蜕皮激素对顺铂诱导小鼠肝损伤的保护作用及机制研究

Protective effect and mechanism of ecdysone on cisplatin induced liver injury in mice

目的:探讨蜕皮激素对顺铂导致小鼠肝损伤的保护作用及机制。方法:采用昆明小鼠和AML12细胞构建顺铂肝损伤体内及体外模型,应用蜕皮激素对顺铂组进行干预,通过HE染色观察肝脏组织病理形态,ELISA和荧光染色观测细胞内ROS的产生及细胞凋亡,RT-qPCR检测组织及细胞内氧化应激、炎症和凋亡相关生物标志物的表达。转录组测序测定其作用靶点,RT-qPCR和Western blot验证靶基因mRNA和蛋白表达。结果:与空白组相比,顺铂组小鼠肝细胞排列疏松,部分细胞轻度肿胀,凋亡细胞数量、血清内ALT、AST、肝细胞内ROS、MDA含量明显增多(P<0.01),GSH-PX、SOD含量明显降低(P<0.01),提示顺铂对肝功能造成明显损伤;与顺铂组相比,蜕皮激素组小鼠肝细胞损伤程度明显减轻,上述各项指标均有不同程度改善,表明其对肝功能具有保护作用。RT-qPCR结果显示,蜕皮激素组NF-κB、TNF-α、IL-1β、Caspase-3、Caspase-9、Hmox1、PI3K、Nqo1 mRNA相对表达量均明显降低,Nrf2 mRNA相对表达量明显升高(P<0.01)。转录组测序及验证实验表明,蜕皮激素通过调控PDK1低表达,Dnmt3b、MEK5高表达,进而保护肝功能。结论:蜕皮激素能够通过炎症、氧化应激和凋亡抑制途径共同作用于肝细胞,并直接影响Dnmt3b、MEK5、PDK1的表达从而可能对顺铂导致的肝损伤具有保护作用。

Objective:To investigate the protective effect and mechanism of ecdysone on cisplatin induced liver injury in mice.Methods:Kunming mice and AML12 cells were used to construct cisplatin induced liver injury model in vivo and in vitro.Ecdysone was used to intervene cisplatin group.The pathological morphology of liver tissue was observed by HE staining,the production of intracellular reactive oxygen and apoptosis were observed by ELISA and fluorescence staining,and the expressions of biomarkers related to oxidative stress,inflammation and apoptosis were detected by RT-qPCR.The target was determined by transcriptome sequencing,RT-qPCR and Western blot were used to verify the target genes'expressions of mRNA and protein.Results:Compared with the blank group,the hepatocytes in cisplatin group were loosely arranged,some cells were slightly swollen.The contents of ALT and AST in serum,ROS and MDA in cells increased significantly.And the contents of GSH-PX and SOD in cells decreased significantly,suggesting that cisplatin caused obvious damage to liver function.Compared with cisplatin group,the degree of hepatocyte injury in ecdysone group was significantly reduced,and the above indexes were improved in varying degrees,indicating that ecdysone had a protective effect on liver function.The results of RT-qPCR showed that,in ecdysone group,the mRNA expressions of NF-κB,TNF-α,IL-1β,Caspase-3,Caspase-9,Hmox1,PI3K,Nqo1 decreased significantly and Nrf2 increased(P<0.01).Transcriptome sequencing and validation experiments showed that ecdysone protects liver function by regulating the low expressions of PDK1 and the high expressions of Dnmt3b and MEK5.Conclusion:Ecdysone has a protective effect on cisplatin induced liver injury through the synergistic regulation of inflammation,oxidative stress and apoptosis.And directly affect the expressions of Dnmt3b,MEK5 and PDK1,which may have a protective effect on liver injury caused by cisplatin.