MicroRNA-148a Ameliorate Hepatic Ischemia/Reperfusion Injury Via Targeting CAMK?? Thus Regulating TAK1 And IRF3

Objective:1.To detect expression of miR-148 a,CAMK?? and downstream inflammatory cytokines in hepatic I/R in vitro model,thus disclosing the correlation of expression levels between miR-148 a and CAMK??.2.To observe influence of miR-148 a and CAMK?? in hepatic I/R in vivo model so as to clarify the immune regulation function of both.3.To explore the internal molecular mechanism by which miR-148 a relieves hepatic I/R injury,and identify the targeting relationship of miR-148 a towards CAMK??.Methods:1.The classical succedaneous cell line of Kupffer cell,RAW 264.7cells,was used to establish mouse liver I/R in vitro model,namely cell hypoxia/reoxygenation(H/R)model.q RT-PCR was used to evaluate m RNA levels of miR-148 a,CAMK??,IL-6,TNF-? and IL-1?,meanwhile the H/R time combination was selected out for subsequent experiments.Western blot was used to determine the protein expression ofCAMK??,IRF3,p-IRF3,TAK1,p-TAK1,I?B?,p-I?B? and IL-1? in cell H/R.2.Mouse hepatic I/R in vivo model was constructed.miR-148 a agomir,antagomir and CAMK?? si RNA were transfected into mouse liver.HE staining was used to detect pathological lesion of liver.IL-6,TNF-?and IL-1? were measured by ELISA.Serum ALT and AST were measured by microplate methods.3.miR-148 a mimic and inhibitor,CAMK?? si RNA,IRF3 si RNA and TAK1 si RNA were transfected into cells.After H/R treatment,protein expression of CAMK??,IRF3,p-IRF3,TAK1 and p-TAK1 were determined by Western blot.IL-6,TNF-? and IL-1? in cell supernate were detected by ELISA.Double luciferase reporter gene experiment was used to determine the relationship between miR-148 a and CAMK??.Results:1.In cell H/R model,expression of miR-148 a was down regulated(p<0.05),while m RNA levels of CAMK??,IL-6(p<0.05),TNF-?(p<0.01)and IL-1?(p<0.05)were enhanced and peaked at the time node that hypoxia for 6 followed by 12 reoxygenation.Expression of miR-148 a and CAMK?? was negatively correlated.Simultaneously,protein levels of CAMK??,p-IRF3,p-TAK1,p-I?B? and IL-1? were increased(p<0.05).2.Expression of miR-148 a slided down with the time of reperfusion prolonged,1/6 was selected out for the follow-up experiments.miR-148 aagomir could significantly ameliorate mouse hepatic I/R injury(p<0.05),whilst miR-148 a antagomir exacerbated the pathological changes in the liver(p<5)and no significant difference was observed in NC groups.Serum IL-6,TNF-?(p<0.001),IL-1?(p<0.01),ALT and AST(p<0.001)were improved after I/R treatment,and miR-148 a agomir was able to inhibit their expression.On the contrary,miR-148 a antagomir elevated levels of serum IL-6,TNF-?,IL-1?,ALT and AST(p<0.05).It was worth mentioning that CAMK?? si RNA could reverse the effect derived from miR-148 a antagomir,including hepatic damage,production of inflammatory factors and release of liver enzyme.3.In cell H/R model,after reducing miR-148 a,protein expression of CAMK??,p-IRF3 and p-TAK1 was raised(p<0.05).On this basis,after interference of CAMK??,protein expression of CAMK??,p-IRF3 and p-TAK1 was cut down(p<0.05).When inhibiting IRF3 and TAK1 at the same time,expression of CAMK?? showed no alterations.At last,results of the double luciferase reporter gene experiment showed that miR-148 a could significantly inhibit luciferase activity in CAMK?? WT groups(p<0.01),but had no impacts in MUT groups.Conclusion:1.In hepatic I/R model,miR-148 a was reduced and CAMK?? within downstream inflammatory protein were increased.A negative correlation between miR-148 a and CAMK?? was observed.2.miR-148 a played a protect role in mouse hepatic I/R injury.Knocking down of miR-148 a aggravated liver injury and liver dysfunction,while inhibition of CAMK?? changeovered its efficacy,demostraing that miR-148 a might regulated hepatic immune in a CAMK??-dependent manner.3.Downstream transduction of miR-148 a and CAMK?? associated inflammatory signals contained mutual activation of IRF3 and TAK1.The results of the double luciferase reporter gene experiment showed that CAMK?? was the direct target of miR-148 a.Above all,miR-148 a targeted CAMK?? in liver I/R,inhibiting the activation of downstream IRF3 and TAK1 inflammatory signals,eventually alleviated hepatic I/R injury.mir-148 a is a potential therapy target for the clinical treatment of hepatic I/R injury.This study provides the research basis and ideas for the diagnosis and remedy of hepatic I/R injury and the prevention of acute rejection of liver transplantation.