| Objective: To investigate the optimal treatment window of thioredoxin in flap ischemiareperfusion injury,and to provide a reference for the timing of drug therapy for ischemiareperfusion injury with similar mechanisms.Methods:1、Sixty-three Sprague-Dawley male rats were randomly divided into six treatment groups,including blank control group A,group B,group C,group D,group E,group F and group G,according to the relationship between the reperfusion time after ischemia(the time when microvascular clamps are placed at the blood supply artery)and the time of intraperitoneal injection of thioredoxin.The treatment group was intraperitoneally injected with thioredoxin(20μg/kg)solution 12 h and 4 h before node and 4 h and 36 h after node.2、Using male Sprague-Dawley rats(SD rats),the 6 cm×3 cm surgical area was marked and hair removal was removed,and the blood supply arteriovenous was located from the body surface,that is,the skin of the marked site was separated from the hypogastric wall arteriovenous position,and the blood supply arteriovenous was clamped with a microvascular clamp,the clamping time was recorded,the skin was sutured,and the vascular clamp was placed outside the body,and the ischemia-reperfusion injury model could be successfully established by removing the vascular clip after 4 hours.3、48 hours after reperfusion,we randomly took 3 rat from each group,and after sacrifice,cut a 2cm×2cm full-thickness island flap near the clamping position,that is,near the root position of the arteriovenous clamping position under the abdominal wall,froze it in the-80 °C refrigerator,and then extracted the tissue protein solution by grinding,centrifugation and Western blot protein electrophoresis to detect the expression of thioredoxin.4、Then,3 rat were randomly selected in each group to cut the skin at the 2cm×2cm size ischemia reperfusion site near the clamping position,divided into 4 equal parts,fixed in the specimen fixative solution,and then paraffin embedding and section staining were carried out to make hematoxylin-eosin and IL-1 immunohistochemical stained tissue sections.5、The remaining rats in each group were photographed 7 days after ischemiareperfusion treatment,and the remaining rats in each group were sacrificed.Results:There were significant differences in flap necrosis rate and pathological manifestations of flap tissue in different treatment groups.The average flap necrosis rate of rats in the control group was 27.50%,the average flap necrosis rates of intraperitoneal thioredoxin injection 4 h and 12 h before ischemia-reperfusion were 5.36% and 17.65%,and the average flap necrosis rates of intraperitoneal thioredoxin injection 0h,4h,12 h and 36 h after ischemiareperfusion were 4.76%,2.61%,7.14% and 9.26%,respectively.The inflammatory cell counts of rats in the control group were 79(175.3)/200 x HPF,the inflammatory cell counts of rats injected intraperitoneally with thioredoxin 4 h and 12 h before ischemia-reperfusion were 21.5(13)/ 200 x HPF and 54.5(36)/ 200 x HPF,respectively,and the inflammatory cell counts of intraperitoneally injected thioredoxin in 0h,4h,12 h and 36 h after ischemiareperfusion were 10.5(5.5)/200 x HPF,respectively.0.5(2.3)/200 x HPF,6.5(7.8)/200 x HPF,34(22)/200 x HPF,and the difference between groups was statistically significant(P <0.001);The neovascular counts of rats in the control group were 0(0)/200 x HPF,the neovascular counts of rats injected intraperitoneally 4 h and 12 h before ischemia-reperfusion were 2(2)/200 x HPF and 0.5(2.3)/200 x HPF,respectively,and the neovascular counts of rats injected intraperitoneally with thioredoxin at 0h,4h,12 h,and 36 h after ischemiareperfusion were 3.5(4.5)/200 x HPF and 4.5(2.3),respectively.200 x HPF,0.5(2.3)/200 x HPF,0(1.3)/200 x HPF,the differences between groups were statistically significant(P<0.001);The fused necrotic adipocytes counts of rats in the control group were 14.5(10)/200 x HPF,the fused necrotic adipocytes counts of intraperitoneally injected thioredoxin 4 h and 12 h before ischemia-reperfusion were 5(4.8)/200 x HPF and 9(3)/200 x HPF,respectively,and the fused necrotic adipocytes of rats injected intraperitoneally at 0h,4h,12 h,and 36 h after ischemia-reperfusion were 2.5(4.3)/200 x HPF,respectively.0(1.3)/200 x HPF,8.5(5.8)/200 x HPF,9.5(3)/200 x HPF,the difference between groups was statistically significant(P<0.001).Conclusions:Thioredoxin reduces the necrosis rate of ischemia-reperfusion flap,reduces inflammation,inhibits cell necrosis,and improves blood perfusion,and its optimal therapeutic window within 4 hours after ischemia-reperfusion. |
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