The Role Of ATF4 In Mice Liver Injury Induced By LPS/D-GalN And CCl4

Objective: The role of ATF4 in liver injury was studied by mouse model.Methods: By raising C57BL/6 mice,healthy male mice aged 6-8 weeks(20-22 g)were selected,The protein and mRNA expression levels of ATF4 in liver,heart,kidney and lung were analyzed by Western blot and reverse transcription-polymerase chain reaction(RT-PCR);The mice were treated with intraperitoneal injection of tunicin(Tun),RT-PCR was used to detect the activation and shearing of the signature molecule XBP1.In addition,Western blot analysis was used to analyze the effect of Tun on phosphorylation,Bip,and ATF4 protein levels in the hepatic and lung tissues.Acute and chronic liver injury models were established by intraperitoneal injection of LPS/D-GalN and CCl4,Western blot and RT-PCR were used to detect the protein and mRNA expression levels of ATF4 in mouse liver tissues.The mouse model was constructed by injecting crisper/cas9-ATF4 plasmid(ATF4-cri plasmid)into mice to construct liver ATF4,Intraperitoneal injection of LPS/D-GalN and CCl4 treated mice,Western blot and RT-PCR were used to detect the level of protein and mRNA expression levels of ATF4 and serum transaminase(AST,ALT)in mouse liver tissues.Results: 1.ATF4 protein was highly expressed in mouse liver tissues: Western blot results showed that the expression of ATF4 in mouse liver tissues was very high compared with other important organ tissues.In addition,RT-PCR results showed no significant differences in mRNA expression levels of ATF4 in mice liver,heart,kidney and lung tissues.The results indicated that ATF4 protein expression was high in mouse liver tissues.2.The high expression of ATF4 protein in mouse liver tissues does not depend on the eIF2 alpha pathway: Western blot results showed no obvious activation of eIF2 alpha in liver,heart,kidney and lung tissues,and there was no significant change between them.The results indicated that the high expression of ATF4 protein in mouse liver tissues was not dependent on the eIF2 alpha pathway.3.The high expression of ATF4 in mouse liver tissue was not dependent on endoplasmic reticulum stress: The results of RT-PCR showed that Tun induced the activation and shearing of the signature molecule XBP1.This indicated that Tun induced the activation of the endoplasmic reticulum stress in mouse liver tissue.At the same time,Western blot results showed that the expression of p-eIF2 alpha and Bip in the liver tissues after the treatment of mice was up-regulated,and the expression of ATF4 was down-regulated.In addition,the expression of eIF2 alpha phosphorylation,Bip and ATF4 of the endoplasmic reticulum stress molecule in the lung tissues of the rats after treatment was significantly up-regulated.These results suggest that the highexpression of ATF4 in mouse liver tissues does not depend on endoplasmic reticulum stress.4.ATF4 protein was reduced in CCl4 induced chronic liver injury in mice: Western blot results showed that ATF4 protein was significantly lower than normal liver tissue in CCl4 induced chronic liver injury model;RT-PCR results showed that CCl4 had no effect on the mRNA of ATF4 in mouse liver tissue.5.ATF4 protein was down-regulated in LPS/D-GalN induced acute liver injury in mice: Western blot results showed that ATF4 protein was significantly lower in LPS/D-GalN induced acute hepatic injury model than normal liver tissue;RT-PCR results showed that LPS/D-GalN also had no effect on the mRNA of ATF4 in mouse liver tissues.6.The successful establishment of the mouse model of liver ATF4: Real-time fluorescence quantitative and PCR results showed that the mRNA of ATF4 in normal liver tissues was significantly lower than that in normal liver tissues after intravenous injection of ATF4-cri plasmid.(P<0.05)In addition,Western blot results showed that the expression of ATF4 protein in hepatic tissue was significantly reduced by intravenous injection of ATF4-cri plasmid.These results indicated that after the injection of ATF4-cri plasmid in the tail vein,the mouse model was successfully constructed.7.Inhibition of ATF4 aggravated liver injury induced by LPS/D-GalN and CCl4: The results of serum aminotransferase(AST,ALT)showed that inhibition of ATF4 expression in mouse liver tissues could aggravate liver damageinduced by LPS/D-GalN and CCl4.This suggests that ATF4 protects liver damage.