The Role Of STAT3 Signal In The Regeneration Of Acetaminophen-induced Liver Cell Injury In Mice

Objective:Drug-induced liver injury(DILI)has various clinical manifestations,which makes early identification and diagnosis difficult,and has become a global concern.Drug-induced liver injury((DILI))has a variety of clinical manifestations,which makes it difficult to identify and diagnose in the early stage,which has become an issue of global concern.Official agency data display that DILI has become the fifth leading cause of death all the globe.DILI poses a great threat to human health,so it is of great significance to study it.Acetaminophen-induced liver injury accounts for more than 50%of DILI cases.The mechanism of acetaminophen-induced liver injury is a hot spot.N-acetylcysteine,as a specific antidote to APAP-induced liver injury,can prevent further deterioration of liver function.Though,a large proportion of patients are in the middle and late stages of liver injury when they go hospital,and eventually develop into liver failure or even death.Liver regeneration is critical to the survival of patients with liver failure after DILI.Therefore,understanding the physiological and pathological mechanisms of liver regeneration,and developing methods to control this process and applying it to the clinic will open up new directions for the treatment of DILI patients,and the mortality rate will also be significantly reduced.The previous experiment of the research group found that TLR4is involved in APAP-induced liver programmed necrosis in acute liver injury,and it is not clear whether TLR4 and its downstream signal molecules are involved in liver regeneration after APAP-induced acute liver injury.Therefore,this study explored the role of TLR4 downstream signaling molecule STAT3 in the proliferation of liver cells after APAP-induced hepatocyte injury in mice through in vitro experiments.Methods:Culture normal mouse hepatocytes AML12 to logarithmic growth phase in vitro,add trypsin digestion,resuspend the cells,and control the cell density to1×10~5/ml.1.CCK8 experiment to detect the effect of APAP on the viability of AML12 cells:add the above cell suspension to a 96-well plate at 100μl per well.After 24 hours of culture,the cells adhered completely,and the upper cell culture medium was discarded.Add 100μl of pre-prepared cell culture medium containing APAP at different concentrations(1,2.5,5,10,20 mmol/L)to the experimental wells,and add an equal volume of AML12 cell culture medium to the control wells.Incubate for 12h,24 h,48 h in the incubator,then add 10μl of CCK8 reagent,continue to incubate for 90 min,place it on the microplate reader,and measure the absorbance of each well at a wave length of 450 nm.2.RT-PCR experiment to detect the effect of APAP on the expression of proliferation-related genes in AML12 cells:aspirate the cell suspension and add it to a T25 culture flask,set up a control group and an APAP(2.5mmol/l)group for 24 h and48 h,respectively.RT-PCR was used to detect the m RNA expression of PCNA,Cyclin D1,Ki67 in AML12.3.CCK8 experiment to detect the effect of tyrosine kinase inhibitor AG490 on the viability of AML12 cellsTake the cell suspension and add it to a 96-well plate,add 0(control group)and 10,50,100μmol/l AG490 respectively,add 2.5 mmol/l APAP after acting for 2 hours and continue acting for 48 hours,add CCK8 reagent to detect AG490 on AML12 cells and the impact of APAP injury on the viability of AML12 cells.(The specific steps are the same as Method 1)4.WB experiment to detect the effect of tyrosine kinase inhibitor AG490 on the expression of STAT3,p-STAT3 and proliferation-related proteins in AML12 cellsTake the above cell suspension and add it to a 10 cm2 petri dish,set up a control group,APAP group,AG490 group,APAP+AG490 group,APAP action for 48 hours,and detect the expression levels of STAT3,p-STAT3,PCNA,and Cyclin D1.5.RT-PCR experiment to detect the effect of tyrosine kinase inhibitor AG490 on the expression of proliferation-related genes in AML12 cellsTake the above cell suspension and add it to T25 culture flask,set up a control group,APAP group,APAP+AG490 group,APAP action for 48 hours,detect the expression of PCNA,Cyclin D1,Ki67 in AML12.Results:1.The CCK8 experiment found that APAP had an effect for 12 hours.Compared with the control group,the APAP concentration group(1?2.5?5?10?20 mmol/L)had no significant change in AML12 cell viability(P>0.05);APAP acts for 24 h and 48 h.Compared with the control group,the viability of AML12 cells in each concentration group is reduced(P<0.05);when APAP is treated at a concentration of 2.5 mmol/L,24 h and 48 h AML12 The cell viability was 0.717±0.027 and 0.752±0.014,respectively.It is speculated that AML12 cells may regenerate after injury when treated with 2.5 mmol/l APAP concentration for 48 hours,so 2.5 mmol/L was selected.2.RT-PCR experiments showed that compared with the control group,the expressions of PCNA,Cyclin D1 and Ki67 m RNA in the 24 h APAP(2.5 mmol/l)group were all decreased(P<0.01)The expression levels of PCNA and Cyclin D1 m RNA in the 48 h APAP(2.5 mmol/l)group did not change significantly(P>0.05),and the expression of Ki67 m RNA in the 48 h APAP(2.5 mmol/l)group increased(P<0.01)Compared with the 24 h APAP group,the expressions of PCNA,Cyclin D1 and Ki67 m RNA in the 48 h APAP(2.5 mmol)group were increased(P<0.01),so APAP 2.5 mmol/l and stimulation time 48 h were chosen to simulate in vitro damage to AML12 Model of proliferation and regeneration of hepatocytes after cell.3.Through the CCK8 experiment,it was found that compared with the control group,the cell viability of the 10,50μmol/l AG490 group was not statistically significant(P>0.05),and the cell viability of the remaining groups decreased(P<0.01);compared with the APAP group,AG490 The cell viability of the(10μmol/l)+APAP group did not change significantly(P>0.05),and the cell viability of the AG490(50,100μmol/l)+APAP group decreased(P<0.01).4.The effect of tyrosine kinase inhibitor AG490 on the expression of STAT3 and proliferation-related proteins in AML12 cells was found by WB experiments.Compared with the control group,the protein expression level of p-STAT3 in the APAP group increased(P<0.01),while the AG490 group and APAP+AG490 group were all decreased(P<0.05);compared with the APAP group,the expression of PCNA and Cyclin D1 protein in the APAP+AG490 group decreased(P<0.05).5.The effect of tyrosine kinase inhibitor AG490 on the expression of proliferation-related genes in AML12 cells was found by RT-PCR.Compared with the control group,the expression of PCNA and Cyclin D1 m RNA in APAP group had no significant changes in the expression levels of PCNA and Cyclin D1 m RNA.(P>0.05),the expression of Ki67 m RNA in the APAP group increased(P<0.01);compared with the APAP group,the expression of PCNA,Cyclin D1,and Ki67m RNA in the APAP+AG490 group decreased(all P values<0.01).Conclusions:STAT3 is involved in APAP-induced cell proliferation after liver cell injury in mice,and AG490 as a STAT3 inhibitor inhibits the proliferation of liver cells after APAP liver injury by inhibiting STAT3 phosphorylation.