Protective Effect And Possible Mechanism Of Astaxanthin On Renal Ischemia-reperfusion Injury

Objective:1.To verify the protective effect of astaxanthin?ATX?against renal ischemia-reperfusion injury.2.To explore the mechanism of ATX's protective effect in renal ischemia reperfusion?RIRI?injury.Methods:Research in vitro:1.H₂O₂ toxicity research is done to select appropriate H₂O₂ concentration.We preprocessed HK2 cells with different ATX concentration for 24 hours.And then the H₂O₂ would be added for 2hours.CCK-8?Annexin V-FITC/PI flow cytometry were used to measure the cell vitality and to certificate the RIRI protective effect of ATX.2.Cell model was divided into four groups containing control?ATX?H₂O₂?H202+ATX group.MiRNA array was used to select different expression between control group and ATX group.Real-time PCR and western blot were used to explore the relationship between miRNA 221-3p and PERK-CHOP signal pathway.Dual luciferase activity was performed to identify the direct regulation of mi221-3p on PERK.3.After transfection of miRNA 221-3p mimic or inhibitor into HK2 cell,we process the HK2 with H₂O₂.And the cell vitality was detected by CCK-8?Annexin V-FITC/PI flow cytometry.The expression of PERK?p-elf2??ATF4?CHOP was detected as above mentioned.Research in vivo:1.32 male B6 mice were divided into four groups:the sham group?ATX group?RIRI group?RIRI+ATX group.Mice serum and renal were collected 24h post RIRI.The level of Cr and BUN were detected to evaluate the mice renal function.SOD and MDA were used to measured the degree of RIRI.Mice renal cell apoptosis was assessed by HE and Tunel staining.The expression of miRNA221-3p?PERK?eIF2??p-eIF2??CHOP in renal was detected by real-time PCR and western blot.2.24 male B6 mice were divided into four groups:sham group?agomir group?RIRI group and RIRI+agomir group.Caliper IVIS Lumina ? was used to measure the accumulation of miR221-3p agomir in mice kidney.Cr?BUN?SOD?MDA were also detected.HE and Tunel staining were also used to assess the apoptosis of mice renal cell.The expression of miRNA221-3p?PERK?eIF2??p-eIF2??CHOP in renal was similarly detected by real-time PCR and western blot.Results:1.ATX + H₂O₂ group had higher cell viability than H₂O₂ group?P<0.05?.2.The expression of PERK and CHOP in ATX + H₂O₂ group was lower than that in H₂O₂ group,and the expression of miRNA221-3p was increased in H₂O₂ group compared with control group and ATX group?P<0.05?.3.HK2 was transfected with miRNA 221-3p mimic and then treated with H₂O₂,their cell viability was higher than that treated with H₂O₂ only?P<0.05?.But,the cell viability of HK2 which treated was treated with H₂O₂ only was lower than that transfected with miRNA 221-3p inhibitor?P<0.05?.4.PERK?CHOP expression of HK2 which was transfected with miRNA 221-3p mimic and then treated with H₂O₂,was significantly lower than that treated with H₂O₂ only?P<0.05?.But,the PERK?CHOP expression of HK2 which treated was treated with H₂O₂ only was lower than that transfected with miRNA 221-3p inhibitor?P<0.05?.5.In vivo research,Cr,BUN and PERK?CHOP expression were higher in the RIRI group than in the sham and ATX group,and the histological manifestations were more severe?P<0.05?.Compared with IR group,the levels of Cr and BUN decrease and PERK?CHOP expression in RIRI + ATX group were lower than those in IR group?P<0.05?.6.In the animal model of miR221-3p agomir,Cr,BUN and PERK?CHOP expression were higher in the RIRI group than in the sham and agomir group,and the histological manifestations were more severe?P<0.05?.the levels of Cr and BUN decreased,PERK?CHOP expression in RIRI + agomir group was lower than that in RIRI group?P<0.05?.Conclusion:1.ATX plays a protective role in oxidative stress injury which simulated with H₂O₂ and mouse renal ischemia-reperfusion injury,which can effectively reduce the degree of oxidative stress injury.2.PERK-CHOP signaling pathway plays an important role in renal reperfusion injury and regulates apoptosis.While ATX can attenuate the PERK-CHOP signal pathway.3.miRNA221-3p alleviates renal ischemia-reperfusion injury by down-regulating the downstream target gene PERK,which may be one of the mechanisms of ATX on protecting renal ischemia-reperfusion injury.