Objective： To test whether tanshinone ⅡA （Tan ⅡA）, a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin （lipopolysaccharides, ...Objective： To test whether tanshinone ⅡA （Tan ⅡA）, a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin （lipopolysaccharides, LPS）-induced endothelial injury. Methods： Endothelial cell injury was induced by treating human umbilical vein endothelial cells （HUVECs） with 0.2 μg/mL LPS for 24 h. Y27632 and valsartan were used as positive controls. The effects of tanshinone Ⅱ A on the LPS-induced cell viability and apoptosis rate of HUVECs were tested by flow cytometry, cell migration by transwell, adhesion by a 96-well plate pre-coated with vitronectin and cytoskeleton reorganization by immunofluorescence assay. Rho/Rho kinase （ROCK） pathway- associated gene and protein expression were examined by microarray assay; quantitative real-time polymerase chain reaction and Western blotting were used to confirm the changes observed by microarray. Results： Tan ][ A improved cell viability, suppressed apoptosis and protected cells from LPS-induced reductions in cell migration and adhesion at a comparable magnitude to that of Y27632 and valsartan. Tan II A, Y27632 and valsartan also normalized LPS-induced actomyosin contraction and vinculin protein aggregation. A microarray assay revealed increased levels of fibronectin, integrin A5 （ITG A5）, Ras homolog gene family member A （RheA）, myosin light chain phosphatase, phosphatidylinositol-4,5-bisphosphate 3-kinase （PI3K, or PIP2 in Western blotting）, focal adhesion kinase, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in the damaged HUVECs, which were attenuated to different degrees by Tan ⅡA, Y27632 and valsartan. Conclusion： Tan ⅡA exerted a strong protective effect on HUVECs, and the mechanism was caused, at least in part, by a blockade in the Rho/ROCK pathway, presumably through the down-regulation of ITG A5.展开更多
Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protei...Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1αmRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells;while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.展开更多
AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) an...AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) and type I collagen as induced by connective tissue growth factor(CTGF) or transforming growth factor-β (TGF-β) in a human retinal pigment epithelial cell line, ARPE-19. METHODS:The effect of Y27632 on the CTGF or TGF-β induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. ARPE-19 cells were treated with CTGF (1, 10, 100ng/mL)and TGF-β (10ng/mL) in serum free media, and analyzed for fibronectin, laminin, and MMP-2 and type I collagen by RT-qPCR and immunocytochemistry. Cells were also pretreated with an ROCK inhibitor, Y27632, to analyze the signaling contributing to ECM production. ·RESULTS:Treatment of ARPE-19 cells in culture with TGF-β or CTGF induced an ECM change from a cobblestone morphology to a more elongated swirl pattern indicating a mesenchymal phenotype. RT-qPCR analysis and different gene expression analysis demonstrated an upregulation in expression of genes associated with cytoskeletal structure and motility. CTGFor TGF-β significantly increased expression of fibronectin mRNA (P =0.006, P =0.003 respectively), laminin mRNA (P =0.006, P =0.005), MMP-2 mRNA (P =0.006, P =0.001), COL1A1 mRNA (P =0.001, P =0.001), COL1A2 mRNA (P = 0.001, P =0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF-β induced fibronectin (P=0.005, P=0.003 respectively), MMP-2 (P = 0.003, P =0.002), COL1A1 (P =0.006, P =0.003), and COL1A2 (P =0.006, P =0.004) gene expression, but not laminin (P =0.375, P =0.516). CONCLUSION:Our study demonstrated that both TGF-β and CTGF upregulate the expression of ECM components including fibronectin, laminin, MMP-2 and type I collagen by activating the RhoA/ROCK signaling pathway. During this process, ARPE-19 cells were shown to change from an epithelial to a mesenchymal phenotype in vitro. Y27632, a ROCK inhibitor, inhibited the transcription of fibronectin, MMP-2 and type I collagen, but not laminin. The data from our work suggest a role for CTGF as a profibrotic mediator. Inhibiting the RhoA/ROCK pathway represents a potential target to prevent the fibrosis of retinal pigment epithelial (RPE) cells. This might lead to a novel therapeutic approach to preventing the onset of early proliferative vitreoretinopathy(PVR).展开更多
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 ce...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.展开更多
Hippocampal neurons of neonatal rats were cultured in serum-free culture medium for 5 days in vitro, and treated with the Rho-kinase inducer lysophosphatidic acid. Atomic force microscopy revealed that the numbers of ...Hippocampal neurons of neonatal rats were cultured in serum-free culture medium for 5 days in vitro, and treated with the Rho-kinase inducer lysophosphatidic acid. Atomic force microscopy revealed that the numbers of level-1, -2 and -3 neurites protruding from rat hippocampal neurons was significantly reduced. After treatment with the Rho kinase inhibitor Y27632, a significant increase in the numbers of these neurites was observed. Our experimental findings indicate that the Rho-kinase pathway is closely associated with the neurites of hippocampal neurons.展开更多
Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydroc...Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydrochloride on claudin-5 protein expression has not been reported after cerebral ischemia/reperfusion. Therefore, this study sought to explore the effects of fasudil hydrochloride on blood-brain barrier permeability, growth-associated protein-43 and claudin-5 protein expression, and to further understand the neuroprotective effect of fasudil hydrochloride. A focal cerebral ischemia/reperfusion model was established using the intraluminal suture technique. Fasudil hydrochloride （15 mg/kg） was intraperitoneally injected once a day. Neurological deficit was evaluated using Longa＇s method. Changes in permeability of blood-brain barrier were measured using Evans blue. Changes in RhoA, growth-associated protein-43 and claudin-5 protein expression were detected using immunohistochemistry and western blotting. Results revealed that fasudil hydrochloride noticeably contributed to the recovery of neurological function, improved the function of blood-brain barrier, inhibited RhoA protein expression, and upregulated growth-associated protein-43 and claudin-5 protein expression following cerebral ischemia/reperfusion. Results indicated that Rho kinase exhibits a certain effect on neurovascular damage following cerebral ischemia/reperfusion. Intervention targeted Rho kinase might be a new therapeutic target in the treatment of cerebral ischemia/reperfusion.展开更多
A spinal cord injury refers to an injury to the spinal cord that is caused by a trauma instead of diseases. Spinal cord injury includes a primary mechanical injury and a much more complex secondary injury process invo...A spinal cord injury refers to an injury to the spinal cord that is caused by a trauma instead of diseases. Spinal cord injury includes a primary mechanical injury and a much more complex secondary injury process involving inflammation, oxidation, excitotoxicity, and cell death. During the secondary injury, many signal pathways are activated and play important roles in mediating the pathogenesis of spinal cord injury. Among them, the Rho A/Rho kinase pathway plays a particular role in mediating spinal degeneration and regeneration. In this review, we will discuss the role and mechanism of Rho A/Rho kinase-mediated spinal cord pathogenesis, as well as the potential of targeting Rho A/Rho kinase as a strategy for promoting both neuroprotection and axonal regeneration.展开更多
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 ...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.展开更多
AIM To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODS Crypts were isolated from jejunu...AIM To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODS Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1(ENR) or ENR/CHIR 99021/VPA(ENR-CV). F o rsubculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using Ed U staining, methyl thiazolyl tetrazolium assay, q PCR and time-lapse live cell imaging.RESULTS We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5^+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5^+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSION The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.展开更多
基金Supported by Science and Technology Development Plan of Shandong Province(No.2012G0021837)
文摘Objective： To test whether tanshinone ⅡA （Tan ⅡA）, a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin （lipopolysaccharides, LPS）-induced endothelial injury. Methods： Endothelial cell injury was induced by treating human umbilical vein endothelial cells （HUVECs） with 0.2 μg/mL LPS for 24 h. Y27632 and valsartan were used as positive controls. The effects of tanshinone Ⅱ A on the LPS-induced cell viability and apoptosis rate of HUVECs were tested by flow cytometry, cell migration by transwell, adhesion by a 96-well plate pre-coated with vitronectin and cytoskeleton reorganization by immunofluorescence assay. Rho/Rho kinase （ROCK） pathway- associated gene and protein expression were examined by microarray assay; quantitative real-time polymerase chain reaction and Western blotting were used to confirm the changes observed by microarray. Results： Tan ][ A improved cell viability, suppressed apoptosis and protected cells from LPS-induced reductions in cell migration and adhesion at a comparable magnitude to that of Y27632 and valsartan. Tan II A, Y27632 and valsartan also normalized LPS-induced actomyosin contraction and vinculin protein aggregation. A microarray assay revealed increased levels of fibronectin, integrin A5 （ITG A5）, Ras homolog gene family member A （RheA）, myosin light chain phosphatase, phosphatidylinositol-4,5-bisphosphate 3-kinase （PI3K, or PIP2 in Western blotting）, focal adhesion kinase, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in the damaged HUVECs, which were attenuated to different degrees by Tan ⅡA, Y27632 and valsartan. Conclusion： Tan ⅡA exerted a strong protective effect on HUVECs, and the mechanism was caused, at least in part, by a blockade in the Rho/ROCK pathway, presumably through the down-regulation of ITG A5.
基金supported by National Natural Science Foundation of China(No.81560124)Hainan Key Research and Development Projects(ZDYF2018131,ZDYF2017113,ZDYF2017114)+1 种基金Hainan Science and Technology Planned Project of Youth Outstanding Ability of Innovation(201704)Hainan Health Family Planning Industry Project(13A210277)
文摘Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1αmRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells;while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.
文摘AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) and type I collagen as induced by connective tissue growth factor(CTGF) or transforming growth factor-β (TGF-β) in a human retinal pigment epithelial cell line, ARPE-19. METHODS:The effect of Y27632 on the CTGF or TGF-β induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. ARPE-19 cells were treated with CTGF (1, 10, 100ng/mL)and TGF-β (10ng/mL) in serum free media, and analyzed for fibronectin, laminin, and MMP-2 and type I collagen by RT-qPCR and immunocytochemistry. Cells were also pretreated with an ROCK inhibitor, Y27632, to analyze the signaling contributing to ECM production. ·RESULTS:Treatment of ARPE-19 cells in culture with TGF-β or CTGF induced an ECM change from a cobblestone morphology to a more elongated swirl pattern indicating a mesenchymal phenotype. RT-qPCR analysis and different gene expression analysis demonstrated an upregulation in expression of genes associated with cytoskeletal structure and motility. CTGFor TGF-β significantly increased expression of fibronectin mRNA (P =0.006, P =0.003 respectively), laminin mRNA (P =0.006, P =0.005), MMP-2 mRNA (P =0.006, P =0.001), COL1A1 mRNA (P =0.001, P =0.001), COL1A2 mRNA (P = 0.001, P =0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF-β induced fibronectin (P=0.005, P=0.003 respectively), MMP-2 (P = 0.003, P =0.002), COL1A1 (P =0.006, P =0.003), and COL1A2 (P =0.006, P =0.004) gene expression, but not laminin (P =0.375, P =0.516). CONCLUSION:Our study demonstrated that both TGF-β and CTGF upregulate the expression of ECM components including fibronectin, laminin, MMP-2 and type I collagen by activating the RhoA/ROCK signaling pathway. During this process, ARPE-19 cells were shown to change from an epithelial to a mesenchymal phenotype in vitro. Y27632, a ROCK inhibitor, inhibited the transcription of fibronectin, MMP-2 and type I collagen, but not laminin. The data from our work suggest a role for CTGF as a profibrotic mediator. Inhibiting the RhoA/ROCK pathway represents a potential target to prevent the fibrosis of retinal pigment epithelial (RPE) cells. This might lead to a novel therapeutic approach to preventing the onset of early proliferative vitreoretinopathy(PVR).
基金supported by the Natural Science Foundation of Jilin Province of China,No.200705272,20140414028GH
文摘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.
文摘Hippocampal neurons of neonatal rats were cultured in serum-free culture medium for 5 days in vitro, and treated with the Rho-kinase inducer lysophosphatidic acid. Atomic force microscopy revealed that the numbers of level-1, -2 and -3 neurites protruding from rat hippocampal neurons was significantly reduced. After treatment with the Rho kinase inhibitor Y27632, a significant increase in the numbers of these neurites was observed. Our experimental findings indicate that the Rho-kinase pathway is closely associated with the neurites of hippocampal neurons.
基金funded by the National Natural Science Foundation of China,No.30870855the Natural Science Foundation of Beijing,No.7082028Beijing Municipal Health System High-Level Technician Cultivation Project,No.2009-3-07
文摘Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydrochloride on claudin-5 protein expression has not been reported after cerebral ischemia/reperfusion. Therefore, this study sought to explore the effects of fasudil hydrochloride on blood-brain barrier permeability, growth-associated protein-43 and claudin-5 protein expression, and to further understand the neuroprotective effect of fasudil hydrochloride. A focal cerebral ischemia/reperfusion model was established using the intraluminal suture technique. Fasudil hydrochloride （15 mg/kg） was intraperitoneally injected once a day. Neurological deficit was evaluated using Longa＇s method. Changes in permeability of blood-brain barrier were measured using Evans blue. Changes in RhoA, growth-associated protein-43 and claudin-5 protein expression were detected using immunohistochemistry and western blotting. Results revealed that fasudil hydrochloride noticeably contributed to the recovery of neurological function, improved the function of blood-brain barrier, inhibited RhoA protein expression, and upregulated growth-associated protein-43 and claudin-5 protein expression following cerebral ischemia/reperfusion. Results indicated that Rho kinase exhibits a certain effect on neurovascular damage following cerebral ischemia/reperfusion. Intervention targeted Rho kinase might be a new therapeutic target in the treatment of cerebral ischemia/reperfusion.
基金supported by NIH NS050243,NS059622,NS073636,DOD CDMRP W81XWH-12-1-0562,DVA 1I01BX002356-01A1Craig H Neilsen Foundation#296749+2 种基金Wallace H.Coulter FoundationIndiana Spinal Cord and Brain Injury Research FoundationMari Hulman George Endowment Funds
文摘A spinal cord injury refers to an injury to the spinal cord that is caused by a trauma instead of diseases. Spinal cord injury includes a primary mechanical injury and a much more complex secondary injury process involving inflammation, oxidation, excitotoxicity, and cell death. During the secondary injury, many signal pathways are activated and play important roles in mediating the pathogenesis of spinal cord injury. Among them, the Rho A/Rho kinase pathway plays a particular role in mediating spinal degeneration and regeneration. In this review, we will discuss the role and mechanism of Rho A/Rho kinase-mediated spinal cord pathogenesis, as well as the potential of targeting Rho A/Rho kinase as a strategy for promoting both neuroprotection and axonal regeneration.
基金supported by the National Natural Science Foundation of China, Nos.81473577 (to CGM), 81903596 (to QW), 82004028 (to LJS)China Postdoctoral Science Foundation, No.2020M680912 (to LJS)+2 种基金Open Project of The Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, The Ministry of Education of China,No.2019004 (to CGM)Science and Technology Innovation Project of Shanxi Colleges of China, Nos.2019L0728 (to QW)Cultivation Project of Shanxi Universtity of Chinese Medicine of China, No.2019PY130 (to QW)
文摘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.
基金a grant of the Korea Institute of Radiological and Medical Sciences,funded by Ministry of Science,ICT and Future Planning,South Korea,No.1711031810/50586-2016 and No.1711031808/50581-2016
文摘AIM To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODS Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1(ENR) or ENR/CHIR 99021/VPA(ENR-CV). F o rsubculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using Ed U staining, methyl thiazolyl tetrazolium assay, q PCR and time-lapse live cell imaging.RESULTS We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5^+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5^+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSION The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.
