The Effects Of Nrf-2 Signaling Pathway On Sepsis Induced Acute Liver Injury In Rats

Objective :By cecal ligation and perforation(CLP),the model of sepsis acute liver injury(ALI)in rat is established.Intervening CLP model with sulforaphane(SFN),which is the agonist of nrf-2,and inhibitors of all trans retinoic acid(ATRA)validates nrf-2 transcription activity.It can be identified whether the Nrf-2 signaling pathway has a protective effect on liver function in patients with acute liver injury which can provide the theoretical basis for the new target in the treatment of acute liver injury in sepsis.Methods :A total of 128 healthy male SD rats in SPF grade were selected.Arranged in four groups,A: sham operation group,B: cecal ligation and perforation(CLP)Group,C: sulforaphane(SFN)intervented group and D: all trans retinoic acid(ATRA)intervented group,Each group have 32 rats.Four groups of rats were divided according to the average time again 6h,12 h,18h and survival analysis group of four subgroups.After the rats in sham operation group were anesthetized with a 1ml syringe press 5ml / kg intraperitoneal injection of solvent corn oil,open their abdominal cavity and find the appendix,then close the abdominal cavity.After CLP rats' abdominal cavity is opened and the cecum is found,ligate the cecum at the 1/4 point of the cecum with silk.Then pierce the opposite side of 1/2 and 1/4 of cecum mesentery with venous retained needle,extrude part of intestinal contents,Press 5ml / kg intraperitoneal injection of corn oil,and close the abdominal cavity.At SFN intervented group and ATRA intervented group,close the abdominal cavity after inject SFN(5mg/kg),ATRA(1mg/kg).Celect the heart blood at each time point,centrifuge and take the serum,detect the level of aminotransferase(ALT,AST)and total bilirubin.After the rats were sacrificed,remove the liver in order to determine the levels of inflammatory factors(TNF-?,IL-6)and antioxidant enzymes(GSH-Px,SOD).Western-Blot method assay Nrf2 protein level,and use RT-PCR to detect the gene of Nrf-2 and the anti-oxidative enzyme GSH-Px,SOD mRNA.Results:(1)The rat AST,ALT,and total bilirubin lecel changes:6 Hours after the surgery,the AST,ALT,and Tbi levels of CLP,SFN and ATRA group were higher compared to the SHAM group.The total bilirubin levels of SFN group were lower than the CLP group.Whereas the total bilirubin levels of ATRA group were higer than the CLP group.the AST,ALT,and total bilirubin levels of ATRA group were higher compared to the SFN group.12 Hours and 18 Hours after the surgery,the AST,ALT,and Tbi levels of CLP,SFN and ATRA group were higher compared to the SHAM group.The AST,ALT,and Tbi levels of SFN group were lower than the CLP group.Whereas the levels in ATRA group were higer than the CLP group.At the same time,compared to the SFN group,the AST,ALT,and Tbi levels of ATRA group were significantly higher.(2)The appearance of rat's liver and the pathology of HE staining: For the SHAM group,the appearance of liver tissues was normal,the structure outline was clear,and no clear pathology was found.In the CLP,SFN and ATRA groups,the appearance and the pathology of liver tissues had varying degrees of damage compared to the SHAM group,the liver damage in SFN group was slighter than the CLP group,and the liver damage in ATRA group was severe than that of the CLP group At each experiment time point.(3)The inflammatory mediator detection of liver tissue homogenate: At each experiment time point,the inflammatory mediator levels of CLP,SFN group and ATRA group were higher compared to the SHAM group.The inflammatory mediator levels of SFN group were lower than the CLP group.Whereas the inflammatory mediator levels of ATRA group were higer than the CLP group.At the same time the inflammatory mediator levels of ATRA group were significantly higher compared to the SFN group.(4)The antioxidant enzyme detection of liver tissue homogenate:6 Hours after the surgery,the SOD levels of CLP group was higher compared to the SHAM group,but the SOD levels became to decrease in 18 Hours,and lower than SHAM group.At each experiment time point,the SOD levels of SFN group was higher than SHAM and CLP group,whereas the SOD levels of ATRA group was lower than SHAM and CLP group.Meanwhile,compared with the SFN group,ATRA group SOD levels decreased more significantly.At six and Twelve hour time point,the GSH-Px levels of CLP group was higher compared to the SHAM group,but at Eighteen time point it became lower than SHAM group.At each experiment time point,the GSH-Px levels of SFN group was higher than SHAM and CLP group,but compared to the SHAM and CLP group,the GSH-Px levels of ATRA group was lower.At the same time,compared to the SFN group ATRA group were significantly lower.(5)The Expression in rat liver tissue protein of Nrf-2: At each experiment time point,compared to the sham group,The Nrf-2 protein expression level of the CLP group and SFN group was increased;the Nrf-2 protein expression level of ATRA group increased than the CLP group;the Nrf-2 protein expression level of CA group decreased than the CLP group at 18 hours.And also the Nrf-2 protein expression level of ATRA group decreased than the SFN group at each time point.(6)The expressions of Nrf-2,GSH-Px,and SOD mRNA:Nrf-2 mRNA: compared with the SHAM group,The Nrf-2 mRNA expressions of the CLP and SFN group were higher at each time point.While compared with the SHAM group,The Nrf-2 mRNA expressions of the SFN group was higher.The Nrf-2 mRNA expressions of the ATRA group was lower than CLP and SFN group.GSH-Px mRNA: At six and Twelve hours time point The GSH-Px mRNA expressions of the CLP group was higher than that of the SHAM group.and The GSH-Px mRNA expressions of the SFN group was higher than that of SHAM and CLP group at each time point.The GSH-Px mRNA expressions of the ATRA group was lower than that of the SHAM group at Eighteen hours points.The GSH-Px mRNA expressions of the ATRA group was significantly lower than that of the SFN group at six,twelve and Eighteen hours time point.The SOD mRNA expressions: At six and Twelve hour time point,The SOD mRNA expressions of the CLP rats was higher than that of the SHAM rats.The SOD mRNA expressions of the SFN group was higher than that of the SHAM and CLP group at each experimental time point.The SOD mRNA expressions of the ATRA group was lower than SHAM group at twelve and Eighteen hours time point.Meanwhile the SOD mRNA expressions of the ATRA group rats was lower than that of the SFN group at each experimental time point.Conclusion :After the Nrf-2 signaling pathway Activated it can increase the expression of antioxidant enzymes such as Superoxide dismutase and Glutathione peroxidase,thus to exert anti-inflammatory and anti-oxidative stress effect,reduce liver damage and improve liver function.It has a protective effect on sepsis induced acute liver injury.