| Objective:Free flap grafting is one of the important surgical methods in the treatment of skin and soft tissue repair in clinical practice.Although microsurgery technique and equipment have greatly improved,there are still flap necrosis in many cases.Ischemia reperfusion injury(IRI)is an important cause of flap failure.Proliferation of vascular endothelial cells is regarded to act protective effects against IRI.Irisin has been proved to be an agent that could facilitate proliferation of endothelial cells.In the present study,both clinical and experimental studies were carried out to preliminarily verify the protective effect of endogenous and exogenous irisin against IRI of flaps.We also investigated its relationship with vascular endothelial cell proliferation and underlying mechanisms and path.It may provide theoretical basis for the clinical use of irisin in relieving IRI of flaps in future.Methods:1.Clinical study(Part I):(1)Patients who received transplantation of anterolateral femoral skin flap in the treatment of skin and soft tissue defect were included in the study.Indices of the patients,including sex,age,height,weight,waist,hip were recorded.The activity intensity,activity time,skeletal muscle mass,and metabolic indices were also recorded.(2)The serum concentration of irisin were measured before and on the 1st,3rd,and 7th day after the surgery.Blood flow of the flaps were measured before pedicle division,immediately after recanalization and on the 1st,3rd,and 7th day after the surgery,respectively.Association of blood flow of the flap and related indices were analyzed.Relationship between blood flow recovery rate and irisin increase rate were also analyzed.This part of study aimed at revealing the relationship between endogenous irisin and IRI of flaps.2.Experimental study(Part II):(1)A total of 150 female Sprague Dawley(SD)rats were randomly assigned to 5 groups(30 rats in each group).Group A(Saline group):flap IRI+saline administration;Group B(Low dose irisin group):flap IRI+irisin(1ng/g);Group C(Medium dose irisin group):flap IRI+irisin administration(2ng/g);Group D(High dose irisin group):flap IRI+ irisin administration(4ng/g);Group E(Sham surgery group):flap dissociation+saline administration.(2)The following measures were conducted before and on the 1st,3rd,5th,and 7th day after the surgery:surviving area of the flap,blood flow perfusion of the flap,flap angiography,HE staining(microvessel),immumohistochemical staining(ErG),Western blot(p-Akt/Akt),RT-PCR(CCNB?CCND1?CCN1?PLK?BuB?Ki67?MYBL2 gene expression).The surviving rate of flap,blood flow recovery,vessels distribution,microvessel proliferation,vascular endothelial cell proliferation,PI3K/Akt signal pathway activation and endothelial cell proliferation related gene expression were analyzed.This part of study aimed at exploring the relationship between exogenous irisin and IRI of flaps.Results:1.Clinical study(Part I):(1)A total of 40 patients were recruited in the study,including 30 men and 10 women.They were 18-56 years old,averaged 35.9±11.2.The average Body Mass Index(BMI)was(22.8±4.8)kg/m2,Waist Hip Rate(WHR)was 0.84±0.07 and skeletal muscle mass was(25.32±4.82)kg.The triglycerides(TG)was(1.16±0.44)mmol/L,total cholesterol was(4.16±0.92)mmol/L,low density lipoprotein cholesterin(LDC)was(1.49±0.93)mmol/L,high density lipoprotein cholesterin(HDL-C)was(1.41±0.35)mmol/L,fasting blood-glucose(FBG)was(5.38±1.11)mmol/L,and glycosylated hemoglobin(HbA1c)was(5.28±0.89)%.(2)During the 7 days after flap IRI,the blood flow generally increased.The blood flow recovery rate was(51.7±50.7)%on the 7th day after the flap grafting surgery.The serum concentration of irisin also significantly increased after the surgery,peaked on the 3rd day and slightly declined on the 7th day.(3)No significant difference in the irisin level was observed between the male and female groups.The irisin concentration was negatively related with age.(4)Medium and high intensity of physical activity were related with higher level of irisin.The time of physical activity did not influence the irisin level greatly.(5)The endogenous irisin level was negatively related with BMI and WHR,and positively related with skeletal muscle mass.(6)The irisin level was negatively related with FBG and HbA1c,but not significantly related with TG,TC,LDL-C or HDL-C.(7)The irisin level before surgery was positively related with blood flow recovery rate on the 7th day after the surgery.The blood flow recovery rate on the 1st and 3rd days after the surgery was positively related with the irisin increase rates.On the 7th day after the surgery,there was no significant relationship between the blood flow recovery rate and irisin increase rate.2.Experimental study(Part II):(1)Surviving area of the flap:the necrosis area was greatest in Group A and smallest in Group E.In the irisin groups(Groups B,C and D),the necrosis area was significantly greater than those in Groups B and D.No significant difference was found between Groups B and D.(2)Blood flow perfusion:After the surgery,the blood flow of ROIs in the angiosome zones of deep circumflex iliac,posterior intercostal and thoracodorsal vessels were tested.The blood flow was highest in Group E.Since the 5th day after the surgery,blood flow in Group C improved significantly and were all higher than that in Groups A,B and D.(3)Angiography:The vascular imaging was best in Group E on the 7th day and worst in Group A.Angiography in Groups B,C and D was between Groups A and E.Compared with Group A,the deep circumflex iliac arteries in Group B,C and D were significantly greater in length,and higher in quantity,especially in the distal area of the flap.And more micovessels were observed in the choke zones in Group B,C and D.(4)HE staining:On the 7th day after the surgery,the density of microvessels in the choke zones were highest in Group E among the 5 groups.The densities of microvessels in Groups B,C,and D significantly increased on the 5th day,and were higher than that in Group A.However,no significant difference was found between Groups D and A on the 7th day.(5)Immunohistochemical staining:The ErG expression in the choke zone was highest in Group E.Since the 5th day after the surgery,the expression of ErG in the choke zones in Groups B,C,and D significantly increased and higher than that in Group A.But no significant difference was found between Groups D and A.(6)Western blot testing:On the 1st and 3rd days after the surgery,no significant difference was found among the 5 groups in the level of p-Akt/Akt ratio.On the 5th and 7th days,the p-Akt/Akt ratios in Groups B,C,and D were significantly higher than that in Groups A and E.On the 7th day,the p-Akt/Akt ratio in Group C was higher than that in Group D.No significant differences were found either between Groups B and D or between Groups A and E.(7)RT-PCR testing:The expression of proliferation related genes in Group C was the highest in the 5 groups,including CCNB,CCND1,CCN1,PLK,BuB,Ki67,and MYBL2.The differences between Groups A,B,D,E were without significant regulation.Conclusion:(1)Endogenous irisin was related with relieve of IRI following free flaps grafting and improvement of blood flow perfusion.(2)In IRI flap model,administration of exogenous irisin could improve blood supply and increase the surviving area of the flap.The underlying mechanism was related with facilitation of vascular endothelial cell proliferation. |
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