Protective Effects And Mechanism Of Ajuga Concentrated Solution And Ecdysone On Cisplatin Induced Liver Injury In Mice

Objective:To investigate the protective effects and mechanism of Ajuga(AdT)concentrated solution and Ecdysone(20E)on Cisplatin(DDP)induced liver injury in mice.Methods:Kunming mice and AML12 cells were used to construct the in vivo(10mg/kg DDP)and in vitro(19μg/mL DDP)models of liver injury.The mice were treated by intragastric administration of AdT(the intragastric concentration was 1.206mg/kg based on its main pharmacodynamic component Acetylhabaside)or 20E(5mg/kg,20mg/kg,100mg/kg).DDP was added with AdT(120μg/mL)or 20E(75μg/mL)to reduce the injury of AML 12 cells.The histopathological changes of liver were observed by Hematoxylin-eosin staining(HE);the activities of Alanine aminotransferase(ALT)and Aspartate aminotransferase(AST)were detected by automatic biochemical analyzer;the production of Active oxygen(ROS)in hepatocytes was detected by Enzyme linked immunosorbent assay(ELISA);and the production of ROS and apoptosis were observed by fluorescence staining.The activity of Superoxide dismutase(SOD)in liver tissue and AML 12 cells was measured by WST-1;the content of Malondialdehyde(MDA)in liver tissue and AML 12 cells was measured by TBA;the content of Glutathione peroxidase(GSH-PX)in AML 12 cells was measured by spectrophotometry;and the factors of oxidative stress,inflammation and apoptosis in liver and AML 12 cells were detected by Real time fluorescence quantitative PCR(RT-qPCR).The different genes between the blank control group,120μg/mL AdT group,75μg/mL 20E group and DDP group were screened by transcriptome sequencing technology.And then the mRNA and protein expressions were detected by RT-qPCR and Western blot in the blank control group,DDP group,120μg/mL AdT group and 75μg/mL 20E group.Results:1.HE staining showed that the cells in DDP group were slightly swollen,the connections between cells were damaged,the arrangement of cells was disordered.After treated with AdT or 20E,the pathological changes were reduced in varying degrees.In the morphological observation of AML 12 cell culture,the number of cells in DDP group was reduced.In AdT or 20E group,the cell growth was improved and the number of cells was increased.2.The liver function of mice in AdT or 20E group was significantly improved,which showed that the activities of serum ALT and AST were significantly lower than those in DDP group(P<0.01).3.Compared with DDP group,the results of ELISA showed that the production of ROS in liver tissue of mice treated with AdT or 20E decreased(P<0.01).4.The results of fluorescence staining showed that in AML 12 cells in vitro,compared with DDP group,the production of ROS and the number of apoptotic cells decreased in AdT group and 20E group.5.The results of lipid peroxide detection showed that in vivo,compared with DDP group,the level of SOD in liver tissue of mice treated with AdT or 20E increased(P<0.01),and the level of MDA decreased(P<0.01).In vitro,compared with group DDP,the levels of GSH-PX and SOD in AML12 cells in AdT group and 20E group increased(P<0.01),and the level of MDA decreased(P<0.01).6.The results of RT-qPCR showed that in vivo and in vitro,compared with DDP group,the inflammatory factors,apoptosis and oxidative stress of liver tissue and AML 12 cells in AdT group and 20E group were reduced.The mRNA and protein expressions of inflammatory factors such as Nuclear factor kappa B(NF-κB),Tumor Necrosis Factor α(TNF-α),Interleukin-1β(IL-1β),apoptosis factors such as Cysteine aspartate protease 3(Caspase-3),Cysteine aspartate protease 9(Caspase-9)and oxidative stress related signal pathway factors such as Heme oxygenase 1(Hmox 1),Phosphatidylinositol-3-kinase(PI3K),NAD(P)H dehydrogenase,quinone 1(Nqo1)were lower than DDP group,the mRNA expressions of NF-E2-related Factor 2(Nrf2)were higher than DDP group(P<0.01).7.After transcriptome sequencing,we found that there were 262 different genes between AdT group and DDP group(44 genes were up-regulated and 218 genes were down-regulated),and 227 different genes between 20E group and DDP group(55 genes were up-regulated and 172 genes were down-regulated).8.Transcriptome sequencing validation experiment showed that AdT promotes hepatocyte survival by high expression of Cd2ap and Rab7b.20E promotes hepatocyte survival by low expression of PDK1,high expression of Dnmt3b and MEK5.Conclusion:1.AdT has a protective effect on DDP induced liver injury through the synergistic regulations of inflammation,oxidative stress and apoptosis.The effector molecules may be Cd2ap and Rab7b,which may be related to oxidative stress and inflammation related signal pathway of PI3K/Akt,NF-κB,TLR4.2.20E has a protective effect on DDP induced liver injury through the synergistic regulations of inflammation,oxidative stress and apoptosis.The effector molecules may be Dnmt3b,MEK5 and PDK1,which may be related to mitochondrial function,and signal pathway of PI3K/Akt,NF-κB,MAPK related with oxidative stress and inflammation.