The Anti-Inflammatory Effects And Mechanisms Of Myrrha Based On The Gene Expression Profile In Stroke With Blood Stasis Syndrome

BackgroundIschemic stroke,also known as cerebral infarction,has high morbidity,mortality and disability.The clinical treatment,however,is limited.Traditional Chinese medicine(TCM)is extensive and profound,it has unique advantages in the prevention and treatment of stroke.According to the theory of TCM,ischemic stroke belongs to the category of “Zhong Feng”,and the main pathogenesis is blood stasis.Qi-deficiency blood stasis(QDBS)and Yin-deficiency blood stasis(YDBS)are the two most common syndromes.Syndrome differentiation and treatment is the basic principle of TCM treatment.Syndrome differentiation is the premise and key for treatment of stroke with blood stasis syndrome.It is of great theoretical and practical significance to further explore the material basis and scientific connotation of TCM syndrome differentiation of blood stasis syndrome and find relevant key targets in syndrome differentiation and treatment.With the implementation of the human genome project and the rapid development of modern molecular biology technology,functional genomics is becoming more and more mature.Genomics has the characteristics of high efficiency,rapid and automatic.It has attracted much attention in clinical diagnosis and basic scientific research.The high throughput and whole gene level of genomics technology coincides with the complex system of multiple channels and multiple targets in TCM.The application of genomics technology in the study of TCM syndromes could provide scientific basis for TCM theory by revealing the material basis of TCM syndrome,and it could also provide a reference for the treatment by screening potential key syndromes targets.Myrrha,dry resin from myrrha exudation,was first recorded in the book “Song Dynasty Materia Medica” as Chinese medicine.The property of myrrha is flat,and the taste is bitter.It belongs to the heart,liver and spleen meridian with the effects of invigorating blood circulation,relieving pain,reducing swelling and promoting tissue regeneration.It was commonly used in the treatment of stroke,chest pain,rheumatism,traumatic injury and postpartum blood stasis etc.Modern pharmacological studies show that myrrha has a significant anti-inflammatory effect.Myrrha and the active ingredient Z-guggulsterone(Z-GS)could improve the memory impairment and inhibit neuroinflammation.Inflammation is one of the main factors of nerve injury after ischemic stroke,suggesting that myrrha can be used as a therapeutic drug for ischemic stroke.Objectives1.The essence of TCM syndrome: To investigate the differential gene expression profiles between QDBS and YDBS of ischemic stroke based on functional genomics,and to screen the key targets for treatment of stroke with blood stasis syndrome.2.The treatment of TCM syndrome: To explore the neuroprotective and anti-inflammatory mechanism of myrrha and Z-GS on stroke with blood stasis syndrome.Methods1.Methods of syndrome essence research:(1)Male SD rats were randomly divided into three groups: QDBS group(QDBS/MCAO model),YDBS group(YDBS/MCAO model)and control group(sham operation).Middle cerebral artery occlusion(MCAO)was used to simulate ischemic stroke.(2)After 24 hours of reperfusion,the neural function score and TTC staining were performed to verify the cerebral apoplexy model.(3)The hemorrheology and coagulation parameters were measured with abdominal aorta blood,so as to verify the model of blood stasis syndrome(BSS).(4)The thoracic aorta and brain tissue were stained with H&E to observe the histopathological changes.(5)Total RNA of blood was extracted,and the differential genes were analyzed by the gene-chip after quality examination.(6)The qRT-PCR was performed to verify the data reliability form the genomic.(7)GO and KEGG pathway enrichment analysis were carried out to study the functions and pathways of the genes.(8)The construction of pathway network and gene co-expression network were constructed to screen target genes and core pathways.2.Methods of syndrome treatment research:(1)Male SD rats were randomly divided into six groups: sham operation group,model group,positive control group,myrrha group,Z-GS low dosage group(Z-GS-L,30 mg/kg)and high dosage group(Z-GS-H,60 mg/kg).(2)Rats in model and treatment group undertook MCAO operation,the corresponding dose of Z-GS was injected intraperitoneally after the operation,and the neurologic score was scored 3 days after the administration.(3)TTC,H&E,TUNEL and immunofluorescence staining,as well as Western blot and qRT-PCR were detected in the brain tissue.(4)The primary cultured astrocytes were isolated and divided into control group,model group,low dosage group(Z-GS-L,30 μM)and high dose group(Z-GS-H,60 μM).(5)Oxygen glucose deprivation(OGD)model was used to simulate the pathological state of cerebral ischemia.The treatment groups were given the Z-GS before OGD treatment.(6)MTT,ELISA,FACS were used to detect cell viability,apoptosis rate and LDH level,while western blot and qRT-PCR were used to detect the RNA and protein expression levels in cells.Results1.Results of syndrome essence research:(1)There are significant differences in neurological function score,cerebral infarction volume,hemorrheology index,coagulation parameters and histopathology between the model group and control group,which indicates that the models are successfully established.(2)The results of microarray analysis showed that there were 2121 differentially expressed genes(DEGs)in QDBS group,including 1849 up-regulated genes and 272 down-regulated genes.There were 2166 DEGs in YDBS group,including 1989 up-regulated genes and 177 down-regulated genes.(3)Three groups of genes were obtained by intersecting of these DEGs,they are: 445 QDBS-specific genes,490 YDBS-specific genes and 1676 BSS common genes.(4)GO analysis showed that the DEGs were related to the functions of aging,immune response,inflammatory response and apoptosis regulation.(5)Pathway analysis showed that the DEGs were involved in T cell receptor pathway,metabolic pathway and cell cycle pathway.(6)The network analysis showed that T cell receptor pathway,MAPK pathway and apoptosis pathway were the core pathway of YSBS,YDBS and BSS,while Nfκb1,Egfr and Casp3 were the core genes.(7)The results of western blot were consistent with the data from genomic and bioinformatics.2.Results of syndrome treatment research:(1)There was no significant difference between Z-GS group and the myrrha group on the neurological score,infarct volume and pathological changes.(2)Compared with the sham group,the neurological scores and infarct volume of rats in the model group were increased.Z-GS could significantly improve the MCAO induced neurological impairment and cerebral infarction.(3)H&E and TUNEL staining showed that disordered neurons arrangement,neuronal loss,karyopyknosis and inflammatory cell infiltration were demonstrated after ischemia,and the number of apoptotic cells in infarction area was significantly increased.The pathological changes were significantly relieved after Z-GS treatment.(4)The apoptosis rate and LDH level in astrocytes were increased after OGD,and the cell viability was decreased.Compared with the OGD group,the apoptosis rate in Z-GS group was decreased,the cell viability was increased and the LDH level was decreased.(5)The expressions of GFAP in rat brain tissues and astrocytes were increased significantly after MCAO and OGD stimulation,which indicated that the astrocytes were activated.However,Z-GS significantly decreased the expression of GFAP.(6)The PCR results showed that TNF-α,IL-1β,IL-6,IFN-γ and MCP-1 levels were significantly increased in MCAO and OGD groups,and the levels were decreased after administration with Z-GS.This indicated that Z-GS can inhibit inflammatory reaction.(7)The Western blot results showed that OGD induced the increase of TLR4,p-IκBα,p-P65(ser536)and nuclear-P65 in astrocytes.The protein expressions were inhibited after administration of Z-GS.(8)The immunofluorescence results showed that the number and fluorescence intensity of TLR4/GFAP and p-P65/GFAP double stained cells in brain tissue were increased significantly after MCAO,and they were significantly decreased after Z-GS treatment.ConclusionsThe results of syndrome essence research showed that genomics technology is an effective and feasible tool for TCM syndrome differentiation study.The differential gene expression profiles revealed the pathological characteristics of ischemic stroke with QSBS and YDBS,as well as the material basis of TCM syndrome differentiation.The selected core genes can be used as biomarkers and potential targets for the diagnosis and treatment of stroke with blood stasis syndrome.The results of syndrome treatment research showed that Z-GS could significantly alleviate ischemia induced brain damage,astrocyte activation and inflammation,the mechanism may be by blocking the activation of TLR4/NF-κB pathway.Z-GS can be used as a potential candidate for the prevention or treatment of stroke with blood stasis syndrome.