The Role Of LRRK2 In The Secondary Brain Injury Induced By Intracerebral Hemorrhage By Regulating P38 MAPK/Drosha Pathway And Potential Mechanism

Part ?:The expression of LRRK2 in brain tissues of rats after ICHObjectiveTo investigate the expression of LRRK2 in brain tissues of rats after ICH t.Methods1.Animal groups:54 health male Sprague-dawley(SD)rats(250-300 g)were numbered and randomly divided into 9 groups(6 rats/group):Normal group,Sham group and 7 experimental groups arranged by time points:3,6,12,24,48,72 h,and 7 d after ICH.2.Establishing the ICH model in rats:Rats were fixed in a stereotaxic apparatus after they were anesthetized.Autologous whole blood from the heart was infused slowly into the right basal ganglia through a micropipettor.All rats were killed at the indicated time point following ICH.All brain cortexes of the rats in each group were extracted for analyse.3.Indicators:The expression of LRRK2 brain tissues of rats after ICH at different time points was evaluated by western blot method.Double immunofluorescence assays was used to measure the neuronal LRRK2 protein levels in rat brain tissues after ICH.Results1.The results of western blot analysis indicated that ICH caused an increase in expression of LRRK2 compared with the Sham group.After induction of ICH,levels of LRRK2 increased,peaked at 6 h,subsequently decreased sharply,and then increased gradually but remained below the level at 6 h.2.Double immunofluorescence assays confirmed increases in neuronal LRRK2 protein levels after ICH.More LRRK2-positive neurons were detected in brain tissues at 6 h after ICH,whereas there was no significant difference between the normal and Sham groupsConclusionsICH induced increases in expression levels of neuronal LRRK2 protein in rat brain tissuesPart ?:The role of LRRK2 in SBI after ICHObjectiveTo study the role of LRRK2 in SBI after ICH in rats.Methods1.Animal groups:96 health male Sprague-dawley(SD)rats were randomly assigned to the following 8 groups(12 rats/group):Sham,ICH,ICH+Vehicle,ICH+GNE7915,ICH+Standard Negtive Control Reagents(Si-NC),ICH+Si-LRRK2,ICH+Vector,and ICH+ Over-LRRK2.2.Establishing the ICH model in rats:Rats were fixed in a stereotaxic apparatus after anesthetized.Autologous whole blood from the heart was infused slowly into the right basal ganglia through a micropipettor.3.Drug Administration:First,GNE-7915 was dissolved in dimethylsulphoxide(DMSO)to 88 mg/ml and injected through intraperitoneal at the dosage of 25 mg/kg 6 h before blood injection.500 pmol scramble siRNA and 500 pmol LRRK2 siRNA were dissolved separately in 5?l RNase-free water.Next,10 ?l Entranster-in vivo-RNA transfection reagent was added to 5 ?l scramble siRNA or LRRK2 siRNA solution,and the solution was mixed for 15 min.Finally,the Entranster-in vivo-siRNA mixtures were injected intracerebroventricularly 24 h before ICH induction.Over-LRRK2 and Vector were diluted to 0.5 mg/ml and injected intracerebroventricularly 24h before blood injection.4.Detection methods and indicators:At 6 hours after ICH,behavioral activity was examined in all groups,blood samples were collected from the heart prior to sacrifice to measure concentrations of TNF-? and IL-1? with specific ELISA kits.Levels of ROS in brain tissues were measured using the Reactive Oxygen Species Assay Kit.LDH Levels were measured using LDH Assay Kit.Neuronal degeneration was detected by fluoro-jade B staining(FJB)staining and Nissl staining.Cell apoptosis in brain tissues was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)staining.Western blot method was used to evaluate the expression of Albumin to determine the role of LRRK2 in BBB injury.,Results1.The neurological scores of rats in the ICH group were significantly higher compared with the sham group,which suggested remarkable neurological deficits induced by ICH.Inhibitor vehicle,scramble siRNA,and Vector treatments did not affect neurological outcome.However,inhibiting LRRK2 with GNE7915 and genetic knockdown could significantly alleviate ICH-induced neurological deficits.Conversely,overexpression of LRRK2 aggravated ICH-induced impairment of neurological behavior.2.Brain water content significantly increased in brain tissues in the ICH group compared with the Sham group.However,there was lower brain water content in rats subjected to inhibition or genetic knockdown of LRRK2 than that in the ICH group.Conversely,overexpression of LRRK2 elevated brain water content.3.FJB,TUNEL staining showed that there was a significant increase in the cell death ratio and neuronal degeneration ratio in brain tissues of rats in the ICH group compared with those in the Sham group.Downregulation of LRRK2 decreased the cell death ratio,while more TUNEL-positive neurons were observed following overexpression of LRRK2.Results of Nissl staining indicated fewer surviving neurons in the CA1 region of the hippocampus and cortex in the ICH group compared with the Sham group.Upregulation of LRRK2 decreased the number of surviving neurons,while there was no clear effect in the Vector treatment group.4.Compared with the Sham group,concentrations of IL-1?,TNF-?,ROS,and LDH significantly increased in the ICH group.Furthermore,overexpression of LRRK2 elevated levels of IL-1?,TNF-?,ROS,and LDH,while Vector had no effect.In contrast,inhibition or genetic knockdown of LRRK2 had opposite effects on inflammatory responses and oxidative stress,which suggested pro-inflammatory and pro-oxidant roles of LRRK2.5.The results of Western blot analysis showed that there were markedly higher levels of albumin in brain tissues of rats in the ICH group than that in the Sham group.Moreover,both inhibition and knockdown of LRRK2 attenuated ICH-induced albumin extravasation,whereas upregulation of LRRK2 enhanced ICH-induced albumin extravasationConclusionsLRRK2 plays an important role in SBI after ICH.Inhibition of LRRK2 reduced neuronal apoptosis and degeneration,inflammatory responses brain edema and BBB injury,promoted neuronal survival and induced a significant suppression in neurological behavior scoring.Part ?:The mechanism of LRRK2 in the SBI after ICH by regulating P38 MAPK/Drosha pathwayObjectiveTo investigate the potential mechanism of LRRK2 in the SBI after ICH by regulating P38 MAPK/Drosha pathway.Methods1.Experimental animal groups:48 health male Sprague-dawley(SD)rats were randomly assigned to the following 8 groups(6 rats/group):Sham,ICH,ICH+Vehicle,ICH+GNE7915,ICH+Si-NC,ICH+Si-LRRK2,ICH+Vector,and ICH+Over-LRRK2.2.Establishing the ICH model in rats and detection methods:Rats were fixed in a stereotaxic apparatus after anesthetized.Autologous whole blood from the heart was infused slowly into the right basal ganglia through a micropipettor.All rats were killed at the indicated time point following ICH.All brain cortexes of the rats in each group were extracted for analyses.3.Drug Administration:First,GNE-7915 was dissolved in dimethylsulphoxide(DMSO)to 88 mg/ml and injected through intraperitoneal at the dosage of 25 mg/kg 6 h before blood injection.500 pmol scramble siRNA and 500 pmol LRRK2 siRNA were dissolved separately in 5 ?l RNase-free water.Next,10?l Entranster-in vivo-RNA transfection reagent was added to 5 ?l scramble siRNA or LRRK2 siRNA solution,and the solution was mixed for 15 min.Finally,the Entranster-in vivo-siRNA mixtures were injected intracerebroventricularly 24 h before ICH induction.Over-LRRK2 and Vector were diluted to 0.5 mg/ml and injected intracerebroventricularly 24h before blood injection.4.The detection index in vivo:Western blot was used to analyze the level of LRRK2 protein,p38 MAPK protein and phosphorylation,Drosha protein and phosphorylation.5.ICH model in vitro:Cortical tissues were isolated from fetal rats at 18 d of gestation to prepare primary neuron-enriched cultures.After a week of incubation,neurons were divided into 4 groups for as follows:Control,OxyHb,OxyHb+Vehicle,and OxyHb+GNE7915.6.The detection index in vitro:Western blot was used to analyze the level of LRRK2 protein,p38 MAPK protein and phosphorylation,Drosha protein and phosphorylation.Cell apoptosis was detected by Hoechst 33258 staining,and Annexin V and PI stainingResults1.In vivo,results of western blot analysis showed that knockdown and inhibition of LRRK2 attenuated phosphorylation of p38 MAPK and Drosha compared with the Sham group,while phosphorylation of p38 MAPK and Drosha could be augmented by overexpression of LRRK2.There was no clear change in phosphorylation of p38 MAPK and Drosha in inhibitor vehicle,scramble siRNA,and Vector groups compared with the ICH group.2.In vitro,results of western blot analysis showed that OxyHb treatment promoted phosphorylation of p38 MAPK and Drosha,which was attenuated when LRRK2 was inhibited.3.In vitro,Hoechst 33258 staining showed that significantly more apoptotic cells were detected in the OxyHb group compared with the Control group,and the number of apoptotic cells was remarkably decreased by GNE7915 treatment.The results of Annexin V and PI staining showed that there was a higher apoptotic ratio after OxyHb treatment relative to the Control group.In addition,inhibition of LRRK2 with GNE7915 significantly decreased the OxyHb-induced increase in the apoptotic ratio of neurons compared with the vehicle group.ConclusionLRRK2 can play an important role in SBI after ICH by regulating p38MAPK/Drosha pathway.Inhibition of LRRK2 can inhibit p38MAPK/Drosha pathway,reduce neuronal apoptosis after ICH,and protect the brain from SBI after ICH.