| Objective: The aim of the study was to screen the therapeutic target and related signal pathway of Salvia Miltiorrhiza for acute COP brain injury by network pharmacology,and explore the network pharmacological mechanism of multi-component,multi-target and multi-signal pathway in Salvia Miltiorrhiza,furthermore,Tanshinone IIA,the main effective component of Salvia miltiorrhiza,was selected as an intervention measure to clarify the role of MAPK/ERK1/2signaling pathway in the pathogenesis of acute brain injury in COP rats,and to explore the molecular mechanism of Tanshinone IIA in the treatment of acute brain injury in COP ratsMethods: The main compounds and drug target genes of Salvia Miltiorrhiza were screened by Traditional Chinese Medicine Systems Pharmacolog y(TCMSP).Gene Cards,Drugbank and Online Mendelian Inheritance in Man(OMIM)were used to establish the target genes of COP and acute brain injury induced by COP.STRING database was used to constructed protein-protein interaction(PPI)network.Drug-compound-target-disease network was constructed by Cytoscape 3.7.2software.GO enrichment analysis and KEGG pathway analysis were performed by Metascape database.Based on the results of network analysis,to further uncover the underlying molecular mechanism of Tanshinone IIA in the treatment of acute brain injury induced by COP through MAPK/ERK1/2 signaling pathway,a total of 216 male healthy Sprague Dawley rats were collected in the present study and randomly divided into 3 groups: a normal control group(NC group,n =72),a carbon monoxide poisoning group(COP group,n =72)and a Tanshinone IIA sulfonate treatment group(Tanshinone IIA group,n =72).The rat model of acute severe COP was established by the secondary inhalation in a hyperbaric oxygen chamber.The pathological injury of brain tissues was detected via hematoxylin-eosin(HE)staining.Nissl staining was used to observe the morphological characteristics of neurons and the quantitative changes of Nissl bodies in the cerebral cortex.The apoptosis of cerebral cortex cells was detected by TUNEL assay.The expression levels of VEGF-A,phosphorylated mitogen extracellular kinase1/2(P-MEK1/2)and phosphorylated extracellular signal-regulated protein kinase1/2(P-ERK1/2)were observed in brain tissue by immunohistochemistry and Western blotting.Results: A total of 202 active compounds were retrieved from TCMSP database,and71 intersecting genes of acute brain injury induced by COP and Salvia Miltiorrhiza were obtained by comparing with Gene Cards,Druggbank and OMIM databases,including 13 key genes such as cellular tumor antigen p53(TP53),transcription factor AP-1(JUN),interleukin-6(IL-6),tumor necrosis factor(TNF),mitogenactivated protein kinase 1(MAPK1)and VEGF-A.The possible mechanism is mainly involved in biological processes such as reaction to toxic substances,reaction to reactive oxygen species,response to inorganic substance;and the signal pathway involves hormone metabolism,inflammatory reaction,apoptosis and so on.The results of the rat experiment indicated that the brain edema was the most serious in COP group on 1 d(85.92±2.20),and gradually decreased after that [7 d(80.25±1.51),14 d(74.81±0.87),30 d(73.07±1.63)].Under light microscope,nerve cells were swollen and deformed,the number of normal nerve cells was reduced,and the nucleus was pyknotic.Tanshinone IIA could significantly reduce the degree of brain edema in rats [1 d(83.50±1.21),7 d(76.76±0.99),14 d(73.09±0.70),30 d(72.59±0.73),P<0.05].Under a microscope,the arrangement of nerve cells tended to be orderly,the structure was relatively intact and the number of apoptotic cells in Tanshinone IIA group was significantly lower than that in COP group(P<0.05).There were a few expressions of VEGF-A,P-MEK1/2 and P-ERK1/2 in brain tissue of NC group rats [VEGF-A: 1 d(0.0109±0.0024),7 d(0.0117±0.0036),14 d(0.0112±0.0029),30 d(0.0105±0.0024);P-MEK1/2: 1 d(0.0189±0.0034),7 d(0.0170±0.0042),14 d(0.0173±0.0033),30 d(0.0179±0.0033);P-ERK1/2: 1 d(0.0102±0.0016),7 d(0.01±0.002),14 d(0.0107±0.0017),30 d(0.0105±0.0023)].The staining intensity and protein expression levels of VEGF-A,P-ERK1/2 and PMEK1/2 positive cells in the COP group increased at the early stage(<7 d)and decreased gradually at the later stage [VEGF-A: 1 d(0.0192±0.0029),7 d(0.0315±0.0038),14 d(0.0179±0.0023),30 d(0.0159±0.0035);P-MEK1/2: 1 d(0.0297±0.0041),7 d(0.0490±0.0038),14 d(0.0369±0.0058),30 d(0.029±0.0047);P-ERK1/2: 1 d(0.0144±0.0027),7 d(0.0307±0.0016),14 d(0.0211±0.0023),30 d(0.0171±0.0024)],but they were significantly higher than those in the NC group at the same time points(P<0.05).Although the expression trend of VEGF-A,PMEK1/2 and P-ERK1/2 in the Tanshinone ⅡA group was consistent with that in the COP group,the expression levels of these three proteins in Tanshinone IIA group was significantly higher than that in COP group at the same time [VEGF-A:1 d(0.0259±0.0030),7 d(0.0373±0.0030),14 d(0.0229±0.0027);30 d(0.0212±0.003);P-MEK1/2: 1 d(0.0369±0.0076),7 d(0.0785±0.0059),14 d(0.0485±0.0074),30 d(0.039±0.0069);P-ERK1/2: 1 d(0.0177±0.0022),7 d(0.0411±0.0033),14 d(0.0299±0.0023),30d(0.0212±0.0015).P< 0.05].Conclusion: Salvia Miltiorrhiza has multiple active components,and plays a role in treating acute brain injury induced by COP through multiple targets and multiple pathways,among them,MAPK/ERK1/2 signaling pathway is one of the most important.Carbon monoxide poisoning can activate t he MAPK/ERK1/2 signaling pathway,and promote VEGF-A protein expression.Tanshinone IIA can effectively inhibit apoptosis and alleviate brain edema,and may up-regulate the expressions of VEGF-A,P-MEK1/2 and P-ERK1/2 proteins,thereby protect endothelial cells,promote angiogenesis and microcirculation,and further alleviate brain injury caused by COP. |
