Involvement of Rho-kinase/IκB-α/NF-κB activation in IL-1ß-induced inflammatory response and oxidative stress in human chondrocytes.

It has been clearly indicated that osteoarthritis (OA) is an inflammatory and degenerative disease that could be promoted by Rho-kinase (ROCK); however, little is known about the role of ROCK/inhibitor κB alpha (IκB-α)/nuclear factor-κB (NF-κB) p65 pathway activation in interleukin-1ß (IL-1ß) induced inflammatory response and oxidative stress in primary human chondrocytes. To test this hypothesis, we focused on determining ROCK-II, IκB-α, p-IκB-α, NF-κB p65, p-NF-κB p65, IL-6, tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), p22phox, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subtype 4 (NOX4) protein expression, ROCK-II activity, NADPH oxidase levels, and total antioxidant capacity (TAC) in the presence and absence of ROCK-inhibitor fasudil. IL-1ß (2 ng·mL-1, 24 h) increased the expression of ROCK-II, p-IκB-α, NF-κB p65, p-NF-κB p65, IL-6, TNF-α, COX-2, and p22phox proteins, and decreased the expression of IκB-α, and the NOX4 protein level did not alter. ROCK activity and NADPH oxidase levels were increased, whereas the TAC was decreased by IL-1ß. Fasudil (10-5-10-7 M) reversed all these changes induced by IL-1ß. These results demonstrate that ROCK/IκB-α/NF-κB p65 pathway activation contributes to the IL-1ß-induced inflammatory response and oxidative stress, and thus, ROCK inhibition might be a beneficial treatment option for OA patients mainly based on its anti-inflammatory and antioxidant effects.

The effect of testosterone replacement therapy on bladder functions, histology, apoptosis, and Rho-kinase expression in bladder outlet obstruction and hypogonadism rat model

Background/aim: The effect of testosterone replacement therapy was investigated on bladder functions, histology, apoptosis as well as Rho-kinase expression in the rat bladder outlet obstruction (BOO) and hypogonadism models. Materials and methods: 30 mature male rats divided into 4 groups: sham group (n = 8), BOO group (n = 8), BOO + orchiectomy group (n = 7), BOO + orchiectomy + testosterone (T) treatment group (n = 7). Cystometric findings, apoptosis index, Rho-kinase (ROCK-2) expression, and smooth muscle/collagen ratio were compared. Results: BOO did not change ROCK-2 expression level, compared to sham group (P > 0.05). However, when compared to BOO group (P < 0.01), BOO + orchiectomy led ROCK-2 increase. The testosterone treatment failed to reverse the up-regulation of ROCK-2 induced by orchiectomy although it tended to lower ROCK-2 level. Compared to sham group (P = 0.002), changes in maximal bladder capacity and leak point pressure were higher (P = 0.026, P = 0.001), and bladder compliance was lower in BOO group. Also, the apoptosis index was different between the two groups (P = 0.380). Smooth muscle/collagen ratio was higher in BOO + orchiectomy + T group than in BOO + orchiectomy group (P = 0.010). Conclusions: The research draws attention to alternating treatment approaches in case of the presence of hypogonadism and BOO.

Effects of 17ß-estradiol and progesterone on the production of adipokines in differentiating 3T3-L1 adipocytes: Role of Rho-kinase.

Effect of female sex hormones on the production/release of adipocyte-derived cytokines has been debatable. Furthermore, whether the cellular signaling triggered by these hormones involve Rho-kinase has not been investigated yet. Therefore, in this study, effects of 17ß-estradiol and progesterone as well as the Rho-kinase inhibitor, Y-27632 on the level of adipokines such as resistin, adiponectin, leptin, TNF-α and IL-6 were investigated in 3T3-L1-derived adipocytes. Differentiation was induced in the post-confluent preadipocytes by the standard differentiation medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum together with the mixture of isobutylmethylxanthine, dexamethasone and insulin) in the presence of 17ß-estradiol (10(-8)-10(-7)M), progesterone (10(-6)-10(-5)M), the Rho-kinase inhibitor, Y-27632 (10(-5)M) and their combination for 8days. Measurements of the adipokines were performed in the culturing medium by ELISA kits using specific monoclonal antibodies. 17ß-estradiol elevated resistin but decreased adiponectin and IL-6 levels; however, it did not alter the concentration of leptin and TNF-α. Y-27632 pretreatment inhibited the rise of resistin and the fall of adiponectin by 17ß-estradiol without any effects by its own. Progesterone did not change resistin, leptin and TNF-α level; however, it elevated adiponectin and decreased IL-6 production. Neither 17ß-estradiol nor Y-27632 was able to antagonize the increase of adiponectin and the reduction of IL-6 levels by progesterone. While Y-27632 alone lowered IL-6 level, it increased leptin and TNF-α concentration without altering resistin and adiponectin. In conclusion, 17ß-estradiol could modify adipokine production in 3T3-L1 adipocytes with the actions some of which involve Rho-kinase mediation.

Association of angiotensin-converting enzyme gene insertion/deletion polymorphism with allergic contact dermatitis.

Angiotensin-converting enzyme (ACE) plays an important role in the physiological control of blood pressure and inflammation. Insertion/deletion (I/D) polymorphism of the gene for ACE was investigated in relation to cardiovascular, cerebrovascular, neurodegenerative and inflammatory diseases. The purpose of the present study was to investigate the possible association between allergic contact dermatitis and insertion/deletion polymorphism of the ACE gene. A total of 90 patients with allergic contact dermatitis and 160 control persons were enrolled in the present study. ACE I/D genotypes were determined by the polymerase chain reaction. Allelic frequencies and genotype distribution of the ACE I/D polymorphism in the patient group were significantly different from control group (ACE II genotype 30.0% versus 17.5%, P = 0.022; ACE I allele 51.7% versus 39.4%, P = 0.008). Our data suggest that the ACE polymorphism could be a risk factor for patients with allergic contact dermatitis.

Rho kinase expression and its central role in ovine gallbladder contractions elicited by a variety of excitatory stimuli.

Rho kinase has contractile activity, which induces Ca2+ sensitization in various cells. Several receptors are linked to the Rho/Rho-kinase pathway. Therefore, in this study we aimed to demonstrate the central importance of this novel pathway for diverse excitatory stimuli in the smooth muscle of the sheep gallbladder. Accordingly, the effects of a Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-8)-3 x 10(-5) M), were investigated on cholecystokinin-8 (CCK-8, 10(-8) M), endothelin-1 (10(-8) M), carbachol (10(-6)-10(-5) M), 5-hydroxytryptamine (5-HT, 10(-6)-10(-5) M), histamine (10(-6)-10(-5) M), phenylephrine (10(-5)-10(-4) M), neurokinin A (10(-7)-10(-6) M), electrical field stimulation (40 V, 0.5 ms, 2, 4, 8, 16, 32 Hz, 15 s, 3 min intervals) and potassium chloride (KCl, 25-50 mM)-induced contractions as well as spontaneous contractile activity. Electrical field stimulation evoked tetrodotoxin (3 x 10(-6) M)-sensitive reproducible contractions, which were inhibited by atropine (2 x 10(-6) M) and potentiated by eserine (5 x 10(-7) M). EFS-induced contraction was significantly inhibited by Y-27632 (10(-5) M). In addition, spontaneous contractile activity was suppressed in the presence of the compound (10(-6)-10(-5) M). This Rho kinase inhibitor also dramatically decreased the contractions elicited by 5-HT, neurokinin A and carbachol. KCl-induced contraction, which was not atropine-sensitive, was also conspicuously attenuated by Y-27632. Moreover, Y-27632 (10(-8)-3 x 10(-5) M) relaxed gallbladder strips that were contracted by histamine, endothelin-1, CCK-8 and phenylephrine in a concentration-dependent manner. pEC50 values for Y-27632 were 6.25+/-0.10, 5.79+/-0.12, 5.83+/-0.09 and 5.70+/-0.13 for the contraction elicited by histamine, CCK-8, endothelin-1 and phenylephrine, respectively. Furthermore, we also demonstrated Rho kinase protein expression (ROCK-1 and ROCK-2) by Western blot analysis. In conclusion, ROCK is expressed in the smooth muscle of the ovine gallbladder, and it has a central role in the contractile activity induced by diverse excitatory stimuli.

Rho-kinase (ROCK-1 and ROCK-2) upregulation in oleic acid-induced lung injury and its restoration by Y-27632.

The possible contribution of Rho/Rho-kinase signalling in oleic acid (100 mg kg-1, i.v., for 4 h)-induced lung injury was investigated in rats. Furthermore, the possible protective effect of the administration of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 0.5-5 mg kg-1, i.v., 15 min before the administration of oleic acid), was also examined. Western blot analysis as well as histopathological examination revealed that Rho-kinase (ROCK-1 and ROCK-2) was upregulated in lungs obtained from oleic acid-administrated rats. In addition, the markers of oxidative and nitrosative stress, i.e., malondialdehyde, myeloperoxidase, 3-nitro-L-tyrosine and nitrite/nitrate, in serum and lung tissue were also increased in the injury group. Treatment of rats with 5 mg kg-1 Y-27632 reversed the oleic acid-induced lung damage, which was demonstrated by histopathological assessment and confirmed in Western blot experiments: ROCK-blots were more intense in the oleic acid group than in control and Y-27632 treatment reversed ROCK upregulation. In addition, malondialdehyde, myeloperoxidase, 3-nitro-L-tyrosine and nitrite/nitrate were also normalized after the administration of Y-27632 (0.5 mg kg-1 and 5 mg kg-1). These findings suggest that ROCK-1 and ROCK-2 are involved in oleic acid-induced lung damage in rats, and that inhibition of this enzyme by Y-27632 may have a protective effect against such damage. Consequently, Rho kinase inhibitors may be potential therapeutic agents in the treatment of acute respiratory distress syndrome (ARDS).