Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke,...Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway changes the pathogenic processes of the blood–brain barrier(BBB) after intracerebral hemorrhage(ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. Methods: Scalp acupuncture(SA) therapy was performed on rats with ICH at the acupuncture point “Baihui”-penetrating “Qubin,” and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the Rho A/ROCK Ⅱ/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. Results: We found that ROCK Ⅱ acts as a promoter of the Rho A/ROCK Ⅱ/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the preintervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK Ⅱ, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at “Baihui”-penetrating “Qubin” and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. Conclusion: This study found that these experimental data indicated that SA at “Baihui”-penetrating “Qubin” could preserve BBB integrity and neurological function recovery after ICH by inhibiting Rho A/ROCK Ⅱ/MLC 2 signaling pathway activation and by regulating endothelial cell–related proteins.展开更多
Rho-associated kinase (ROCK) is a serine/threonine kinase and one of the major downstream effectors of the small GTPase RhoA. The Rho/ROCK pathway is closely related to the pathogenesis of several central nervous syst...Rho-associated kinase (ROCK) is a serine/threonine kinase and one of the major downstream effectors of the small GTPase RhoA. The Rho/ROCK pathway is closely related to the pathogenesis of several central nervous system (CNS) disorders, and involved in many aspects of neuronal functions including neurite outgrowth and retraction. In the adult CNS, the damaged neuron regeneration is very difficult due to the presence of myelin-associated axon growth inhibitors such as Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (Omgp), etc. The effects of these axon growth inhibitors are reversed by blocking the Rho/ROCK pathway 47 vitro, and the inhibition of Rho/ROCK pathway can promote axon regeneration and functional recovery in the injured CNS in viva In addition, the therapeutic effects of the Rho/ROCK inhibitors have also been demonstrated in some animal models and the Rho/ROCK pathway becomes an attractive target for the development of drugs for treating CNS disorders. In this review, we summarized on the effect of the Rho and the downstream factor ROCK in neural regeneration, and the potential therapeutic effect of Rho/ROCK inhibitors in the survival and axonal regeneration of retinal ganglion cell was also discussed.展开更多
Mesenchymal stem cell （MSC）-based treatments have shown promise for improving tendon healing and repair. MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical st...Mesenchymal stem cell （MSC）-based treatments have shown promise for improving tendon healing and repair. MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical stimuli, including into tenocytes. Cell elongation and cytoskeletal tension have been shown to be instrumental to the process of MSC differentiation. Previous studies have shown that inhibition of stress fiber formation leads MSCs to default toward an adipogenic lineage, which suggests that stress fibers are required for MSCs to sense the environmental factors that can induce differentiation into tenocytes. As the Rho/ROCK signal transduction pathway plays a critical role in both stress fiber formation and in cell sensation, we examined whether the activation of this pathway was required when inducing MSC tendon differentiation using rope-like silk scaffolds. To accomplish this, we employed a loss-of-function approach by knocking out ROCK, actin and myosin （two other components of the pathway） using the specific inhibitors Y-27632, Latrunculin A and blebbistatin, respectively. We demonstrated that independently disrupting the cytoskeleton and the Rho/ ROCK pathway abolished the expression of tendon differentiation markers and led to a loss of spindle morphology. Together, these studies suggest that the tension that is generated by MSC elongation is essential for MSC teno-differentiation and that the Rho/ROCK pathway is a critical mediator of tendon differentiation on rope-like silk scaffolds.展开更多
Objective:The effect of QingguanganⅡon the transcription of RhoA mRNA,ROCK mRNA,Caspase-3 mRNA and Bcl-2 mRNA in the retina of DBA/2J mice was observed.Methods:Forty-eight DBA/2J mice were randomly divided into six g...Objective:The effect of QingguanganⅡon the transcription of RhoA mRNA,ROCK mRNA,Caspase-3 mRNA and Bcl-2 mRNA in the retina of DBA/2J mice was observed.Methods:Forty-eight DBA/2J mice were randomly divided into six groups:model groups,Qingguangan II decoction group,low concentration,medium concentration and high concentration group of Qingguangan II effective ingredient and positive control group(Yimaikang tablet group),and eight C57BL/6 mice were used as blank group,DBA/2J mice were fed until 38 weeks before forming a glaucoma model,The transcription of RhoA mRNA,ROCK mRNA,Caspase-3 mRNA and Bcl-2 mRNA in the retinal of DBA/2J mice was detected using real-time fluorescence quantitative PCR(Quantitative Real-time PCR)after 4 weeks of intervention.Results:Four weeks after the intervention,In the transcription of the RhoA mRNA,ROCK mRNA and the Caspase-3 mRNA,Compared to the blank groups,Relative expression was increased in the other 6 groups,There are statistical differences in the model group,Yimaikang tablet group and low concentration group(P<0.05);In comparison to the model groups,The other 6 groups were lower than the model group,Among them,there are statistical differences between the effective groups of Qingguangan II decoction and high concentration group of Qingguangan II effective ingredient in RhoA mRNA transcription(P<0.05);In the transcription of the ROCK mRNA and the Caspase-3 mRNA,Statistics have differences between the model group and the effective component of the medium and high concentration group(P<0.05);In the Bcl-2 mRNA transcription,Compare them to blank groups,Relexpression expression decreased in the other 6 groups,Statistics have differences between model group,Qingguangan II decoction group and low concentration groups(P<0.05);The relative expression of Bcl-2 mRNA in high concentration group of effective component is higher than that of the model group,There are differences in statistics(P<0.05).Conclusion:The high concentration of QingguanganⅡprescription probably attenuated Caspase-3 transcription in retinal ganglion cells by inhibiting the Rho/ROCK signaling pathway and activated Bcl-2 expression by inhibiting ROCK signaling,which attenuated apoptosis in retinal ganglion cells.展开更多
目的探究竹沥对肝纤维化小鼠Ras同源基因-Rho相关螺旋卷曲蛋白激酶(Ras homologousgene-Rho-associatedcoiled-coilcontainingkinases,Rho-ROCK)信号转导通路的作用,分析竹沥抗肝纤维化的分子机制。方法将40只KM小鼠随机分为空白组、模...目的探究竹沥对肝纤维化小鼠Ras同源基因-Rho相关螺旋卷曲蛋白激酶(Ras homologousgene-Rho-associatedcoiled-coilcontainingkinases,Rho-ROCK)信号转导通路的作用,分析竹沥抗肝纤维化的分子机制。方法将40只KM小鼠随机分为空白组、模型组、竹沥组和丹参酚酸B组,每组10只,模型组、竹沥组和丹参酚酸B组小鼠给予40%CCl4花生油混合液(剂量2μl/g),空白组给予等量生理盐水,腹腔注射,2次/周,连续8周。模型构建成功后,竹沥组以竹沥灌胃,每日1次,每次30 ml/100 g;丹参酚酸B组以丹参酚酸B溶液灌胃,每日1次,每次用量0.25 mg/100 g;其余2组给予等量生理盐水灌胃,持续4周。末次给药后对小鼠眼球取血并处死,采用HE染色观察肝组织病理并进行Ishak评分,采用γ放射免疫全自动双探头计数器检测肝纤维化指标,包括透明质酸、层粘连蛋白、Ⅲ型前胶原和Ⅳ型胶原,采用实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,q RT-PCR)检测Rho A和ROCK2m RNA的相对表达量,采用Western blot检测Rho A和ROCK2蛋白的相对表达量。结果空白组、模型组、竹沥组和丹参酚酸B组小鼠Ishak评分分别为(0.40±0.05)分、(4.80±0.84)分、(3.20±0.45)分和(2.80±0.83)分,差异有统计学意义(F=34.807,P<0.001),其中空白组显著低于模型组、竹沥组和丹参酚酸B组(t=8.690,P<0.001;t=5.674,P<0.001;t=4.768,P=0.001),模型组显著高于竹沥组和丹参酚酸B组(t=3.016,P=0.017;t=3.922,P=0.004),竹沥组和丹参酚酸B组差异无统计学意义(t=0.809,P=0.464)。空白组小鼠肝纤维化指标均显著低于模型组、竹沥组和丹参酚酸B组[透明质酸:(26.14±2.69)μg/L vs(53.69±5.87)μg/L vs(37.21±3.04)μg/L vs(32.88±3.60)μg/L,层粘连蛋白:(120.37±15.57)μg/L vs(214.48±21.39)μg/L vs(162.54±19.22)μg/L vs(161.66±14.23)μg/L,Ⅲ型前胶原:(4.04±1.01)μg/L vs(11.70±3.09)μg/L vs(8.12±1.87)μg/L vs(7.80±1.72)μg/L,Ⅳ型胶原:(12.96±2.82)μg/L vs(31.34±4.44)μg/L vs(23.72±3.69)μg/L vs(22.85±3.00)μg/L;P均<0.05],模型组小鼠肝纤维化指标均显著高于竹沥组和丹参酚酸B组(P均<0.05),竹沥组和丹参酚酸B组差异无统计学意义(P均>0.05)。空白组Rho A和ROCK2m RNA相对表达量均显著低于模型组、竹沥组和丹参酚酸B组(Rho A m RNA:1.05±0.18 vs 2.61±0.37 vs 1.62±0.21 vs 1.50±0.14,ROCK2 m RNA:1.04±0.17vs2.32±0.29vs1.46±0.08vs1.45±0.09;P均<0.05),模型组Rho A和ROCK2m RNA相对表达量均显著高于竹沥组和丹参酚酸B组(P均<0.05),竹沥组和丹参酚酸B组差异无统计学意义(P均>0.05)。空白组Rho A和ROCK2蛋白相对表达量均显著低于模型组、竹沥组和丹参酚酸B组(Rho A蛋白:0.14±0.03 vs 0.43±0.05 vs 0.26±0.02 vs 0.30±0.15;ROCK2蛋白:0.28±0.03 vs 0.76±0.09 vs 0.38±0.04 vs 0.49±0.03;P均<0.05),模型组Rho A和ROCK2蛋白相对表达量均显著高于竹沥组和丹参酚酸B组(P均<0.05),竹沥组和丹参酚酸B组差异无统计学意义(P均>0.05)。结论竹沥可抑制Rho-ROCK信号转导通路的活动,可能是其抗肝纤维化的作用机制。展开更多
基金supported by the National Natural Science Foundation of China(numbers:81774416 and 81473764)。
文摘Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway changes the pathogenic processes of the blood–brain barrier(BBB) after intracerebral hemorrhage(ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. Methods: Scalp acupuncture(SA) therapy was performed on rats with ICH at the acupuncture point “Baihui”-penetrating “Qubin,” and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the Rho A/ROCK Ⅱ/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. Results: We found that ROCK Ⅱ acts as a promoter of the Rho A/ROCK Ⅱ/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the preintervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK Ⅱ, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at “Baihui”-penetrating “Qubin” and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. Conclusion: This study found that these experimental data indicated that SA at “Baihui”-penetrating “Qubin” could preserve BBB integrity and neurological function recovery after ICH by inhibiting Rho A/ROCK Ⅱ/MLC 2 signaling pathway activation and by regulating endothelial cell–related proteins.
基金Supported by National Nature Science Foundation of China (No.81070728)Shanghai "Science and Technology Innovation Action Plan" Basic Research Key Project,China (No.11JC1407700 and 11 JC1407701)+1 种基金Shanghai Nature Science Foundation, China (No.08ZR1413900)Shanghai Leading Academic Discipline Project, China(No.S30205)
文摘Rho-associated kinase (ROCK) is a serine/threonine kinase and one of the major downstream effectors of the small GTPase RhoA. The Rho/ROCK pathway is closely related to the pathogenesis of several central nervous system (CNS) disorders, and involved in many aspects of neuronal functions including neurite outgrowth and retraction. In the adult CNS, the damaged neuron regeneration is very difficult due to the presence of myelin-associated axon growth inhibitors such as Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (Omgp), etc. The effects of these axon growth inhibitors are reversed by blocking the Rho/ROCK pathway 47 vitro, and the inhibition of Rho/ROCK pathway can promote axon regeneration and functional recovery in the injured CNS in viva In addition, the therapeutic effects of the Rho/ROCK inhibitors have also been demonstrated in some animal models and the Rho/ROCK pathway becomes an attractive target for the development of drugs for treating CNS disorders. In this review, we summarized on the effect of the Rho and the downstream factor ROCK in neural regeneration, and the potential therapeutic effect of Rho/ROCK inhibitors in the survival and axonal regeneration of retinal ganglion cell was also discussed.
文摘Mesenchymal stem cell （MSC）-based treatments have shown promise for improving tendon healing and repair. MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical stimuli, including into tenocytes. Cell elongation and cytoskeletal tension have been shown to be instrumental to the process of MSC differentiation. Previous studies have shown that inhibition of stress fiber formation leads MSCs to default toward an adipogenic lineage, which suggests that stress fibers are required for MSCs to sense the environmental factors that can induce differentiation into tenocytes. As the Rho/ROCK signal transduction pathway plays a critical role in both stress fiber formation and in cell sensation, we examined whether the activation of this pathway was required when inducing MSC tendon differentiation using rope-like silk scaffolds. To accomplish this, we employed a loss-of-function approach by knocking out ROCK, actin and myosin （two other components of the pathway） using the specific inhibitors Y-27632, Latrunculin A and blebbistatin, respectively. We demonstrated that independently disrupting the cytoskeleton and the Rho/ ROCK pathway abolished the expression of tendon differentiation markers and led to a loss of spindle morphology. Together, these studies suggest that the tension that is generated by MSC elongation is essential for MSC teno-differentiation and that the Rho/ROCK pathway is a critical mediator of tendon differentiation on rope-like silk scaffolds.
基金The National Natural Science Foundation of China(No.81874492,81904260)The Natural Science Foundation of Hunan Provin cial(No.2020JJ5436,2018JJ3389)+4 种基金The open Foundation of Hunan Provincial Key Laboratory for Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chin Med(No.2018YZD03)The open Foundation of Hunan Provincial Key Laboratory for Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chin Med,National Administration of Traditional Chin Med Key Discipline Construction Project of Ophthalmology of Traditional Chin MedProject funded by the Domestic First-class Discipline Construction Project of Hunan University of Chin MedKey Disciplines in Local Universities Supported by Central Government Funds the Construction Projects of TCM Ophthalmology Innovation TeamHunan Provincial Construction Project of Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chin Med and Visual Function Protection Engineering and Technological Research Center.
文摘Objective:The effect of QingguanganⅡon the transcription of RhoA mRNA,ROCK mRNA,Caspase-3 mRNA and Bcl-2 mRNA in the retina of DBA/2J mice was observed.Methods:Forty-eight DBA/2J mice were randomly divided into six groups:model groups,Qingguangan II decoction group,low concentration,medium concentration and high concentration group of Qingguangan II effective ingredient and positive control group(Yimaikang tablet group),and eight C57BL/6 mice were used as blank group,DBA/2J mice were fed until 38 weeks before forming a glaucoma model,The transcription of RhoA mRNA,ROCK mRNA,Caspase-3 mRNA and Bcl-2 mRNA in the retinal of DBA/2J mice was detected using real-time fluorescence quantitative PCR(Quantitative Real-time PCR)after 4 weeks of intervention.Results:Four weeks after the intervention,In the transcription of the RhoA mRNA,ROCK mRNA and the Caspase-3 mRNA,Compared to the blank groups,Relative expression was increased in the other 6 groups,There are statistical differences in the model group,Yimaikang tablet group and low concentration group(P<0.05);In comparison to the model groups,The other 6 groups were lower than the model group,Among them,there are statistical differences between the effective groups of Qingguangan II decoction and high concentration group of Qingguangan II effective ingredient in RhoA mRNA transcription(P<0.05);In the transcription of the ROCK mRNA and the Caspase-3 mRNA,Statistics have differences between the model group and the effective component of the medium and high concentration group(P<0.05);In the Bcl-2 mRNA transcription,Compare them to blank groups,Relexpression expression decreased in the other 6 groups,Statistics have differences between model group,Qingguangan II decoction group and low concentration groups(P<0.05);The relative expression of Bcl-2 mRNA in high concentration group of effective component is higher than that of the model group,There are differences in statistics(P<0.05).Conclusion:The high concentration of QingguanganⅡprescription probably attenuated Caspase-3 transcription in retinal ganglion cells by inhibiting the Rho/ROCK signaling pathway and activated Bcl-2 expression by inhibiting ROCK signaling,which attenuated apoptosis in retinal ganglion cells.
文摘目的探究竹沥对肝纤维化小鼠Ras同源基因-Rho相关螺旋卷曲蛋白激酶(Ras homologousgene-Rho-associatedcoiled-coilcontainingkinases,Rho-ROCK)信号转导通路的作用,分析竹沥抗肝纤维化的分子机制。方法将40只KM小鼠随机分为空白组、模型组、竹沥组和丹参酚酸B组,每组10只,模型组、竹沥组和丹参酚酸B组小鼠给予40%CCl4花生油混合液(剂量2μl/g),空白组给予等量生理盐水,腹腔注射,2次/周,连续8周。模型构建成功后,竹沥组以竹沥灌胃,每日1次,每次30 ml/100 g;丹参酚酸B组以丹参酚酸B溶液灌胃,每日1次,每次用量0.25 mg/100 g;其余2组给予等量生理盐水灌胃,持续4周。末次给药后对小鼠眼球取血并处死,采用HE染色观察肝组织病理并进行Ishak评分,采用γ放射免疫全自动双探头计数器检测肝纤维化指标,包括透明质酸、层粘连蛋白、Ⅲ型前胶原和Ⅳ型胶原,采用实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,q RT-PCR)检测Rho A和ROCK2m RNA的相对表达量,采用Western blot检测Rho A和ROCK2蛋白的相对表达量。结果空白组、模型组、竹沥组和丹参酚酸B组小鼠Ishak评分分别为(0.40±0.05)分、(4.80±0.84)分、(3.20±0.45)分和(2.80±0.83)分,差异有统计学意义(F=34.807,P<0.001),其中空白组显著低于模型组、竹沥组和丹参酚酸B组(t=8.690,P<0.001;t=5.674,P<0.001;t=4.768,P=0.001),模型组显著高于竹沥组和丹参酚酸B组(t=3.016,P=0.017;t=3.922,P=0.004),竹沥组和丹参酚酸B组差异无统计学意义(t=0.809,P=0.464)。空白组小鼠肝纤维化指标均显著低于模型组、竹沥组和丹参酚酸B组[透明质酸:(26.14±2.69)μg/L vs(53.69±5.87)μg/L vs(37.21±3.04)μg/L vs(32.88±3.60)μg/L,层粘连蛋白:(120.37±15.57)μg/L vs(214.48±21.39)μg/L vs(162.54±19.22)μg/L vs(161.66±14.23)μg/L,Ⅲ型前胶原:(4.04±1.01)μg/L vs(11.70±3.09)μg/L vs(8.12±1.87)μg/L vs(7.80±1.72)μg/L,Ⅳ型胶原:(12.96±2.82)μg/L vs(31.34±4.44)μg/L vs(23.72±3.69)μg/L vs(22.85±3.00)μg/L;P均<0.05],模型组小鼠肝纤维化指标均显著高于竹沥组和丹参酚酸B组(P均<0.05),竹沥组和丹参酚酸B组差异无统计学意义(P均>0.05)。空白组Rho A和ROCK2m RNA相对表达量均显著低于模型组、竹沥组和丹参酚酸B组(Rho A m RNA:1.05±0.18 vs 2.61±0.37 vs 1.62±0.21 vs 1.50±0.14,ROCK2 m RNA:1.04±0.17vs2.32±0.29vs1.46±0.08vs1.45±0.09;P均<0.05),模型组Rho A和ROCK2m RNA相对表达量均显著高于竹沥组和丹参酚酸B组(P均<0.05),竹沥组和丹参酚酸B组差异无统计学意义(P均>0.05)。空白组Rho A和ROCK2蛋白相对表达量均显著低于模型组、竹沥组和丹参酚酸B组(Rho A蛋白:0.14±0.03 vs 0.43±0.05 vs 0.26±0.02 vs 0.30±0.15;ROCK2蛋白:0.28±0.03 vs 0.76±0.09 vs 0.38±0.04 vs 0.49±0.03;P均<0.05),模型组Rho A和ROCK2蛋白相对表达量均显著高于竹沥组和丹参酚酸B组(P均<0.05),竹沥组和丹参酚酸B组差异无统计学意义(P均>0.05)。结论竹沥可抑制Rho-ROCK信号转导通路的活动,可能是其抗肝纤维化的作用机制。
