Reduction of hepatic ischemia/reperfusion-induced injury by a specific ROCK/Rho kinase inhibitor Y-27632

BACKGROUND: The low-molecular-weight GTPase Rho is known to act as a molecular switch by activating several downstream effectors, one of which is Rho-associated coiled-coil forming protein kinase (ROCK). ROCK/Rho kinase mediates cytoskeleton-dependent cell functions, such as actomyosin-based smooth muscle contraction and integrin-mediated cell adhesion. A specific inhibitor of ROCK/Rho kinase, Y-27632, was recently developed. The present study examined whether Y-27632 could provide a beneficial effect on hepatic ischemia/reperfusion (I/R)-induced injury through the attenuation of microcirculatory disturbance. MATERIALS AND METHODS: In male Sprague-Dawley rats, normothermic partial ischemia was induced by clamping the hepatic pedicle to the left and median lobes for 90 min, followed by 2 h of reperfusion. In the treatment group, Y-27632 was intravenously administered prior to ischemic insult. Hepatic microcirculation was investigated by using intravital fluorescence microscopy. Liver enzyme release and histological changes of the liver tissue were also evaluated. RESULTS: Y-27632 significantly improved sinusoidal perfusion and reduced the number of leukocytes sticking in hepatic sinusoids and adhering in postsinusoidal venules. The postischemic narrowing of both sinusoids and postsinusoidal venules was also markedly suppressed. Consequently, liver enzyme release was reduced and postischemic histological damage was suppressed. CONCLUSIONS: A specific ROCK/Rho kinase inhibitor, Y-27632, was useful to alleviate hepatic I/R-induced injury through ameliorating postischemic microcirculation. The administration of Y-27632 may be a novel strategy for conquering hepatic I/R-induced injury.     

A novel inhibitor of Rho-associated protein kinase,Y-27632, ameliorates hepatic ischemia and reperfusion injury in rats

BACKGROUND: A Rho-ROCK signal system induces vascular contraction and neutrophil migration, both of which are characteristic features found with ischemia and reperfusion injury of the liver. We tested our hypothesis that a novel ROCK I inhibitor, Y-27632, attenuates hepatic ischemia and reperfusion injury. METHODS: Rats underwent 70% partial hepatic ischemia for 120 minutes and subsequent reperfusion. Y-27632 of 10mg/kg was given orally 1 hour before ischemia, while distilled water was given to the control animals. One week animal survival, systemic hemodynamics, hepatic tissue blood flow, liver function tests, plasma endothelin-1, serum hyaluronic acid levels, myeloperoxidase activity and malondialdehyde level in liver tissue, membrane attack complex-1 and intracellular adhesion molecule-1 staining, and histological architecture were analyzed. RESULTS: Y-27632 prolonged 1-week animal survival from 25% of untreated animals to 75% accompanied with significant amelioration of hepatic tissue blood flow, liver function tests and histological architecture without any adverse effects on systemic hemodynamics. In addition, plasma endothelin-1 and serum hyaluronic acid levels decreased markedly compared to the control, concomitant with remarkable suppression of membrane attack complex-1 stain positive neutrophils infiltration, myeloperoxidase activity and malondialdehyde level. CONCLUSION: Present study suggests that activation of a Rho-ROCK signal system is associated with ischemia and reperfusion injury of the liver, and that Y-27632 may be an attractive agent for application in major liver resection using temporary inflow occlusion and hepatic preservation.     

Therapeutic significance of Y-27632, a Rho-kinase inhibitor,on the established liver fibrosis

BACKGROUND: Recently, we demonstrated that Y-27632, a Rho-kinase inhibitor, inhibited hepatic stellate cells (HSCs) activation in terms of cellular morphology, improved the progression of carbon tetrachloride (CCl(4))-induced rat liver fibrosis. The objective of the present study was to investigate the effects of Y-27632 on the established liver fibrosis. METHODS AND METHODS: Liver cirrhosis was induced by intragastric administration of CCl(4) once a week for 12 weeks. After the first 6 weeks of CCl(4) injection, Y-27632 (30 mg/kg body weight) or saline was continuously administered to the rats via an intraperitoneally implanted osmotic pump during the final 6 weeks of CCl(4) injection. Two days after the last CCl(4) injection, 70% hepatectomy was performed. RESULTS: Y-27632 prevented the development of CCl(4)-induced liver fibrosis and improved the fibrotic changes, hydroxyproline content, and serum hyaluronic acid level in the liver. Moreover, Y-27632 reduced the number of smooth muscle alpha-actin- and transforming growth factor beta1-positive cells, and inhibited the expression of Na(+)/Ca(2+) exchanger mRNA which was reported to be an indicator of HSCs activation and liver fibrosis. Further, the Y-27632-treated group showed markedly increased survival rate after hepatectomy. CONCLUSIONS:These findings indicated that Y-27632 may be useful therapeutically in liver cirrhosis.     

A highly selective inhibitor of Rho-associated coiled-coil forming protein kinase, Y-27632, prolongs cardiac allograft survival of the BALB/c-to-C3H/He mouse model.

BACKGROUND: Current studies provide evidence that a small G protein, RhoAp21, and its target protein, Rho-associated coiled-coil forming protein kinase (ROCK), regulate not only cell shape but also cell migration. However, contribution of Rho/ROCK signaling to graft rejection is unknown. The purpose of this study was to evaluate the inhibitory effect of Y-27632, a highly selective ROCK inhibitor, on rejection of heterotopic cardiac transplantation in mice. METHODS: BALB/c (H-2(d)) hearts were transplanted into C3H/He (H-2(k)) as allografts that were full histoincompatibility combinations. The recipients received several doses of Y-27632, commencing 1 day before cardiac transplantation until rejection. We used immunohistochemical study to detect the expression of myocardial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and we immunoenzymatically measured serum interleukin (IL)-6. Furthermore, we evaluated cardiac allograft vasculopathy treated with either FK506 or Y-27632 at Day 100. RESULTS: The Y-27632-treated (2 mg/kg/day) allografts prolonged the mean survival time (49.6 +/- 10.1 days, n = 12) as compared with the untreated allografts (8.1 +/- 0.4 days, n = 7, p < 0.001). Histologic examinations of the Y-27632-treated allografts at Day 7 showed greatly reduced leukocyte infiltration compared with the untreated allografts. The Y-27632-treated allografts revealed faint expression of myocardial ICAM-1 and VCAM-1 at Day 7. The serum IL-6 levels also decreased in the Y-27632-treated mice. In the long-surviving Y-27632-treated allografts at Day 100, we saw neither active rejection nor apparent thickening of vascular intima. CONCLUSION: Our results suggest that ROCK plays a major role in cardiac rejection in the BALB/c-to-C3H/He mouse model. Inhibition of this Rho/ROCK signaling may be an alternative therapeutic option for managing acute and chronic rejection.     

Suppression of alloreactivity and allograft rejection by SP600125, a small molecule inhibitor of c-Jun N-terminal kinase

BACKGROUND: c-Jun N-terminal kinase (JNK) is reported to play crucial roles in T-cell activation and differentiation, and SP600125 is a small molecule that inhibits JNK. The aim of this study was to examine immunosuppressive action of this compound. METHODS: Rat heterotopic heart transplantation, popliteal lymph node (PLN) hyperplasia bioassay and lymphocyte proliferation assay. RESULTS: SP600125 treatment reduced histological rejection, and dose-dependently extended median survival time of cardiac allografts from 7 days (vehicle) up to 20 days (40 mg/kg/day). Alloantigen-induced PLN hyperplasia was also inhibited by SP600125 in a similar fashion. SP600125 suppressed mixed lymphocyte reaction and OX52-positive lymphocyte proliferation (IC50: 1.5-5.7 microM). Thus, SP600125 inhibits both T-lymphocyte expansion in vitro and T-cell-mediated alloimmune responses in vivo. In addition, SP600125 interacted with cyclosporine additively to prolong cardiac allograft survival. CONCLUSION: Our data provide the first evidence indicating the potential for JNK as a therapeutic target to inhibit the alloimmune response.     

Inhalation of stable dust extract prevents allergen induced airway inflammation and hyperresponsiveness

BACKGROUND: Recent epidemiological studies have shown that growing up on a traditional farm provides protection from the development of allergic disorders such as hay fever and allergic asthma. We present experimental evidence that substances providing protection from the development of allergic diseases can be extracted from dust collected in stables of animal farms. METHODS: Stable dust was collected from 30 randomly selected farms located in rural regions of the Alps (Austria, Germany and Switzerland). The dust was homogenised with glass beads and extracted with physiological sodium chloride solution. This extract was used to modulate immune response in a well established mouse model of allergic asthma. RESULTS: Treatment of mice by inhalation of stable dust extract during sensitisation to ovalbumin inhibited the development of airway hyperresponsiveness and airway eosinophilia upon challenge, as well as the production of interleukin 5 by splenocytes and of antigen specific IgG(1) and IgE. Dust extract also suppressed the generation of human dendritic cells in vitro. The biological activity of the dust extract was not exclusively mediated by lipopolysaccharide. CONCLUSIONS: Stable dust from animal farms contains strong immune modulating substances. These substances can interfere with the development of both cellular and humoral immunity against allergens, thus suppressing allergen sensitisation, airway inflammation, and airway hyperresponsiveness in a murine model of allergic asthma.     

Ventilation heterogeneity is a major determinant of airway hyperresponsiveness in asthma, independent of airway inflammation

BACKGROUND: Airway hyperresponsiveness is the ability of airways to narrow excessively in response to inhaled stimuli and is a key feature of asthma. Airway inflammation and ventilation heterogeneity have been separately shown to be associated with airway hyperresponsiveness. A study was undertaken to establish whether ventilation heterogeneity is associated with airway hyperresponsiveness independently of airway inflammation in subjects with asthma and to determine the effect of inhaled corticosteroids on this relationship. METHODS: Airway inflammation was measured in 40 subjects with asthma by exhaled nitric oxide, ventilation heterogeneity by multiple breath nitrogen washout and airway hyperresponsiveness by methacholine challenge. In 18 of these subjects with uncontrolled symptoms, measurements were repeated after 3 months of treatment with inhaled beclomethasone dipropionate. RESULTS: At baseline, airway hyperresponsiveness was independently predicted by airway inflammation (partial r2 = 0.20, p<0.001) and ventilation heterogeneity (partial r2 = 0.39, p<0.001). Inhaled corticosteroid treatment decreased airway inflammation (p = 0.002), ventilation heterogeneity (p = 0.009) and airway hyperresponsiveness (p<0.001). After treatment, ventilation heterogeneity was the sole predictor of airway hyperresponsiveness (r2 = 0.64, p<0.001). CONCLUSIONS: Baseline ventilation heterogeneity is a strong predictor of airway hyperresponsiveness, independent of airway inflammation in subjects with asthma. Its persistent relationship with airway hyperresponsiveness following anti-inflammatory treatment suggests that it is an important independent determinant of airway hyperresponsiveness. Normalisation of ventilation heterogeneity is therefore a potential goal of treatment that may lead to improved long-term outcomes.