Date Published: February 14, 2019
Publisher: Sociedade Brasileira para o Desenvolvimento da Pesquisa em
Author(s): Jiangying Zhu, Xinyuan Pan, Bojie Lin, Guanyu Lin, Rohan Pradhan, Feiwen Long, Guoqian Yin.
To investigate whether hirudin exerts its antithrombin action to decrease the
ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis.
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.
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. 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.
Natural hirudin is a polypeptide isolated from the salivary glands of the leech, with
a molecular weight of 7000 Dalton and composed of 64 to 66 amino acids. The tight
structure formed by the disulfide bond at the N end of leech element can bind to the
thrombin active site. The c-end is rich in acidic amino acid residues, and then
binds to the fibrinogen binding site of thrombin, thus having a highly specific
inhibition of thrombin and inhibiting the anticoagulation of thrombin (Fig. 1).
The HMVEC apoptosis rate of each group was detected by flow-cytometric analysis,
after 24 hours of drug treatment. The evaluation of the results indicated no
significant difference of cell apoptosis rate between normal group and natural
hirudin group (P>0.05, Fig. 2 A,B). Thus,
natural hirudin concentration of 2ATU/ml was considered not to be cytotoxic to
HMVECs. However, the cell apoptosis rate in the thrombin group was 4-fold higher
than that in the normal group (P <0.05, Fig. 2 A,B). In the T+H group, the cell apoptosis rate was decreased compared with the thrombin group (P <0.05, Fig. 2 A,B), but it was larger than the normal group (P <0.05, Fig. 2 A,B). To investigate whether the hirudin could attenuate HMVEC apoptosis via antagonizing thrombin by blocking the JAK2 signaling pathway, 50μmol/L of AG490 was added 30min before thrombin treatment. There was no significant difference of cell apoptosis rate between the normal group and the AG490 group (P>0.05, Fig. 2 A,C). This indicates
that AG490 has no toxic effects on cells. The treatment of cells with AG490 produced
a significant inhibition of apoptosis (P <0.05, Fig. 2 A,C), which was even larger than that of the T+H group (P <0.05, Fig. 2 A,C). These results suggest the possibility that hirudin could attenuate HMVEC apoptosis via antagonizing thrombin, not only by blocking the JAK2 signaling pathway, but also by other signaling pathways. Random pattern skin flaps are used to repair skin defects during plastic and reconstructive surgery, however, flap necrosis remains a challenging problem. Our previous studies have shown that natural hirudin can increase flap viability in animal experiments8. Flap necrosis can be induced by many conditions such as ischemia , hypoxia, activation of the coagulation system, vascular thrombosis, venous congestion and inflammation13. The individual causes can’t be studied separately in vivo experiments. The work presented here aimed to study the molecular mechanism that mediates the anti-apoptosis effect of natural hirudin in human endothelial cells by in-vitro experiments. The results show that natural hirudin does protect HMVECs and decrease cell apoptosis via antagonizing thrombin, by blocking the JAK2 signaling pathway. The natural hirudin can inhibit the apoptosis of vascular endothelial cells by blocking antithrombin and blocking the JAK2 pathway. The JAK2 pathway is a significant, but not the only signal pathway that plays a critical role in regulation of apoptosis after natural hirudin treatment: our results of the experiments with added AG490 indicate the presence of other signaling pathways. This study provides an experimental basis and elucidates some of the active pathways for the clinical application of natural hirudin to prevent the flap ischemia in humans. Source: http://doi.org/10.1590/s0102-865020190010000006