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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.
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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.
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Animals , Mice , Brain Injuries , Reperfusion Injury , Brain Ischemia , Oxidative StressABSTRACT
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.
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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.
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Objective:To evaluate the curative effect and safety of fasudil hydrochloride injection in the prevention and treatment of aneurysm postoperative cerebral vasospasm by meta-analysis. Methods: The randomized controlled trials were retrieved from PubMed, EMBASE, Cochrane Library, VIP, Wangfang, CNKI and so on. Meta-analysis was conducted using RevMan 5. 0 software. Results:Totally 418 reference studies were screened, from which 11 ones were chosen including 786 patients in all. In the treatment of cerebral vasospasm (CVS), there was no significant difference between the groups (OR=1. 56, 95%CI:0. 95-2. 58, P>0. 05). While in the prevention of CVS, the incidence rate of CVS in fasudil group was significantly lower than that in nimodipine group ( OR=0. 43, 95%CI:0. 23-0. 81, P=0. 008). However, the incidence rate of ADR in fasudile group was higher than that in nimodipine group (OR=0. 43, 95%CI:0. 25-0. 75,P=0. 003). Conclusion:In the prevention of CVS, fasudil may be better than nimodipine, while the incidence of ADR is higher.
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Objective:To establish an HPLC method for the determination of isomeric impurity in fasudil hydrochloride. Meth-ods:The chromatographic method was carried out on a Kromasil 100-5 Phenyl C18 column with phenyl bonded silica as the filler (250 mm × 4. 6 mm, 5 μm), and phosphate buffer (10 mmol· L-1 ammonium dihydrogen phosphate, adjusting pH to 4. 0 with 1% phos-phoric acid) -acetonitrile (80∶ 20) was used as the mobile phase. The detection wavelength was 275nm and the column temperature was 40℃. The flow rate was 1. 0 ml· min-1 , and the injection volume was 10 μl. Results:Fasudil hydrochloride and its derivative was linear within the range of 0. 148-2. 960 μg·ml-1(r=1. 0000) and 0. 101-2. 014μg·ml-1(r=0. 999 9), respectively. The av-erage recovery of isomer impurity in fasudil hydrochloride was 101. 9% with RSD of 0. 98%(n=9). Conclusion:The method is sim-ple,accurate and reproducible,which can be used for the quality control of fasudil hydrochloride and its isomer.
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OBJECTIVE@#To observe the protective effect of fasudil hydrochloride against acute renal injury in septicopyemia rats.@*METHODS@#A total of 60 Wister rats were included in the study and divided into control group (n = 10), model group (n = 25) and treatment group (n = 25). Model group and treatment group received intraperitoneal injection of endotoxin (ET) to establish acute renal injury models while the control group only received daily intraperitoneal injection of normal saline 1 mL. Five rats were taken out of model group and treatment group respectively at 1 h (T1), 6 h (T2), 12 h (T3), 24 h (T4) and 48 h (T5), for intraperitoneal injection of ET 30 mg/kg. Treatment group received intraperitoneal injection of fasudil hydrochloride 30 mg/kg 1 h before injection of ET. For three groups, 5 mL blood samples were collected from postcava for determination of serum creatinine and urea nitrogen levels at different time points. Concentrations of serum tumor necrosis factor α and ET-1 were determined by using ELISA. The renal pathologic changes were observed under the microscope.@*RESULTS@#Serum creatinine levels in both model group and treatment group were significantly higher than control group at T2-T5 (P < 0.05) while the levels in treatment group were significantly lower than control group at T3-T5 (P < 0.05). At T2-T5, blood urea nitrogen levels in model group and treatment group were significantly higher than control group (P < 0.05) while the levels in treatment group were significantly lower than model group at T3-T5 (P < 0.05). Concentrations of serum tumor necrosis factor α in model group and treatment group were significantly higher than control group at T1-T5 (P < 0.05) while the levels in treatment group were significantly lower than model group at T1-T5 (P < 0.05). Serum ET-1 concentrations in model group and treatment group were significantly higher than control group at T1-T5 (P < 0.05) while the levels in treatment group at T1-T4 were significantly lower than model group (P < 0.05). Rats in control group showed no swelling or hyperemia in kidney cells but normal structure and normally arranged renal tubular epithelial cells. Obvious injury was observed in model group at T3 and renal tubular epithelial cells in disorder and at swelling condition, hyperemia and angiectasis in glomerulus, degenerative opacities and vacuolar degeneration, and maximized injury were observed at T4. Injury in renal tissue in treatment group was significantly milder than model group.@*CONCLUSIONS@#Fasudil hydrochloride has the significantly protective effect against acute renal injury in septicopyemia rats.
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AIM:To explore the protective effect of fasudil hydrochloride against cisplatin (CP)-induced renal tubular epithelial cell apoptosis via Akt activation and PTEN inhibition .METHODS:Healthy male Sprague-Dawley ( SD) rats were randomly divided into control group , CP group and CP+fasudil group .All animals were sacrificed 96 h after in-jection of 0.9%saline or CP .Blood samples and kidney tissues were collected to evaluate levels of blood urea nitrogen (BUN), serum creatinine (sCr) and morphological alteration of the kidneys , respectively.The apoptosis of renal tubular epithelium cells was detected by TUNEL.Protein levels of Rho-associated protein kinase 1 (ROCK1), PTEN and Akt were measured by Western blotting and immunohistochemistry .The protein level of p-Akt was analyzed by Western blotting . RESULTS:Compared with control group , the sCr and BUN levels , the expression of ROCK 1 and PTEN and TUNEL-posi-tive cells were increased , while the level of p-Akt was decreased in CP group and CP +fasudil group .The histological structure of the kidneys observed by PAS staining was developed marked structural damage in CP group (P<0.05).Com-pared with CP group, sCr level, the expression of ROCK1 and PTEN and TUNEL-positive cells were decreased, while the level of p-Akt was increased in CP+fasudil group (P<0.05).Very little structural damage was detected in fasudil-treated groups .CONCLUSION:Fasudil hydrochloride has a protective effect on CP-induced renal tubular epithelial cell apoptosis via Akt activation and PTEN inhibition 1.
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Objective: To observe the protective effect of fasudil hydrochloride against acute renal injury in septicopyemia rats. Methods: A total of 60 Wister rats were included in the study and divided into control group (n = 10), model group (n = 25) and treatment group (n = 25). Model group and treatment group received intraperitoneal injection of endotoxin (ET) to establish acute renal injury models while the control group only received daily intraperitoneal injection of normal saline 1 mL. Five rats were taken out of model group and treatment group respectively at 1 h (T1), 6 h (T2), 12 h (T3), 24 h (T4) and 48 h (T5), for intraperitoneal injection of ET 30 mg/kg. Treatment group received intraperitoneal injection of fasudil hydrochloride 30 mg/kg 1 h before injection of ET. For three groups, 5 mL blood samples were collected from postcava for determination of serum creatinine and urea nitrogen levels at different time points. Concentrations of serum tumor necrosis factor α and ET-1 were determined by using ELISA. The renal pathologic changes were observed under the microscope. Results: Serum creatinine levels in both model group and treatment group were significantly higher than control group at T2-T5 (P < 0.05) while the levels in treatment group were significantly lower than control group at T3-T5 (P < 0.05). At T2-T5, blood urea nitrogen levels in model group and treatment group were significantly higher than control group (P < 0.05) while the levels in treatment group were significantly lower than model group at T3-T5 (P < 0.05). Concentrations of serum tumor necrosis factor α in model group and treatment group were significantly higher than control group at T1-T5 (P < 0.05) while the levels in treatment group were significantly lower than model group at T1-T5 (P < 0.05). Serum ET-1 concentrations in model group and treatment group were significantly higher than control group at T1-T5 (P < 0.05) while the levels in treatment group at T1-T4 were significantly lower than model group (P < 0.05). Rats in control group showed no swelling or hyperemia in kidney cells but normal structure and normally arranged renal tubular epithelial cells. Obvious injury was observed in model group at T3 and renal tubular epithelial cells in disorder and at swelling condition, hyperemia and angiectasis in glomerulus, degenerative opacities and vacuolar degeneration, and maximized injury were observed at T4. Injury in renal tissue in treatment group was significantly milder than model group. Conclusions: Fasudil hydrochloride has the significantly protective effect against acute renal injury in septicopyemia rats.
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Objective To investigate the neuralprotective effect of Rho kinase inhibitor fasudil hydrochloride in cerebral ischemia/reperfusion injury in rats.Methods The SD rats were randomly divided into four groups:the sham group,the ischemia/reperfusion group,the fasudil hydrochloride group and the physiological saline group.Fasudil hydrochloride were injected intraperitoneally 30 minutes before ischemia.And the physiological saline group were treated with the intraperitoneal injection of the same volume of saline.The phosphorylation and protein expression of GluR6 at 6 hours during reperfusion were detected using immunoprecipitation and immunoblotting analysis to examine the effect of Fasudil hydrochloride.Furthermore,TUNEL staining was used to examine the apoptosis of neurons in rat hippocampal CA1 regions after 3 days reperfusion.Results 1.Immunoprecipitation and immunoblotting analysis were used to analyze the phosphorylation of GluR6 in serine site.The results showed that the GluR6 serine phosphorylation level increased significantly at 6h of reperfusion compared with the sham group (P＜0.05).Fasudil hydrochloride group could inhibit the increased phosphorylation of GluR6 at 6h of reperfusion compared with the ischemia/reperfusion group and saline group,respectively (P ＜ 0.05).2.TUNEL staining was used to examine the apoptosis of neurons in 3 days after reperfusion in CA1 regions of hippocampus.The results indicated that significant numbers of TUNEL positive cells (40.20 ± 2.77) were observed 3 days after ischemia/reperfusion.The numbers of viable neurons per 1 mm length of CA1 pyramidal cells were quantitatively analyzed.Fasudil hydrochloride markedly decreased the neuronal loss compared with the ischemia/reperfusion group (19.80 ± 2.86) (P＜0.05).Conclusion Fasudil hydrochloride can inhibit induced phosphorylation of GluR6 by the ischemia/reperfusion.Fasudil hydrochloride can reduce the neurons apoptosis in hippocampal CA1 regions,and perform a neuralprotective effect on ischemia/reperfusion injury in rats.
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Objective:To evaluate the clinical effects of fasudil hydrochloride on patients with acute cerebral infarction. Methods:100 cases of acute cerebral infarction patients were randomly divided into treatment group(n=50) and control group (n=50). All the cases were given enteric-coated tablets aspirin,ligustrazine hydrochloride injection, piracetam and sodium chlorideand injection and mannitol injection, and so on. Additionally,in the treatment group fasudil hydrochloride injection 30 mg,dissolved in 100 ml 5% glucose or saline were injected intravenously. Results:Basic recovery rate and total effective rate of treatment control were significantly higher(P