The Mechanism Of Gastrodin Regulating The Inflammatory Response Of Cerebral Palsy

BackgroundCerebral Palsy(CP)is a group syndrome of permanent disorders of movement and posture causing lirmited activity,mostly due to the non-progressive damage to the development of the brains of fetus or infants.The main manifestations are dyskinesia,writh or without perception and mental retairdation.The prevalence is about 2.0v3.5%O worldw-ide.The incidence of China is I.8-4.0%o depends on the regions,and also there is a tendency of rise year by year.CP is one of tlhe most serious diseases result in children with disabilities,and also a disease along with the patient's life long,which brought heavy burden to the family and society mentally and financially,thus looking for treatment wlhich can improve CP syndrome is particularly urgent.The brain pathological changes of CP are mainly periventricular white matter damage,including periventricular leukomalacia(PVL),intraventiricular hemorrhage and diffuse myelination disorders caused by the damages to the development of brain in prenatal,intrapartum or/and postpartum stage.The patlhogenesis of CP is thought to be the combination of inflammatory and hypoxia-ischemia response.Therefore,we speculate that the key of treatment of CP is to alleviate the inflanmmatory response,promote angiogenesis,and repair damaged tissue.The early stage is sub-emergency repair stage,macrophages from tlhe pro-inflammatory state Ml type differentiation into anti-inflammatory repair of M2 and play a role in this stage in promoting anti-inflammatory repair;The second stage is pro-angiogenesis,vascular endothelial cells at this stage induced by growth factor promote migration and proliferation,and thus accelerate the formation of new blood vessels which performs the establishment of collateral circulation to alleviate ischemia and hypoxia condition.Macrophages play an important role in regulating the inflammation and anti-irnflammatory responses of tlhe immune system by regulating their M1/M2 differentiation.Studies have shown that M2 macrophages can promote the repair of nerve cells and secrete vascular endothelial growth factor(VEGFA),then promote vascular endothelial cell proliferation and migration,But the mechanism of the regulation of anti-inflammatory and angiogenesis is still unknown.We found that acupuncture points injection of traditional Chinese medicine Gastrodin for CP patients have significantly improved with clinical indicators.In this study,we use the microarray technology to screen the changes of the gene expression of CP,and also explore the mechanism of Gastrodin induces macrophage polarization in the inflammatory response and VEGFA promotes the proliferation and migration of vascular endothelial cells,which provide scientific theoretical basis for Gastrodin in the clinical application.ObjectiveIn this study,we screened genes that were highly correlated with CP by gene chip technology,and discussed the importance of BCL6 in the regulation of macrophage(RAW264.7)in irnflammatory response with Gastrodin,and the involvement of AP-1 family in vasculair endotlhelial cell(HUVEC)with VEGFA.Explore the mechanism of Chinese:medicine Gastrodin regulates the immune response and the treatment of CP.Methods1.mRNA expression profiles of CP Patients.We recruited 44 cases of congenital cerebral palsy,18 cases of acquired cerebral palsy and 30 cases of normal children.The peripheral blood of all participants were collected and subjected to RNA extraction and purification.After qualitative examination,differential mRNA expression was assessed using a GeneChip?PrimeViewr?Human Gene Expression Array develloped by AfiEymetrix.The results were analyzed to identify the gene expression profiles associated with CP disease.2.Effect of Gastrodin on CP and the related action targets.The changes of adductor angle,gross motor function score(GMFM-66)and fine motor ftunction score(FMFM-45)were measured by analyzing the clinical data of CP patients.Real time-PCR was used to detect the difference of gene expression induced by LPS and differentiated into M1 type macrophage,and then determine tlhe role of Gastrodin in macrophage involved in inflammatory response.3.Effects of BCL6 on the effect of Gastrodin in macrophage.Explore the mechanism of BCL6 on anti-inflammatory and anti-apoptotic effect by observing the effect of Gastrodin on Ml and M2 type macrophage with or without BCL6 gene knockout.4.Signaling pathway of VEGFA induced HUVEC on expression of AP-1 family.M2 macrophages stimulate the activation of vascular endothelia cells by secreting VEGFA and promote neovascularization.Explore the signaling pathway of transcription mechanism of AP-I family regulated by VEGFA induced HUVEC.5.Effect of VEGFA induced HUVEC on cell function regulated by AP-1 Family.The changes of cell migration,proliferation and cell cycle of HUVEC was observed by inhibition of AP-1 family activity with or without AP-1 knockout.Resultsl1 Compared with the normal control group;there were significant differences in the expression of 1008 genes in the cerebral palsy group(both congenital and acquired),among which 214 genes were elevated and 794 gene expression was decreased.The expression of SRSF3,ELF2,BCL2L113 BCL6 and CSNKIAl increased significantly more tihan 4 folds,while the expression of' UQCRB,PFDN4,LSM3 and CLEC2B was significantly decreased by more than 5 folds.Gene RAD23B was only significantly reduced in the acquired cerebral palsy group.2.The changes of clinical indexes before and after treatment were cormpared with those of CP group and non-CP group.The results showed that the acupuncture point injection of Gastrodin in the patients with CP improved adductor angle release and GMFM-66 and FMFM-45 scores,and the curative effect was significantly better than that of non-CP neurological disease patients.Furthermore,macrophage experiment showed that Gastrodin could be adjusted to participate in inflammatory reaction and the expression of BCL6 gene in Ml macrophage increased significantly.3.Gastrodin treatment significantly upregulated BCL6 mRNA expression levels in Ml macrophage,but not in M2 macrophage.In addition,Gastrodin could induce Ml macrophage to differentiate into M2,while the cell viability of macrophage stimulated by oxidative stress was increased and the degree of apoptosis was decreased.However,when BCL6 gene was knockout,the ability of inducing and regulating cell activity and apoptosis is inhibited.4.VEGFA significantly increased the expression of AP-1 family mRNA and protein expression in HUVEC and changed in the time manner.The effect of VEGFA up-regulation of AP-1 expression was mainly through MEKI,p38-MAPK,PKC and IGFlR pathway,but not through PI3K pathway.5.VEGFA can promote the migration and proliferation of HUVEC,as well as Cyclin Dl expression in cell cycle.Houever,when AP-1 protein activity was blocked,the migration and proliferation of cells are inlhibited and Cyclin D1 expression decreased.When JunB or JunD gene was knockout,tlhe rmigration of cells is inhibited,but not the cell proliferation,in addition,Cyclin D1 expression decreased.Conclusions1.Compared witih the normal control group,genes witlh abnormal expression of congenital CP and acquired CP are related to the maintenance of inflammation,hypoxia,ischemia,angiogenesis,cell proliferation and vascular integrity,suggesting that the pathogenesis of CP is related to inflammatory response and interaction of hypoxia and ischemia.2.Gastrodin acupoint injection therapy for the treatment of CP shows significant improvement on adduction angle release,GMFM-66 and FMFM-45 score performances;,and the effect is significantly higher than non-CP patients,suggesting that the mechanism of Gastrodin and the pathogenesis of CP have common pathway or action tairgets.3.Gastrodin can induce macrophage polarize from M1 type,which is mainly composed of ilnflammatory reaction,to M2 type,vvlhich involved in anti-inflammatory and cell repair.Gastrodin can also significantly improve the activity of macrophage stimulated by oxidat:ive stress,and reduce the extent of apoptosis by regulating BCL6 gene expression.4.M2 macrophages stimulate the acti,vation of vascular endothelia cells by secreting VEGFA and promote neovascularization.VEGFA can significantly increase the expression of AP-1 family members in HUVEC cells through MEKI,p38-MAPK,PKC and IGFIR pathway,and regulate the cell migration,proliferation and cell cycle of HUVEC through AP-1 family,and then affecting angiogenesis and tlhe establishment of collateral circulation.