| Ischemic encephalopathy(HIE)of newborns is a result of systemic hypoxemia and/or decreased cerebral blood flow from prenatal,intrapartum and/or neonatal asphyxia that can ischemic encephalopathy(HIE)of newborns and result in death and long-term sequelae.Such as cerebral palsy,epilepsy,intellectual disability and behavioural disorders.HIE treatment aims to reduce the loss of nerve cells,protect nerve function and improve the prognosis of the disease.Currently,Western medicine is the main treatment.Traditional Chinese medicine has a deep understanding of the pathophysiological characteristics of children,and believes that children "Zang Qi Qing Ling,easy to recover".Children’s brains are in a period of rapid development,sensitive to ischemia and hypoxia.Timely and effective treatment can minimize the damage of the nervous system of children,and has great significance for the recovery of nervous system function and the reduction of sequelae.Neonates with HIE correspond to the symptoms of neonates in traditional Chinese medicine."The Iron Mirror of Baby Science" said,"Crying comes from the lung,and the silent lung is absolutely sad",which considered the absence of crying as "lung" or "phlegm cover"."Doctor Zong Jin Jian" said: "qi closed can not sound out,Yan Yan breathing life for a moment";"Prepare urgent Qianjin Fang" said: "Son born silent,this by dystocia,less gas so also","Medical Emperor Jin Jian" is considered to be "Qi closed".With the deepening understanding of diseases,modern Chinese medicine has a more comprehensive understanding of neonatal HIE than predecessors.The causes of this disease are mainly parents’ seminal blood deficit,or improper care during pregnancy,which damages fetal qi;Or delivery is not good,resulting in asphyxia,fetal brain injury,the main pathogenesis is insufficient qi and blood,blood is not full,heart and brain loss.Although there are also clinical reports of TCM intervention for HIE,due to the physical and physiological characteristics of newborn children,the experience of TCM interventional therapy is limited at present,and there is no large-scale clinical application.Created by Professor Zhou Desheng’s team from the perspective of "Rongqi theory" and "Rongqi deficiency and stagnation",Huoxue Rongluo formula has the effect of activating blood circulation,removing blood stasis and regulating collaterals and replenishing qi,treating Rongqi deficiency and blood stasis and collaterals obstruction,and has been clinically used in the treatment of ischemic stroke diseases for more than ten years.According to traditional Chinese medicine,the main pathogenesis of HIE neonates is deficiency of qi and blood,insufficiency of blood vessels,and loss of heart and brain,which is similar to ischemic stroke.However,whether Huoxue Rongluo formula can be applied to the treatment of HIE neonates has not been reported.By establishing HIE model of lactating mice,this study observed the therapeutic effect of Huoxue Rongluo prescription and explored the mechanism to enrich the modern connotation of "Rongqi theory" and expand the therapeutic scope of Huoxue Rongluo prescription.Part ⅠObjective: This chapter mainly evaluates the protective effect of Huoxue Rongluo prescription on the neurological function and brain structure of HIE rats.Methods: Neonatal SD rats were bred after conception in the same cage,and neonatal rats with normal delivery were fed normally.At 10 days of age,neonatal rats were randomly divided into control group,HIE group and HXRLF group,with 25 rats in each group.HIE model was made by carotid artery ligation + anoxic bottle anoxia and then carotid artery ligation was removed.The HXRLF group was given gavage 2 hours before surgery and every 12 hours after surgery,and the control group and the ischemic anoxic group were given equal volume of normal saline for 3 consecutive days.Racine grading criteria were used to evaluate the degree of convulsion and HIE.Longa score,suspension test and Grip test were used to evaluate the neural motor function of model rats.The learning and memory ability of model rats was detected by water maze experiment.TTC was used to detect the degree of cerebral infarction.The water content of brain tissue was determined.The structural changes of brain tissue were observed by HE staining.The permeability of blood-brain barrier was detected by EB method.Ultrastructural changes of neurons were observed by transmission electron microscopy.Results:1.Racine score was performed in each group after operation.The control group had no convulsion,HIE group had different degrees of convulsion,and HXRLF group had significant improvement in convulsion(P<0.001).2.Compared with the control group,Longa score of HIE group was significantly higher on day 3 after surgery(P13),grip strength test score was lower on day 5 after surgery and suspension time was shorter(P<0.01).Compared with HIE group,Longa score of HXRLF group was significantly decreased after surgery,while grip strength test score and hanging time of HXRLF group were significantly increased on day 10 after surgery(P<0.01).Compared with the control group,the HIE group had obvious cognitive and memory deficits(P<0.01),while the HXRLF group significantly improved the cognitive and memory abilities compared with the HIE group(P<0.01).3.Compared with the control group,tongue stasis purple in HIE group was significantly improved in HXRLF group compared with HIE group(P<0.01).4.In HIE group,there were liquefaction necrotic foci in the central depression with white ischemic area around it,and obvious brain tissue atrophy occurred 5 days after surgery.HXRLF group showed small area edema in the brain tissue after surgery,which basically returned to normal 5 days after surgery.5.TTC results showed that large white infarcts appeared in HIE group compared with control group(P< 0.01).The cerebral infarction volume of rats in HXRLF group was significantly decreased compared with HIE group(P<0.01).6.Compared with the control group,the brain water content in HIE group was significantly increased(P<0.01),while the brain water content in HXRLF group was significantly decreased compared with HIE group(P<0.01).7.HE staining results showed that the HIE group had local brain tissue necrosis,volume reduction,structure destru-ction,hippocampal CA3 structure disordered,neuron arrangement was disorderly and irregular,and neuron loss was obvious.The morphology and structure of brain tissue in HXRLF group were complete,and the damage was significantly less than that in HIE group.Nith staining showed that,compared with the control group and HXRLF group,the loss of neurons in HIE group was significant(P<0.05).8.EB staining showed that,compared with the control group and HXRLF group,the blood-brain barrier function was still impaired in HIE group on the 5th day after surgery((P<0.05).9.The results of electron microscopy showed that the morphology of hippocampal cells in HIE group was damaged,more vacuolar structures appeared in neurons,and a large number of lysosomes and autophagosomes were observed,while the situation of HIXRLF group was significantly better than that of HIE group.Conclusion: Huoxuerongluo formula can obviously reduce the brain injury of neonatal rats during HIE,and has a positive promoting effect on the recovery of neurological function and tissue morphology of neonatal rats.Part ⅡObjective: The purpose of this part is to screen miRNAs expression profiles in the brain tissues of rats in each group by high-throughput whole transcriptome sequencing technology,and summarize the therapeutic network of Huoxuerongluo formula,so as to provide the molecular basis for the "Rongqi theory".Methods: miRNAs were extracted from the hippocampus of each group,and the 18 ~32nt small RNA fragments were sequenced and a library was constructed.The original sequence data were collected and quality-controlled to analyze the composition,expression and difference of miRNAs.Western Blot and quantitative PCR confirmed the expression changes of miRNA-21-5p,AMPK,mTOR and ULK1 in the hippocampus of each group.Results:1.The results of RNA-seq sequencing of each sample showed that 227.58 M Raw Reads were obtained,and Raw Reads of each sample were more than 10.08 M,and the percentage of Q30 base was 91.58% or above.2.Differential expression of reads among samples was concentrated on chromosomes 1 to 3,6 to 8,10,11,17 and X.The first three digits of differentially expressed reads were up-regulated on chromosome 2,1and 8,while the first three digits of differentially expressed reads were down-regulated on chromosome 7,17 and 8.3.Correlation analysis showed that there were significant differences in miRNA expression between the HXRLF group and HIE group and the control group,while there were little differences in miRNA expression between the HXRLF group and the control group.4.A total of 488 mirnas were expressed among the three groups,among which 59 mirnas were up-regulated and 7 mirnas down-regulated in HIE group compared with control group.Compared with HIE group,6mirnas were up-regulated and 34 mirnas down-regulated in HXRLF group.Compared with the control group,18 mirnas were up-regulated and 10 were down-regulated in the HXRLF group.5.A total of 77 differentially expressed genes were analyzed,including59 differentially expressed genes between HIE group and control group,20 differentially expressed genes between HXRLF group and control group,and 34 differentially expressed genes between HXRLF group and HIE group.Then,the differential genes of HIE vs Ctl group,HXRLF vs Ctl group and HXRLF vs HIE group were differentially screened according to different regulatory changes,and 23 genes were found to be up-regulated in HIE vs Ctl group and down-regulated in HXRLF vs HIE group.HIE vs Ctl group down-regulated,HXRLF vs HIE up-regulated and had differences in 1 gene,HIE vs Ctl group down-regulated,HXRLF vs HIE down-regulated and had differences in2 genes.6.The top five GO terms were TORC1 signal negative regulation,TOR signal negative regulation,lipid phosphorylation,axon occurrence negative regulation,and growth and development negative regulation.Compared with HIE group,the top five GO terms with different gene enrichment were TORC1 signal negative regulation,TOR signal negative regulation,TORC1 signal regulation,synaptic vesicle transport regulation and receptor-mediated entosis regulation.Compared with the control group,the top five GO terms with different gene enrichment in HXRLF group were negative chemotaxis,organ growth,transmembrane transport,potassium ion transport and cellular potassium ion transport.The top five GO terms were TORC1 signal negative regulation,TOR signal negative regulation,lipid phosphorylation,axon occurrence negative regulation,and growth and development negative regulation.Compared with HIE group,the top five GO terms with different gene enrichment were TORC1 signal negative regulation,TOR signal negative regulation,TORC1 signal regulation,synaptic vesicle transport regulation and receptor-mediated entosis regulation.Compared with the control group,the top five GO terms with different gene enrichment in HXRLF group were negative chemotaxis,organ growth,transmembrane transport,potassium ion transport and cellular potassium ion transport.7.The sample components were mainly located in cells and organelles,and the main molecular functions were related to binding and catalytic activities.The main biological processes involved were cellular processes,biological regulation,metabolic processes,etc.The number of enriched gene sets between groups was in descending order: HIE group vs control group,HXRLF group vs control group,HIE group vs HXRLF group.8.Compared with the control group,the top five KEGG pathways with different gene enrichment in HIE group were hippocampal signaling pathway,p53 signaling pathway,phosphatidylinositol signaling system,glycerol phospholipid metabolism and glycolide metabolism in sequence.Compared with the control group,the top five KEGG pathways with different gene enrichment were sphingolipid biosynthesis-globulin and isoglobulin series,Hedgehog signaling pathway,basal cell carcinoma,axon-directed pathway and AMPK signaling pathway.Compared with HIE group,the top five KEGG pathways with different gene enrichment were hippocampal signaling pathway,cell apoptosis,Hedgehog signaling pathway,basal cell carcinoma,and phosphatidylinositol signaling system.9.KEGG classification statistics showed that the function of the samples in the process of environmental information processing mainly focused on signal transduction and signal molecule interaction.In the process of human disease,the functions are mainly concentrated in infection,cancer,neurodegenerative changes,cardiovascular diseases,endocrine and metabolic diseases.According to the classification of organ systems,the functions are mainly concentrated in the immune system,endocrine system,sensory system,nervous system,development and regeneration,digestive system;During the cellular program,the functions are concentrated in transport and catabolism,cell growth and death,and cell colony.In the process of gene expression,the functions focus on folding,sorting and degradation.In terms of metabolism,function is concentrated in lipid metabolism.10.The results of miRNA analysis were further verified,and the results showed that miRNA21-5p,AMPK,mTOR,ULK1 and LC3-2/1 were expression changed in the pathological process of HIE and the therapeutic process of Huoxue Rongluo formulas.Conclusion:1.miRNA omics studies have confirmed that Huoxue Rongluo formula has a complex network regulation mechanism in treating HIE in neonatal rats,which is consistent with the "holistic view" of TCM treatment and the previous research conclusions of our research group.2.Under the guidance of miRNA omics research results,in-depth research on the mechanism of Huoxue Rongluo prescription can improve the accuracy and efficiency of the research;3.miRNA21-5p,AMPK,mTOR and autophagy may play an important role in the occurrence of HIE and the treatment process of Huoxue Rongluo formula.Part ⅢObjective: Further demonstrated that miRNA21-5p,AMPK,mTOR and autophagy play a role in the signaling regulation of HIE and the treatment of Huoxue Rongluo formula.Methods: PC12 cells were cultured in vitro and divided into control group,HIE group,HXRLF group,AMPK inhibitor group and mTOR inhibitor group.After pretreatment,cells were treated with oxygen glucose deprivation/reoxygenated glucose.Western Blot and quantitative PCR confirmed the expression changes of miRNA-21-5p,AMPK,mTOR,ULK1,LC3-2/1 in each group.Results:1.In PC12 cells,the expression of miRNA-21-5p was increased,and the gene expression and protein phosphorylation of AMPK,ULK1 and LC3-2/1 were increased,while the gene expression and protein phosphorylation of mTOR were decreased.2.Huoxue Rongluo formula can alleviate the above changes caused by ischemia,hypoxia and reperfusion.mTOR plays an active role in the regulation of autophagy by miRNA-21-5p and AMPK/ ULK1 pathway.Conclusion: By reducing miRNA-21-5p expression,Huoxue Rongluo formula regulates AMPK/mTOR/ULK1 signaling pathway and reduces neuronal autophagy,thus protecting the brain of young mice during HIE. |
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