Protective Effect Of Ischemic Postconditioning On Flap Ischemia And Reperfusion Injufy And The Mirna Based Mechanism

Background:Free skin flap grafting is an important and regular surgical operation for wound repair and reconstruction.Ischemia and reperfusion injury?IRI?is an inevitable pathophysiological process during microsurgical free tissue transfer,especially when a vascular crisis occurs and a secondary surgical exploration is required.It seriously threatens the graft survival.Recently,ischemic postconditioning?IPo?and remote ischemic postconditioning?RIPo?have become a hot point in basic and clinical research for IRI.Protective effect of IPo and RIPo has been confirmed in the basic researches on IRI models of heart,brain,spinal cord,lung and kidney,and it has been partly applied in clinical prevention and treatment for ischemic heart disease.However,the mechanism of IPo and RIPo is still unclear.In the past few years,miR-21 has been discovered as an important miRNA with protective effect against IRI.Previous researches have proposed that hypoxia-inducible factor-1a?Hif-1a?might promote the cellular expression of miR-21 in the early phase of hypoxia,which can repress pro-apoptotic gene expression through complementarity to the 3'untranslated regions?3'-UTRs?of mRNAs.Objective:First,we aim to investigate the protective effect of IPo and RIPo against IRI,and their effect on the expression level of Hif-1a and a group of ischemia-related miRNAs,in a rat abdominal skin flap model.Second,we aim to investigate the anti-apoptotic effects of miR-21 on primary rat vascular smooth muscle cells?VSMCs?,and its regulation of cellular expression of PTEN in a hydrogen peroxide?H₂0₂?-induced oxidative stress damage cell model.Third,we aim to investigate the expression level of a group of ischemia-related miRNAs in human I/R flap samples.Methods:1.Rat abdominal skin flap model:?1?The superficial inferior epigastric artery flap I/R model was constructed.Fifty SD rats were randomly divided into 5groups according to ischemic duration?0h,4h,6h 8h,12h?;?2?Thiobarbituric acid?TBA?method and xanthine oxidase method were used to determine free malondialdehyde?MDA?content and superoxide dismutase?SOD?activity in the tissues after 24-hour reperfusion;?3?The flap survival region was measured and the survival rate of each group was calculated one week after reperfusion;?4?The expression level of Rno-miR-1,Rno-miR-133,Rno-miR-210,Rno-miR-21,Rno-miR-200c and Rno-miR-92a in flap and vascular tissues were detected by real-time reverse transcription-polymerase chain reaction?real-time RT-PCR?,after2-hour,12-hour,and 24-hour reperfusion;2.In vivo experiment:?1?The superficial inferior epigastric artery flap I/R model was constructed,and 40 SD rats were randomly divided in to Sham group,negative control?NC?group,ischemic postconditioning?Isc?group,and remote ischemic postconditioning?Rem-Isc?group;?2?Thiobarbituric acid?TBA?method and xanthine oxidase method were used to determine free malondialdehyde?MDA?content and superoxide dismutase?SOD?activity in the tissues after 24-hour reperfusion;?3?Immunohistochemistry?IHC?was used to detect Hif-1a protein expression in the flap tissues after 72-hour reperfusion;?4?Western Blot was used to detect Hif-1a protein expression in the vascular tissue after 3-day and 7-day reperfusion;?5?The expression level of Rno-miR-21 and Rno-miR-200c were detected by real-time RT-PCR,after 2-hour,12-hour,and 24-hour reperfusion;?6?The flap survival region was measured and the survival rate of each group was calculated one week after reperfusion;3.In vitro experiment:?1?A H₂0₂-induced oxidative cell damage model was constructed in rat vascular smooth muscle cells?VSMCs?,which were primarily cultured using explants-attached method.?2?VSMCs were randomly divided into NC group;H₂0₂group;miR-21 mimics+H₂0₂ group;NC₁ mimics+H₂0₂ group;miR-21 inhibitor+H₂0₂ group;NC₂+H₂0₂ group;miR-21 mimics group;NC₁ mimics group;miR-21inhibitor group;NC₂ inhibitor group;The VSMCs were transfected with miR-21mimics and inhibitors using lipofectine2000?;?3?The viability of VSMCs was detected by CCK-8 test at time-point of 24 hours,and 48 hours;?4?The cellular protein expression of PTEN,Pre-CASP3,Pre-CASP3 were detected using Western Blot at time-point of 48 hours;?5?The cellular protein expression of a-actin was detected using Immunofluorescence?IF?at time-point of 24 hours;?6?The apoptosis rate of VSMCs was detected by PI/HO staining at time-point of 24 hours;4 Clinical data analysis of the patients with free flap breast reconstruction:?1?126 women undergoing breast reconstruction with free DIEP flap were asked to complete the Rosenberg Self-Esteem Scale,the Patient Health Questionnaire Depression Scale,the Generalized Anxiety Disorder 7-item Scale and were followed and reassessed with the same questionnaires at 6 months postoperatively;?2?Nonischemic flap tissues,flap tissues after 1.5 h-ischemia,and flap tissues after 1.5 h-reperfusion of 6 patients undergoing breast reconstruction with free DIEP flap were collected and the expression level of miR-210,miR-21,miR-200c and miR-92a in the tissues were detected by real-time RT-PCR.Results:1.Rat abdominal skin flap model:?1?At seven days after reperfusion,the average flap survival rate of 0h ischemia group,4h ischemia group,6h ischemia group,8h ischemia group,12h ischemia group were 98.1±1.6%,92.6±7.4%,87.1±14.0%,54.6±13.4%and 24.2±11.1%,respectively.Analysis of variance prompted significant difference?P<0.05?in the five groups;?2?At 24 hours after reperfusion,the tissue MDA content of 0h ischemia group,4h ischemia group,6h ischemia group,8h ischemia group,12h ischemia group were 38.0±5.8 nmol/g,72.9±17.0 nmol/g,103.8±10.9 nmol/g,171.7±4.7 nmol/g and 165.1±10.5 nmol/g,respectively,and the tissue SOD activity were 93.2±12.8 U/g,76.9±9.9 U/g,56.7±7.3 U/g,37.6±6.0 U/g and 26.1±6.5 U/g,respectively.Analysis of variance prompted significant difference?P<0.05?in the five groups;?3?The expression level of Rno-miR-21and Rno-miR-200c in flap and vascular tissues were significantly up-regulated at 2 hours,12 hours and 24 hours after reperfusion,compared to before ischemia;.2.In vivo experiment:?1?Compared to NC group,Isc group and Rem-Isc group showed a significantly higher flap survival rate?P=0.034,P=0.049,respectively?at seven days after reperfusion;?2?Compared to NC group,Isc group and Rem-Isc group showed a significantly lower level of MDA content?P=0.020,P<0.01 respectively?and a significantly higher level of SOD activity?P=0.011,P=0.014,respectively?in flap tissues;?3?Compared to NC group,IHC evaluation revealed higher expression level of Hif-1a in flap tissue in Isc group and Rem-Isc group,at 72 hours after reperfusion;?4?Compared to NC group,Western Blot evaluation revealed higher expression level of Hif-1a in vascular tissue after 3-day and 7-day reperfusion,in Isc group and Rem-Isc group?Isc vs.NC,P<0.01,P<0.01,respectively;Rem-Isc vs.NC,P<0.01,P<0.01,respectively?;?5?Compared to NC group,the expression level of miR-21 in flap tissue was up-regulated at 12 hours and24 hours after reperfusion,in Isc group and Rem-Isc group?Isc vs.NC,P=0.049,P=0.032,respectively;Rem-Isc vs.NC,P=0.045,P=0.007,respectively?,and the expression level of miR-21 in vascular tissue was up-regulated at 12 hours and 24hours after reperfusion,in Isc group and Rem-Isc group?Isc vs.NC,P=0.02,P<0.01,respectively;Rem-Isc vs.NC,P<0.01,P<0.01,respectively?;Compared to NC group,the expression level of miR-200c in vascular tissue was up-regulated 12hours in Isc group?P=0.01?;3.In vitro experiment:?1?Compared to NC group,the expression level of?-actin in VSMCs in the H₂0₂ group was significantly down-regulated?P<0.01?.Compared to NC₂+H₂0₂ group,the cellular expression level of?-actin in VSMCs in miR-21 inhibitor+H₂0₂ group was significantly lower?P=0.049?;?2?Compared to NC group,the cellular protein expression of Pro-CASP and cleaved CASP3 was significantly higher in H₂0₂ group?P=0.011,P<0.01?;Compared to NC₁+H₂0₂ group,the cellular protein expression of cleaved CASP3was significantly lower in miR-21 mimics+H₂0₂ group?P<0.01?,and the expression of PTEN was significantly lower?P<0.01?;Compared to NC₂+H₂0₂group,the cellular protein expression of cleaved CASP3 was significantly higher in miR-21 inhibitor+H₂0₂ group?P=0.027?,and the expression of PTEN was significantly higher?P<0.01?;?3?The CCK-8 evaluation revealed higher cell activity in NC group than H₂0₂ group at time-point of 24 hours and 48 hours?P<0.01,P<0.01?;The CCK-8 evaluation revealed higher cell activity in miR-21mimics+H₂0₂ group than NC₁ mimics+H₂0₂?P=0.013?,and higher cell activity in inhibitors NC₂ inhibitor+H₂0₂ group than miR-21 inhibitor+H₂0₂ group?P=0.016?at time-point of 48 hours;?4?Compared to NC group,the cell apoptosis rate in the H₂0₂group was significantly higher at time-point of 24 hours?P<0.01?;Compared to NC₁+H₂0₂ group,the cell apoptosis rate in the miR-21 mimics+H₂0₂ group was significantly lower at time-point of 24 hours?P=0.048?;4.Clinical data analysis of the patients with free DIEP flap breast reconstruction:?1?Compared to preoperatively,BR patients scored significantly higher for RSES?P<0.01?and scored significantly lower for PHQ-9?P<0.01?,but the sores for GAD-7 was not significantly changed;The average Alderman scores self-assessed by the patients undergoing DIEP free flap breast reconstruction at 6 months postoperatively was 27.6±6.3;?2?The Alderman scores had linear positive correlation with postoperative GAD-7 scores?P<0.01?,postoperative reduction of GAD-7 scores?P<0.01?,postoperative RSES scores?P<0.01?and postoperative increase in RSES scores?P<0.01?;?3?Compared to nonischemic flap tissues,flap tissues after 1.5 h-reperfusion showed significantly higher level of miR-21?P=0.02?and miR-200c?P=0.03?,while the expression level of miR-210 and miR-92a was not significantly changed.Conclusion:?1?Ischemic postconditioning and remote ischemic postconditioning reduced the tissue damage caused by oxygen free radicals after flap I/R injury,and increased the survival rate of flap;?2?The up-regulation of miR-21 level and protein expression level of Hif-1a may play a role in the mechanism of RIP and RIPo protecting flap tissues against I/R injury;?3?H₂0₂ could mimic the oxidative stress damage in VSMCs by up-regulating cellular MOD level,down-regulating cellular SOD activity,reducing cell activity and increasing cell apoptosis;?4?Over-expression of miR-21 in VSMCs could increase cell activity,and decrease cell apoptosis induced by H₂0₂,probably by directely targeting PTEN.