The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells

Abstract Purpose: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. Methods: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. Results: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. Conclusion: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.

Impact of hirudin on the numbers of CD34 positive microvessel and RCA-1 positive cell and neutrophil in perihematoma tissue after intracerebral hemorrhage in rats / 国际脑血管病杂志

Objective To observe the dynamic changes of the numbers of CD34 positive microvessel and RCA-1 positive cell and neutrophil in perihematoma tissue after intracerebral hemorrhage (ICH) in rats at different time points and the impact of thrombin-specific inhibitor hirudin on the above different indicators and to investigate the protective mechanisms of hirudin for brain injury following ICH. Methods An experimental model was made with autologous whole blood injecting into the rat brain basal ganglia by using the stereotactic method. The rats were randomly assigned to sham operation, ICH, hirudin intervention, and normal saline groups.CD34 and RCA-1 immunobistochemistry stainings and conventional HE staining were used to observe CD34-positive microvessel, RCA-1 positive cell and neutrophil.Results The numbers of CD34-positive microvessel began to decrease at 12 hours after ICH, it decreased to the lowest at 72 hours, and it gradually returned to normal levels at day 7. The RCA-1 positive cells could be observed at 6 hours after ICH. It reached the peak at 48 hours. A small amount could persist for two weeks. Neutrophil could be observed at 12 hours after ICH. It reached the peak at 48 hours and disappeared at week 2. The administration of hirudin significantly reduced the numbers of RCA-1 positive cell and neutrophil in the early stage of ICH (5 min). At the same time, it significantly inhibited the decreased numbers of CD34-positive microvessel (all P ＜0. 01). The administration of hirudin during the edema formation also significantly reduced the numbers of RCA-1 positive cell and neutrophil (all P＜ 0.05), however, it could not significantly increase the numbers of CD34-positive microvessel. Conclusions Thrombinmediated inflammatory response has involved in the process of brain injury after ICH, and early administration of hirudin may significantly relieve perihematoma tissue injury.