibitor(SB203580)were added in advance,respectively.Apoptosis rate,malondialdehyde(MDA)content,and caspase3 activity were detected.Western blot was used to detect p38 mitogen-activated protein kinase(p38 MAPK),c-jun amino terminal kinase(JNK),cytochrome oxidase P450 2E1(CYP2E1),glucose regulatory protein 78(GRP78),activated caspase 3,B cell lymphoma associated Protein X(Bax),B cell lymphoma 2(Bcl-2),and expression of C/EBP homologous protein(CHOP)protein.LSD or Dunnett’s T3 test were used to compare the mean of multiple samples.Results Palmitic acid increased the phosphorylation of p38 MAPK and JNK in HepG2 cells(P<0.05).Furthermore,it increased the expression of GRP78,CHOP,CYP2E1,MDA,Bax,caspase3 and apoptosis rate,but inhibited the expression of Bcl-2(Pvalue<0.05).SP600125 and SB203580 had inhibited oxidative stress and apoptosis induced by palmitic acid(including CYP2E1,MDA,Bax,Bcl-2,caspase3,CHOP)(P<0.05).The phosphorylation level of p38 MAPK and JNK was reduced with liraglutide and the expression of apoptosis-related proteins(Bax,Bcl-2,caspase3,CHOP)(P<0.05)was regulated.There was no significant difference in the effect of liraglutide on apoptotic proteins(Bax,Bcl-2,caspase-3,CHOP)(P>0.05)after pretreatment with those two inhibitors.Conclusion Palmitic acid has strong lipotoxicity to HepG2 cells and induces apoptosis.Glucagon-like peptide-1 analogue,liraglutide may improve lipotoxicity of palmitic acid by mediating p38 MAPK and JNK pathways.