1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Hypertension, Pulmonary ; drug therapy ; Hypoxia ; pathology ; Protein Kinase Inhibitors ; pharmacology ; Rats ; rho-Associated Kinases ; antagonists & inhibitors

OBJECTIVETo study the expression of Rho/Rho kinase of cardiac muscle in heart failure rats caused by pressure overload and the effects of fasudil on heart failure.

METHODSThe heart failure models were successfully induced by coarctation of ascending aorta after 20 weeks in this study. Thirty female Wistar operated rats were divided randomly into three groups (n = 10) for 4 week treatment. (1) Sham operation group: normal saline, 0.1 ml, i.p,Bid. (2) Heart failure group: normal saline, 0.1 ml,i.p,Bid. (3) Fasudil group: fasudil 5 mg/kg, i.p, Bid. The hemodynamic parameters, the ratio of LV weight to body weight, the expressions of RhoA and Rho kinase mRNA, and the concentration of calcium ion same as [Ca(2+)](i) were investigated in the three groups.

RESULTSHemodynamic parameters were significantly changed in heart failure group than those in sham operation group, such as left ventricular diastolic end pressure increased [(13.00 +/- 0.30) mm Hg vs (3.78 +/- 0.31) mm Hg, P < 0.01], left ventricular systolic pressure decreased [(97.20 +/- 7.21) mm Hg vs (129.45 +/- 7.52) mm Hg, P < 0.01]. Those results could be significantly changed by use of fasudil, P < 0.01. The ratio of LV weight to body weight was significantly increased in heart failure group than that in sham operation group [(4.77 +/- 0.08) mg/g vs (2.51 +/- 0.12) mg/g, P < 0.01]. Fasudil could significantly decrease the ratio of LV weight to body weight compared with that in heart failure group [(4.05 +/- 0.08) mg/g vs (4.77 +/- 0.08) mg/g, P < 0.01]. Cardiac muscle RhoA, Rho kinase mRNA level and [Ca(2+)](i) were higher in heart failure group than those in sham operation group [Ca(2+)](i) (475.93 +/- 28.22) nmol/L vs (79.25 +/- 3.33) nmol/L, P < 0.01. Compared with those in heart failure group, the expressions of RhoA, Rho kinase mRNA level decreased significantly, P < 0.01, and the levels of cardiomyocyte [Ca(2+)](i) had no change in fasudil group [(462.78 +/- 16.72) nmol/L vs (475.93 +/- 28.22) nmol/L, P > 0.05].

CONCLUSIONSThese results indicated that heart failure was probably related to activating of RhoA, Rho kinase. Fasudil may contribute to the observed beneficial effects on heart failure such as the decrease of RhoA, Rho kinase mRNA expression and not increase of [Ca(2+)](i) level. Rho/Rho kinase may be a novel, potent signaling of heart failure.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Female ; Heart Failure ; metabolism ; physiopathology ; Myocardium ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Rats ; Rats, Wistar ; rho-Associated Kinases ; metabolism

The study aims to identify the related substances in fasudil hydrochloride by hyphenated techniques. A WondaSil C18 (250 mm x 4.6 mm, 5 microm) column was used for the separation of the related substances with a mixture of methanol and ammonium acetate buffer solution as the mobile phase by gradient elution. The structures of the related substances were speculated by electrospray positive ionization LC-TOF/MS accurate ion mass and MS/MS determination and elucidation, and verified further through synthesis and spectroscopic analysis. Fasudil hydrochloride and the related substances were separated under the established HPLC condition. Three related substances in fasudil hydrochloride were characterized by hyphenated techniques. The hyphenated LC-MS method is useful for the identification of related substances in fasudil hydrochloride and the results obtained are valuable for its manufacturing process and quality control.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; chemistry ; Calcium Channel Blockers ; chemistry ; Chromatography, Liquid ; Drug Contamination ; Quality Control ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry ; Vasodilator Agents ; chemistry

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Cardiomyopathy, Hypertrophic ; drug therapy ; metabolism ; Connexin 43 ; metabolism ; Disease Models, Animal ; Male ; Myocardium ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of fasudil, an inhibitor of Rho kinase, on the erectile function of hypertensive rats and its action mechanism.

METHODSTwenty 12-week-old healthy male Sprague-Dawley rats were randomly divided into groups A (control), B (hypertension) and C (fasudil treatment). After establishment of the hypertension model, group C received intraperitoneal injection of fasudil at 30 mg/(kg x d), while A and B normal saline only. At 10 weeks after surgery, we measured the corpus cavernosum pressure/mean carotid arterial pressure (ICPmax / MAP), and the expression levels of ROCK1 and ROCK2 proteins in the corpus cavernosum of the rats by Western-blot.

RESULTSThe systolic blood pressure (mmHg) and the expressions of ROCK1 and ROCK2 proteins were significantly increased in group B (190.39 +/- 5.07, 0.048 +/- 0.002 and 0.143 +/- 0.011) as compared with A (124.81 +/- 4.01, 0.036 +/- 0.001 and 0.101 +/- 0.011) (P<0.05), but markedly decreased in group C (182.03 +/- 4.32, 0.044 +/- 0.001 and 0.126 +/- 0.007) in comparison with B (P<0.05). ICPmax /MAP was significantly lower in group B (36.82 +/- 5.47) than in A (59.99 +/- 5.69) (P<0.05), but remarkably higher in group C (51.1 +/- 5.63) than in B (P<0.05).

CONCLUSIONFasudil can improve erectile function in hypertensive rats by inhibiting the expression of RhoA / Rho kinase signaling and its possible attenuating effect on hypertension.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; therapeutic use ; Animals ; Hypertension ; physiopathology ; Male ; Penile Erection ; drug effects ; Rats ; Rats, Sprague-Dawley ; Vasodilator Agents ; pharmacology ; therapeutic use

OBJECTIVETo investigate the effect of fasudil on in vitro cultured cardiomyocytes (CMs) exposed to omethoate and its possible mechanism.

METHODSCardiomyocytes were isolated from male SD rats and were then cultured in DMEM conventionally. The CMs were divided into different groups based on the doses of omethoate and fasudil in culture media. After 3, 6, 12, and 24 h of culture, the survival rate of CMs in each group was measured, the CMs in the medium-dose omethoate and medium-dose fasudil groups were subject to shortening amplitude measurement , and the content of lactate dehydrogenase (LDH) in culture media and expression of Bcl-2 and Bax in CMs were measured.

RESULTSCompared with the normal control group, each omethoate group showed significantly lower survival rate of CMs, which was negatively correlated with the dose of omethoate (P < 0.01). Compared with the normal control group, the medium-dose omethoate and medium-dose fasudil groups showed significantly decreased shortening amplitudes of CMs at all time points (P < 0.01), and the shortening amplitudes of CMs were significantly higher in the medium-dose fasudil group than in the medium-dose omethoate group after 12 h and 24 h of culture (P < 0.01). The LDH level was significantly higher in the medium-dose omethoate and medium-dose fasudil groups than in the normal control group, and the medium-dose fasudil group showed significantly lower LDH level than the medium-dose omethoate group (P < 0.01). Compared with those in the normal control group, the Bcl-2 expression in the medium-dose omethoate and medium-dose fasudil groups was decreased significantly, and the Bax expression in the medium-dose omethoate group was increased significantly (P < 0.01). Compared with the medium-dose omethoate group, the medium-dose fasudil group had significantly increased Bcl-2 expression and significantly decreased Bax expression (P < 0.01).

CONCLUSIONFasudil can inhibit the abnormal expression of apoptosis regulatory proteins (Bcl-2 and Bax) induced by omethoate, which might be one of the factors that reduce the toxic effect of omethoate on CMs.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Cells, Cultured ; Dimethoate ; analogs & derivatives ; toxicity ; Male ; Myocytes, Cardiac ; drug effects ; metabolism ; Pesticides ; poisoning ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism

This paper provides an overview of the current state of pharmacotherapy in children with pulmonary arterial hypertension (PAH) and a brief introduction to the potentially novel pharmacologic targets for PAH. Currently, 3 classes of drugs including prostacyclin analogues, endothelin receptor antagonists and phosphodiesterase-5 inhibitors are approved for the treatment of PAH in children, which has led to improved hemodynamics, increased exercise capacity and prolonged survival. Despite these improvements, there is still a need to carry out well-designed, randomized, controlled studies with larger samples. In addition, novel drugs targeting other molecular pathways should be developed.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; therapeutic use ; Calcium Channel Blockers ; therapeutic use ; Child ; Epoprostenol ; analogs & derivatives ; therapeutic use ; Familial Primary Pulmonary Hypertension ; Humans ; Hypertension, Pulmonary ; diagnosis ; drug therapy ; Iloprost ; therapeutic use ; Sulfonamides ; therapeutic use

The present study was designed to test whether Rho-kinase (ROCK) specific inhibitor fasudil (HA-1077) could contribute to migration and vasculogenesis of endothelial cells differentiated from rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. rBMSCs were separated by gradient centrifugation on lymphocytes separation medium from bone marrow of Sprague-Dawley rats, and were cultured, purified and expanded in vitro. Cells of passage 2 to 3 were induced to differentiate into endothelial lineage cells by HG-DMEM plus EGM-2. These cells were identified as endothelial cells with positive factor VIII and Ulex europaeus agglutinin-1 expressions and DiI-Ac-LDL uptake. HA-1077 and VEGF synergistically promoted cell migration, especially in response to transwell chamber assay. When the cells were cultured on ECMatrix™, they showed cellular protrusions and/or cords of aligned cells resembling primitive capillary-like structures at 8 to 12 h of incubation. HA-1077 promoted cell migration and formation of capillary-like tubes. The length of the total capillary tubes was longer than that in the control group (P<0.05). When the cells were exposed to a combination of VEGF and HA-1077, the number of the capillary-like networks and the stability of tube increased. The results obtained suggest that HA-1077 can promote migration and vasculogenesis of endothelial cells differentiated from rBMSCs in vitro.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Movement ; drug effects ; Endothelial Cells ; cytology ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; rho-Associated Kinases ; antagonists & inhibitors

OBJECTIVETo investigate the effect of notch signaling on differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons induced by fasudil hydrochloride.

METHODSThe experiments were divided into non-transfected group, transfected group (transfected with Rn-Notch1-siRNA), positive control group (transfected with Rn-MAPK-1 Control siRNA) and negative control group (transfected with negative control siRNA). Fasudil hydrochloride induced MSCs differentiating into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The expression of notch1 mRNA, Hes1 mRNA and MAPK1 mRNA in MSCs was detected by RT-PCR. The expression of Notch1 protein, NSE, neurofilament M (NF-M) and glial fibrillary acidic protein(GFAP)was detected by immunocytochemical method. The viability of MSCs was detected by MTT.

RESULTS(1) The fluorescence of MSCs was mostly displayed after transfection for 72 h and the efficiency of transfection was up to 91.3% +/- 4.2%. Meanwhile, the notch1 mRNA and Hes1 mRNA expressed by MSCs of transfected group were significantly decreased (P < 0.05) and MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). (2) Fasudil hydrochloride could induce MSCs differentiate into neurons and the best efficiency of induction was observed in the transfected group. There was higher expression of NSE and neurofilament-M (NF-M) than the other groups (P < 0.05).

CONCLUSIONThere may be notch1 signaling and Rho/Rho GTPase signaling synergy on differentiation of rat bone marrow stromal cell into neurons induced by fasudil hydrochloride and they jointly promote the differentiation of MSCs into neurons.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Neurons ; cytology ; Rats ; Rats, Wistar ; Receptor, Notch1 ; metabolism ; Signal Transduction

ObjectiveTo investigate the potential role of the RhoA/Rock signaling pathway in the formation of prostate cancer and the effects of the Rock inhibitor fasudil on the invasion, migration and apoptosis of human prostate cancer cells.

METHODSHuman prostate cancer cell lines PC3 and DU145 were treated with fasudil at the concentrations of 5, 10, 20, 40, 80, and 160 μmol/L, respectively, and those as negative controls cultured in the Ham's-F12 medium, all for 24 hours. Then, MTT assay was used to measure the cell inhibition rate and half maximal inhibitory concentration (IC50) value of fasudil, with 1/4 of IC50 as the medication dose for further investigation. The expressions of RhoA, RockⅠ, and RockⅡ proteins in the PC3 and DU145 cells were detected by Western blot and immunohistochemistry, and the invasion, migration and apoptosis of the cells were determined using the Transwell chamber, scratch wound healing assay and flow cytometry.

RESULTSFasudil inhibited the proliferation of the PC3 cells from (9.29±1.23)% at 5 μmol/L to (81.37±3.97)% at 160 μmol/L and that of DU145 from (7.59±1.54)% to (76.53±2.67)%, both in a dose-dependent manner (P<0.05 ). Significantly fewer PC3 and DU145 cells migrated into the lower compartment in the experimental group (39.2±8.4 and 34.2±6.7) than in the negative control (116.8±9.3 and 112.5±10.8) (P<0.05 ). The wound healing rates of the PC3 and DU145 cells were remarkably lower in the former (［37.26±1.17］% and ［32.38±2.73］%) than in the latter (［78.12±4.16］% and ［69.47±6.71］%) (P<0.05 ). Annexin V-FITC/PI double staining showed markedly increased apoptosis rates of PC3 and DU145 cells treated with fasudil (［31.88±2.49］% and ［28.65±2.99］%) as compared with the negative controls (［7.51±2.28］% and ［7.13±1.61］%) (P<0.05 ). The expressions of RockⅠ and RockⅡ were significantly reduced in the fasudil-treated cells in comparison with those of the control group (P<0.05 ) while that of RhoA showed no significant difference between the two groups (P>0.05 ).

CONCLUSIONSThe RhoA/Rock signaling pathway may play an important role in the formation of prostate cancer. Fasudil can significantly inhibit the proliferation, migration, and invasion and promote the apoptosis of human prostate cancer PC3 and DU145 cells by reducing RhoA/Rho kinase activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Male ; Prostatic Neoplasms ; drug therapy ; pathology ; Signal Transduction ; rho-Associated Kinases ; antagonists & inhibitors