Study Of Effect Of Natural Hirudin On Inflammation And Apoptosis In Postoperative Rat Super-long Random Flap And Its Mechanisms

Background: Random pattern skin flaps are commonly used to repair skin defects during plastic and reconstructive surgery, although flap necrosis remains a problem. Many medications, such as sympatholytics, vasodilators, calcium channel blockers, prostaglandin inhibitors, glucocorticoids and free radical scavengers, have been reported to be effective for increasing flap viability [1, 2]. However, these drugs cannot entirely prevent flap necrosis. Additionally, many side effects could occur or are associated with systemic application of these drugs at high doses, which are often required to produce satisfactory effects.Some studies have shown that natural hirudin can increase flap viability in animal experiments. The possible mechanism may involve improving microcirculation and anti-hyperoxidation. However, whether hirudin can influence inflammation and apoptosis following ischaemia and congestion isstill unknown. The aim of the present study was to evaluate the effect of hirudin on survival rate, inflammation and apoptosis in rat random pattern skin flaps and to explore its underlying mechanism.The research can be divided to three parts:Part One ESTABLISHMENT OF AN EXPERIMENTAL MODEL OF RAT DORSAL SUPER-LONG SKIN FLAPObjective: To observe of congestion and necrosis of rat super-long random flaps after operation and the effect of natural hirudin subcutaneous injection on viability of rat super-long flap. Method: Forty-eight healthy SD rats were randomly divided into 2 groups. After the dorsal skin flap operation(3 cm ×10 cm in size), group H(n=24) was administered subcutaneous injections of 6 ATU natural hirudin every 12 h, while group C(n=24) received an equal volume of 0.9% normal saline. Continually observe and record flaps after operation,and calculate flap viabilities on the 7th postoperative day.Result: The survival area of group H is 23.56±0.84cm2,and the survival rate is 78.54±2.80%. the survival area of group C is 19.39±0.96cm2,and the survival rate is 64.62±3.20%. The flap survival rate in group H was higher than that in group C(78.54±2.8% vs. 64.62±3.20%, respectively, p<0.05).Conclusion: The distal of super-long random flap will occur congestion and necrosis after operation. Natural hirudin subcutaneous injection could alleviate congestion and necrosis and increase super-long random pattern skin flap viability.Part Two THE EFFECT OF HIRUDIN ON THE EXPRESSION OF IL-6, TNFα AND ICAM-1 VIA p38 MAPK/IKK/NF-κB PATHWAY IN POSTOPERATIVE CONGESTED FLAPSObjective: The present study aimed to evaluate the effect of natural hirudin on inflammation in rat super-long random skin flap and to determine the mechanism.Method: Full skin samples were collected from rats in the part one during operation and on the 1st, 2nd, 4th, and 7th day after the operation, to observe infiltration of inflammatory mediators in tissue via hematoxylin and eosin(HE)stain and to measure the p38 MAPK, pp38 MAPK, IKK,NF-κB p65, p NF-κB p65, TNFα, IL-6 and ICAM-1 levels via western blot(WB) assays.Result:(1)HE stain:In the normal skin flap, the structure of skin and subcutaneous tissue was normal, interstitial occasionally scattered neutrophil distribution, cytoplasm was pale red or pale purple,and nuclear was round, dark blue or purple. In group C, the rats flap occur edema, gradually increased inflammatory exudation after surgery. On the fourth day, a lot of neutrophils aggregated and infiltrated. In group H, inflammatory exudation and neutrophil aggregation were less than group C.(2)IL-6 level:The IL-6 levels of group C were significantly increased from postoperative day 2 to day 7(p<0.05 vs. day 0). In contrast, there were no significant changes in the IL-6 levels of group H, which were lower than those of group C(p<0.05 vs. group C).(3)TNFα level:After the operation, the TNFα levels of group C were significantly increased(p<0.05 vs. day 0), peaking on postoperative day 4. The TNFα levels were also significantly increased in group H(p<0.05 vs. day 0),although the levels were lower than those of group C(p<0.05 vs. group C).(4)ICAM-1 level:The ICAM-1 level trend in group C was similar to that of TNFα. However, no significant increases in ICAM-1 levels were observed in group H, and the levels were lower than those in group C(p<0.05 vs. group C).(5)pp38/p38:The pp38 MAPK /p38 MAPK levels in group C were significantly increased after the operation(p<0.05 vs. day 0) and peaked on postoperative day 4. A similar trend was observed in group H, although the increase was lower than that in group C(p<0.05 vs. group C).(6)IKK level:After the operation, the IKK levels of group C were significantly increased(p<0.05 vs. day 0). There were no significant changes in the IKK levels of group H, which were lower than those of group C(p<0.05 vs. group C).(7)p NF-κB/NF-κB:The p NF-κB /NF-κB levels in group C were significantly increased starting on postoperative day 1(p<0.05 vs. day 0). In group H, these levels were increased after the operation and were significantly increased on postoperative days 2 and 4. However, no significant differences were observed between postoperative day 0 and day 7. The p NF-κB /NF-κB levels were lower in group H than in group C after surgery(p<0.05 vs. group C).Conclusion:(1)There were inflammation in rat super-long random skin flap after surgery, the possible mechanism maybe that thrombin upregulate TNFα, IL-6 and ICAM-1 expression via the PARs/p38 MAPK /NF-κB pathway.(2) Natural hirudin subcutaneous injection could reduce inflammation in rat super-long random skin flap after surgery, and this function of hirudin is likely mediated by mechanisms that involve the downregulation of TNFα, IL-6 and ICAM-1 expression via the PARs/p38 MAPK/IKK/NF-κB pathway.Part Three THE EFFECT OF HIRUDIN ON THE EXPRESSION OF BAX, BCL-2 AND CASPASE3 AND APOPTOSIS VIA p38 MAPK/IKK/NF-κB PATHWAY IN POSTOPERATIVE CONGESTED FLAPSObjective: The present study aimed to observe apoptosis in rat super-long random skin flap after surgery and evaluate the effect of natural hirudin on apoptosis, and to determine the mechanism.Method: Full skin samples were collected from rats in the part one during operation and on the 1st, 2nd, 4th, and 7th day after the operation, to observe apoptosis in flap tissue via terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate(d UTP) biotin nick end labeling( TUNEL) technique, to measure the Bax and Bcl-2 levels via western blot(WB) assays and to measure the Caspase3 levels via absorptiometry assay.Result:(1) TUNEL: In group C, the amounts of positive cell were significantly increased from postoperative day 1 to day 7(p<0.05 vs. day 0). In group H, there were no significant changes in postoperative day 1 to day 2, but there were significantly increases from postoperative day 4 to day 7. The amounts of positive cell in group H were less than in group C after surgery(p<0.05 vs. group C).(2)Bax level:The Bax levels of group C were significantly increased from postoperative day 2 to day 7(p<0.05 vs. day 0). While The Bax levels of group H were significantly increased from postoperative day 4 to day 7(p<0.05 vs. day 0), which were lower than those of group C from postoperative day 2 to day 7(p<0.05 vs. group C).(3)Bcl-2 level: There were no significant changes in the Bcl-2 levels of group C after operation(p>0.05 vs. day0).The Bcl-2 levels of group H weresignificantly increased from postoperative day 4 to day 7(p<0.05 vs. day 0), which were lower than those of group C on postoperative day 4 and day 7(p<0.05 vs. group C).(4)Bax/Bcl-2 :The Bax/Bcl-2 levels of group C were significantly increased from postoperative day 2 to day 7(p<0.05 vs. day 0). In contrast, the Bax/Bcl-2 levels of group H were significantly decreased from postoperative day 1 to day 2(p<0.05 vs. day 0). But the Bax/Bcl-2 levels of group H were significantly increased from postoperative day 4 to day7(p<0.05 vs. day 0). The Bax/Bcl-2 levels of group H were always lower than those of group C after surgery(p<0.05 vs. group C).(5)Caspase3 activity:The Caspase3 activity of group C were significantly increased from postoperative day 1 to day 7(p<0.05 vs. day 0), peaking on postoperative day 4. The Caspase3 activity of group H were also significantly increased from postoperative day 1 to day 7(p<0.05 vs. day 0), peaking on postoperative day 7. The Caspase3 activity of group H were always lower than those of group C after surgery(p<0.05 vs. group C).Conclusion:(1)There were apoptosis in rat super-long random skin flap after surgery, the possible mechanism maybe that thrombin upregulate Bax expression via the PARs/p38 MAPK/IKK/NF-κB pathway, and then enhance Caspase3 activity.(2) Natural hirudin subcutaneous injection could reduce apoptosis in rat superlong random skin flap after surgery, and this function of hirudin is likely mediated by mechanisms that involve the downregulation of Bax expression via the PARs/p38 MAPK/IKK/NF-κB pathway, and then attenuate Caspase3 activity.