Study On Mechanisms Of Molecular Disorders In The Acute Intracerebral Hemorrhage And Pharmacodynamics Of Liangxue Tongyu Prescription Based On RNA-seq And Network Pharmacology

Background:TCM has been widely used in the prevention and treatment of diseases for more than 2000 years.The disease of cerebral hemorrhage is brutal and its condition changes quickly,with high rate of death and disability.At present there is no clear and effective treatment for this disease in the world.The pathogenesis of cerebral hemorrhage still needs to be further studied.TCM compound has the characteristics of multi-component,multi-target and multi-pathway,and has the the innate advantage of systematic and holistic regulation to improve disease.Prior studies have shown that the effect of LXTYF can treat cerebral hemorrhage through a variety of signaling pathways.Since it is still a huge challenge to systematically study the scientific basis of Chinese medicine prescriptions at the molecular level,we used network pharmacology,transcriptomic data and WGCNA method to study the pathological mechanism of cerebral hemorrhage diseases and the multi-effect pharmacological mechanism of LXTYF in the treatment of cerebral hemorrhage.The first part is the molecular pathologic mechanisms of cerebral hemorrhage based on RNA-seq.Objective:To study regulatory factors(the key mRNA and lncRNA)and the affected signaling pathways in the pathogenesis of intracerebral hemorrhage,and construct a weighted gene co-expression network to reveal the pathogenesis of intracerebral hemorrhage.Method:Model of cerebral hemorrhage in hypertensive SHR rats was established by collagenase.The treatment group was treated with LXTYF.Four experimental groups were set as CG in normal SHR group,EG1 in normal SHR group,EG2 in cerebral hemorrhage SHR group,and EG3 in cerebral hemorrhage group with LXTYF.Firstly genes significantly differentially expressed in EG2 group and CG group were analyzed,and the signaling pathways affected by cerebral hemorrhage were preliminarily analyzed.Then WGCNA method was used to analyze the weighted gene co-expression network of genes with large difference coefficient in samples of the four experimental groups,and comprehensively explore the interaction between genes and the co-expression regulation mode.Cytohubba plugin was used to analyze the network to identify the key mRNA and lncRNA in the pathogenesis of intracerebral hemorrhage and to predict the function of hub IncRNA.Finally the correlation between the module genes and clinical information was analyzed to identify the modules with strong correlation and statistical significance with cerebral hemorrhage,and the functions of these modules were analyzed to reveal their important role in the pathogenesis of cerebral hemorrhage.Result:1.We collect significantly differentially expressed genes through the EG2 and CG.Findings showed that the top five pathway that cerebral hemorrhage interfering with is the Cytokine-cytokine receptor interaction,IL-17 signaling pathways,chemokine signaling pathways,TNF signaling pathways,PI3K-Akt signaling pathway and MAPK signal pathways.2.WGCNA method was used to analyze the weighted gene co-expression network of all samples of 4 experimental groups,and explore the interaction between genes and the co-expression regulation model.From Cytohubba plugin analysis of WGCNA coexpression network,we obtained 7 key mRNA such as Ccl7,Resp18,Steap4.Sparc,Clic4,Ptx3.Stxla and 5 key lncRNA such as MSTRG.7544.7,ENSRNOT00000091284,ENSRNOT00000076965,MSTRG.7544.5.MSTRG.1053.1.Among the 5 key lncRNAs,3 of them have coexpression regulatory relationship.The five lncRNAs play an important role in neural activity ligand receptor interaction,glycolysis/sugar dysplasia,citrate(TCA cycle).glycerides metabolism,lipid metabolism,scabbard cholesterol metabolism,pyruvate metabolism,beta alanine metabolism,arginine and proline metabolism,adjust the reabsorption of sodium,aldosterone NOD receptors signaling pathways,IL-17 signaling pathways,apoptotic pathways,such as in energy metabolism and lipid metabolism,inflammatory reaction and apoptosis.3.The correlation analysis between module genes and clinical information(cerebral hemorrhage)revealed that there were 6 modules with strong correlation and statistical significance.Four modules such as pink,skyblue3,darkred and sienna3 are of positive correlation regulation,which are related to PI3k-Akt signaling pathway,Purine metabolism,IL-17 signaling pathway and MAPK signaling pathway respectively.Two modules are of negative correlation regulation include saddlebrown and darkgrey modules,which interact with Adrenergic signaling in cardiomyocytes,MAPK signaling pathway and Toll-like receptor pathway respectively.All these have important effects on cell proliferation and apoptosis,inflammatory response and immune response in the pathogenesis of cerebral hemorrhage.Conclusion:There is a complex pathogenesis on intracerebral hemorrhage,among which IncRNA and mRNA have an important effect.Multiple signaling pathways are regulated,involving cell proliferation and apoptosis,inflammation,immune response and other aspects.The second part is mechanisms of network pharmacology in the treatment of cerebral hemorrhage with LXTYF.Objective:To analyze and verify the key components,targets and pathways of LXTYF in the treatment of intracerebral hemorrhage,and construct the TCM-component-target-pathway network and explore its multi-effect pharmacological mechanism.Method:Network pharmacology was used to identify the active components and potential targets of LXTYF in the treatment of cerebral hemorrhage.PPI network was used to explore protein-protein interaction.R language package was used to analyze the GO function and KEGG pathway of potential targets.Then collagenase was used to build the SHR cerebral hemorrhage model EG2 group,and the treatment group EG3 was given LXTYF.RNA-seq datas of the brain tissue samples in the two groups were detected and the significantly differentially expressed genes were analyzed.Comparing with the potential targets obtained by the network pharmacology method and the significant differentially expressed genes,the important genes of LXTYF on the treatment of cerebral hemorrhage were verified.The PPI network was analyzed for the genes with significant differences in expression,and KOBAS was used to enrich the GO function and KEGG pathway of the genes with differences,so as to find out the relevant pathways regulated by LXTYF in the treatment of cerebral hemorrhage,and explain its multi-effect pharmacological mechanism.Finally combined with the WGCNA correlation analysis of module gene and clinical information in the first part,the comprehensive expression genes were analyzed,and the modules with strong correlation and statistical significance with the treatment of LXTYF were identified.The function of these modules were analyzed to reveal the regulatory mechanisms of the treatment on cerebral hemorrhage with LXTYF.Results:1.76 active ingredients(quercetin,alanine,kaempferol,etc.)and 376 potential targets(PTGS2,PTGS1,ESR1,etc.)were identified by network pharmacology in the treatment of intracerebral hemorrhage.The results of the active component-target network of LXTYF for the treatment of cerebral hemorrhage showed that the quercetin of MDP/SQ,the alanine of SNJ and the xanthol of MDP/SCP had the largest number of targets,which may be the key active compounds.They have anti-inflammatory,antioxidant and anti-apoptotic effects.The results of the topological parameter evaluation showed that among all potential targets of the treatment on cerebral hemorrhage with LXTYF,PTGS2,Bax,TNF,JUN,IL1B and NOS2 are important targets.PPI network results showed the important role of target STAT3 in protein-protein interactions.The potential targets collected by network pharmacology were significantly enriched to 72 KEGG signaling pathways,among which the MAPK and PI3k-akt signaling pathways enriched the largest number of genes,which were the most likely signaling pathways to be regulated by LXTYF in the treatment of cerebral hemorrhage.The cerebral hemorrhage model of SHR rats was constructed by collagenase and treated with LXTYF.2.RNA-seq datas were detected and the expression of significant differences were analyzed.Compared with the 376 potential targets obtained by network pharmacology,14 important genes were verified.Among them,PTEN,CTH,PTGS2,PTAFR,FOS and NOS1 were up-regulated,while NOS2,PGR,MMP3,PON 1,NEDD4,ACADSB,KL and CASP3 were down-regulated.The results of enrichment analysis also showed that LXTYF mainly treated cerebral hemorrhage by regulating MAPK,calcium signal,cell apoptosis,tumor necrosis factor and other signaling pathways,playing its antioxidant,anti-inflammatory,anti-apoptosis and lowering blood pressure pharmacological effects.3.Weighted gene express network analysis of all four experimental samples with WGCNA method is conducted.The module genes associated with clinical information(LXTYF)have a statistically significant negative-related correlation with darkgrey and white modules,respectively with Adrenergic signaling in cardiomyocytes,MAPK signaling pathway,MAPK signal pathways,Glycerophospholipid metabolism,Inositol phosphate metabolism,Oxidative phosphorylation.The treatment of LXTYF on ICH is involved in cell proliferation and apoptosis,lipid metabolism,antioxidant and anti-inflammatory,etc.Conclusion:LXTYF exerts a variety of pharmacodynamic function to treat cerebral hemorrhage,and multiple signaling pathways are regulated,including cell proliferation and apoptosis,lipid metabolism,antioxidant and anti-inflammatory,etc.MAPK signaling pathway is also the most likely signaling pathway to be regulated by LXTYF in the treatment of cerebral hemorrhage.