Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

Multiple sclerosis is characterized by demyelination and neuronal loss caused by inflammatory cell activation and infiltration into the central nervous system.Macrophage polarization plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis,a traditional experimental model of multiple sclerosis.This study investigated the effect of Fasudil on macrophages and examined the therapeutic potential of Fasudil-modified macrophages in experimental autoimmune encephalomyelitis.We found that Fasudil induced the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type,as shown by reduced expression of inducible nitric oxide synthase/nitric oxide,interleukin-12,and CD16/32 and increased expression of arginase-1,interleukin-10,CD14,and CD206,which was linked to inhibition of Rho kinase activity,decreased expression of toll-like receptors,nuclear factor-κB,and components of the mitogen-activated protein kinase signaling pathway,and generation of the pro-inflammatory cytokines tumor necrosis factor-α,interleukin-1β,and interleukin-6.Crucially,Fasudil-modified macrophages effectively decreased the impact of experimental autoimmune encephalomyelitis,resulting in later onset of disease,lower symptom scores,less weight loss,and reduced demyelination compared with unmodified macrophages.In addition,Fasudil-modified macrophages decreased interleukin-17 expression on CD4^(+)T cells and CD16/32,inducible nitric oxide synthase,and interleukin-12 expression on F4/80^(+)macrophages,as well as increasing interleukin-10 expression on CD4^(+)T cells and arginase-1,CD206,and interleukin-10 expression on F4/80^(+)macrophages,which improved immune regulation and reduced inflammation.These findings suggest that Fasudil-modified macrophages may help treat experimental autoimmune encephalomyelitis by inducing M2 macrophage polarization and inhibiting the inflammatory response,thereby providing new insight into cell immunotherapy for multiple sclerosis.

Fasudil在EAM小鼠中通过抑制NOTCH信号通路下调IL-6表达

目的探究法舒地尔(Fasudil)在实验性自身免疫性心肌炎(experimental autoimmune myocarditis,EAM)小鼠治疗中发挥的作用及机制,为临床上Fasudil治疗心肌炎提供理论依据。方法以Balb/c雄性小鼠为研究对象,使用MyHC-α614-629多肽构建EAM小鼠模型。单个核细胞分离和培养检测小鼠脾脏中单个核细胞的数量变化,HE染色、Masson染色和免疫组化分别检测小鼠心肌组织的炎症浸润情况、纤维化情况和IL-6的表达,Western blotting检测Notch1和白细胞介素-6(IL-6)蛋白的表达,qRT-PCR检测促炎因子(IL-1α、IL-1β和IL-6)、TLRs和NOTCH信号通路的关键基因表达。结果EAM小鼠心脏明显增大发白、HW增加、BW下降和HW/e-BW增大,心肌组织炎症浸润和纤维化加重。EAM小鼠经过Fasudil治疗后,上述的症状或者病理特征得到改善。进一步分析发现,EAM小鼠心肌组织中促炎因子IL-1α、IL-1β和IL-6明显增加,但是经过Fasudil治疗后只有IL-6的表达下调具有统计学差异,此外,还发现TLRs信号可能也在Fasudil治疗EAM发挥重要作用。IL-6和Notch1的表达具有一致性,经过Fasudil治疗后,NOTCH信号通路的关键基因(Notch1、Hes1和Jag2)显著下调。结论Fasudil在EAM小鼠中通过抑制NOTCH信号通路下调IL-6的表达,从而发挥保护作用。

Fasudil对实验性自身免疫性脑脊髓炎小鼠小胶质细胞和星形胶质细胞的作用

目的:确认法舒地尔(Fasudil)对实验性自身免疫性脑脊髓炎(EAE)的治疗效果,观察法舒地尔对小胶质细胞和星形胶质细胞的作用。方法:成年雌性C57BL/6小鼠用MOG35-55肽免疫制作慢性EAE模型,分别随机在免疫后第3天(Fasudil早期治疗组)和发病时给予Fasudil(Fasudil晚期治疗组),以同样方式给予生理盐水作为对照。观察临床症状和体质量变化;采用免疫荧光组织化学染色、Western blot法检测脊髓小胶质细胞和星形胶质细胞iNOS和p-NF-κB/p65的表达,ELISA测定脊髓匀浆中IL-1β和TNF-α水平。结果:Fasudil推迟EAE起病,减轻EAE症状,抑制脊髓中小胶质细胞iNOS以及星形胶质细胞p-NF-κB/p65表达,并伴随炎性因子IL-1β和TNF-α释放降低。结论:Fasudil可抑制EAE小鼠小胶质细胞和星形胶质细胞炎性分子的释放。

Fasudil促进体外培养骨髓源神经干细胞的增殖与存活

目的探讨法舒地尔（Fasudil）对体外培养骨髓源神经干细胞（BMNSC）增殖和存活的影响。方法取3～4周龄的C57BL／6小鼠，分离骨髓基质细胞并诱导产生细胞球，用免疫荧光染色鉴定细胞类型。羧基荧光素二乙酸盐琥珀酰亚胺酯（CFSE）标记细胞，检测Fasudil促进BMNSC增殖能力。利用脂多糖（LPS）和γ干扰素（IFN-γ）联合处理BV-2小胶质细胞制备炎性条件培养液处理BMNSC，Fasudil干预，通过流式细胞术检测线粒体膜电位、碘化丙啶（PI）荧光值，探讨Fasudil对BMNSC的保护效果。结果免疫荧光染色证明骨髓源细胞球为神经上皮干细胞蛋白（nestin）阳性的神经干细胞。Fasudil处理明显降低CFSE平均荧光值。在LPS和IFN-γ联合作用下，BV-2小胶质细胞释放更多的白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）等炎性细胞因子，显著高于PBS处理组。该炎性条件培养液导致BMNSC线粒体膜电位明显降低并诱导更多的细胞死亡，而Fasudil处理则显著恢复线粒体膜电位接近正常值，减少细胞死亡，并且具有明显的剂量依赖性。结论Fasudil对体外培养BMNSC的增殖和存活具有显著的促进作用。

Rho激酶抑制剂Fasudil对Aβ_(1-42)诱导AD模型大鼠抗氧化作用研究

目的探讨Rho激酶抑制剂Fasudil对β淀粉样蛋白1-42(Aβ1-42)诱导阿尔茨海默病大鼠学习、记忆功能和海马区氧化应激指标的影响。方法 80只成年雄性Wistar大鼠随机分成对照组(侧脑室模拟注射+生理盐水腹腔注射)、模型组(侧脑室Aβ1-42注射+生理盐水腹腔注射)、Fasudil低剂量组[侧脑室Aβ1-42注射+Fasudil 5 mg/(kg·d)腹腔注射]、Fasudil高剂量组[侧脑室Aβ1-42注射+Fasudil 10 mg/(kg·d)腹腔注射],每组各20只。采用侧脑室注射Aβ1-42制备AD模型,Fasudil腹腔注射14 d干预。Morris水迷宫实验进行大鼠的行为学测试,采用分光光度法测定海马组织匀浆中谷胱苷肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量。结果模型组大鼠水迷宫实验的逃避潜伏期明显较对照组延长(P<0.01),目标象限活动时间和穿台次数明显少于对照组(P<0.01);海马组织匀浆中SOD、GSH-Px活性明显下降(P<0.05,P<0.01),MDA的含量增加(P<0.05)。Fasudil高剂量组逃避潜伏期明显短于模型组(P<0.01),目标象限活动时间和穿台次数明显多于模型组(P<0.01);海马组织匀浆中SOD、GSH-Px活性较模型组增加(P<0.05),MDA含量变化不明显。结论 Fasudil能够剂量依赖性的减少Aβ1-42诱导AD大鼠模型海马区的氧化应激反应,改善其学习、记忆功能。

法舒地尔(Fasudil)抑制脂多糖诱导的小鼠星形胶质细胞活化和炎性反应及其机制

目的探讨法舒地尔(Fasudil)对脂多糖(LPS)诱导的星形胶质细胞活化和炎症反应及Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路的影响。方法体外培养新生C57BL/6小鼠大脑皮质星形胶质细胞,细胞分为PBS对照组、1μg/m L LPS刺激组、1μg/m L LPS联合15μg/m L盐酸法舒地尔处理组,Griess法检测培养细胞上清液一氧化氮(NO)的水平,ELISA检测肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-10和IL-4的水平,免疫荧光细胞化学染色检测星形胶质细胞胶质原纤维酸性蛋白(GFAP)及TLR4的表达,Western blot法检测GFAP、TLR4和磷酸化的NF-κBp65(p-NF-κBp65)蛋白水平。结果与PBS组比较,LPS组NO、TNF-α和IL-6水平显著升高,IL-10和IL-4水平降低;法舒地尔能抑制LPS诱导的NO、TNF-α和IL-6的分泌,增加IL-10和IL-4的分泌。法舒地尔处理组星形胶质细胞GFAP表达显著降低,同时TLR4和NF-κB蛋白的水平也降低。结论法舒地尔阻断TLR4/NF-κB信号通路抑制LPS诱导的星形胶质细胞活化及炎性反应。

Fasudil通过TLR4通路抑制脂多糖诱导的小鼠BV-2小胶质细胞TNF-α和IL-1β的表达

目的探讨Fasudil对脂多糖(LPS)诱导BV-2小胶质细胞系促炎细胞因子表达中的作用。方法体外培养BV-2小胶质细胞系,实验分为PBS对照组、LPS刺激组、LPS联合Fasudil干预组,ELISA检测细胞TNF-α、IL-1β的释放,Griess法检测NO释放水平,流式细胞术检测Toll样受体4(TLR4)、TLR2蛋白表达。结果 LPS刺激BV-2细胞可导致TNF-α、IL-1β和NO的释放明显增加,还可导致炎性信号通路中的受体TLR4表达明显增加。Fasudil能明显抑制炎性因子的释放和TLR4的表达。结论 Fasudil可抑制LPS诱导的小胶质细胞NO、TNF-α和IL-1β释放,其作用机制可能与Fasudil下调TLR4通路有关。

Fasudil对实验性自身免疫性脑脊髓炎小鼠致脑炎性T细胞的免疫调节作用

目的:探讨法舒地尔(Fasudil)修饰的脾单个核细胞(mononuclear cells,MNCs)治疗实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)的疗效及可能的分子机制。方法:雌性C57BL/6小鼠采用髓鞘少突胶质细胞糖蛋白35–55诱导,建立主动免疫EAE模型,免疫后第9天制备脾MNCs,和/无Fasudil培养。作用72 h后收集上述细胞,检测T细胞亚群的变化、Rho激酶(Rho kinase,ROCK)活性和细胞因子水平;以5×107个细胞/只小鼠腹腔注射诱导,建立被动转移EAE模型,并将小鼠分为PBS-MNCs组和Fasudil-MNCs组,每组8只。观察各组小鼠临床评分和体质量变化。结果:免疫第9天EAE小鼠脾致脑炎性MNCs可通过被动转移实验诱导EAE模型的建立,而体外Fasudil修饰的MNCs不能诱导EAE的发生;与PBS-MNCs组相比,Fasudil-MNCs组小鼠体质量减轻较少(P<0.05)。体外实验显示:Fasudil处理的MNCs ROCK活性降低(P<0.01);Fasudil可抑制IFN-γ和IL-17的CD4+T细胞比例(IFN-γ:P<0.01;IL-17:P<0.05),并增强TGF-β和IL-10的CD4+T细胞分泌(均P<0.001);此外,Fasudil可降低细胞因子IL-17水平(P<0.001)和增强细胞因子IL-10分泌(P<0.05)。结论:Fasudil介导的细胞治疗通过抑制辅助性T细胞1(helper T cell 1,Th 1)和Th 17炎性反应,促进Th 2免疫调节作用,影响EAE的发生和发展。

The role of Rho/Rho-kinase pathway and the neuroprotective effects of fasudil in chronic cerebral ischemia

The Rho/Rho-kinase signaling pathway plays an important role in cerebral ischemia/reperfusion injury. However, very few studies have examined in detail the changes in the Rho/Rho-kinase signaling pathway in chronic cerebral ischemia. In this study, rat models of chronic cerebral ischemia were established by permanent bilateral common carotid artery occlusion and intra- gastrically administered 9 mg/kg fasudil, a powerful ROCK inhibitor, for 9 weeks. Morris water maze results showed that cognitive impairment progressively worsened as the cerebral ischemia proceeded. Immunohistochemistry, semi-quantitative RT-PCR and western blot analysis showed that the expression levels of Rho-kinase, its substrate myosin-binding subunit, and its relat- ed protein alpha smooth muscle actin, significantly increased after chronic cerebral ischemia. TUNEL staining showed that chronic cerebral ischemia could lead to an increase in neuronal apoptosis, as well as the expression level of caspase-3 in the frontal cortex of rats subjected to chronic cerebral ischemia. Fasudil treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, and decreased the expression level of Rho-kinase, myosin-binding subunit and alpha smooth muscle actin. Furthermore, fasudil could regulate cerebral injury by reducing cell apoptosis and decreasing caspase-3 expression in the frontal cortex. These findings demonstrate that fasudil can protect against cognitive impairment induced by chronic cerebral ischemia via the Rho/Rho-kinase signaling pathway and anti-apoptosis mechanism.

Fasudil促进大鼠损伤脊髓神经功能恢复的研究

目的研究Rho激酶抑制剂Fasudil对大鼠脊髓损伤的促修复作用。方法用健康成年SD大鼠建立脊髓损伤模型,通过腹腔注射盐酸法舒地尔,并设立对照。术后1、2、4周,运用BBB功能评分进行后肢运动功能评价;损伤后4周,脊髓损伤局部行SABC法ROCK2免疫组织化学染色。结果术后1、2、4周,Fasudil治疗组与对照组比较,评分明显增高(P<0.05);术后4周Fasudil治疗组损伤局部组织内ROCK2的表达较对照组明显减少(P<0.05)。结论腹腔注射Fasudil能够减少脊髓损伤局部ROCK2表达,改善大鼠的运动功能。

Fasudil hydrochloride differentiates bone marrow mesenchymal stem cells into neurons via notch signaling

BACKGROUND： Notch signaling regulates bone marrow mesenchymal stem cell （MSC） proliferation, differentiation, and apoptosis, Notch signaling and Rho kinase signaling exhibit a crosstalk phenomenon with JAK/STAT, and both participate in the neuronal dendritic spine development. Inhibition of RhoA/Rho kinase signaling may regulate MSC differentiation into neuronal-like cells. OBJECTIVE： To investigate the effect of Notch1 signaling on the differentiation of rat MSCs into neurons induced by fasudil hydrochloride （C14H17N3O2S-HCI）, a Rho kinase inhibitor, through a siRNA approach. DESIGN, TIME AND SETTING： An in vitro cytological experiment was performed in the Cell Laboratory of Henan Academy of Medical and Pharmaceutical Sciences between December 2007 and May 2009. MATERIALS： MSCs were obtained from Wistar rat femoral bone, fasudil hydrochloride was provided by -Tianjin Chase Sun Pharmaceutical Co., Ltd. Rn-notchl-siRNa, negative control siRNA （Cy3 label） and Rn-MAPK1 control siRNA were provided by QIAGEN, Coloqne, German. METHODS： The cultured MSCs were divided into non-transfected, transfected group （transfected with Rn-Notchl-siRNA）, positive control （transfected with Rn-MAPK-1 control siRNA）, and negative control （transfected with negative control siRNA） groups. Fasudil hydrochloride was applied to induce MSCs to differentiate into neurons. MAIN OUTCOME MEASURES： The fluorescence expression by the transfected MSCs was observed under an inverted fluorescence microscope; the expression of Notch1 mRNA, Hesl mRNA, and MAPK1 mRNA in MSCs was detected by reverse transcription polymerase chain reaction; the expression of Notch1 protein, nestin, neurofilament M, and glial fibrillary acidic protein was detected by immunocytochemistry. The viability of MSCs was detected by tetrazolium bromide assay. RESULTS： MSC fluorescence increased following a 72-hour siRNA transfection, with transfection efficiencies of up to （0.91 ± 0.04）; the Notch1 mRNA and Hesl mRNA expressed by transfected MSCs was significantly decreased （P 〈 0.05） compared with non-transfected cells. Fasudil hydrochloride induced MSCs to differentiate into neurons with greater efficiency in the transfected group （P 〈 0.05）. CONCLUSION： Fasudil hydrochloride induces rat MSCs to differentiate into neurons; inhibition of Notch1 signaling and Hesl expression may jointly promote the differentiation of MSCs into neurons.

The Rho-associated kinase inhibitors Y27632 and fasudil promote microglial migration in the spinal cord via the ERK signaling pathway

Rho-associated kinase（ROCK） is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system.Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase（ERK） signaling pathway,but its effect on microglial migration was unknown.Therefore,in this study,we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord,and we examined the underlying mechanisms.The microglia were treated with Y27632,fasudil and/or the ERK inhibitor U0126.Cellular morphology was observed by immunofluorescence.Transwell chambers were used to assess cell migration.ERK levels were measured by incell western blot assay.Y27632 and fasudil increased microglial migration,and the microglia were irregularly shaped and had many small processes.These inhibitors also upregulated the levels of phosphorylated ERK protein.The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil.These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.

Combination of fasudil and celecoxib promotes the recovery of injured spinal cord in rats better than celecoxib or fasudil alone

Resistance mechanisms of rho-associated kinase（ROCK） inhibitors are associated with the enhanced expression of cyclooxygenase-2（COX-2）. The therapeutic effects of ROCK on nervous system diseases might be enhanced by COX-2 inhibitors. This study investigated the synergistic effect of the combined use of the ROCK inhibitor fasudil and a COX-2 inhibitor celecoxib on spinal cord injury in a rat model established by transecting the right half of the spinal cord at T11. Rat models were orally administrated with celecoxib（20 mg/kg） and/or intramuscularly with fasudil（10 mg/kg） for 2 weeks. Results demonstrated that the combined use of celecoxib and fasudil significantly decreased COX-2 and Rho kinase II expression surrounding the lesion site in rats with spinal cord injury, improved the pathomorphology of the injured spinal cord, and promoted the recovery of motor function. Moreover, the effects of the drug combination were better than celecoxib or fasudil alone. This study demonstrated that the combined use of fasudil and celecoxib synergistically enhanced the functional recovery of injured spinal cord in rats.

In vitro inhibition of proliferation,migration and epithelial-mesenchymal transition of human lens epithelial cells by fasudil

AIM： To study the potential role of fasudil as a treatment for posterior capsular opacification（PCO） of the human crystalline lens.METHODS： Human lens epithelial cells（HLECs; line SRA01/04） was exposed to transforming growth factor-β2（TGF-β2） to induce the process of epithelial-mesenchymal transition（EMT）. Fasudil was applied to the cell samples. Its effect on overall HLECs proliferation and migration was studied, as was its influence on EMT induction by TGF-β2 using cell migration assay, MTT colorimetric assay and Western blot assay.RESULTS： Fasudil inhibited the proliferation of SRA01/04. Its effect was time-and concentration-dependent. The migration of SRA01/04 cells was significantly reduced 24-72 h after fasudil treatment, and the half maximal inhibitory concentration（IC50） was 22.37 μmol/mL at 72 h. Reversal of the elongated, fibroblast-like shape changes induced by TGF-β2 in SRA01/04 cells was observed. Fasudil up-regulated the expression of Connexin43 protein and down-regulated the expression of α-SMA protein compared with the cells treated with TGF-β2. Furthermore, when exposed to fasudil, the phosphorylation of Rhoassociated protein kinase（Rock） and myosin light chain（MLC） could not be activated in the cell preparations.CONCLUSION： Fasudil suppresses the proliferation and migration of SRA01/04 cells, and inhibits the process of EMT induced by TGF-β2. These results suggest that fasudil may serve as a therapeutic agent for PCO.

Protective effect of fasudil hydrochloride against acute renal injury in septicopyemia rats

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 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 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 man model group(P <0.05).Rats in control group showed no swelling or hyperaemia 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,hyperaemia 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.

Environmental enrichment combined with fasudil promotes motor function recovery and axonal regeneration after stroke

Fasudil,a Rho-associated protein kinase(ROCK)inhibitor,has a protective effect on the central nervous system.In addition,environmental enrichment is a promising technique for inducing the recovery of motor impairments in ischemic stroke models.The present study aimed to explore whether environmental enrichment combined with fasudil can facilitate motor function recovery and induce cortical axonal regeneration after stroke.First,a mouse model of ischemic cerebral stroke was established by photochemical embolization of the left sensorimotor cortex.Fasudil solution(10 mg/kg per day)was injected intraperitoneally for 21 days after the photothrombotic stroke.An environmental enrichment intervention was performed on days 7-21 after the photothrombotic stroke.The results revealed that environmental enrichment combined with fasudil improved motor function,increased growth-associated protein 43 expression in the infarcted cerebral cortex,promoted axonal regeneration on the contralateral side,and downregulated ROCK,p-LIM domain kinase(LIMK)1,and p-cofilin expression.The combined intervention was superior to monotherapy.These findings suggest that environmental enrichment combined with fasudil treatment promotes motor recovery after stroke,at least partly by stimulating axonal regeneration.The underlying mechanism might involve ROCK/LIMK1/cofilin pathway regulation.This study was approved by the Institutional Animal Care and Use Committee of Fudan University,China(approval No.20160858A232)on February 24,2016.

Fasudil alleviates LPS-induced lung injury by restoring aquaporin 5 expression and inhibiting inflammation in lungs

Fasudil, a selective rho kinase(ROCK) inhibitor, has been reported to play a beneficial role in systemic inflammation in acute lung injury, but its mechanism for ameliorating pulmonary edema and inflammation remains unclear. Using hematoxylin-and-eosin(H&E) staining, immunohistochemistry, enzyme-linked immunosorbent assay,quantitative real time PCR and Western blotting, we found that fasudil attenuated LPS-induced lung injury, decreased lung edema, and suppressed inflammatory responses including leukocyte infiltration and IL-6 production. Further,fasudil upregulated LPS-induced aquaporin 5 reduction and inhibited NF-κB activation in the lungs of mice. Our results suggest that fasudil could restore the expression of aquaporin 5 to eliminate LPS-induced lung edema and prevent LPS-induced pulmonary inflammation by blocking the inflammatory pathway. Collectively, blockade of the ROCK pathway by fasudil may be a potential strategy for the treatment of acute lung injury.

Advantages of Rho-associated kinases and their inhibitor fasudil for the treatment of neurodegenerative diseases

Ras homolog(Rho)-associated kinases(ROCKs)belong to the serine-threonine kinase family,which plays a pivotal role in regulating the damage,survival,axon guidance,and regeneration of neurons.ROCKs are also involved in the biological effects of immune cells and glial cells,as well as the development of neurodegenerative disorders such as Alzheimer’s disease,Parkinson’s disease,and multiple sclerosis.Previous studies by us and others confirmed that ROCKs inhibitors attenuated the symptoms and progression of experimental models of the abovementioned neurodegenerative diseases by inhibiting neuroinflammation,regulating immune imbalance,repairing the blood-brain barrier,and promoting nerve repair and myelin regeneration.Fasudil,the first ROCKs inhibitor to be used clinically,has a good therapeutic effect on neurodegenerative diseases.Fasudil increases the activity of neural stem cells and mesenchymal stem cells,thus optimizing cell therapy.This review will systematically describe,for the first time,the effects of abnormal activation of ROCKs on T cells,B cells,microglia,astrocytes,oligodendrocytes,and pericytes in neurodegenerative diseases of the central nervous system,summarize the therapeutic potential of fasudil in several experimental models of neurodegenerative diseases,and clarify the possible cellular and molecular mechanisms of ROCKs inhibition.This review also proposes that fasudil is a novel potential treatment,especially in combination with cell-based therapy.Findings from this review add support for further investigation of ROCKs and its inhibitor fasudil for the treatment of neurodegenerative diseases.

Fasudil prevents liver fibrosis via activating natural killer cells and suppressing hepatic stellate cells

BACKGROUND Fasudil,as a Ras homology family member A(RhoA)kinase inhibitor,is used to improve brain microcirculation and promote nerve regeneration clinically.Increasing evidence shows that Rho-kinase inhibition could improve liver fibrosis.AIM To evaluate the anti-fibrotic effects of Fasudil in a mouse model of liver fibrosis induced by thioacetamide(TAA).METHODS C57BL/6 mice were administered TAA once every 3 d for 12 times.At 1 wk after induction with TAA,Fasudil was intraperitoneally injected once a day for 3 wk,followed by hematoxylin and eosin staining,sirius red staining,western blotting,and quantitative polymerase chain reaction(qPCR),and immune cell activation was assayed by fluorescence-activated cell sorting.Furthermore,the effects of Fasudil on hepatic stellate cells and natural killer(NK)cells were assayed in vitro.RESULTS First,we found that TAA-induced liver injury was protected,and the positive area of sirius red staining and type I collagen deposition were significantly decreased by Fasudil treatment.Furthermore,western blot and qPCR assays showed that the levels of alpha smooth muscle actin(α-SMA),matrix metalloproteinase 2(MMP-2),MMP-9,and transforming growth factor beta 1(TGF-β1)were inhibited by Fasudil.Moreover,flow cytometry analysis revealed that NK cells were activated by Fasudil treatment in vivo and in vitro.Furthermore,Fasudil directly promoted the apoptosis and inhibited the proliferation of hepatic stellate cells by decreasingα-SMA and TGF-β1.CONCLUSION Fasudil inhibits liver fibrosis by activating NK cells and blocking hepatic stellate cell activation,thereby providing a feasible solution for the clinical treatment of liver fibrosis.

A Rho-kinase Inhibitor, Fasudil, Attenuates Progressive Glomerulosclerosis Induced by Daunorubicin in Rats

Accumulating evidence suggests that the small G protein Rho and its downstream effec-tor Rho kinase may play important roles in kidney biology. The present study examined the effects of a Rho-kinase inhibitor, fasudil, on daunorubicin-induced progressive glomerulosclerosis and explored the underlying mechanism by which fasudil ameliorates glomerulosclerosis. Thirty-six male SD rats were randomly allocated into sham-operation group （sham group, n=12）, unilateral nephrectomy （UNX）＋daunorubicin （DRB） group （model group, n=12）, UNX＋DRB＋Fasudil group （treatment group, n=12）. Two to four weeks after the establishment of the animal model, 6 rats in each group were taken randomly for the detection of 24-h urine protein excretion. Kidney sections were exam-ined by HE and PAS staining, immunohistochemistry and transmission electric microscopy （TEM）. The expression of Rho-kinase mRNA and P27 mRNA in kidney were detected by RT-PCR. It was found that the 24-h urine protein excretion in model group was increased significantly as compared with sham group （P〈0.01）. But this increase was significantly suppressed by fasudil （P〈0.05）. At 4 week, the foot process effacement in podocytes, mesangial proliferation and ECM accumulation were observed in model group, presenting as focal segmental glomerulosclerosis. But in the treatment group, the fasudil alleviated glomerular injury, with proliferating cell nuclear antigen （PCNA）-positive cell infiltration ameliorated and the expression of P27 increased. The expression of Rho-kinase mRNA was significantly enhanced in model group and was suppressed in treatment group. Moreover, fasudil up-regulated the mRNA expression of P27. Our study demonstrated that the glomerulosclerosis was substantially ameliorated by inhibiting the expression of Rho-kinase. It is suggested that Rho-kinase pathway is involved in the renal injury and the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury.