Role Of RhoA/ROCK Signaling Pathway In The Enhanced Permeability Of Blood-brain Barrier Induced By Methamphetamine

Background:Methamphetamine(METH),which is known as crystal Meth,is a psychostimulant with severe neurotoxicity,potential of addiction and widely spread worldwide.China has become a METH abused disastrous due to the large population of drug addicts,which are increasing every year and the age of drug used population has shown a descending trend.Meth abused is a huge threat to the public safety and social order.Plenty of drug crime present in forensic work.Not only because of the hallucinogenic effects,but also one-time overdose and long time abused.Although the mechanisms of neurotoxicity are currently fully understood,but a considerable research has been conducted on the direct effect of METH on central neuronal system(CNS)and cardiovascular system(CVS).Traditionally,major study showed that oxidative stress,mitochondrial dysfunction,neuronal apoptosis,astrocytes activation are the possible mechanisms of neurotoxicity on dopaminergic neurons.Moreover,the direct effect of METH on Cardiovascular system(CVS)is based on METH induces cardiomyocyte apoptosis,which caused severe cardiovascular diseases.Besides the direct effects of METH on CNS and CVS,METH-induced neurotoxicity can also compromise the blood brain barrier(BBB)function and increase the BBB permeability indirectly.Blood-brain barrier plays a critical role in the regulation of metabolism and transport,which controls cellular traffic between the brain and the circulation in order to preserve the internal environment homeostasis of CNS.The integrity of the BBB is maintained by multiple components,including the capillary endothelial cell,tight junction(TJ),astrocyte endfeet,pericytes,basement membrane and the extracellular matrix.METH has small molecular weight and lipid solubility,which can easily cross the BBB to the CNS and affect the CNS indirectly.In addition,potential consequences of METH-induced BBB disruption have become the next research focus.Recently,considerable data indicates that METH have been shown to produce disruption of BBB by decreasing the expression of Tight Junction Protein and rearrange the distribution of cytoskeletal protein to increase BBB permeability in various molecular pathways.The Rho family of GTPases is a family of small(?21 kDa)signaling G proteins,and is a subfamily of the Ras superfamily.The members of the Rho GTPase family are under the responsibility of intracellular actin dynamics regulation in many aspects.Three of the family members have been studied in thoroughly:Racl,Cdc42,and RhoA.Rho proteins are responsible for regulating organelle development,cytoskeletal dynamics,cell movement,and other common cellular functions.RhoA is one of the most important members in Rho GTPases family which not only involved in spine morphogenesis,but also modulates the regulation and timing of cell division.As the downstream signaling molecules,ROCK plays a role in cancer and in particular cell motility,metastasis,and angiogenesis.The major molecular components of the TJ include the transmembranous and structural proteins,Occludin,junctional adhesion molecule(JAM)and claudins and the submembranous peripheral Zona Occluden(ZO)proteins.RhoA/ROCK signaling pathway potentially participates in the increase process of METH-induced BBB permeability by decreasing the expression and distribution of the tight junction protein and rearrange the F-actin.We intended to establish METH poisoning model both in vivo and in vitro.The BBB vitro model uses RBMEC to simulate BBB structure.After treated with METH,TJ associated proteins were measured by molecular biological detection and morphological observation.SD rats were used to establish vivo model of BBB.Furthermore,inhibitor of RhoA/ROCK was utilized to demonstrate the role of RhoA/ROCK signaling pathway.Overall,the goal of this research is to reveal that RhoA/ROCK regulates BBB disruption under METH exposed.It will be important in order to develop new treatments to improve the long-term neurological damage and possibly METH addiction.Objectives:The present study aimed to substantiate the effect of METH on BBB and the role of RhoA/ROCK signaling pathway both in vivo and in vitro by molecular biotechnology detection,morphology observation and functional assessment.Furthermore,inhibitors of RhoA and ROCK were chosen to suppress the expression of upper stream signaling molecules in order to determine whether the RhoA/ROCK signaling pathway is one of the regulatory mechanism or not.Method:1.Establishment of the METH poisoned BBB model in vitro by cultured RBMEC.(1)Primary cultured RBMEC were maintained in DMEM media containing 15%fetal bovine serum in a 37? incubator with 5%CO2.Subcultured cell divided into 96-well plates and treatments were performed after the RBMECs were confluent while cell density reaches 80%-85%.METH was dissolved in DMEM media containing 2%fetal bovine serum at Ommol/L,0.4mmol/L,0.8mmol/L,1.2mmol/L,1.6 mmol/L,2.0 mmol/L,2.4 mmol/L,2.8 mmol/L,3.2 mmol/L,3.6mmol/L and cultured RBMEC for 6h.Detected the LC25,LC50 of RBMEC in different concentrations of METH by CCK-8.(2)Subcultured cell was divided into 6-well plate and treated with 0 mmol/L,0.5 mmol/L,1.0mmol/L,1.5 mmol/L,2.0 mmol/L and 2.5 mmol/L of METH for 6 hours.Total protein was extracted from RBMECs after METH treatment,and the protein concentrations were measured using the bicinchonic acid(BCA)protein assay.Western-Blot was used to detect the expression changes of TJ proteins and MMP-9 after exposed to METH,in order to choose a suitable concentration for METH poisoned model.(3)RBMEC was divided into 6-well plate and treated with 1.5 mmol/L of METH for 0 hours,4 hours,6 hours,8 hours,12 hours and 24 hours.Total protein was extracted from RBMECs after METH treatment,and Western-Blot was used to detect the expression changes of TJ proteins and MMP-9 after exposed to METH,in order to choose an appropriate action time for METH poisoned model.(4)RBMEC was cultured in Transwell chambers and exposed to 1.5 mmol/L of METH for 6 hours.Assessed changes in BBB permeability by using the fluorescent tracer,Lucifer Yellow(LY).(5)RBMEC was cultured in confocal petri and treated with 1.5 mmol/L of METH for 6 hours.Using standard immunofluorescence staining method to assess the change of TJ protein distribution and expression.2.Role of RhoA/ROCK signaling pathway in methamphetamine-induced BBB permeability increasing.(1)Establish a METH poisoned BBB model in vitro by cultured RBMEC.Using Western-blot to analyze the expression of signal molecules of RhoA/ROCK pathway.(2)Pretreated the RBMEC with RhoA specific inhibitor clostridium botulinum C3 exoenzyme and ROCK specific inhibitor Y-27632 respectively in order to inhibit the expression of RhoA and ROCK.(3)RBMEC was divided into blank control group(Ctrl),METH treated group(METH),C3 exoenzyme treated group(C3),C3 exoenzyme pretreated and METH treated group(METH+C3),Y-27632 treated group(Y)and Y-27632 pretreated and METH treateded group(METH+ Y).Total protein was extracted from RBMECs after administered respectively,and Western-Blot was used to detect the expression changes of signal molecules of RhoA/ROCK pathway.(4)Use Lucifer Yellow assay to measure the permeability of RBMEC and immunofluorescence assay to determine the distribution of ZO-1 and F-actin.3.Detected the change of TJ protein expression in the midbrain of rats treated with METH by Western-blot and observed the signaling molecules expression changes of the RhoA/ROCK signaling pathway.(1)Establishment of sub-acute METH poisoned model in vivo.(2)Evaluate the BBB permeability by intravenous injection of Evans blue.(3)Frozen sections of midbrain tissue using immunofluorescence staining to observe the rearrangement of F-actin.(4)Total proteins were collected from midbrain of rats to measure the level of TJ protein and signal molecules of RhoA/ROCK pathway.Compare METH treated group with blank control group.Results:1.METH poisoned BBB model in vitro by cultured RBMEC.(1)CCK-8 result indicates that with the increase in concentration of METH,survival rates of RBMEC was decreased.LC25 and LC50 of RBMEC cells were 1.63mmol/L and 3.26mmol/L respectively.(2)RBMEC was treated by METH of a concentration gradient of 0 mmol/L,0.5 mmol/L,1.0 mmol/L,1.5 mmol/L,2.0 mmol/L,2.5 mmol/L.With the increase of METH concentration,Western-blot shows a decrease of Occludin,Claudin-5 and ZO-1,and a increase of MMP-9 on the contrary.The expression of Occludin,Claudin-5 and ZO-1 reduced by 50%between the control group and 1.5mmol/L group.The expression of MMP-9 was 7 times increase between the control group and 1.5mmol/L group.(3)RBMEC was treated by METH under different time gradient.With increasing exposure time,Western-blot shows the tendency of Occludin,Claudin-5 and ZO-1 goes down first,and then up and the turning point is between 4 to 6 hours.MMP-9 goes up at first,and then down and the turning point is at 4 to 6 hours as well.(4)Lucifer Yellow was added to the upper chamber of the Transwell system that contains co-cultured RBMEC monolayers.(5)Using immunofluorescence analysis,we found that F-actin and ZO-1 was localized at cell-cell boundaries,with continuous distribution in co-cultured RBMECs in blank control group.METH treatment induced an apparent loss of F-actin from the membrane regions of RBMECs and stress fiber formation.ZO-1 as well.2.RhoA/ROCK signaling pathway involved in regulation of METH-induced BBB hyperpermeability.(1)Western-blot shows a two times increases of RhoA and ROCK in METH treated group.Total MLC and Cofilin were significantly increased and phosphorylation MLC and Cofilin as well.Under the regulation of RhoA/ROCK,downstream signaling pathway PI3K/PKB increased in METH treated group.(2)Inhibition experiments were performed using pharmacological inhibitors C3 Exoenzyme and Y-27632 to either block RhoA activity or inhibit ROCK activity.Both C3 Exoenzyme and Y-27632 can reverse the down-regulation of TJ related proteins by METH.(3)Both C3 Exoenzyme and Y-27632 can reverse the up-regulation of permeability by METh.(4)Immunofluorescence staining indicates that both C3 Exoenzyme and Y-27632 can reduce stress fiber formation and reverse F-actin rearrangement.3.RhoA/ROCK signaling pathway involved in the regulation of METH induced BBB permeability increase in vivo.(1)Sub-acute METH poisoned model in vivo established successfully.Tail injection of Evans blue shows a hyperpermeability effect.(2)Immunofluorescence staining indicates that METH caused loosen of brain endothelial cell and rearrangement of F-actin 1.(3)Western-blot shows a decrease of Occludin,Claudin-5 and ZO-1,and an increase of MMP-9 on the contrary.Moreover,RhoA and ROCK increases in METH treated group.Total MLC and Cofilin were significantly increased and phosphorylation MLC and Cofilin as well.Under the regulation of RhoA/ROCK,downstream signaling pathway PI3K/PKB increased in METH treated group.Conclusion:1.After administration of METH,the protein expression levels of Occludin,Claudin-5 and ZO-1 decreased,besides,MMP-9,RhoA,ROCK,MLC,Cofilin,p-MLC,p-Cofilin,PI3K,PKB expression levels increased significantly in vitro.After the pretreatment of C3 Exoenzyme and Y-27632,all the aforementioned changes were reversed.2.Tight junctions between brain membrane endothelial cell were disrupted by METH,permeability of intracellular and intercellular were both elevated.The hyperpermeability effect of METH can be reversed by the inhibitor of RhoA and ROCK.3.METH treatment induced an apparent loss of F-actin from the membrane regions of RBMECs and stress fiber formation.ZO-1 as well.4.Intravenous injection of Evans blue shows a permeability increase effect.5.The protein expression levels of Occludin,Claudin-5 and ZO-1 decreased,besides,MMP-9,RhoA,ROCK,MLC,Cofilin,p-MLC,p-Cofilin,PI3K,PKB expression levels increased significantly in midbrain of SD rats after METH treatment.