Rho/Rock signal transduction pathway is required for MSC tenogenic differentiation

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.

Effect of QingguanganⅡon Rho/ROCK associated factors in the retina of DBA/2J mice

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.

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

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.

Houshiheisan and its components promote axon regeneration after ischemic brain injury

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.

ROCK inhibitor:Focus on recent updates

Rho-associated coiled-coil-containing protein kinase(RoCK)belongs to the serine-threonine family,and RoCK is involved in a variety of biological processes including cell migration,adhesion,proliferation and differentiation through phosphorylation of different downstream substrates.The aberrant activation of ROCK is associated with the pathological conditions in different systems including various diseases,including cancer,neurological diseases,inflammation,cardiovascular diseases and glaucoma.Therefore,the ROCK inhibitors have potential applicability for treating the aforementioned diseases.Four small molecule ROCK inhibitors have been approved for clinical use:fasudil,ripasudil,netarsudil and belumosudil.In recent years,more small molecule ROCK inhibitors have been identified.This paper reviews the ROCK inhibitors reported in past seven years.We mainly focused on the summarization of the structure-activity relationships,inhibitory efficacy,pharmacological mechanisms and the relevant clinical studies of the reported ROCK inhibitors.Besides the small molecular inhibitors,the peptides and biological extracts which exhibit ROCK inhibitory effects are also included.We also provide suggestions for the future development of the potent ROCK inhibitors.

Effects of Bunao-Fuyuan decoction serum on proliferation and migration of vascular smooth muscle cells in atherosclerotic

Atherosclerosis(AS)is a chronic inflammatory disease,the main causes of which include abnormal lipid metabolism,endothelial injury,physical and chemical injury,hemodynamic injury,genetic factors and so on.These causes can lead to inflammatory injury of blood vessels and local dysfunction.Bunao-Fuyuan decoction(BNFY)is a traditional Chinese medicine compound that can treat cardiovascular and cerebrovascular diseases,but its effect on AS is still unknown.The aim of this study was to investigate the effect and mechanism of BNFY in proliferation and migration of vascular smooth muscle cells(VSMCs)on AS.At first,the expression ofα-SMA protein in ox-LDL-induced VSMCs,which was detected by immunofluorescence staining and western blot.CCK-8 technique and cloning technique were used to detect the cell proliferation of ox-LDL-induced VSMCs after adding BNFY.Meanwhile,the expression of proliferating protein Ki67 was detected by immunofluorescence staining.Western blot was also used to detect the expression of proliferation-related proteins CDK2,CyclinE1 and P27.Flow cytometry was used to detect the effect of BNFY on cell cycle.The effects of BNFY on proliferation and migration of cells were detected by cell scratch test and Transwell.Western blot was used to detect the expression of adhesion factors ICAM1,VCAM1,muc1,VE-cadherin and RHOA/ROCK-related proteins in cells.We found that the expression of AS markerα-SMA protein increased significantly and cells shriveled and a few floated on the medium after induction of ox-LDL on VSCMs.The proliferation rate of ox-LDL VSMCs decreased significantly after adding different doses of BNFY,and BNFY can inhibit cell cycle.Meanwhile,we also found that cell invasion and migration rate were significantly inhibited and related cell adhesion factors ICAM1,VCAM1,muc1 and VE-cadherin were inhibited too by BNFY.Finally,we found that BNFY inhibited the expression of RHOA,ROCK1,ROCK2,p-MLC proteins in the RHOA/ROCK signaling pathway.Therefore,we can summarize that BNFY may inhibit the proliferation and migration of atherosclerotic vascular smooth muscle cells by inhibiting the activity of RHOA/ROCK signaling pathway.