Fasudil mediates neuroprotection in ischemia/reperfusion by modulating the ROCK-PPARα-NOX axis

Purpose: Cerebral ischemia-reperfusion (I/R) is a neurovascular disorder that leads to brain injury. In mice, Fasudil improves nerve injury induced by I/R. However, it is unclear if this is mediated by increased peroxisome proliferator-activated receptor-α (PPARα) expression and reduced oxidative damage. This study aimed to investigate the neuroprotective mechanism of action of Fasudil. Methods: MCAO (Middle cerebral artery occlusion) was performed in male C57BL/6J wild-type and PPARα KO mice between September 2021 to April 2023. Mice were treated with Fasudil and saline; 2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to analyze cerebral infarction. PPARα and Rho-associated protein kinase (ROCK) expression were detected using Western blot, and the expression of NADPH subunit Nox2 mRNA was detected using real-time polymerase chain reaction. The NADPH oxidase activity level and reactive oxygen species (ROS) content were also investigated. Results: After cerebral ischemia, the volume of cerebral necrosis was reduced in wild-type mice treated with Fasudil. The expression of PPARα was increased, while ROCK was decreased. Nox2 mRNA expression, NADPH oxidase activity, and ROS content decreased. There were no significant changes in cerebral necrosis volumes, NADPH oxidase activity, and ROS content in the PPARα KO mice treated with Fasudil. Conclusions: In mice, the neuroprotective effect of Fasudil depends on the expression of PPARα induced by ROCK-PPARα-NOX axis-mediated reduction in ROS and associated oxidative damage.

Fasudil exerts a protective effect on down-regulation of IL-6 expression by inhibiting the NOTCH signaling pathway in EAM mice / 西安交通大学学报(医学版)

【Objective】 To explore the effect and mechanism of Fasudil in the treatment of experimental autoimmune myocarditis (EAM) in mice so as to provide a theoretical basis for the clinical use of Fasudil in treating myocarditis. 【Methods】 Balb/c male mice were used as the research objects, and the EAM mice model was constructed using MyHC-α614-629 polypeptide. Mononuclear cells were isolated and cultured to detect the number of mononuclear cells in mouse spleen. Inflammation infiltration, fibrosis and IL-6 expression in mouse myocardial tissue were detected by HE staining, Masson staining and immunohistochemistry, respectively. The protein expressions of Notch1 and IL-6 were detected by Western blotting. qRT-PCR was used to detect the expressions of pro-inflammatory factors (IL-1α, IL-1β and IL-6) as well as key genes of TLRs and NOTCH signaling pathway. 【Results】 EAM mice showed increased HW, decreased BW, increased HW/e-BW, and increased inflammatory infiltration and fibrosis in myocardial tissue. The above-mentioned symptoms or pathological features were improved in EAM mice treated with Fasudil. The analysis showed that the pro-inflammatory factors IL-1α, IL-1β and IL-6 in the myocardial tissue of EAM mice were significantly increased, but only the expression of IL-6 was statistically different after Fasudil treatment compared with the control group. In addition, TLRs signaling pathway might also play an important role in the EAM mice treated with Fasudil. The expressions of IL-6 and Notch1 were consistent, and the expressions of the key genes of NOTCH signaling pathway (Notch1, Hes1 and Jag2) were down-regulated after Fasudil treatment. 【Conclusion】 Fasudil exerts a protective effect on down-regulation of IL-6 expression by inhibiting the NOTCH signaling pathway in EAM mice.

Effects of fasudil hydrochloride on ROCK2 protein and ferroptosis in hippocampus during early brain injury in rats with subarachnoid hemorrhage / 中华行为医学与脑科学杂志

Objective:To investigate the effects of fasudil hydrochloride(FH) on Rho-associated kinase 2(ROCK2) protein and ferroptosis in hippocampal area during early brain injury in rats with subarachnoid hemorrhage(SAH).Methods:Total 36 SPF grade Sprague-Dawley rats were divided into 3 groups by random number table method: Sham group, SAH group and SAH+ FH (a ROCK2 protein inhibitor) group (FH goup) with 12 rats in each group.SAH animal model was established by internal carotid artery perforation.The rats in FH group were injected intraperitoneally with FH(15 mg/kg) 30 minutes after successful modeling, and rats in Sham group and SAH group were injected intraperitoneally with the same volume of 0.9% sodium chloride solution.Twenty-four hours after the intervention, shuttle box test was used to observe the learning and memory ability of rats.The Fe 2+ content in rat hippocampus tissue was detected by colorimetry, and the protein levels of ROCK2 and ferroptosis-related long-chain acyl-CoA synthetase 4(ACSL4) and glutathione peroxidase 4(GPX4) in hippocampus were detected by immunohistochemistry and Western blot.Statistical analysis was performed by SPSS 20.0 software.One-way ANOVA was used for multigroup comparison, and LSD test was used for further pairwise comparison. Results:(1)In the shuttle box test, there were statistically significant differences in the number of avoidance reactions and avoidance reaction time of rats among the three groups( F=20.348, 22.316, both P<0.05). The number of avoidance reaction in SAH group was less than that in Sham group ((17.92±2.94) times, (27.13±3.48) times, P<0.05), the time of avoidance reaction in SAH group was longer than that in Sham group ((9.15±2.87) s, (3.68±1.09) s, P<0.05), while the number of avoidance reaction in FH group ((21.63±4.11) times) was more than that in SAH group, and the time of avoidance reaction ((6.08±1.76) s) was shorter than that in SAH group (both P<0.05). (2) The colorimetry results showed that there was a statistically significant difference in the content of Fe 2+ in hippocampus of rats among the three groups( F=7.965, P<0.05). The Fe 2+ content in SAH group was significantly higher than that of Sham group((0.091±0.032) nmol/mg, (0.038±0.024) nmol/mg, P<0.05), and the Fe 2+ content in the FH group ((0.065±0.021) nmol/mg) was lower than that of SAH group ( P<0.05). (3) There were significant differences in the number of ROCK2, ACSL4 and GPX4 positive cells in hippocampus of rats among the three groups in immunohistochemistry ( F=7.602, 14.171, 36.077, all P<0.05). The positive cells of ROCK2 and ACSL4 in SAH group ((21.63±4.72), (55.13±19.41)) were significantly higher than those of Sham group ((11.63±3.62), (23.38±3.74)) (both P<0.05), and the positive cells of ROCK2 and ACSL4 in FH group ((15.88±6.64), (44.75±8.29)) were both lower than those of SAH group(both P<0.05), while the number of GPX4 positive cells in SAH group (25.38±6.30) was significantly lower than that of Sham group (60.25±10.36) ( P<0.05), and the number of GPX4 positive cells in FH group (45.13±7.51) was higher than that of SAH group( P<0.05). (4)The results of Western blot showed that there were significant differences in the expression levels of ROCK2, ACSL4 and GPX4 proteins in the hippocampus of rats among the three groups( F=4.812, 12.573, 10.849, all P<0.05). The protein expression levels of ROCK2 and ACSL4 in SAH group were significantly higher than those in Sham group(both P<0.05), and the protein expression levels of ROCK2 and ACSL4 in FH group were lower than those in SAH group (both P<0.05), while the expression level of GPX4 protein in SAH group (0.27±0.09) was significantly lower than that in Sham group( P<0.05), and the expression level of GPX4 protein in FH group was higher than that of SAH group ( P<0.05). Conclusion:FH can inhibit ferroptosis in the hippocampus and improve the learning and memory ability of rats, and the mechanism may be related with down-regulation of ROCK2 protein.

Fasudil alleviates lung injury via targeting Rhoa/ROCK signal pathway in septic mice / 中华急诊医学杂志

Objective:To determine the protective effect of fasudil on acute lung injury in septic mice.Methods:Forty-five 4-6-week-old male C57BL mice were randomly(random number) assigned to three groups ( n=15 each group): control group, lipopolysaccharide (LPS) group and Fasudil intervention group (FAS+LPS). Acute lung injury model of septic mice was established with an intraperitoneal injection and intratracheal infusion of LPS. The mice in the FAS+LPS group were injected with fasudil hydrochloride intraperitoneally 30 min before intraperitoneal LPS injection and 1 h after intratracheal LPS infusion, respectively. All mice were sacrificed at 4 h after modeling, and lung tissues were collected. Hematoxylin-eosin staining was preformed to observe the morphological changes in the lung tissue. The wet /dry weight (W/D) ratio, malondialdehyde (MDA) content and the activity of myeloperoxidase (MPO) in the lung tissues were detected. Caspase-3 expression was examined by immunohistochemical (IHC) staining. Western blot was employed to detect the expression of RhoA, ROCK1, endothelial nitric oxide synthase (eNOS), and p-eNOS. Results:Inflammatory cell infiltration and erythrocyte exudation were significantly reduced, and the degree of interstitial oedema and derangement of alveolar structure appeared in a decreasing degree after FAS intervention. Compared with the LPS group, the W/D ratio, MDA content, MPO activity and the expression of Caspase-3 in the FAS+LPS group were significantly reduced (all P<0.01). Meanwhile, the expression of RhoA and ROCK1 of the LPS group were obviously higher than those in the control group ( P<0.05), and p-eNOS was obviously lower than that in the control group ( P<0.05). Furthermore, the expression of RhoA and ROCK1 of the FAS+LPS group were obviously lower than those in the LPS group, and p-eNOS was obviously higher than that in the LPS group. There was no significant difference on the expression of eNOS among the three groups. Conclusions:Fasudil can alleviate the degree of inflammatory cell infiltration, reduce apoptosis in lung tissue, inhibit the RhoA/ROCK1 signaling activity, and promote the phosphorylation expression of eNOS in septic mice.

Effect of fasudil on experimental autoimmune neuritis and its mechanisms of action

This study aimed to investigate the therapeutic effect of fasudil on treating experimental autoimmune neuritis (EAN). Twenty-four EAN mice were randomly assigned to fasudil treatment (Fasudil group) or saline treatment (EAN model group) for 28 days. Clinical symptom score was evaluated every other day; inflammatory cell infiltration, demyelination, anti-myelin basic protein (MBP), inflammatory cytokines, inducible nitric oxide synthase (iNOS), and arginase-1 were detected in sciatic nerves at day 28. Th1, Th2, Th17, and Tregs proportions in splenocytes were detected at day 28. Clinical symptom score was found to be attenuated in the Fasudil group compared to the EAN model group from day 12 to day 28. Sciatic nerve inflammatory cell counts by HE staining and demyelination by luxol fast blue staining were both reduced, while MBP was increased in the Fasudil group compared to the EAN model group at day 28. Interferon γ (IFN-γ) and interleukin (IL)-17 were reduced, while IL-4 and IL-10 were elevated in the Fasudil group at day 28. Sciatic nerve M1 macrophages marker iNOS was decreased while M2 macrophages marker arginase-1 was increased in the Fasudil group at day 28. CD4+IFN-γ+ (Th1) and CD4+IL-17+ (Th17) cell proportions were both decreased, CD4+IL-4+ (Th2) cell proportion was similar, while CD25+FOXP3+ (Treg) cell proportion in splenocytes was increased in the Fasudil group. In summary, fasudil presented a good therapeutic effect for treating EAN by attenuating Th1/Th17 cells and promoting Tregs activation as well as M2 macrophages polarization.

Clinical effect of fasudil combined with cattle encephalon glycoside and ignotin injection in the treatment of diabetic deafness and tinnitus / 中国基层医药

Objective@#To study the clinical effect of fasudil plus cattle encephalon glycoside and ignotin injection in treating diabetic deafness and tinnitus.@*Methods@#From January 2016 to December 2016, 60 patients with diabetic deafness and tinnitus in the Fifth People's Hospital of Datong were selected and randomly divided into two groups according to the digital table, with 30 cases in each group.The treatment group received fasudil injection plus cattle encephalon glycoside and ignotin injection (iv gtt), and the control group received cattle encephalon glycoside and ignotin injection.The effective rate after treatment for 10 days and 20 days were compared between the two groups.@*Results@#Compared with the control group, the effective rate of hearing in the treatment group was not higher at 10 days after treatment (26.7% vs.16.7%, χ2=0.884, P=0.347), while it was higher in the treatment group at 20 days after treatment (53.3% vs.26.7%, χ2=4.444, P=0.035). The effective rate of tinnitus in the treatment group at 10 days after treatment was not higher compared with the control group (30.0% vs.20.0%, χ2=1.491, P=0.222), while it was higher in the treatment group at 20 days after treatment (56.7% vs.30.0%, χ2=4.344, P=0.037). As far as hearing threshold: there were no statistically significant differences between the control group and the observation group before treatment and 10 days after treatment(all P>0.05). The hearing threshold of the observation group was significantly better than that of the control group at 20 days after treatment (t=-2.511, P=0.02).@*Conclusion@#The combination therapy of fasudil injection plus cattle encephalon glycoside and ignotin injection has better efficacy for diabetic deafness and tinnitus.

Clinical effect of fasudil combined with cattle encephalon glycoside and ignotin injection in the treatment of diabetic deafness and tinnitus / 中国基层医药

Objective To study the clinical effect of fasudil plus cattle encephalon glycoside and ignotin injection in treating diabetic deafness and tinnitus.Methods From January 2016 to December 2016,60 patients with diabetic deafness and tinnitus in the Fifth People＇s Hospital of Datong were selected and randomly divided into two groups according to the digital table ,with 30 cases in each group.The treatment group received fasudil injection plus cattle encephalon glycoside and ignotin injection (iv gtt),and the control group received cattle encephalon glycoside and ignotin injection.The effective rate after treatment for 10 days and 20 days were compared between the two groups.Results Compared with the control group ,the effective rate of hearing in the treatment group was not higher at 10 days after treatment (26.7%vs.16.7%,χ2 ＝0.884,P＝0.347),while it was higher in the treatment group at 20 days after treatment (53.3%vs.26.7%,χ2 ＝4.444,P＝0.035).The effective rate of tinnitus in the treatment group at 10 days after treatment was not higher compared with the control group (30.0%vs.20.0%,χ2 ＝1.491,P＝0.222),while it was higher in the treatment group at 20 days after treatment (56.7% vs.30.0%,χ2 ＝4.344,P＝0.037).As far as hearing threshold :there were no statistically significant differences between the control group and the observation group before treatment and 10 days after treatment ( all P ＞0.05).The hearing threshold of the observation group was significantly better than that of the control group at 20 days after treatment (t＝－2.511,P＝0.02 ).Conclusion The combination therapy of fasudil injection plus cattle encephalon glycoside and ignotin injection has better efficacy for diabetic deafness and tinnitus.

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.

The neuroprotective effects of Fasudil on brain ischemia/reperfusion injury through maintaining blood-brain barrier function / 中华行为医学与脑科学杂志

Objective To investigate the neuroprotective effects of Fasudil in cerebral I/R injury in mice.Methods 51 C57BL/6J mice was divided into two groups,CMC treated group (n=26) and Fasudil treated group (n=25),randomly.The mice were treated with Fasudil (10 mg/kg) or CMC (0.5％ CMC 10 ml/kg) separately.Then the treated mice were subjected to 60 min of focal ischemia and 18 h reperfusion.The infarct volume of brain was analyzed by TTC staining with MCID image system.BBB permeability was assessed by Evans blue extravasation and albumin leakage which was detected by immuno-blotting assay.The activity of MMP9 was analyzed by zymography.Results The infarct volume in CMC group ((99.07±6.53) mm3) was larger than that in Fasudil group ((57.02±8.93) mm3),the difference was statistically significant (P＜0.01).The activity of MMP9 in the mice treated with Fasudil was lower than that in CMC group.Compared with the CMC group(albumin (2.95±0.77),Evans blue (5.15±0.24)),the albumin and Evans blue content in the Fasudil treated group (albumin (1.04±0.18),Evans blue (1.96±0.31))reduced significanctly(all P＜0.01).Conclusion Fasudil protects I/R damage by inhibiting the activity of MMP9 to maintain blood-brain barrier permeability.

Clinical effect of fasudil in heart failure induced by chronic pneumocardial disease and the influence in serum level of NO and ET-1 / 药物评价研究

Objective To investigate the clinical effect of fasudil in heart failure induced by chronic pneumocardial disease and the influence in serum level of NO and ET-1.Methods 100 cases of patients with heart failure induced by chronic pneumocardial disease in our hospital from September 2013 to September 2016 were selected and divided into observation group and control group,50 cases in each group.Patients in the control group were treated with conventional therapy,and in the observation group were treated with fasudil based on the conventional therapy.Compared the clinical effect,blood oxygen partial pressure (PaO2),CO2 partial pressure (PaCO2),tricuspid regurgitation speed,fight ventricular outflow tract inside diameter(RVOT),left ventricular ejection fraction (LVEF) and serum biochemical indexes (NO and ET-1) levels.Results After treatment,the total effective rate of the observation group was significantly higher than control group (P ＜ 0.05).PaO2,LVEF,serum level of NO of the two groups were significantly higher,PaCO2,tricuspid regurgitation speed,RVOT,serum level of ET-1 significantly lower than before treatment (P ＜ 0.05),and the PaO2,LVEF,serum level of NO of observation group were significantly higher,PaCO2,tricuspid regurgitation speed,RVOT,serum level of ET-1 of observation group were significantly lower than control group (P ＜ 0.05).Conclusion Fasudil had remarkable clinical effect in heart failure induced by chronic pneumocardial disease,and improve the patient's clinical symptoms,adjust the serum level of NO and ET-1.

Neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells on mice with experimental autoimmune encephalomyelitis / 中国病理生理杂志

AIM: To explore the neuroprotective effect of fasudil combined with bone marrow -derived neural stem cells ( BM-NSCs) on the mice with experimental autoimmune encephalomyelitis ( EAE).METHODS: Female C57BL/6 mice (8~10 weeks old, n=32) were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) to establish chronic EAE model .The mice were randomly divided into control ( ddH2 O ) group, fasudil group , BM-NSCs group , and fasudil+BM-NSCs group .The clinical score and body weight were recorded every other day .The expression of neurotrophic factors was determined by immunofluorescence staining .RESULTS:In comparison with ddH2O group, fasud-il combined with BM-NSCs delayed onset and ameliorated severity of EAE .The numbers of brain-derived neurotrophic fac-tor, glial cell-derived neurotrophic factor , nerve growth factor , neurotrophin-3 and ciliary neurotrophic factor positive cells in fasudil group, BM-NSCs group and fasudil +BM-NSCs group were all increased in various extents .In particularly, the expression of these neurotrophic factors in fasudil +BM-NSCs group was significantly higher than that in the mice treated with fasudil or BM-NSCs alone (P<0.01).CONCLUSION:Fasudil combined with BM-NSCs promotes the expression of neurotrophic factors and improves microenvironment of central nervous system , thus playing a positive role in neural restora-tion and regeneration through a synergistic and superimposed effect .

Evaluation of the effect of fasudil hydrochloride on outflow facility in enucleated eyes of different animals / 眼科新进展

Objective To evaluate the influence of a selective Rho-associated protein kinase inhibitor (fasudil hydrochloride) on outflow facility in enucleated porcine,rabbit and bovine eyes.Methods At the constant perfusion pressure of 15 mm-Hg (1 kPa =7.5 mmHg),the baseline coefficient of outflow facility (C0) of the isolated porcine,rabbit and bovine eyes was recorded respectively.The enucleated porcine eyes were divided into two groups randomly (n =6),and they were control group and experimental group.The same grouping method was also used-C0 the ribbit and bovine eyes.The control group was subjected to GPBS perfusion,while the experimental group was treated with 100 μmol · L-1 fasudil solution,followed by recording the experimental coefficient of outflow facility (C1),as well as calculating ΔC (ΔC =C1-C0) and ΔC％ (ΔC％ =ΔC/C0).Finally,the paired t test and one-way analysis of variance were performed using SPSS 17.0.Results As for porcine eyes,the ΔC％ of the control group was (17.83 ± 3.84) ％ while the experimental group was (44.00 ± 6.44) ％;as for rabbit eyes,the ΔC％ of the control group was (15.50 ± 2.93) ％,while the experimental group was (31.67 ±6.54)％;as for bovine eyes,the ΔC％ of the control group was (11.67 ± 1.17)％,while the experimental group was (37.17 ± 4.48)％.The ΔC％ in the experimental group was significantly increased when compared with the control group in three animals,with significant difference (all P ＜ 0.05).There was no statistical difference in ΔC％ of three experimental groups among different kinds of animals (all P ＜ 0.05).Conclusion Fasudil can improve outflow facility in enucleated eyes of animals,and it can redistribute aqueous humor drainage to a wider area through directly regulating the cytoskeleton of cells and matrix,resulting in increased coefficient of outflow facility.

Fasudil blocks muscle atrophy and C2C12 myoblasts respiratory dysfunction triggered by ROCK1 / 第二军医大学学报

Objective To confirm whether fasudil can block C2C12 myoblasts respiration dysfunction triggered by Rho-associated coiled-coil containing protein kinase 1 (ROCK1), and whether it can block the occurrence of muscle atrophy. Methods C2C12 myoblasts were cultured in vitro, and 2% horse serum was used to induce cell differentiation and maturation. The obtained mature muscle tubule cells were divided into four groups according to the different stimuli: Ad-GFP group, only transfected GPT-adenovirus vector (Adv) in C2C12 myoblasts; Ad-ROCKl group, transfected ROCKl-Adv in C2C12 myoblasts to induce ROCK1 overexpression; Ad-GFPF group, transfected GFP-Adv and given 10 μmol/L fasudil in C2C12 myoblasts; and Ad-ROCKIF group, transfected ROCKl-Adv and given 10 μmol/L fasudil in C2C12 myoblasts. The oxygen consumption rate (OCR) and extracelluar acidification rate (ECAR) of C2C12 myoblasts under different stimulation conditions were evaluated by cell energy metabolism analyzer (Seahorse), so as to determine the effect of ROCK1 overexpression and fasudil stimulation on the respiratory function of C2C12 myoblasts. Mitochondrial fission was measured by MitoTracker® red fluorescent probes. The expressions of ROCK1, mitochondrial related protein 1 (Drpl) and phosphorylated p-Drpl, E3 ubiquitin ligase muscle RING finger-1 protein (MuRFl) and muscle atrophy F-box (MAFbx, Atrogin 1) was measured by Western blotting analysis. Results Seahorse analysis showed that the OCR, ECAR, basal respiration, maximal respiration and respiration required for coupling ATP of C2C12 myoblasts in the Ad ROCK1 group were significantly increased compared with those in the Ad-GFP group (P<0.01); Meanwhile, MitoTracker® staining showed that the mitochondrial fission was increased and the mitochondrial size frequency distribution shifted left in the Ad-ROCK1 group. After exposed to fasudil. the OCR and EACR of C2C12 myoblasts in the Ad-ROCKIF group were significantly decreased versus the Ad-ROCK1 group, and the basal respiration and maximal respiration were significantly increased (P<0.05). Western blotting analysis showed that p-Drpl/Drpl ratio, and the expressions of ROCK1, MuRFl and Atroginl in Ad-ROCKIF group were significantly reduced compared with Ad-ROCKl group (P<0.05). Conclusion Fasudil, an inhibitor of ROCK1, can block the abnormal cell respiration of C2C12 myoblasts caused by overexpressed ROCK 1 in vitro, and can reduce the activity of mitochondrial kinetic protein and the expression of muscle atrophy-related proteins.

Effects of Rho kinase inhibitor fasudil on paraquat-induced pulmonary fibrosis in rats / 中华急诊医学杂志

Objective To investigate the effect of Rho kinase inhibitor,fasudil,on pulmonary fibrosis induced by paraquat in rats in order to elucidate the underlying mechanisms.Methods A total of 72 SpragueDawley male rats of specific pathogen free (SPF) were randomly (random number) divided into four groups:the normal control group (NS group,n =18),fasudil control group (FS control group,n =18),paraquat poisoning group (PQ poisoning group,n =18) and fasudil intervention group (FS intervention group,n =18).On days 7,14,28 after paraquat exposure,six rats were respectively selected from each group.These rats were anesthetized and sacrificed immediately,and their lung tissues were collected.The hydroxyproline (HYP) in the lung tissue was detected by using alkaline hydrolysis.The expressions of type Ⅰ,Ⅲ collagen protein,connective tissue growth factor (CTGF) and ROCK1 mRNA in Rho/ROCK signaling pathway were assayed by using the real-time quantitative PCR (RT-PCR),and the levels of type Ⅰ,Ⅲ collagen protein,connective tissue growth factor (CTGF) and Rho / ROCK signaling pathway ROCK1 protein were measured by using Western blotting.The pathological changes of lung tissue were observed under light microscope.Results There were no significant differences in the observed biomarkers between FS control group and NS group (P ＞ 0.05).While in PQ poisoning group and FS intervention group on days 7,14,28 (all P ＜ 0.05),the amount of HYP increased obviously (P ＜ 0.05),the expressions of type Ⅰ,Ⅲ collagen protein,CTGF,ROCK1 mRNA and protein levels were increased significantly (P ＜ 0.05).Compared with the PQ poisoning group,the amount of HYP decreased significantly,and the expressions of type Ⅰ,Ⅲ collagen protein,CTGF,ROCK1 mRNA and protein levels were decreased significantly in FS intervention group on days 7,14,28 (all P ＜ 0.05).The pathological changes of lung tissue revealed that the degree of pulmonary fibrosis in the PQ poisoning group were most serious on 28 d after paraquat exposure,and the degree of pulmonary fibrosis were lessened in FS intervention group on days 7,14,28.Conclusions ROCK inhibitor,fasudil,has obvious therapeutic effects on paraquat-induced lung fibrosis,by regulating Rho / ROCK signaling pathway with downregulated expression of CTGF,and decrease in the levels of type Ⅰ,Ⅲ collagen protein,thus reducing protein deposition.

Study for improving effect on ventricular structure and clinic effect of fasudil in patients with heart failure / 心血管康复医学杂志

Objective:To observe clinical therapeutic effect of fasudil hydrochloride in patients with chronic heart failure (CHF).Methods: A total of 134 CHF cases were selected from our department.According to random number table, they were randomly and equally divided into routine treatment group and fasudil group, both groups were treated for three months.Left ventricular ejection fraction (LVEF), left ventricular end-diastolic dimension (LVEDd), left ventricular end-systolic dimension (LVESd), levels of brain natriuretic peptide (BNP), endothelin (ET)-1 and atrial natriuretic peptide (ANP) were compared between two groups before and after treatment.Results: Compared with fasudil group before treatment and routine treatment group after treatment, there were significant reductions in LVEDd [(58.20±8.44) mm, (58.22±10.21)mm vs.(51.24±7.37)mm], LVESd [(47.52±6.51) mm, (47.56±6.54)mm vs.(41.24±5.33)mm], levels of BNP [(381.35±62.48) μg/L, (377.82±61.46) μg/L vs.(294.52±51.33) μg/L], ET-1 [(81.93±13.53) μg/L, (81.32±12.63) μg/L vs.(68.44±11.42) μg/L] and ANP [(215.33±39.72) μg/L, (210.51±36.17) μg/L vs.(172.26±27.54) μg/L], and significant rise in LVEF [(29.69±4.08)%, (29.94±4.25)% vs.(37.47±5.26)%] in fasudil group after three-month treatment, P0.05.Conclusion:Fasudil hydrochloride can significantly reduce levels of BNP, ET-1 and ANP, delay ventricular remodeling and improve cardiac function in CHF patients.The therapeutic effect is significant and safety is good.

Role of Rho-kinase in remote ischemicpostconditioning against myocardial ischemia/reperfusion injury / 中国药理学通报

Aim To explore the role of Rho-kinase in remote ischemic postcondi-tioning and its possible mechanism.Methods Thirty male Sprague-Dawley rats were divided into five groups(n=6): sham group(Sham), ischemia/reperfusion group(I/R), remote ischemic postconditioning group(RIPostC), I/R with Rho-kinase inhibitor fasudil group(I/R+Fas) and RIPostC with Rho-kinase activator lysophosphatidic acid group(RIPostC+LPA).Throughout the whole process of experiment, mean arterial pressure(MAP), heart rate(HR) and Ⅱ lead electrocardiogram were continuously monitored.At the end of the reperfusion, plasma creatine kinase(CK) and lactate dehydrogenase(LDH) were measured.Myocardial histopathologic changes were observed by hematoxylin and eosin(HE) staining.Infarct size was measured using 2,3,5-triphenyltetrazolium chloride(TTC) staining.The expressions of phospho-myosin light chain(p-MLC) were detected with Western blot analysis.Results Compared with Sham group, the MAP and HR of other groups decreased, while the amplitude of ST segment increased.Compared with I/R group, MAP and HR increased, the amplitude of ST segment decreased, plasma CK and LDH activity decreased, myocardial pathological morphology and infarct size were improved significantly, infiltration of inflammatory cells was reduced, and the expression of p-MLC decreased in RIPostC and I/R+Fas group.Compared with RIPostC group, RIPostC+LPA group attenuated the effects of RIPostC, and the recovery of the above indicators were inhibited.Conclusion Rho-kinase signaling pathway might mediate remote ischemia postconditioning against myocardial ischemia/reperfusion injury.

Effects of Fasudil Hydrochloride on Learning and Memory Ability and Autophagy in CA1 Area of Hippocampus in Subarachnoid Hemorrhage Rats / 中国康复理论与实践

Objective To investigate the effects of fasudil hydrochloride on learning and memory, and the autophagy in hippocampal CA1 neurons in subarachnoid hemorrhage (SAH) rats. Methods Fifty-four male Sprague-Dawley rats were randomly divided into sham group (n=18), SAH group (n=18) and drug group (n=18). Subarachnoid hemorrhage model was established with internal carotid artery punc-ture. The drug group was injected fasudil hydrochloride 10 mg/kg intraperitoneally after modeling per 24 hours, while the sham group and SAH group were injected the same volume of saline. They were tested with shuttle box test 6, 24 and 72 hours after intervention, then the hippocampal CA1 area was stained with HE and immunohistochemistry to observe the morphology of cells and the expression of Beclin-1 and microtubule-associated protein 1 light chain 3 II (LC3-II). Results Compared with the sham group, the frequence of avoidance de-creased in SAH group at each time point (P<0.05), while the avoidance reaction time increased (P<0.05);the survival of neurons in hippo-campal CA1 area decreased (P<0.05), and the expression of Beclin-1 and LC3-II increased (P<0.05). Compared with SAH group, the fre-quence of avoidance increased in the drug group at each time point (P<0.05), while the avoidance reaction time decreased (P<0.05);the sur-vival of neurons in hippocampal CA1 area increased (P<0.05) and the expression of Beclin-1 and LC3-II increased further (P<0.05). Con-clusion Fasudil hydrochloride can improve learning and memory ability and protect neurons from damage, which may associate with the ex-cess of autophagy activation in hippocampal CA1 areas in SAH rats.

Effects of fasudil on the expressions of Bcl-2 and Bax in cerebral cortex of rats with subarachnoid hemorrhage / 天津医药

Objective To investigate the effects of fasudil on expressions of Bcl-2 and Bax in cerebral cortex of model rats with subarachnoid hemorrhage (SAH). Methods Thirty rats were divided into sham operation group, SAH group and SAH+fasudil group, 10 rats in each group. Double injection of blood into occipital cistern method was used for SAH model in SAH group and SAH+fasudil group. In the sham operation group, the blood injection was instead by normal saline. In the SAH+fasudil group, at 30 min after the second injection of blood, rats were administrated with intraperitoneal injection of fasudil at a dose of 3 mg/kg. The general situation, neurological score, TUNEL staining for cortex cell apoptosis, immune histochemical staining and Western blotting assay for Bcl-2 and Bax protein expression were compared 24 h after the operation between the three groups. Results Compared with the sham operation group, rats in SAH group and SAH +fasudil group appeared obvious neurological deficits. The neurological score was significantly lower in SAH group ( 2.68 ± 0.31) than that of sham operation group (5.00±0.00). The neurological score was significantly higher in SAH+fasudil group (3.27 ± 0.35) compared with that of SAH group (2.68 ± 0.31, P<0.05). There was obvious cell apoptosis in SAH group and SAH+fasudil group, and the apoptosis was less in SAH+fasudil group than that of SAH group (P<0.05). The level of Bcl-2 expression was significantly lower in SAH group than that of sham operation group, and Bax expression was significantly higher in SAH group than that of sham operation group (P<0.05). The level of Bcl-2 expression was significantly higher in SAH+fasudil group than that of SAH group, but Bax expression was significantly lower in SAH+fasudil group than that of SAH group (P<0.05). Conclusion Fasudil can improve the neurological damage in rats with SAH, which may be related with the regulation of apoptosis related proteins Bcl-2 and Bax.

Fasudil reduces formation of urethral stricture after injury via inhibiting Rho/ROCK pathway activation in rabbit urethra fibroblasts / 中国病理生理杂志

AIM:To investigate the role of Rho-associated kinase ( ROCK) inhibitor fasudil in the formation of rabbit urethral stricture after injury and to observe the cell activity , migration and extracellular matrix synthesis in the rabbit urethra fibroblasts.METHODS:The rabbit model of urethral stricture was established by microsurgical techniques .The rabbits were divided into sham operation group , operation group and fasudil (3 mg/kg, 10 mg/kg, 30 mg/kg) groups.The diameter of the stenosis was measured by retrograde urethrography 3 months after surgery .The fibroblasts were isolated from urethral scar, and then incubated with fasudil (12.5 μmol/L, 25 μmol/L, 50 μmol/L) in the presence of transforming growth factor-β1 (TGF-β1, 10 μg/L).The untreated cells were used for control .The cell activity was measured by MTT assay.The cell migration ability was tested by the method of Transwell chambers .The protein expression of ROCK , α-smooth muscle actin (α-SMA) , collagen I and collagen III was determined by Western blot analysis .RESULTS:Fasudil significantly reduced formation of urethral stricture after injury (P<0.05).Cultured rabbit fibroblasts with different con-centrations of fasudil inhibited the cell activity and cell migration ability (P<0.05).The protein expression of ROCK,α-SMA, collagen I and collagen III was also inhibited by treatment with fasudil in a dose -dependent manner ( P<0.05 ) . CONCLUSION:Fasudil inhibits the formation of extracellular matrix and reduces the incidence of urethral stricture after injury by down-regulating TGF-β1-induced Rho/ROCK pathway activation in the rabbit urethra fibroblasts .

The protective effects of different doses of fasudil on hepatic ischemia/reperfusion injury in rats with cirrhosis / 中华普通外科杂志

Objective To investigate the protective effects of different doses of fasudil on hepatic ischemia/reperfussion (I/R) injury in rats with liver cirrhosis.Methods Cirrhosis was induced in rats by subcutaneous injection of 60％ carbon tetrachloride (CC14) corn oil solution (0.4 ml/100 g) twice a week for twelve weeks.Five percent of alcohol was given intermittently in drinking water.Then 40 cirrhotic rats were randomized into 4 groups.In sham group,sham operation was performed.In I/R group A and B,whole rat livers were subjected to warm ischemia by clamping the hepatic artery and portal vein for 30 min.In group A,the selected rats were pretreated with low-dose fasudil 1 mg/kg (intraperitoneal injection) 30 min before the induction of ischemia,and in group B,with high-dose 10 mg/kg.The serum levels of alanine aminotransferase (ALT) and endothelin-1 (ET-1),and the liver tissue superoxide dismutase (SOD)activity,malondialdehyde (MDA) content,the expression of HIF-1a (hypoxia-inducible fador-1a) were measured after reperfusion for 6 hours.Hepatic pathologic changes were observed under microscope.Results Compared with I/R group,the serum ALT,AST,ET-1 levels,MDA content and the expression of HIF-1 a were markedly decreased in group B,while the SOD activity significantly increased (P ＜ 0.05).And the pathologic changes were less severe in group B.Conclusion The high-dose fasudil markedly lessened the expression of HIF-1a,up-regulated the concentration of SOD,and lowered the levels of MDA and ET-1,protecting against heoatic ischemia/reperfusion injury in rats with liver cirrhosis.