Effect and mechanism of Poria cocos polysaccharides on myocardial cell apoptosis in rats with myocardial ischemia-reperfusion injury by regulating Rho-ROCK signaling pathway / 中国中药杂志

This study aimed to investigate the effect and underlying mechanism of Poria cocos polysaccharides(PCP) on myocardial cell apoptosis in the rat model of myocardial ischemia-reperfusion injury(MI/RI). Male SPF-grade SD rats were randomly divided into a sham group(saline), a model group(saline), low-and high-dose PCP groups(100 and 200 mg·kg~(-1)), and a fasudil group(10 mg·kg~(-1)), with 16 rats in each group. Except for the sham group, the other four groups underwent left anterior descending coronary artery ligation for 30 min followed by reperfusion for 2 h to establish the MI/RI model. The myocardial infarct area was assessed by TTC staining. Histological changes were observed through HE staining. Myocardial cell apoptosis was evaluated using TUNEL staining. Serum lactate dehydrogenase(LDH), creatine kinase MB(CK-MB), interleukin-1β(IL-1β) and IL-18 levels, myocardial superoxide dismutase(SOD) activity and malondialdehyde(MDA) levels were detected by ELISA. Protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved caspase-3, Ras homolog gene A(RhoA), myosin phosphatase target subunit 1(MYPT-1), phosphorylated MYPT-1(p-MYPT-1), and Rho-associated coiled-coil forming kinase 1(ROCK 1) were measured by Western blot. Pathological staining of myocardial tissue revealed that in the model group, there was focal necrosis of myocardial tissue, myocardial cell swelling, unclear boundaries, and neutrophil infiltration. These pathological changes were alleviated in the low-and high-dose PCP groups and the fasudil group. Compared with the model group, the low-and high-dose PCP groups and the fasudil group showed significantly reduced myocardial infarct area and myocardial cell apoptosis rate. Compared with the sham group, the model group exhibited elevated serum LDH, CK-MB, IL-1β and IL-18 levels, increased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and decreased myocardial SOD levels and Bcl-2 protein expression. Compared with the model group, the PCP groups and the fasudil group showed lowered serum LDH, CK-MB, IL-1β and IL-18 levels, decreased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and increased myocardial SOD levels and Bcl-2 protein expression. PCP exhibited a certain preventive effect on myocardial tissue pathological damage and myocardial cell apoptosis in MI/RI rats, possibly related to the inhibition of the Rho-ROCK signaling pathway activation, thereby reducing oxidative stress and inflammatory responses.

La inhibición de Rho quinasa post infarto mejora el remodelado y la función ventricular: mecanismos involucrados a nivel preclínico / Rho-kinase inhibition post myocardial infarction improves remodeling and systolic function: a preclinical study of intervening factors in a pre-clinical study

Resumen: Objetivo: Determinar algunos mecanismos moleculares por los cuales la activación de ROCK cardíaca post infarto del miocardio (IAM) participa en el remodelado y en deterioro de la función sistólica. Métodos: Determinación simultánea de niveles de proteínas blanco de ROCK cardíaca, de función sistólica in vivo del ventrículo izquierdo (VI) y de fibrosis e hipertrofia cardíaca en ratas con IAM en condiciones de inhibición de ROCK con fasudil. Resultados : Siete días post IAM la masa ventricular relativa aumentó significativamente en un 30% en el grupo MI y se redujo con fasudil. La disfunción sistólica VI mejoró significativamente con fasudil mientras que la activación de ROCK cardíaca se redujo a niveles del grupo control. El inhibidor de ROCK también redujo significativamente los niveles cardíacos elevados de las isoformas ROCK1 y ROCK2, de MHC-β y del colágeno miocárdico. En el grupo con IAM aumentaron significativamente los niveles de fosforilación de ERK 42 y ERK 44 (en 2 veces y en 63%, respectivamente), mientras que en el grupo IAM tratado con fasudil estos niveles fueron similares a los del grupo control. El IAM aumentó significativamente los niveles fosforilados del factor de transcripción GATA-4, que se normalizaron con el inhibidor de ROCK. Conclusiones: La disfunción sistólica post IAM se asoció fuertemente con la activación del ROCK cardíaca y con la fosforilación de proteínas río abajo de ROCK que promueven remodelado cardíaco como β-MHC y la vía ERK / GATA-4.

Abstracts: Objective: to determine some molecular mechanisms by which cardiac ROCK activation after myocardial infarction (MI) intervene in cardiac systolic function decline and remodeling. Methods: simultaneous measurement of different cardiac ROCK target proteins levels, in vivo left ventricular (LV) systolic function, myocardial fibrosis, and hypertrophy in rats with MI under ROCK inhibition with fasudil were performed. Results: seven days after MI the relative ventricular mass increased significantly by 30% in the MI groupand was reduced with fasudil. LV systolic dysfunction improved significantly with fasudil whereas at the same time cardiac ROCK activation was reduced to sham levels. The ROCK inhibitor also reduced increased cardiac levels of both ROCK1 and ROCK2 isoforms, β-MHC levels and myocardial collagen volume fraction decline. MI significantly increased phosphorylation levels of ERK 42 and ERK 44 by 2-fold and 63% respectively whereas in the fasudil-treated MI group these levels were similar to those in the sham group. MI significantly increased phosphorylated levels of the transcription factor GATA-4 which were normalyzed by the ROCK inhibitor. Conclusion: LV systolic dysfunction after MI was strongly associated to cardiac ROCK activation and subsequent phosphorylation of ROCK target proteins that promote ventricular remodeling, such as β-MHC and the ERK/GATA-4 pathway. ROCK inhibition with fasudil significantly improved systolic function, diminished myocardial fibrosis, and normalized β-MHC and ERK/GATA-4 phosphorylation levels.

Effects of the Rho-kinase inhibitor fasudil on the invasion, migration, and apoptosis of human prostate cancer PC3 and DU145 cells / 中华男科学杂志

<p><b>Objective</b>To 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.</p><p><b>METHODS</b>Human 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.</p><p><b>RESULTS</b>Fasudil 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 ).</p><p><b>CONCLUSIONS</b>The 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.</p>

Immunoregulative effect of Fasudil on encephalomyelitic T cells in experimental autoimmune encephalomyelitis mice / 中南大学学报(医学版)

OBJECTIVE@#To explore the therapeutic effect of Fasudil-modified splenic mononuclear cells (MNCs) in experimental autoimmune encephalomyelitis (EAE) and the possible mechanisms.
@*METHODS@#C57BL/6 female mice were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to establish active immunity EAE model. Splenic MNCs were isolated on the 9th day after immunization and treated with or without Fasudil for 72 h in vitro. These cells were collected for analysis of the variance of T cell subtypes, the level of cytokines and the activity of Rho kinase (ROCK). MNCs (5×107 cells) were resuspended in 500 µL of phosphate buffer solution (PBS) and transferred into EAE model (intraperitoneal injection), which was divided into a PBS-MNCs group and a Fasudil-MNCs group. Changes of body weight and clinical symptom scores were observed.
@*RESULTS@#Splenic encephalitogenic MNCs from EAE mice on the 9th day after immunization could establish passive transfer EAE model. But Fasudil-treated MNCs did not trigger EAE development. Compared with the PBS-MNCs group, the loss of body weight was less in the Fasudil-MNCs group. The in vitro experiment indicated that Fasudil could suppress the activity of ROCK on MNCs (P<0.01), decrease the percentage of CD4+ T cells with the expression of interferon-γ (IFN-γ) and interleukin-17 (IL-17) (IFN-γ: P<0.01; IL-17: P<0.05), while increase the secretion of CD4+ T cells with the expression of transforming growth factor-β (TGF-β) and IL-10 (all P<0.001) . Furthermore, Fasudil could inhibit the release of IL-17 (P<0.001) and enhance the level of IL-10 (P<0.05).
@*CONCLUSION@#Fasudil-modified cell therapy affects the occurrence and development of EAE by inhibiting the inflammatory reaction of helper T cell 1 (Th1) and Th17 while enhancing the immunoregulative effect of Th2.

ROCK inhibition with fasudil promotes early functional recovery of spinal cord injury in rats by enhancing microglia phagocytosis / 华中科技大学学报（医学）（英德文版）

Emerging evidence indicates that microglia activation plays an important role in spinal cord injury (SCI) caused by trauma. Studies have found that inhibiting the Rho/Rho-associated protein kinase (ROCK) signaling pathway can reduce inflammatory cytokine production by microglia. In this study, Western blotting was conducted to detect ROCK2 expression after the SCI; the ROCK Activity Assay kit was used for assay of ROCK pathway activity; microglia morphology was examined using the CD11b antibody; electron microscopy was used to detect microglia phagocytosis; TUNEL was used to detect tissue cell apoptosis; myelin staining was performed using an antibody against myelin basic protein (MBP); behavioral outcomes were evaluated according to the methods of Basso, Beattie, and Bresnahan (BBB). We observed an increase in ROCK activity and microglial activation after SCI. The microglia became larger and rounder and contained myelin-like substances. Furthermore, treatment with fasudil inhibited neuronal cells apoptosis, alleviated demyelination and the formation of cavities, and improved motor recovery. The experimental evidence reveals that the ROCK inhibitor fasudil can regulate microglial activation, promote cell phagocytosis, and improve the SCI microenvironment to promote SCI repair. Thus, fasudil may be useful for the treatment of SCI.

Effect of p65 gene inhibited by siRNA on differention of rat marrow mesenchymal stem cells into neurons / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of p65 gene inhibited by siRNA on neuronic differentiation in the marrow mesenchymal stem cells (MSCs).</p><p><b>METHODS</b>The MSCs were transfected with Rn-p65-siRNA. Fasudil hydrochloride induced MSCs differentiating into neurons. The non-transfected group and negative control group (transfected with negative control siRNA marked by Cy3) were used as controls. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope at 24 h,48 h and 72 h after transfected with negative control siRNA. The viability of MSCs was detected by MTT at 24 h, 48 h and 72 h after transfected with Rn-p65-siRNA. The expressions of p65 mRNA and protein in MSCs were detected by RT-PCR and Western blot respectively. The expressions of p65 protein, NSE, MAP-2 and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical method after transfection for 6 h.</p><p><b>RESULTS</b>The fluorescence of MSCs was mostly displayed after transfection of 72 hours and the efficiency of transfection was up to 83.3% ± 3.8%. Meanwhile, the p65 mRNA and p65 protein expressed by MSCs of transfected group were significantly decreased (P < 0.05); MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). The best efficiency of induction was observed in the transfected group. There were higher expressions of NSE and MAP-2 than the other group (P < 0.05).</p><p><b>CONCLUSION</b>The p65 gene inhibited by siRNA can promote the marrow mesenchymal stem cells to differentiate into neurons.</p>

Fasudil hydrochloride hydrate, a Rho-kinase inhibitor, ameliorates hepatic fibrosis in rats with type 2 diabetes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hyperglycemia may accelerate liver fibrosis. Currently, there is no effective treatment for liver fibrosis induced by type 2 diabetes. The study aim was to investigate whether RhoA/Rho kinase (ROCK) pathway is involved in liver fibrosis in the rats with type 2 diabetes and define the protective effects of fasudil on livers.</p><p><b>METHODS</b>A rat model of type 2 diabetes was established by high fat diet combined with streptozotocin (30 mg/kg, intraperitoneal injection). Animals were randomly assigned to 3 groups: control rats, untreated diabetic rats that received vehicle and fasudil-treated diabetic rats that received ROCK inhibitor fasudil hydrochloride hydrate (10 mg/kg per day, intraperitoneal injection, for 14 weeks). The morphological features of liver were observed by HE staining. Accumulation of collagen in livers was determined by Masson staining and the measurement of hydroxyproline. The mRNA expression of transforming growth factor-β1 (TGFβ1), connective tissue growth factor (CTGF), type-I, and type-III procollagen was assessed with real-time polymerase chain reaction. The phosphorylation of myosin phosphatase target subunit-1 (MYPT1) and the protein levels of TGFβ1 and α-smooth muscle actin (a-SMA) were evaluated by Western blotting.</p><p><b>RESULTS</b>Compared with control rats, untreated diabetic rats showed higher values of collagen and hydroxyproline in livers (P < 0.01), the phosphorylation of MYPT1 and the protein levels of TGFβ1 and α-SMA were increased (P < 0.01), and the mRNA expression of TGFβ1, CTGF, type-I, and type-III procollagen was upregulated (P < 0.01); compared with untreated diabetic rats, treatment with fasudil signifcantly reduced values of collagen and hydroxyproline (P < 0.01), and decreased the phosphorylation of MYPT1 and the levels of TGFβ1 and α-SMA (P < 0.01), concomitant with the downregulation of TGFβ1/CTGF, type-I, and type-III procollagen mRNA expression (P < 0.01).</p><p><b>CONCLUSIONS</b>Fasudil ameliorates liver fibrosis in rats with type 2 diabetes at least partly by inhibiting TGFβ1/CTGF pathway and α-SMA expression. Inhibition of RhoA/ROCK may be a novel therapeutic target for liver fibrosis in diabetic non-alcoholic steatohepatitis.</p>

Fasudil reverses monocrotaline-induced pulmonary hypertension in rats / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe the effects and related mechanisms of fasudil on monocrotaline-induced pulmonary arterial hypertension (PAH) in rats.</p><p><b>METHODS</b>A total of 56 healthy male Sprague-Dawley rats were randomly divided into 5 groups: 4 weeks control group (N4), 4 weeks PAH group (M4), 8 weeks control group (N8), 8 weeks PAH group (M8), 8 weeks PAH and fasudil group (F8). PAH was induced by subcutaneous injection of monocrotaline (50 mg/kg). Animals in F8 group received intraperitoneal injection of fasudil hydrochloride (15 mg×kg(-1)×d(-1)) from the end of the 4th week to the end of the 8th week. Rats in control groups and PAH groups received equal volume saline injection. Polyethylene catheters were inserted into the RV through the jugular vein for right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) measurements after various treatment protocols. RV hypertrophy index [RV/(LV+S)] was also measured. Arteries of 50 to 150 µm were evaluated for the median wall thickness and wall area by HE staining as follows: percent wall thickness (WT%) = [(medial thickness×2/external diameter)]×100 and percent wall area (WA%) = (wall area/total area)×100%. The mRNA expression of ROCK-1 in lung tissue was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of ROCK-1 and MYPT-1 in lung tissue were analyzed by Western blot and MYPT-1 phosphorylation, respectively.</p><p><b>RESULTS</b>Forty-one rats survived and mortality rate was zero in N4, N8 and M4 groups. Survival rate was significantly higher in F8 group compared to M8 group (75.00% vs. 31.25%, P < 0.05). At the end of the 4th week, RVSP [(62.25 ± 3.24) vs. (31.33 ± 2.35) mm Hg(1 mm Hg = 0.133 kPa)], mPAP [(36.38 ± 2.31) vs.(20.32 ± 1.81) mm Hg], [RV/(LV+S)] (0.5648 ± 0.0580 vs. 0.3458 ± 0.0455), WT% [(25.63 ± 5.35)% vs.(13.38 ± 3.45)%], WA% [(60.36 ± 2.51)% vs. (38.42 ± 2.84)%] were all significantly higher in M4 group than in N4 group (all P < 0.01). RVSP [(54.64 ± 4.11) vs. (67.37 ± 4.68) mm Hg], mPAP [(26.25 ± 2.32) vs. (39.83 ± 1.83) mm Hg], and markedly relieve [RV/(LV+S)] (0.3985 ± 0.0210 vs. 0.7600 ± 0.0341), WT% [(15.64 ± 2.81)% vs. (28.26 ± 4.38)%], WA% [(40.35 ± 2.82)% vs. (68.83 ± 1.63)%] were all significantly lower in F8 group than in M8 group (all P < 0.05) while the expression of ROCK-1 mRNA (1.2139 ± 0.1778 vs. 1.6839 ± 0.3251, P < 0.01), and the protein expression of ROCK-1 and MYPT-1 as well as the extent of MYPT-1 phosphorylation were all downregualted in F8 group compared to M8 group (all P < 0.01).</p><p><b>CONCLUSIONS</b>Fasudil can effectively reverse the MCT-induced PAH in rats via downregulating ROCK-1 and MYPT-1.</p>

Identification of the related substances in fasudil hydrochloride with hyphenated techniques / 药学学报

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.

Effect of fasudil of on rat cardiomyocytes acutely exposed to omethoate / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effect of fasudil on in vitro cultured cardiomyocytes (CMs) exposed to omethoate and its possible mechanism.</p><p><b>METHODS</b>Cardiomyocytes 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.</p><p><b>RESULTS</b>Compared 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).</p><p><b>CONCLUSION</b>Fasudil 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.</p>

Progress in pharmacotherapy of pulmonary arterial hypertension in children / 中国当代儿科杂志

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.

Fasudil improves erectile function of hypertensive rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effect of fasudil, an inhibitor of Rho kinase, on the erectile function of hypertensive rats and its action mechanism.</p><p><b>METHODS</b>Twenty 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Fasudil can improve erectile function in hypertensive rats by inhibiting the expression of RhoA / Rho kinase signaling and its possible attenuating effect on hypertension.</p>

Rat bone marrow mesenchymal stem cells-differentiated endothelial like cells facilitate angiogenesis and the Rho kinase contribution / 生理学报

The aim of this study was to investigate the influence of endothelial like cells differentiated from rat bone marrow mesenchymal stem cells (rBMSC-ECs) on angiogenesis and the effect of Rho kinase (ROCK) inhibitor using an in vitro model of cells co-cultured with rat aorta ring. Cell proliferation capability was detected by MTT method. The rBMSC-ECs were co-cultured with rat aorta ring in rat tail collagen and endothelial medium. A ROCK specific inhibitor, HA-1077 at different concentrations (0, 10, 30 and 60 mmol/L, respectively) was added into the medium of ring-cell co-culture. The protein expression of ROCK I and ROCK II were detected by Western blot. On the third day of cultivation, the branch number of neogenetic microvessels increased by 34.5% in ring-cell co-culture group compared with that in simple aorta ring group (P < 0.01). Compared with that in ring-cell co-culture group, it was significantly decreased by 57.70%, 64.13% and 48.23% respectively in three concentrations of HA-1077 groups (all P < 0.01). However, on the sixth day, rBMSC-ECs proliferated and migrated to the nearby aorta ring, and the growth of microvessels became slow. On the ninth day, some of neogenetic microvessels were retracted, some became thicken, coarsen and lengthen, and some of rBMSC-ECs were sprouting and forming capillary like picture. The protein expression of ROCK I/II was slightly higher in ring-cell co-culture group than that in simple aorta ring group. But, in three concentrations of HA-1077 groups, it was slightly lower than that in ring-cell co-culture group. By using rhodamine-phalloidin staining and laser scanning confocal fluorescence microscope, it showed that there were a lot of the F-actin cytoskeletons in neogenesis microvessels of aorta ring, and there were a lot of thick and long stress fibers in the cells. F-actin-rich surface protrusions at the leading edge of the cell were also shown. Another ROCK inhibitor, Y-27632 (10 μmol/L) induced the actin cytoskeleton reorganization: F-actins appeared to be peripheral fibers at outer area of cell; stress fiber and filopodia disappeared. These results suggest that rBMSC-ECs themselves can be differentiated into new microvessels and facilitate angiogenesis when they are co-cultured with rat aorta ring. The mechanisms involve ROCK activation and F-actin cytoskeleton recombination.

Fasudil inhibits platelet-derived growth factor-induced human pulmonary artery smooth muscle cell proliferation by up-regulation of p27kip¹ via the ERK signal pathway / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>RhoA/Rho kinase (ROCK) pathway is involved in pulmonary arterial hypertension (PAH) and pulmonary artery smooth muscle cell (PASMC) proliferation. Inhibition of ROCK has been proposed as a treatment for PAH. But the mechanism of RhoA/ROCK pathway and its downstream signaling in proliferation of human PASMCs is unclear. We investigated the effect of fasudil, a selective ROCK inhibitor, on platelet-derived growth factor (PDGF) induced human PASMC proliferation, and the possible association between RhoA/ROCK and extracellular signal-regulated kinase (ERK), p27(Kip1).</p><p><b>METHODS</b>Human PASMCs were cultured with the stimulation of 10 ng/ml PDGF, and different concentrations of fasudil were added before the addition of mitogen. Cell viability and cell cycle were determined with MTT and flow cytometry respectively. ROCK activity, ERK activity and protein expression of proliferating cell nuclear angigen (PCNA) and p27(Kip1) were measured by immunoblotting.</p><p><b>RESULTS</b>By MTT assay, PDGF significantly increased the OD value that represented human PASMC proliferation, and pretreatment with fasudil significantly reversed this effect in a dose-dependent manner. After PDGF stimulation, the percentage of cells in S phase increased dramatically from 15.6% to 24.3%, while the percentage in G(0)/G(1) phase was reduced from 80.6% to 59%. And pretreatment with fasudil reversed the cell cycle effect of PDGF significantly in a dose-dependent manner. PDGF markedly induced ROCK activity and ERK activity with a peak at 15 minutes, which were significantly inhibited by fasudil. In addition, fasudil significantly inhibited PDGF-induced PCNA expression and fasudil also upregulated p27(Kip1) expression in human PASMCs, which decreased after PDGF stimulation.</p><p><b>CONCLUSION</b>RhoA/ROCK is vital for PDFG-induced human PASMC proliferation, and fasudil effectively inhibited PDGF-induced human PASMC proliferation by up-regulation of p27(Kip1), which may be associated with inhibition of ERK activity.</p>

Effect of notch signaling on differentiation of rat marrow mesenchymal stem cells into neurons induced by fasudil hydrochloride / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of notch signaling on differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons induced by fasudil hydrochloride.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>(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).</p><p><b>CONCLUSION</b>There 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.</p>

CaMKII Inhibitor KN-62 Blunts Tumor Response to Hypoxia by Inhibiting HIF-1alpha in Hepatoma Cells

In rapidly growing tumors, hypoxia commonly develops due to the imbalance between O2 consumption and supply. Hypoxia Inducible Factor (HIF)-1alpha is a transcription factor responsible for tumor growth and angiogenesis in the hypoxic microenvironment; thus, its inhibition is regarded as a promising strategy for cancer therapy. Given that CamKII or PARP inhibitors are emerging anticancer agents, we investigated if they have the potential to be developed as new HIF-1alpha-targeting drugs. When treating various cancer cells with the inhibitors, we found that a CamKII inhibitor, KN-62, effectively suppressed HIF-1alpha specifically in hepatoma cells. To examine the effect of KN-62 on HIF-1alpha-driven gene expression, we analyzed the EPO-enhancer reporter activity and mRNA levels of HIF-1alpha downstream genes, such as EPO, LOX and CA9. Both the reporter activity and the mRNA expression were repressed by KN-62. We also found that KN-62 suppressed HIF-1alpha by impairing synthesis of HIF-1alpha protein. Based on these results, we propose that KN-62 is a candidate as a HIF-1alpha-targeting anticancer agent.

Fasudil promotes migration and vasculogenesis of endothelial cells differentiated from rat bone marrow mesenchymal stem cells in vitro / 生理学报

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.

Effects of Rho/ROCK signal pathway on AGEs-induced morphological and functional changes in human dermal microvascular endothelial cells / 生理学报

The present study aimed to determine the role of Rho/Rho kinase (Rho/ROCK) phosphorylation on advanced glycation end products (AGEs)-induced morphological and functional changes in human dermal microvascular endothelial cells (HMVECs). HMVECs were respectively incubated with different concentrations of AGEs-modified human serum albumin (AGEs-HSA) for different time. In some other cases, HMVECs were pretreated with ROCK inhibitors (H-1152 or Y-27632). The morphological changes of F-actin cytoskeleton were visualized by rhodamine-phalloidin staining and the phosphorylation of Rho and ROCK were determined by Western blot. Endothelial monolayer permeability was assessed by measuring the flux of FITC-albumin across the endothelial cells. The results showed that the distribution of F-actin was significantly altered by AGEs-HSA in time and dose-dependent patterns. These effects were inhibited by ROCK inhibitors. The phosphorylation of Rho and RCOK was remarkably increased by AGEs-HSA treatment while total Rho and ROCK protein levels were not affected. The permeability of endothelial monolayer was dramatically increased by AGEs-HSA, and both ROCK inhibitors (H-1152 or Y-27632) attenuated these hyperpermeability responses. The results obtained suggest that the phosphorylation of Rho/ROCK plays an important role in AGEs-induced morphological and functional alterations in HMVECs.

Effects of chronic hypoxia on the expression of calmodulin and calcicum/calmodulin-dependent protein kinase II and the calcium activity in myocardial cells in young rats / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Calcium plays an important role in the impairment of heart function and arrhythmia under the condition of acute hypoxia, but the mechanism is different from that of chronic hypoxia. This study aimed to evaluate the effect of chronic hypoxia on the expression of calmodulin (CaM) and calcicum/calmodulin-dependent protein kinase II (CaMKII) and the calcium activity in myocardial cells through an animal model of chronic hypoxia in order to get a deeper sight into the mechanism.</p><p><b>METHODS</b>A chronic hypoxia model of the rat was prepared by hypoxia exposure (FiO2=10%). The expression of mRNA and protein of CaM and CaMKIIgamma and CaMKIIdelta in myocardial cells were measured by RT-PCR and Western Blot in normal rats and hypoxia rats 1 and the 3 weeks after exposure. The cardiac cells of the rats from the control group and the 3-week hypoxia group were cultured. Then the intracellular calcium activity was detected using laser confocal equipment. The effect of CaMKII on the calcium activity in myocardial cells was evaluated by the application of KN-62 (CaMKII specific inhibitor).</p><p><b>RESULTS</b>The expression of CaM, CaMKIIgamma and CaMKIIdelta mRNA in myocardial tissues increased in hypoxia rats compared with that in normal controls (P<0.01). The CaM and CaMKIIdelta mRNA expression was different between the 1-week and the 3-week hypoxia groups (P<0.01). The laser confocal demonstrated that the amplitude of calcium wave in hypoxic myocardial cells was not different from that in normal controls, but the duration of calcium wave in hypoxic myocardial cells was longer than that in normal controls (P<0.01). After KN-62 use, the amplitude of calcium wave decreased and the duration of calcium wave prolonged significantly.</p><p><b>CONCLUSIONS</b>The contents of CaM and CaMKII in myocardial cells increased under condition of chronic hypoxia as a compensation to keep calcium homeostasis in a certain time. With more prolonged hypoxia time, abnormal electric activities of heart occurred and the heart function may be impaired.</p>

Inhibition of rho kinase attenuates high flow induced pulmonary hypertension in rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The RhoA/Rho kinase pathway may participate in the pathogenesis of hypoxia and monocrotaline induced pulmonary hypertension. This study tested whether RhoA/Rho kinase pathway is involved in the pathogenesis of high flow induced pulmonary hypertension in rats.</p><p><b>METHODS</b>Male Wistar rats (4 weeks) were randomly divided into 4 shunt groups, 4 treated groups and 4 control groups. Shunt and treated groups underwent left common carotid artery/external jugular vein shunt operation. Control groups underwent sham operation. Treated groups received fasudil treatment and the others received same dose of saline. At weeks 1, 2, 4 and 8 of the study, right ventricular systolic pressure was measured and blood gases were analysed to calculate Qp/Qs. The weight ratio of right ventricle to left ventricle plus septum and the mean percentage of medial wall thickness in moderate sized pulmonary arteries were obtained. RhoA activity in pulmonary arteries was detected using Rho activity assay reagent. Rho kinase activity was quantified by the extent of MYPT1 phosphorylation with Western blot. Proliferating cells were evaluated using proliferating cell nuclear antigen immunohistological staining.</p><p><b>RESULTS</b>Carotid artery/jugular vein shunt resulted in high pulmonary blood flow, both an acute and a chronic elevation of right ventricular systolic pressure, significant medial wall thickening characterized by smooth muscle cells proliferation, right ventricular hypertrophy and increased activation of RhoA and Rho kinase. Fasudil treatment lowered pulmonary artery systolic pressure, suppressed pulmonary artery smooth muscle cells proliferation, attenuated pulmonary artery medial wall thickening and inhibited right ventricular hypertrophy together with significant suppression of Rho kinase activity but not Rho activity.</p><p><b>CONCLUSIONS</b>Activated RhoA/Rho kinase pathway is associated with both the acute pulmonary vasoconstriction and the chronic pulmonary artery remodelling of high flow induced pulmonary hypertension. Fasudil treatment could improve pulmonary hypertension by inhibiting Rho kinase activity.</p>