Effect Of Curcumin On Radiation Injury Brain

Radiation injury brain?RIB?is a direct damage or subsequent secondary damage caused by radiation on brain tissue after exposure in special environment.It is common in patient who have primary or secondary head and?or?neck cancer especially nasopharyngeal carcinoma?NPC?and received radiation therapy.Radiation therapy is the main treatmen method of head and?or?neck tumors,it damage the tumor tissue as well as the normal brain tissue around the tumor,so RIB is one of the most serious complications among the long-term suevivers.According to statistics,the incidence rate of brain tissue necrosis after radiation is up to 28.5%^[3].It is already irreversible if clinical symptoms have appeared.The refractory of RIB affects the quality of patient's life seriously and increasing the burden of patient's family and society.The pathogenesis of RIB is still unclear now,the main researches focus on several aspects such as vascular injury,neurons and neural stem cells?neural stem cells,NSCs?damage,immune response ect.Traditional clinical medications such as dehydration,reduce intracranial pressure,supply nutrition for nerve,symptomatic and supportive treatment are ineffective.Curcumin is polyphenols and be extracted from dry stalks of Curcuma which is a perennial herb of Curcuma Genus.A lot of research shows that curcumin has a variety of moleclular targets and signaling pathways in body and has antioxidant,anti-inflammatory,scavenging free radical and other biological activities.Recently,curcumin has become a hot research topic at home and abroad,research areas are becoming increasingly widespread^[2].It has Significant security and multi nerve protection potential.Though research of the effect of curcumin on RIB are just start,the protective function has been confirmed.Notch signaling system,which is highly conservative in evolutionary process,plays an important role in the process of biological development and decide the fate of cells.Notch signaling pathways regulate cells'function of hyperplasia,differentiate,apoptosis in almost all the tissues and organs through the lateral inhibition effect.It also regulate the cell differentiation indirectly in the process of neurons and endothelial cells.The main mechanism of Notch singnaling pathway is a series of signal transduction after the binding of receptor and ligand which promote cell directional defferentation or keep nondifferentiated status.Many experiments indicated that Notch-hes signal system,found in central nervous system,can inhibitate the convertion from neural stem cells to neuron or neurogliocyte and maintin the growth and hyperplasia of neural stem cells.Activation of Notch signaling pathway can promote the hyperplasia of neural stem cells,and inhibitation can turn neural stem cells into differentiation process.The whole regulat prcess is very complecated and has wildly conection with other patheway.?-secretory enzyme is in a prominent position in Notch signal pathway for its participate in the enzymatic hydrolysis prosess of Notch signal pathway.When the function of?-secretory enzyme is interdicted,activation form of Notch intra-cellular domain will become less,downstream signaling molecules will be in static status,which lead the stopping of NSC hyperplasia and enter differentiation program.The inhibitor of?-secretory-DAPT can inhibitate the activaty of?-secretory and hyperplasia of NSC.Learning and memory are complex and advanced intelligent activity of CNS.The relating structure include hippocampus,temporal lobe,amygdala and the limbic system.Decline of learning and memory ability is the main clinical manifestations of early radiation injury of brian.The more serious the brain damaged and longer survival time,the more obvious decline of learning and memory ability.Declining of learning and memory seriously affect the life quality of patients.There is no specific therapy and finding effective treatment is very urgent.Blood brain barrier?BBB?are barriers between blood and brain cells formed by brain capillary wall and the neuroglial cells,and barriers between blood and CSF formed by the choroid plexus.These barriers can limit the free exchange of matter between blood and CSF or blood and brain cells,make brain tissue less damaged by substances in blood circulation and maintain the stability of internal envirenment.It also clears toxic substances and metabolites of brain tissue.It plays an important role in maintaining normal physiological state of the central nervous system.RIB patients'.In pathological conditions the harmful substances can enter brain and aggravate brain injury for BBB is damaged,the permeability increase and its neuroprotection function is weaken in RIB patients.In order to clarify the protective effects of curcumin on treatment of radiation-induced brain injury,understanding relationship between Notch signaling pathway and protective effect of curcumin,we use C17.2 neural stem cells as tool cell to establish radiation damaged neural stem cells model,treating the C17.2NSCs and model cells with curcumin and DAPT,observing the growth and hyperplasia of cells,detecting the expression of Notch signaling pathways'downstream genes such as Notch1,RBP-K,Hes1,Hash1.It is expected to elucidate the interaction between Notch signaling pathway and the mechanism of curcumin pathway and the complex pathological process of RIB.According to experimental results,animal model of brain radiation injury was established and be given different doses of curcumin to interven.Using Morris water maze and escaping platform to test the change of learning and memory ability,testing Evans blue?EB?content in brain tissue and the expression of GFAP and CNPase in hippocampus to observe the protective effect of curcumin on RIB rats'blood brain barrier and provide basis for clinical treatment.Objective1.Establish NSC C17.2 radiation damage model to observe different expression of Notch downstream molecules of Notch pathway,such as Notch1,Hes1,RBP-K and Hash1,in different periods.2.To observe the expression of nodes molecules in different stages under the protection of curcumin.3.To observe model C17.2 NSC's apoptosis,necrosis and proliferation in different period after the intervention of DAPT.4.To test the effect of curcumin on model rats'learning and memory ability by Morris maze and escape platform test.5.To observe the protection effect of curcumin on BBB by testing the change of EB content in brain tissue and HE staining.To observe the expression of GFAP?CNPase in hippocampal by immunohistgochchemical staining.Research mothod1.Cultivate C17.2NSC.2.Nestin immunohistgochchemical staining of C17.2NSCs.3.Testing cell activity by MTT.4.Induce C17.2NSC apoptosis by radiation and observe the effect of curcumin and DAPT on model cells.5.Protein expression detection.?1?Cellular immune fluorescence staining.?2?Extracte total cellular protein and detecte its content.?3?Extracte total cellular protein and detecte its content.6.Notch signaling pathway involved inhibition of radiation induced C17.2 NSCs apoptosis by curcumin.8.In vivo experiment?1?Establish RIB model rats and give different doses of curcumin?2?Morris water maze test and Platform avoidance test?3?EB content detection in brain tissue Result1.Activity of NSCs in the control group is higher than every irradiated group,the p<0.05 between different concentration of curcumin groups,OD value in the concentration of curcumin at 0.5umol/L,2.5umol/L,12.5umol/L groups are higher than irradiation group,the differences of each group are statistically significant?p<0.05?.2.5umol/L curcumin has the strongest antiapoptosis effection and 62.5umol/L curcumin significantly inhibited the activity of NSCs,suggesting that high consentration of curcumin inhibit NSCs'activity.2.Cell proliferation ability in each concentration of DAPT group is lower than the negtive group,indicating that DAPT ihibited the proliferation of NSCs.OD value decreased with the increase of DAPT concentration.Compared with the corresponding concentration of DAPT group,proliferation ability raise in the protection of curcumin,the comparation of each group are statistically significant.3.Neural stem cell proliferation was significantly decreased in the DAPT+radiation groups,compared with negtive group?p<0.05?.The difference between different concentration of DAPT groups are statistically significant?p<0.05?.Difference between DAPT+radiation+curcumin groups and DAPT+radiation groups are statistically significant?p<0.05?.4.NSCs apoptosis rate is significantly different between treated groups and control group?p<0.05?.Comparied different concentration of DAPT groups,cell apoptosis rate were significantly difference?p<0.05?.Compared radiation+curcumin groups with radiation group,the difference is statistically significant?p<0.05?,curcumin can inhibite NSCs apoptosis caused by radiation.Cell apoptosis rate in DAPT groups are lower than DAPT+radiation groups,the corresponding GAPT concentration groups are statistically significant?p<0.05?.5.Immunofluorescence staining showed that Hes1,RBP-??protein in NSCs.6.Western blot shows that expression of RBP-??protein in 2.5 umol/L curcumin group is the lowest,but gradually increased with the increasing concentration of DAPT.Content of Hes1 protein in 2.5 umol/L curcumin group is the highest,and decrease with the increase of DAPT concentration.Compared the radiation+DAPT+curcumin groups with the radiation+DAPT groups,the expression of RBP-??protein is lower and the Hes1protein is higher in the former groups.7.Rats in negative control group had normal spirit,activities,drinking and eating,acted flexibly,had normal reaction speed;rats in the positive control group had significantly bad spirit,acted less,response slower,drunk and ate significantly less and had irritable,aggressive behaviors.The general situation of rats in curcumin treatment group is better than positive control group after 2-3 days,and return to normal in 7-8 days.8.Morris water maze tests:in the learning ability test,the escape latency time in curcumin treatment group was significantly shorter compared with the positive control group,the number of errors significantly reduced?p<0.05?.In the memory test,the original platform quadrant staying time in curcumin treatment group of is longer than the positive control group?p<0.05?,the total path is shorter?p<0.05?.9.Platform test:in the learning ability test,compared with the positive control group,the escape latency in curcumin treatment group was significantly shorter and the number of errors are significantly reduced?p<0.05?.In the memory test,compared with the positive control group,latency time in curcumin treatment was significantly prolonged?p<0.05?,the number of errors was significantly reduced?p<0.05?10.In BBB permeability test,content of EB in curcumin treatment group is lower than positive control group.?p<0.05?.11.HE staining:In negative control group:there is no edema and deformation,the nucleis are large and round,cells arranged neat,cells'edge are neat and have clear boundaries;In positive control group:there is obvious edema,deformation,and necrosis,nuclear pyknosis,fragment and dissolute;In curcumin treatment group:cells damaged was obviously lighter than the positive control group.12.GFAP immunohistochemical staining:positive astrocytes in hippocampus are brown,high density,plump cell bodies,clear contour,coarse projections and homogeneous hyperchromatic.compared with positive control group,the positive cell number in curcum-in treatment group and negative control group are small,the difference is significant?p<0.05?.14.CNPase immunohistochemical staining,the positive cells are brown,round or oval,concentration and not clear boundaries.The main distution of positive cells are in the corpus callosum,callosal convolution,alveus hippocampi,Ventricular bed,internal capsule,entopeduncular nucleus,striatum and thalamus.Cells in hippocampus are filamentous.Compared with positive control group,positive CNPase gray value in curcumin treatment group and negative control group increase significantly?p<0.05?.Conclusion1.DAPT can inhibit the growth and hyperplasia of C17.2 neural stem cells,the inhibition effect enhance with the increase of DAPT concentration.2.Radiation can inhibit the growth and hyperplasia of neural stem cells.3.Curcumin can inhibit the damage of DAPT and ray to neural stem cells.This protective effect may be achieved through the role of Notch signaling pathway,which may affect the expression of downstream proteins in the signaling pathway.4.Curcumin can increase the learning and memory ability of experimental rats in Morris water maze test and Platform avoidance test.5.Curcumin can reduce the damage of blood brain barrier in RIB rats.6.Curcumin can reduce the expression of GFAP in hipocampus.7.Curcumin can increase the expression of CNPase in hipocampus.