| PART Ι Effects of Rosemary Extract on Short-term & Long-term Spatial Cognition and Affective Behavior in Rats with Multiple Cerebral ConcussionObjective(s): Recently,more and more studies have found that multiple cerebral concussion(MCC)often occurs in athletes,veterans and family abusers.The patients of them develop chronic traumatic encephalopathy(CTE).The pathophysiologic mechanism of CTE is still unknown and the criteria for clinical diagnosis,treatment and prognosis are absent.We have previously found that rosemary extract(RE)can effectively improve the early behavioral damage of MCC rats through a series of experiments.This study was designed to evaluate the evaluate changes of short-term(14 days)and long-term(10 months)spatial cognition and emotion in rats with MCC improved by RE.Methods:(1)Model :3MCC rat model was established by using a metal pendul um impinging device,which was struck once every 24 hours and three times consecut ively(2)Animal groups: A total of 112 healthy adult male SD rats,weighing 300±10g,were randomly divided into Sham group and 3MCC group.Each model group was randomly subdivided based on two time points of 14 d and 10 m,Rosemary extract group(RE)and Tween solvent group(TW)were replicated from 3MCC at each time point.After each impact,eligible rats were randomly assigned to each experimental group,including the 14d-Sham group,14d-3MCC group,14d-TW group,14d-RE group and 10m-Sham group,10m-3MCCgroup,10m-TW group,10m-RE group(n=14).RE and TW were administered by gavage within 12 h after successful establishment of the model.In the 14 d group,the drug was administered continuously for 14 days,and in the 10 m group,the drug was administered continuously for 28 days after injury.(3)Morris water maze was used to evaluate the changes in spatial cognition and learning in rats after injury and elevated plus-maze(EPM)to evaluate the degree of depression after injury.Results:(1)Results of Morris water maze test: I.14 days after injury:Platform experiment(underwater and above-water phase),the escape latency of the injury groups decreased gradually.Platform-free experiment,the time delay of reaching the original platform in injury groups,the time of passing through the original platform were decreased in injury groups,the residence time of rats in quadrant I decreased in injury groups.Reverse platform experiment,the escape latency was delayed for rats in the injury groups.All these changes with statistically significant differences between groups(Sham VS 3MCC,P<0.05;Sham VS TW,P<0.05).Rosemary extract improved except for the number of times of passing through the original platform after intervention,and the difference was statistically significant(TW VS RE,P<0.05).II.10 months after injury: Underwater platform experiment,escape latency of the injured group was prolonged,and the differences were statistically significant(Sham VS 3MCC,P<0.05;Sham VS TW,P<0.05).Escape latency was shortened in the RE intervention group,and the difference was statistically significant(TW VS RE,P<0.05).Above-water platform experiment,escape latency was longer in the injury group,and the difference was statistically significant(Sham VS 3MCC,P<0.05).Platform-free experiment,the time to reach the original platform was delayed in the injury groups,and times of crossing the original platform and residence time in quadrant Ⅰ were decreased,with statistical significance(Sham VS 3MCC,P<0.05;Sham VS TW,P<0.05).Reverse platform experiment,escape latency was prolonged in injury groups,and the difference was statistically significant(Sham VS3 MCC,P<0.05;Sham VS TW,P<0.05).(2)Results of EPM test: I.At 14 d after injury: Overall activity,number of entries into OAs,duration within OAs,number of entries into EAs and times of head-dipping were decreased in the injury groups,and the differences were statistically significant(Sham VS 3MCC,P<0.05;Sham VS TW,P<0.05),which were increased after RE intervention(TW VS RE,P<0.05).The duration within EAs was increased in injury groups,with statistically significant differences(Sham VS 3MCC,P<0.05;Sham VS TW,P<0.05),which was decreased after RE intervention,with statistically significant differences(TW VS RE,P<0.05).II.At 10 m after injury: Overall activity,duration within OAs and times of head-dipping were decreased in injury groups,the differences were statistically significant(Sham VS 3MCC,P<0.05;Sham VS TW,P<0.05).The number of entries into OAs decreased in injury groups,with statistically significant differences(Sham VS 3MCC,P<0.05;Sham VS TW,P<0.05),which was increased after RE intervention,with statistically significant difference(TW VS RE,P<0.05).The duration within EAs increased in injury groups,and the differences were statistically significant(Sham VS 3MCC,P<0.05;Sham VS TW,P<0.05).Percentage of entries into OAs decreased and the percentage of entries into EAs increased in the injury group,with a statistically significant difference(Sham VS TW,P<0.05),which were improved after RE intervention,with a statistically significant difference(TW VS RE,P<0.05).Conclusion(s):(1)The long-term spatial memory and short-term working memory were significantly impaired 14 days and 10 months after injury in rats with MCC.(2)There were significant changes in anxiety-like behaviors in the early 14 days and the long 10 months after injury in rats with MCC.(3)Rosemary extract can significantly improve the cognitive and memory impairment,and anxiety behavior in the early and long-term after injury in rats with MCC.Part II Transcriptome Molecular Markers of Cerebral Concussion and Chronic Traumatic Encephalopathy in RatsObjective(s): Multiple cerebral concussion(MCC)is the initial cause of chronic traumatic encephalopathy(CTE).The mechanism of MCC progression to CTE is unclear,and there is no specific biomarker for prognosis evaluation.This experiment explored the transcriptome biological process,molecular function,cell composition and signal pathway involved in the evolution of MCC to CTE by using RNA-Seq for frontal cortex of MCC rats,and systematically identify key genes as biomarkers for diagnosis and prognosis of MCC/CTE and potential therapeutic targets.Methods:(1)Model :Rat model was established by using metal pendulum imp inging device,struck once every 24 hours,establish pure cerebral concussion(PCC)rat model with one blow,and establish 3MCC rat model with three consecutive blows.(2)Animal groups: A total of 54 healthy adult male SD rats,weighing 300±10g,were randomly divided into Sham group and 3MCC group.Each model group was randomly subdivided based on three time points of 24 h,14d and 10 m.After each impact,eligible rats were randomly assigned to each experimental group,including24h-Sham group,24h-PCC group,24h-3MCC,14d-Sham group,14d-PCC group,14d-3MCC group and 10m-Sham group,10m-PCC group,10m-3MCC group(n=6),(3)Three rats were randomly selected from each experimental group to obtain the prefrontal cortex of rats,and the original database of m RNA and Lnc RNA were established through high-throughput sequencing.(4)Identify differentially expressed genes according to |log FC| > 1.0 and P<0.05,GO function enrichment analysis and KEGG pathway analysis were performed on the obtained differentially expressed m RNA.(5)Analyze the differentially expressed m RNA of 3MCC at three different time points,and analyze the cumulative effect of different injuries at three time points to identify key m RNA.(6)3MCC m RNA was analyzed by time series cluster analysis,GO function enrichment analysis was performed for different clusters,and protein interaction network was established through STRING database.Then,key m RNA was identified through the selected gene combining proteomics data.(7)Download frontal cortex m RNA data of rats with concussion at 24 after injury from GEO database,Obtain key genes by using differential expression analysis and protein interaction network analysis,Identify chemokines among them.(8)Screening the difference gene expression consistency of three different time after injury,screening the key Lnc RNA,and carrying out target gene prediction and WGCNA co-expression analysis.(9)The key difference m RNA and Lnc RNA were verified by Q-PCR.Results:(1)At 24 h after injury,142 m RNAs were up-regulated and 16 genes were down-regulated in the 3MCC group compared with the Sham group.Further GO enrichment analysis and KEGG pathway analysis revealed that the up-regulated genes were mainly involved in the response to injury,the regulation of immune and inflammatory processes through the complement and coagulation cascade pathways,and the down-regulated genes participated primarily in various biological processes such as cell response to tumor necrosis factor and blood pressure regulation through the African trypanosomiasis pathway.(2)At the 14 d after injury,a total of 54 m RNA were up-regulated and 10 m RNA were down-regulated in the 3MCC group compared with the Sham group.The up-regulated genes were mainly involved in biological processes such as injury response,immunity,cell migration,adhesion and inflammation through protein digestion and absorption,PI3K-Akt signaling pathway and ECM-receptor interaction;and the down-regulated genes participated in biological processes such as response to organic substances,protein phosphorylation,endogenous stimulus response,ERK1 and ERK2 cascade and MAPK cascade through the regulation of calcium signaling pathway and the actin cytoskeleton.(3)At 10 months after injury,a total of 298 m RNA were up-regulated and 100 m RNA were down-regulated in 3MCCgroup compared with the Sham group.The up-regulated genes were mainly involved in multiple biological processes such as tissue development,cell proliferation,necrosis,apoptosis and migration through adrenergic signaling in cardiomyocytes,c AMP signaling pathway,protein digestion and absorption,calcium signaling pathway,drug metabolism-cytochrome P450 and ECM-receptor interaction;and the down-regulated genes participated in biological processes such as signal transduction,neuronal differentiation,cell proliferation,and negative regulation of development by stimulating the interaction between neurons,calcium signaling pathway and c AMP signaling pathway.(4)Through consistent screening of the expression of differential genes in 3 rats with MCC at different times and analysis of the cumulative effect of differential genes in rats with different injury levels,it was found that Ada,Ano2,RT1-Bb and Calca may be the candidate key differential genes for MCC.(5)Gnal was found to be the key m RNA by time series clustering analysis of m RNA combined proteomics data.(6)24h after injury in PCC group: IL10,Mmp9,Ccl2,Spp1,Timp1,C3,Cxcl10 and Anxa1 were found to be the key genes at 24 h after injury,these key genes were up-regulated in PCC.Ccl2 and Cxcl10 were chemokines involved in chronic neuritis.(7)Ada,Ano2,RT1-Bb,Calca,Ccl2 and Cxcl10 were verified by Q-PCR.Results:(1)Ada: It was significantly up-regulated in 3MCC group at 24 h,14d and 10 m after injury(P<0.05),with the most obvious up-regulation at 14d(P<0.01).(2)Ano2: It was significantly up-regulated in3 MCC group at 24 h,14d and 10 m after injury(P<0.05).(3)RT1-Bb: The up-regulation of RT1-Bb was obvious at 14 d and 10 m after injury(P<0.05),and the up-regulation was most obvious at 14 d after injury(P<0.01).(4)Calca: In the 3MCC group at 24 h,14d and 10 m after injury,it was significantly down-regulated P<0.05).(5)Ccl2:14th day after injury,the 3MCC group was significantly up-regulated(P<0.01).Compared with PCC group,the up-regulation was statistically significant(P<0.05).(6)Cxcl10:There was a significant increase in both PCC and 3MCC groups at 24 h after injury(P<0.001),among which 3MCC was significantly up-regulated(P<0.05).14 th day after injury,PCC group and 3MCC group were significantly up-regulated(P<0.001).(8)Lnc RNA(AABR07060620.1): Up-regulated at 24 h and 10 m post-injury.The m RNA(Pde1c)was predicted to be the target gene regulated by AABR07060620.1.The co-expression analysis of WGCNA revealed that Pde1 c was the co-expression gene,and Q-PCR showed that Lnc RNA(AABR07060620.1)and Pde1 c were significantly up-regulated in 3MCC at 24 h and 14 d post-injury(P<0.05).Conclusion(s):(1)Bioinformatics analysis of differentially expressed genes,found that the up-regulated genes in the early stage after injury were mainly involved in the biological processes of immunity and inflammation,and the long-term injury was mainly involved in the biological processes of cell proliferation,necrosis and apoptosis.These biological processes are the pathological basis of the evolution from MCC to CTE and affect the final outcome of the disease.(2)Biomarkers of MCC and CTE rats and potential therapeutic targets Ada,Ano2,RT1-Bb,Calca,Ccl2,Cxcl10 and Gnal were identified,which may play an important role in the pathogenesis of MCC and the evolution of CTE.(3)Lnc RNA(AABR07060620.1)of MCC rat was screened which drives MCC to CTE evolution through Pde1 c down-regulation of c AMP Pathway.Part III Mechanism of Rosemary Extract in Improving Spatial Cognition and Affective Behavior in Rats with Multiple Cerebral ConcussionObjective(s): So far,there is no specific effective treatment for patients with chronic traumatic encephalopathy(CTE).Previous experiments suggested that Rosemary extract(RE)can significantly improve cognitive and affective disorders and anti-oxidation effect in rats with multiple cerebral concussion(MCC),indicating that RE is a potential therapeutic drug for CTE.However,However,the mechanism of RE is unclear.To explore the biological process and functional pathway of the differential expression m RNA after RE intervention by using RNA-Seq for frontal cortex of MCC rats after RE intervention.To test the feasibility of the biomarker from part II as the therapeutic target of MCC/CTE through multiple concussion rats intervened by RE and explore the anti-apoptotic and anti-inflammatory effects of RE.Methods:(1)Model:3MCC rat model was established by using a metal pendul um impinging device,which was struck once every 24 hours and three times consecut ively(2)Animal groups: A total of 48 healthy adult male SD rats,weighing 300±10g,were randomly divided into Sham group and 3MCC group.Each model group was randomly subdivided based on two time points of 14 d and 10 m,Rosemary extract group(RE)and Tween solvent group(TW)were replicated from 3MCC at each time point.After each impact,eligible rats were randomly assigned to each experimental group,including 14d-Sham group,14d-TW group,14d-RE group,10m-Sham group,group,10m-TW group,10m-RE group(n=8).RE and TW were administered by gavage within 12 h after successful establishment of the model.In the 14 d group,the drug was administered continuously for 14 days,and in the 10 m group,the drug was administered continuously for 28 days after injury.(3)Three rats were randomly selected from each experimental group and killed at the time point,and the prefrontal cortex was frozen for examination.The m RNA sequencing of prefrontal cortex was sequenced by high-throughput sequencing,and differentially expressed m RNA was identified according to |log FC| >1 and P<0.05.(4)GO functional enrichment analysis and KEGG pathway analysis were performed on the differential m RNA.Integrate the biomarkers obtained from part II,compare the reverse m RNA after RE intervention,Identify the key m RNA after RE intervention with 3MCC rats.It also focuses on the verification of apoptosis and inflammation-related m RNA by Q-PCR.Results:(1)Analysis results of differentially expressed genes in prefrontal cortex of RE group compared with TW group:(1)At the 14 d after injury,it showed that a total of 51 m RNAs were up-regulated and 163 m RNAs were down-regulated.According to GO functional enrichment analysis and KEGG pathway analysis for the differentially expressed m RNAs,these up-regulated genes were mainly concentrated in the extracellular region and participated in cell differentiation,response to external stimuli and regulation of multicellular biological processes,and KEGG pathway analysis of these genes was generally enriched in Axonal guidance and Focal adhesion.The down-regulated genes were mainly concentrated in the cytoplasm,nucleus and plasma membrane,and participated in the regulation of signal transduction,cell response to organic substances,cell surface receptor signaling pathway,response to external stimuli,cell proliferation,cell death and apoptosis,and KEGG pathway analysis of these genes was mainly enriched in MAPK signaling pathway,the regulation of inflammatory mediators on TRP channel and neurotrophin signaling pathway.Furthermore.(2)At 10 m after injury,62 m RNA were up-regulated and 187 m RNA were down-regulated in the RE intervention groups compared with the control group.GO functional enrichment analysis and KEGG pathway analysis revealed that the up-regulated genes were mainly centered at neurons and synapses,which participated in the development of the nervous system,the regulation of synaptic transmission signals,transmembrane transport,oxidative stress response and other biological processes,and were mainly enriched in the calcium signaling pathway.Meanwhile,the down-regulated genes were mainly concentrated in the extracellular matrix,extracellular regions,extracellular vesicles and endoplasmic reticulum,participated in biological processes such as cell differentiation,adhesion,apoptosis and death,and were enriched in the focal adhesion kinase signaling pathway and PI3K-Akt signaling pathway.(2)Analysis of differential expression genes in reversing MCC after RE intervention showed that Ada and Nupr1 are important targets of RE intervention.(3)GO function enrichment analysis was used to identify apoptosis-related m RNA: Nupr1 and Casp12 are key genes in RE intervention.Nupr1 and Casp12 participate in ER stress.To verify ER stress pathway,Chop,Bcl2,Bax and Casp3 were used as Q-PCR verification targets.(1)Nupr1:There was a down-regulated trend in RE group at 14 d and 10 m after injury.(2)Chop: 14 days after injury,the RE group was significantly down-regulated(P<0.05),and the 10 m RE group was down-regulated trend.(3)Casp12: 14 days after injury,the RE group was significantly down-regulated(P<0.05),and the 10 m RE group was down-regulated trend.(4)Casp3: 14 days after injury,the RE group was significantly down-regulated(P<0.05),and the 10 m RE group was down-regulated trend.(5)Bcl2: 14 days after injury,the RE group was significantly up-regulated(P<0.05),and the 10 m RE group was up-regulated trend.(6)Bax:14 days after injury,the RE group was significantly down-regulated(P<0.05),and the 10 m RE group was down-regulated trend.(4)Combined with Part II,the inflammation-related m RNA was verified by Q-PCR.(1)Ada:In 14 d and 10 m RE groups,it was significantly down-regulated(P<0.05).(2)Calca: It was significantly up-regulated in 14 d RE group(P<0.05)and up-regulated trend in 10 m RE group.(3)Ccl2: It was significantly down-regulated in 14 d RE group(P<0.01).(4)Cxcl10:It was significantly down-regulated in 14 d RE group(P<0.01).(5)C3:It was significantly down-regulated in 14 d RE group(P<0.01).Conclusion(s):(1)This study found that after the RE intervened the rats with MCC,a series of differentially expressed m RNAs were involved in many biological processes such as cell proliferation,differentiation,adhesion,death and apoptosis;(2)RE may alleviate the stress reaction of ER and reduce the occurrence of neuronal apoptosis through two pathways in the early post-traumatic period.one is to down-regulate the expression of Casp12 and Casp3,the other is to up-regulate the expression of Bcl-2 and down-regulate the expression of Bax through the Nupr1/Chop pathway to reduce neuronal apoptosis.(3)RE exerts neuroprotective effects by downregulating the expression of Ada,Ccl2,Cxcl10,and C3 in rats with MCC and upregulating the expression of Calca. |
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