大黄素干预PI3K/Akt/mTOR信号通路影响肝癌前病变大鼠铁死亡的作用机制

Mechanism of Emodin on Ferroptosis by Interfering PI3K/Akt/mTOR Signaling Pathway in Rats with Precancerous Lesions of Liver Cancer

目的 探究大黄素对二乙基亚硝胺(diethylnitrosamine, DEN)诱导的肝癌前病变大鼠磷脂酰肌醇3-激酶(phosphoinositide 3-kinase, PI3K)/蛋白激酶B(protein kinase B, Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号转导通路的调控作用及对铁死亡的影响。方法 采用DEN诱发大鼠肝癌前病变进行肝癌前病变造模。随机将50只雄性SD大鼠分为对照组[未造模+10ml/(kg·d)生理盐水灌胃)]、DEN模型组[肝癌前病变造模+10 ml/(kg·d)生理盐水灌胃]、DEN+大黄素组[肝癌前病变造模+80 mg/(kg·d)大黄素浓缩液灌胃]、DEN+护肝片组[肝癌前病变造模+900 mg/kg护肝片灌服]和DEN+大黄素+护肝片组[肝癌前病变造模+80 mg/(kg·d)大黄素浓缩液灌胃+900 mg/kg护肝片灌服],每组各10只大鼠。各组大鼠连续干预治疗12周。生化分析法检测各组大鼠血清丙氨酸氨基转移酶(alanine aminotransferase, ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)、白蛋白(albumin, ALB)水平;比较各组大鼠的肝脏指数。酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)法检测各组大鼠血清白细胞介素1β(interleukin 1 beta, IL-1β)、肿瘤坏死因子α(tumor necrosis factor alpha, TNF-α)、IL-10含量。苏木精-伊红(hematoxylin-eosin, HE)染色法观察各组大鼠肝组织病理形态学改变;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling, TUNEL)分析各组大鼠肝组织细胞凋亡情况。免疫组织化学法检测各组大鼠肝组织中铁死亡中心调节因子谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白表达。马松三色染色(Masson′s trichrome staining, MASSON)法分析各组大鼠肝组织病理改变。逆转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)法检测各组大鼠肝组织中PIK3、Akt、mTOR的mRNA水平。结果 与对照组比较,DEN模型组大鼠ALT水平、AST水平、肝脏指数显著升高,ALB水平显著降低(P均<0.05),血清IL-1β、TNF-α、IL-10含量明显升高(P均<0.05);肝细胞明显炎性浸润,细胞免疫应答增加,肝细胞形态畸变并出现变性死亡;肝组织中GPX4蛋白表达量明显下降(P<0.05),PI3K、Akt、mTOR的mRNA表达量明显上升(P均<0.05)。与DEN模型组比较,DEN+大黄素组、DEN+护肝片组、DEN+大黄素+护肝片组大鼠ALT水平、AST水平、肝脏指数显著降低,ALB显著升高(P均<0.05),血清IL-1β、TNF-α、IL-10含量明显降低(P均<0.05);肝细胞排列趋向正常,炎性浸润减轻,肝细胞弥漫性纤维化病变减弱,血管及胆管周围肝细胞形态逐渐好转,蓝色胶原纤维组织减少;肝组织中GPX4蛋白表达量明显上升(P均<0.05),PI3K、Akt、mTOR的mRNA表达量明显下降(P均<0.05),且上述变化DEN+大黄素+护肝片组改善效果明显优于DEN+大黄素组和DEN+护肝片组(P均<0.05)。结论 大黄素可通过促进组织中铁死亡相关蛋白GPX4表达来改善大鼠肝癌前病变,其作用机制可能与大黄素调控PI3K/Akt/mTOR信号通路有关。

Objective To explore the regulatory effects of emodin on the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of ra-pamycin(mTOR)signaling pathway in diethylnitrosamine(DEN)induced precancerous lesions of liver cancer of rats and its impacts on ferroptosis.Methods Modeling of precancerous lesions of liver cancer was carried out by DEN inducing precancerous lesions of liver cancer on rats.A total of 50 male SD rats were randomly divided into control group[no mod-eling+10 ml/(kg·d)normal saline by gavage],DEN model group[precancerous lesions of liver cancer modeling+10 ml/(kg·d)normal saline by gavage],DEN+emodin group[precancerous lesions of liver cancer modeling+80 mg/(kg·d)emodin concentrated solution by gavage],DEN+liver-protecting tablet group[precancerous lesions of liver canc-er modeling+900 mg/kg liver-protecting tablet by gavage]and DEN+emodin+liver-protecting tablet group[precan-cerous lesions of liver cancer modeling+80mg/(kg·d)emodin concentrated solution by gavage+900 mg/kg liver-pro-tecting tablet by gavage],with 10 rats in each.The rats in each group were treated continuously for 12 weeks.Serum ala-nine aminotransferase(ALT),aspartate aminotransferase(AST)and albumin(ALB)levels of rats in each group were detected by biochemical analysis;the liver indexes of rats in each group was compared.The levels of serum interleukin 1 beta(IL-1β),tumor necrosis factor alpha(TNF-α)and IL-10 contents of rats in each group were detected by enzyme-linked immunosorbent assay(ELISA).The pathological changes of liver tissues were observed by hematoxylin-eosin(HE)staining;the apoptosis of liver tissue cells was analyzed by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling(TUNEL).The protein expression of glutathione peroxidase 4(GPX4),a central regulator of ferropto-sis,in liver tissues of rats in each group was detected by immunohistochemistry.The pathological changes in liver tissues of rats in each group were analyzed by Masson's trichrome staining(MASSON).The mRNA levels of PIK3,Akt and mTOR in liver tissues of rats in each group was detected by reverse transcription polymerase chain reaction(RT-PCR).Results Compared with the control group,ALT level,AST level and liver indexes of the rats in the DEN model group significantly increased,and ALB level significantly decreased(all P<0.05),the serum IL-1β,TNF-αand IL-10 contents significantly increased(all P<0.05);obvious inflammatory infiltration of hepatocytes,increased cellular immune re-sponse,morphological distortion of hepatocytes and degeneration and death of hepatocytes in liver tissues were occured;the protein expression of GPX4 significantly decreased(P<0.05),the mRNA expressions of PI3K,Akt and mTOR sig-nificantly increased(all P<0.05).Compared with the rats in DEN model group,ALT levels,AST levels and liver inde-xes in DEN+emodin group,DEN+liver-protecting tablet group and DEN+emodin+liver-protecting tablet group signifi-cantly decreased,while ALB significantly increased(all P<0.05),the contents of serum IL-1β,TNF-αand IL-10 signifi-cantly decreased(all P<0.05);the arrangement of liver cells tended to be normal,the inflammatory infiltration reduced,the diffuse fibrosis of liver cells weakened,the morphology of liver cells around blood vessels and bile ducts gradually im-proved and the blue collagen fiber tissue reduced;the protein expression of GPX4 in liver tissues significantly increased(all P<0.05),the mRNA expressions of PI3K,Akt and mTOR significantly decreased(all P<0.05),and the improve-ment effect of DEN+emodin+liver-protecting tablet group was obviously better than that of DEN+emodin group and DEN+liver-protecting tablet group(all P<0.05).Conclusion Emodin can improve rat precancerous lesions of liver cancer by promoting the expression of ferroptosis-related proteins GPX4,and its mechanism may be related to the regula-tion of PI3K/Akt/mTOR signaling pathway by emodin.