Protective Effect Of Ferulic Acid On Renal Ischemia Reperfusion Injury In Mice And Its Research Of Correlation With HIF-1? And Adenosine

Objective:To investigate the protective effect of ferulic acid(FA)on renal ischemia-reperfusion injury in mice and its underlying mechanism.Methods:Ninety-six male C57BL/6J mice were randomly divided into 2 parts.The first part was for the testing experiment.They were randomly divided into 6 groups,6 mice in each group: control group,ferulic acid control(FA)group,model(IR)group,low-,medium-,and high-dose ferulic acid intervention(FA+IR)groups.In IR group,after bilateral kidneys exposed,the bilateral renal pedicles were clipped for 35 min and then reperfused for 24 h.Control group mice only exposed bilateral kidneys without clamping renal pedicles.In low,medium,and high doses of FA+IR group,mice were administrated by gavage of 10,30,and 100 mg/kg ferulic acid for three times within 24 h,respectively,then following IR group method.The mice in FA group were administrated by gavage of 100 mg/kg ferulic acid for three times within 24 h.Then each group of mice was sacrificed at the same time to harvest the blood sample and kidney tissues for later analyses.The second part of the mice were for the seven-day survival rate experiment.The grouping method was the same as the first part,10 mice in each group.The renal exposure or ferulic acid pretreatment was performed as above.The bilateral renal pedicles of IR group and 10,30,and 100 mg/kg FA+IR group mice were clipped for 50 minutes,then reperfused.The survival of the mice was observed every 24 h and recorded.The mice were sacrificed after 7 days and then draw a KM survival curve.To demonstrate the role of hypoxia inducible factor-1?(HIF-1?)in renal IR injury in mice by ferulic acid,we performed a HIF-1?interference experiment.Twelve male C57BL/6J mice were randomly divided into two groups.HIF-1? interference group: FA(100 mg/kg)+ Ad-HIF-1? siRNA +IR group and control group: FA(100 mg/kg)+ Ad-RFP+ IR group.The kidneys were transfected with Ad-HIF-1? siRNA or Ad-RFP.After 48 h,the ferulic acid were administrated by gavage of 100 mg/kg ferulic acid for three times within 24 h,then following IR group method.The blood and kidneys of the mice were obtained after 24 h.The serum creatinine(Scr)and blood urea nitrogen(BUN)levels were measured by the kits,hematoxylin-eosin(HE)staining was used to observe the pathological changes of the kidneys,terminal deoxynucleotidly transferased UTP nick-end labeling(TUNEL)assay was used to test the renal cell apoptosis,the caspase-3 activity kit was used to measure the Caspase-3 level in the kidney.Flow cytometry(FCM)and Myeloperoxidase(MPO)kits were used to detect neutrophil infiltration.Enzyme-linked immunosorbent assay(ELISA)of tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)were used to explore serum inflammatory activity.High performance liquid chromatography(HPLC)was used to detect adenosine content in kidney tissue.The expression of hypoxia inducible factor-1?(HIF-1?),CD39 and CD73 in renal tissues were tested by quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot(WB).Results:1.Compared with the Control group,the 7-day survival rate was significantly decreased,Scr and BUN levels were significantly increased(P<0.01),and the degree of renal tubular injury was significantly aggravated(P<0.01)in IR group.2.Compared with the control group,there were no significant differences in 7-day survival rate,Scr and BUN levels,the degree of renal tubular injury,apoptosis of proximal tubular epithelial cells,Caspase-3 activity,neutrophil infiltration,pro-inflammatory cytokines TNF-? and IL-1?,the expression of HIF-1?,CD39,and CD73 at mRNA and protein levels in FA group.3.Compared with IR group,the levels of Scr and BUN in10,30,and 100 mg/kg FA+IR groups were significantly reduced,especially when the dose was 100 mg/kg,Scr and BUN decreased most significantly(P<0.01);the KM survival curve showed that the 7-day survival rate of 10,30,and 100 mg/kg FA+IR groups rised,especially when the dose was 100 mg/kg,the 7-day survival rate was the highest,so this dose was chosen for the later experimental analysis.4.Compared with IR group,the apoptosis of proximal tubular epithelial cells was significantly decreased,and the Caspase-3 activity was significantly reduced(P<0.01),neutrophil infiltration was lowered,and the pro-inflammatory cytokine TNF-? and IL-1? was significantly decreased(P<0.01)in 100 mg/kg FA+IR group.5.Compared with IR group,HIF-1?,CD39,and CD73 were significantly increased in both mRNA and protein levels,and adenosine production was also significantly augmented(P <0.01)in 100 mg/kg FA+IR group.6.Compared with FA(100 mg/kg)+ Ad-RFP + IR group,the protein level of HIF-1?,CD39 and CD73 reduced,adenosine production lessened(P<0.01),the levels of Scr and BUN significantly increased(P<0.01),and renal tubular damage worsened(P<0.01)in FA(100 mg/kg)+ Ad-HIF-1 siRNA + IR group.Conclusion:Ferulic acid could increase the survival rate,improve renal function and allievate renal tubular injury caused by ischemia reperfusion injury in mice.Its possible mechanism is to promote the production of endogenous adenosine by up-regulation HIF-1?,and then reduce the inflammatory response.