Possible involvement of rho kinase 1 (rock 1) in cecal ligation and puncture-induced sepsis

Abstract

We have previously demonstrated that following cecal ligation and puncture (CLP) there is an increase in lung epithelial apoptosis in a rat model [1]. On the other hand, it has been shown that the rho/rho kinase pathway is involved in mechanisms of several aspects such as endothelial cell dysfunction and apoptosis. However, the role of rho kinase in the concept of sepsis-induced apoptosis has yet to be elucidated. In this study, to investigate the possible contribution of rho/rho kinase signalling in CLP-induced lung injury, rho kinase expression and the possible protective effect of the administration of an inhibitor of rho kinase, (+)-(R)-trans-4-1-aminoethyl-N-4-pyridyl cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), has been investigated in rats in this model. Thirty-two male Wistar rats were randomly divided into four groups: (1) sham, (2) CLP, (3) sham + Y27632, (4) CLP + Y27632. Y27632 was administered at 1.5 mg/kg, intaperitoneally, 20 min before performing operations. Twenty-four hours later, histopathology and apoptosis were assessed by H&E and immunohistochemically by caspase-3 to demonstrate septic lung injury. Additionally, expression of rock 1 and rock 2 proteins in lung tissue was analyzed by western blotting, and the contribution of oxidative damage was assessed by measuring the levels of thiobarbituric acid reactive substances (TBARS) and the 3-L-nitrotirozin (3-NT)/total tyrosine ratio. The TBARS and 3-NT/total tirosine ratio levels in lung homogenates were found to be increased (11.05 ± 0.31 vs 29.855 ± 2.87) (0.1485 ± 0.10 vs 0.281 ± 0.05) in the CLP group compared with the sham group, and the administration of Y27632 prevented their increase (8.86 ± 1.87) (0.178 ± 0.02) significantly (P < 0.05). The number of apoptotic cells was significantly lower in the CLP + Y27632 group than CLP group and this finding was supported by caspase-3 expression in the lung. Lung histopathology was also protected by Y27632 in CLP-induced sepsis. Immunoblot experiments revealed the increased expression of active fragment of rock 1 in the CLP group. However, rock 2 expressions were similar in all groups. In conclusion, since CLP induced active fragmentation of rock 1 and rho-kinase inhibitor prevented peroxynitrite-mediated apoptotic lung injury in this CLP-induced sepsis model, this suggests that rho-kinase plays an important role in apoptotic lung injury. Our data indicate that rock 1 or downstream components of this pathway may be potential targets for the development of novel therapies in sepsis.