The Investigation Of Natural Hirudin Against Ischemic Injury In Rat Ischemic Flap And Against Human Microvascular Endothelial Cells Apoptosis

Random skin flap is a flap that does not contain axial blood vessels,but only the dermal vascular network and subdermal vascular network for blood supply.Over dimensioned random flaps refer to excessive ratio of length to width(length:width>3:1).If the length-to-width ratio of the flap exceeds 3:1,the distal tissue of the flap will be congested and eventually lead to necrosis of the flap.The demand for over dimensioned random skin flap is increasing because of the irregular wound surface in clinical practice.However,the use of over dimensioned random flaps in clinic is limited because of the necrosis of the flap caused by venous congestion.The living leech therapy is to place the leech on the congested flap,through the leech sucking effect and secretion of anticoagulant substances to make the flap stasis blood discharge,preventing the blood coagulation to maintain blood flow smooth.Living leech therapy is considered as the standard therapy to treat the complications of venous congestion of flaps,however its popularization is limited due to the living habits of living leech.Living leeches carry many kinds of bacteria,which limits their clinical application.Natural hirudin is a specific thrombin antagonist which plays an anticoagulant role in living leeches.In our previous study,many factors may influence the skin flap viability after operation,such as hypoxia,ischemia,thrombin increasing,inflammatory cascade reaction and so on.Hypoxia leads to inflammation and oxidative stress in endothelial cells,which result in the endothelial injury.Apoptosis plays a key role in post-ischemic angiogenetic pathogenesis.The natural hirudin was applied directly to the congested flap of rats,and it was found that it could improve flaps survival,which is related to the anti-inflammatory effect,antioxidant effect and angiogenesis effect of natural hirudin,however,the mechanisms underlying the anti-apoptotic effects of natural hirudin are not clear.JAK/STAT is a family of intracellular signal transduction pathways that have been studied a lot in recent years.The JAK/STAT pathway is closely related to inflammatory response,oxidative stress,cell injury and apoptosis,among which JAK2/STAT1 signaling pathway is related to apoptosis.In this study,we will study the effect of natural hirudin on postoperative skin flap apoptosis and JAK2/STAT1 pathway through in vivo and in vitro experiments.In vitro experiments were conducted to further study the effect of natural hirudin on antagonizing thrombin on apoptosis of vascular endothelial cells and the effect of natural hirudin on apoptosis of human microvascular endothelial cells induced by ischemia and hypoxia.Part 1:The Investigation of Natural Hirudin Against CongestedInjury in Rat Ischemic FlapObjective:In this part of the study,we will explore the effect of natural hirudin on apoptosis of cells in rat over dimensioned random flap and the possible signal pathways involved.Method:Healthy SD rats were selected in the experiment,which were male or female,with a weight of 250-300g.Twenty-four SD rats were randomly divided into control group(group Control,n=12)and natural hirudin group(group Hirudin,n=12).An over dimensioned random flap was established on the back of the rat.After the dorsal over dimensioned random skin flap operation(2.5cm×9cm in size),In group hirudin,4 ATU natural hirudin were injected subcutaneously every day,and in group control,0.9%normal saline was injected in the same parts in the same way every day.The skin flap tissue samples were collected on the first day and the fifth day after surgery,and the apoptosis of the skin flap tissue was observed via teminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate(dUTP)biotin nick end labeling(TUNEL)technique,to observe the expression of phosphorylation JAK2(P-JAK2)and phosphorylation STAT1(P-STAT1)in the skin flap mediated immunohistochemistry,to mesasure the P-JAK2 and P-STAT1 levels via western blot(WB)assays.Results:1)TUNEL:The amounts of positive cell in group hirudin were less than in group control in postoperative day 1 to day5.In group hirudin,the amounts of positive cell were significantly increased in postoperative day 5(P<0.05 vs.day 1).In group control,the amounts of positive cell were significantly increased in postoperative day 5(P<0.05 vs.day 1).The amounts of positive cell in group hirudin were less than in group control after surgery(P<0.05 vs.group Control).2)Immunohistochemical analysis:At postoperative day 1 and day 5,the expression levels of phosphorylation JAK2 and phosphorylation STAT1 in the flap tissue were higher in group control than the group hirudin(P<0.05).3)Phosphorylation JAK2 level:The phosphorylation JAK2 levels of group hirudin were significantly increased in postoperative day 5(P<0.05 vs.day 1).The phosphorylation JAK2 levels of group control were significantly increased from postoperative day 1 to day 5,and the phosphorylation JAK2 levels of group control were increased in postoperative day 5 than that in postoperative day 1(P<0.05).The phosphorylation JAK2 levels of group hirudin were always lower than those of group control after surgery(P<0.05 vs.group control).4)Phosphorylation STAT1 level:The phosphorylation STAT1 levels of group hirudin were significantly increased in postoperative day 5(P<0.05 vs.day 1).The phosphorylation STAT1 levels of group control were significantly increased from postoperative day 1 to day 5,and the phosphorylation STAT1 levels of group control were increased in postoperative day 5 than that in postoperative day 1(P<0.05).The phosphorylation STAT1 levels of group hirudin were always lower than those of group control after surgery(P<0.05 vs.group control).Conclusion:(1)Apoptosis was observed after rat skin flap operation,the possible mechanism maybe that thrombin or ischemic hypoxia promote the increase of P-JAK2 ang P-STAT1 via the JAK2/STAT1 pathway,and then enhance the cells apoptosis.(2)Natural hirudin subcutaneous injection could reduce apoptosis in rat over dimensioned random skin flap after surgery.The mechanism maybe that natural hirudin antagonized thrombin and blocked the JAK2/STAT1 pathway,thus slowing down the aggravation of apoptosis.Or the mechanism maybe that natural hirudin could induce the changes of P-JAK2 and P-STAT1 levels in ischemic and hypoxic tissues,thus slowing down the increase of apoptosis.Part2:Effect of natural hirudin in antagonisting thrombininduced apoptosis of human microvascular endothelial cells under the normal conditionObjective:To investigat the influences of natural hirudin on the apoptosis of human microvascular endothelial cells induced by thrombinMethods:Human microvascular endothelial cells in logarithmic growth period were used.The cultured human microvascular endothelial cells were divided into eight groups:nor group(cultured in normal condition),thrombin group(group T,2U/ml thrombin),natural hirudin group(group H,2ATU/ml natural hirudin),thrombin and natural hirudin group(group T+H,2U/ml thrombin+2ATU/ml natural hirudin),AG490 group(group AG490,50 μmol/L AG490),thrombin and AG490 group(group T+AG490,2U/ml thrombin+50μmol/L AG490),natural hirudin+AG490 group(group H+AG490,2ATU/ml natural hirudin+50 μmol/L AG490),thrombin and natural hirudin and AG490 group(group T+H+AG490,2U/ml thrombin+2ATU/ml+natural hirudin+50 μmol/L AG490).Each group was added to the corresponding drug intervention for 24 hours,and then the corresponding indicators were detected.Cells apoptosis was detected by Tunel in each group.The expression of P-JAK2 in each group was detected by cellular immunofluorescence assay.Results:When the culture medium contained 2U/ml thrombin,the apoptotic rate of human microvascular endothelial cells was 23.15+0.92%.Adding natural hirudin into the culture medium can reduce apoptosis induced by thrombin(P<0.05).Thrombin could stimulated the phosphorylation of JAK2.However 50 mol/L of AG490 reatment could suppresses apoptosis of human microvascular endothelial cells induced by thrombin,while the apoptosis rate of T+H group was lower than that of T+AG490 group and was not statistically significant compared with T+H+AG490.Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding.Conclusion:Under normal conditions,thrombin can stimulate the phosphorylation of JAK2 and increase the apoptosis of human microvascular endothelial cells.Natural hirudin may exerts its anti-apoptotic effect via inhibits the thrombin-induced JAK2/STATs under normal condition.It seems to exist in parallel with other pathways.Part3:Effect of natural hirudin in antagonisting thrombininduced apoptosis of human microvascular endothelial cells under the hypoxia conditionObjective:The present study aimed to investigate whether natural hirudin can protect cells and reduce the rate of apoptosis under hypoxia condition.Methods:Human microvascular endothelial cells in logarithmic growth period were used.The cultured human microvascular endothelial cells were divided into eight groups:control group(group NO,cultured with nothing),thrombin group(group T,2U/ml thrombin),natural hirudin group(group H,2ATU/ml natural hirudin),thrombin and natural hirudin group(group T+H,2U/ml thrombin+2ATU/ml natural hirudin),AG490 group(group AG490,50 pmol/L AG490),thrombin and AG490 group(group T+AG490,2U/ml thrombin+50μmol/L AG490),natural hirudin+AG490 group(group H+AG490,2ATU/ml natural hirudin+50 pmol/L AG490),thrombin and natural hirudin and AG490 group(group T+H+AG490,2U/ml thrombin+2ATU/ml+natural hirudin+50 μmol/L AG490).Each group was added to the corresponding drug intervention for 24 hours under the hypoxia condition,and then the corresponding indicators were detected.Cells apoptosis was detected by Tunel in each group.The expression of P-JAK2 in each group was detected by cellular immunofluorescence assay.The phosphorylation levels of JAK2 and STAT1 were detected by Western Blot.Results:The apoptosis rate of human microvascular endothelial cells in the hypoxic control group was 43.93±0.44%.When 2U/ml thrombin was added into the culture medium,the apoptosis rate was significantly higher than that in the hypoxic control group(P<0.05).When 2U/ml natural hirudin was added into the culture medium,the apoptosis rate of cells was not different from that of the hypoxia control group.However,when 2U/ml thrombin was added into the culture medium,the apoptosis rate of cells could be reduced by using natural hirudin against thrombin(P<0.05).When 50 μmol/L AG490 was added into the culture medium,the apoptosis rate of cells was reduced,but the difference was not statistically significant compared with the hypoxia control group.When 2U/ml thrombin was added into the culture medium,the application of AG490 could reduce the apoptosis rate of cells(P<0.05),but the apoptosis rate of cells was higher than that of T+H group(P<0.05).Hypoxia could increase the phosphorylation level of JAK2 and STAT1 in human microvascular endothelial cells,and thrombin could further increase the phosphorylation level of JAK2 and STAT1 under the hypoxia environment(P<0.05).Natural hirudin could inhibit the intracellular phosphorylation of JAK2 and STAT1 induced by thrombin(P<0.05),but it could not inhibit the intracellular phosphorylation of JAK2 and STAT1 induced by hypoxia(P>0.05).Conclusion:1)Apoptosis was induced by hypoxia in HMVECs.2)Thrombin can further increase the apoptosis of human microvascular endothelial cells and thrombin can stimulate the phosphorylation of JAK2 and STAT1 under the hypoxia conditon.3)Natural hirudin does not attenuate HUVECs cell apoptosis induced by hypoxia.4)Natural hirudin does attenuate HMVECs cell apoptosis induced by thrombin under the condition of hypoxia.5)Exposure to natural hirudin exerts a protective effect against thrombin-induced apoptosis under the condition of hypoxia among HMVECs and the JAK2/STAT1 signal cascade may be taken part in the hirudin effects on apoptosis.