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
Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially availabl...Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.展开更多
Aim DL0805-2 is a novel Rho-kinases inhibitor which has been found to have potent cardiovascular effects. In the present research, we aimed to study the potential of DL0805-2 in the treatment of pulmonary arterial hyp...Aim DL0805-2 is a novel Rho-kinases inhibitor which has been found to have potent cardiovascular effects. In the present research, we aimed to study the potential of DL0805-2 in the treatment of pulmonary arterial hypertension （PAH） and discuss the underlying mechanisms preliminarily. Methods A classical PAH animal model was used, which was established by single injection of 50 mg · kg^-1 monocrotaline （MCT）. One week later, the rats were administrated with 1, 3, 10 mg · kg^-1 DL0805-2 via intraperitoneal injection for 18 days. At the end of the experiment, the body weight and survival rate were recorded. Meanwhile, the respiration function, heart function, blood pressure and pulmonary artery pressure were detected. Serum was collected for biochemical index analysis. The weight of vital organs was used to calculate the organ index. Histopathology examination was em-ployed to observe the subtle changes in hearts, vessels and lungs. Furthermore, the mechanisms were studied main- ly by the method of western blotting. Results DL0805-2 did not show significant influence on body weight of PAH rats. But the survival rate of PAH rats treated with 3 and 10 mg · kg^-1 DL0805-2 was increased up to 90. 9% com- pared with the model group （68.2%）. DL0805-2 improved the pulmonary artery blood flow especially the maximal -1 -1 velocity （PV max） from 397.2 cm · s^-1 to 506.5, 540. 1 and 574.0 cm · s^-1 respectively. The results of echocar- diography and electrocardiogram show that DL0805-2 had little effect on left ventricle and systemic circulation but attenuated right ventricle injury and decreased the right ventricle pressure from 73.73 mmHg to 47.80, 42.64 and 46.45 mmHg respectively after DL0805-2 intervention. Disease markers of PAH including NT-proBNP in serum and ET-1 in lung tissue homogenate and serum biochemical indicators, ALT, AST and LDH, were reduced by DL0805-2. DL0805-2 also relieved edema of lungs and decreased inflammatory cytokines production. Through the examination on histopathologic slide of pulmonary main artery, right ventricle and lung, DL0805 derivatives were found to have significant protection effect on structural changes of organs induced by pulmonary hypertension. Ac- cording to the preliminary study on the mechanisms of DL0805-2 in PAH, Rho/ROCK pathway was significantly in- hibited by DL0805 derivatives. In addition, DL0805 derivatives showed effect on BMPRII/p-Smad pathway and ap- optosis related pathway. Conclusion DL0805-2 has showed potent treatment effect on the PAH rats. And the un- derlying mechanisms studies also indicated that RhoA/ROCK and BMPRII pathways were involved. This work will provide basis experimental data for the further research and development of DL0805-2.展开更多
Houshiheisan,a classic prescription in traditional Chinese medicine,contains Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari,Radix Platycodonis,Rhizoma Atractylodis m...Houshiheisan,a classic prescription in traditional Chinese medicine,contains Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari,Radix Platycodonis,Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis,Radix et Rhizoma Ginseng,Radix Scutellariae and Concha Ostreae.According to traditional Chinese medicine theory,Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari and Radix Platycodonis are wind-dispelling drugs;Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis and Radix et Rhizoma Ginseng are deficiency-nourishing drugs.A large number of randomized controlled trials have shown that Houshiheisan is effective in treating stroke,but its mechanism of action is unknown.Axonal remodeling is an important mechanism in neural protection and regeneration.Therefore,this study explored the effect and mechanism of action of Houshiheisan on the repair of axons after cerebral ischemia.Rat models of focal cerebral ischemia were established by ligating the right middle cerebral artery.At 6 hours after model establishment,rats were intragastrically administered 10.5 g/kg Houshiheisan or 7.7 g/kg wind-dispelling drug or 2.59 g/kg deficiency-nourishing drug.These medicines were intragastrically administered as above every 24 hours for 7 consecutive days.Houshiheisan,and its wind-dispelling and deficiency-nourishing components reduced the neurological deficit score and ameliorated axon and neuron lesions after cerebral ischemia.Furthermore,Houshiheisan,and its wind-dispelling and deficiency-nourishing components decreased the expression of proteins that inhibit axonal remodeling：amyloid precursor protein,neurite outgrowth inhibitor protein A（Nogo-A）,Rho family small GTPase A（Rho A） and Rho-associated kinase 2（Rock2）,and increased the expression of growth associated protein-43,microtubule-associated protein-2,netrin-1,Ras-related C3 botulinum toxin substrate 1（Rac1） and cell division cycle 42（Cdc42）.The effect of Houshiheisan was stronger than wind-dispelling drugs or deficiency-nourishing drugs alone.In conclusion,Houshiheisan,and wind-dispelling and deficiency-nourishing drugs promote the repair of axons and nerve regeneration after cerebral ischemia through Nogo-A/Rho A/Rock2 and Netrin-1/Rac1/Cdc42 signaling pathways.These effects are strongest with Houshiheisan.展开更多
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.
基金supported by the National Natural Science Foundation of China,No.81171178
文摘Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.
文摘Aim DL0805-2 is a novel Rho-kinases inhibitor which has been found to have potent cardiovascular effects. In the present research, we aimed to study the potential of DL0805-2 in the treatment of pulmonary arterial hypertension （PAH） and discuss the underlying mechanisms preliminarily. Methods A classical PAH animal model was used, which was established by single injection of 50 mg · kg^-1 monocrotaline （MCT）. One week later, the rats were administrated with 1, 3, 10 mg · kg^-1 DL0805-2 via intraperitoneal injection for 18 days. At the end of the experiment, the body weight and survival rate were recorded. Meanwhile, the respiration function, heart function, blood pressure and pulmonary artery pressure were detected. Serum was collected for biochemical index analysis. The weight of vital organs was used to calculate the organ index. Histopathology examination was em-ployed to observe the subtle changes in hearts, vessels and lungs. Furthermore, the mechanisms were studied main- ly by the method of western blotting. Results DL0805-2 did not show significant influence on body weight of PAH rats. But the survival rate of PAH rats treated with 3 and 10 mg · kg^-1 DL0805-2 was increased up to 90. 9% com- pared with the model group （68.2%）. DL0805-2 improved the pulmonary artery blood flow especially the maximal -1 -1 velocity （PV max） from 397.2 cm · s^-1 to 506.5, 540. 1 and 574.0 cm · s^-1 respectively. The results of echocar- diography and electrocardiogram show that DL0805-2 had little effect on left ventricle and systemic circulation but attenuated right ventricle injury and decreased the right ventricle pressure from 73.73 mmHg to 47.80, 42.64 and 46.45 mmHg respectively after DL0805-2 intervention. Disease markers of PAH including NT-proBNP in serum and ET-1 in lung tissue homogenate and serum biochemical indicators, ALT, AST and LDH, were reduced by DL0805-2. DL0805-2 also relieved edema of lungs and decreased inflammatory cytokines production. Through the examination on histopathologic slide of pulmonary main artery, right ventricle and lung, DL0805 derivatives were found to have significant protection effect on structural changes of organs induced by pulmonary hypertension. Ac- cording to the preliminary study on the mechanisms of DL0805-2 in PAH, Rho/ROCK pathway was significantly in- hibited by DL0805 derivatives. In addition, DL0805 derivatives showed effect on BMPRII/p-Smad pathway and ap- optosis related pathway. Conclusion DL0805-2 has showed potent treatment effect on the PAH rats. And the un- derlying mechanisms studies also indicated that RhoA/ROCK and BMPRII pathways were involved. This work will provide basis experimental data for the further research and development of DL0805-2.
基金supported by the National Natural Science Foundation of China,No.81373526
文摘Houshiheisan,a classic prescription in traditional Chinese medicine,contains Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari,Radix Platycodonis,Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis,Radix et Rhizoma Ginseng,Radix Scutellariae and Concha Ostreae.According to traditional Chinese medicine theory,Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari and Radix Platycodonis are wind-dispelling drugs;Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis and Radix et Rhizoma Ginseng are deficiency-nourishing drugs.A large number of randomized controlled trials have shown that Houshiheisan is effective in treating stroke,but its mechanism of action is unknown.Axonal remodeling is an important mechanism in neural protection and regeneration.Therefore,this study explored the effect and mechanism of action of Houshiheisan on the repair of axons after cerebral ischemia.Rat models of focal cerebral ischemia were established by ligating the right middle cerebral artery.At 6 hours after model establishment,rats were intragastrically administered 10.5 g/kg Houshiheisan or 7.7 g/kg wind-dispelling drug or 2.59 g/kg deficiency-nourishing drug.These medicines were intragastrically administered as above every 24 hours for 7 consecutive days.Houshiheisan,and its wind-dispelling and deficiency-nourishing components reduced the neurological deficit score and ameliorated axon and neuron lesions after cerebral ischemia.Furthermore,Houshiheisan,and its wind-dispelling and deficiency-nourishing components decreased the expression of proteins that inhibit axonal remodeling：amyloid precursor protein,neurite outgrowth inhibitor protein A（Nogo-A）,Rho family small GTPase A（Rho A） and Rho-associated kinase 2（Rock2）,and increased the expression of growth associated protein-43,microtubule-associated protein-2,netrin-1,Ras-related C3 botulinum toxin substrate 1（Rac1） and cell division cycle 42（Cdc42）.The effect of Houshiheisan was stronger than wind-dispelling drugs or deficiency-nourishing drugs alone.In conclusion,Houshiheisan,and wind-dispelling and deficiency-nourishing drugs promote the repair of axons and nerve regeneration after cerebral ischemia through Nogo-A/Rho A/Rock2 and Netrin-1/Rac1/Cdc42 signaling pathways.These effects are strongest with Houshiheisan.
