| Objective: Guanxinning Injection(GXNI),composed of Danshen and Chuanxiong,is a kind of traditional Chinese medicine injection.Danshen and Chuanxiong are the two most frequently used Chinese medicines in treatment of cardio-cerebrovascular diseases in China.As a widely prescribed patent Chinese medicine,GXNI is commonly used for coronary artery disease(CAD),while it is rarely used for the treatment of ischemic stroke.Therefore,this study will explore the protective effect of GXNI on cerebral ischemia/reperfusion injury of mice and oxygen-glucose deprivation/reoxygenation(OGD/R)injury of primary neuronal cells,and clarify the underlying key mechanism of its role,so as to provide theoretical basis for clinical application of GXNI.Methods: 1.Mice with cerebral ischemia-reperfusion injury(CIRI)were established by middle cerebral artery occlusion(MCAO)model for 60 min ischemia and 24 h reperfusion,and were injected with drugs or saline 10 min before reperfusion.The experimental animals were randomly divided into six groups: Sham group,CIRI group,Edaravone group,GXNI 2.5 m L/kg group,GXNI 5 m L/kg group and GXNI 10 m L/kg group.The protective effects of GXNI were evaluated by cerebral infarction volume,neurological deficit score,permeability of blood-brain barrier(BBB),brain edema and histopathological changes.2.Transcriptome sequencing(RNA-seq)was performed on the brain tissue of mice in the CIRI group and GXNI 5 m L/kg group,and the data analysis of the RNA-seq results was carried out.Then,the network pharmacological analysis of RNA-seq results was performed by Ingenuity Pathway Analysis(IPA)to perform to explore the potential mechanism and important target of GXNI in alleviating ischemia/reperfusion(I/R)injury in ischemic stroke mice.The results of the analysis were further verified by real-time reverse transcription-polymerase chain reaction(RT-PCR),Western blot(WB),and immunohistochemistry(IHC).3.To simulate cerebral ischemia-reperfusion injury,an oxygen-glucose deprivation/reoxygenation(OGD/R)model of neurons was established.The effect of different concentrations of GXNI on the cell viability of primary neurons was detected by the CCK-8 solution.The protective effects of GXNI on the primary neuron cell injury induced by OGD/R and the effect of SHH signaling pathway inhibitor(cyclopamine)on GXNI were measured via using Operetta high content imaging system and immunofluorescence(ICC).Results: 1.After 24 hours of reperfusion,CIRI could significantly increase the cerebral infarct volume,neurological deficit score,and permeability of blood-brain barrier,and aggravate cerebral edema and histopathological changes.The experimental results showed that Edaravone and GXNI at 2.5,5 and 10 m L/kg significantly ameliorated histopathological change and permeability of BBB,and markedly decreased cerebral infarction volume,neurological deficit score and brain edema.Among them,GXNI at a dose of 5 m L/kg had the most significant effect.2.RNA-seq analysis identified 1867 differentially expressed genes(DEGs)between GXNI(5m L/kg)treatment and CIRI model in brain tissue with fold change ≥ 1.5 and P-value ≤ 0.01.The core analysis conducted by Ingenuity Pathway Analysis(IPA)indicated that the first ranked pathway was axonal guidance signaling,and the 12 most pivotal genes in this pathway were Shh,Ptch1,Hhip,Gli1,Dcc,Vegfa,Wnt3,Fgfr3,Met,Ace,Baiap2 and Bmp7 which regulated by GXNI.Meanwhile,the results confirmed that 5 m L/kg of GXNI significantly improved the levels of m RNA expression of Shh,Ptch1,Hhip,Gli1,Ntn1,Dcc,Unc5 d and Vegfa,while markedly reduced the levels of m RNA expression of Met in brain tissue of CIRI mice.In addition,protein interaction(PPI)analysis of IPA revealed that SHH,PTCH1,and GLI1 are the most important proteins in axonal guidance signaling pathways.WB and IHC results showed that GXNI at 2.5,5 and 10 m L/kg also significantly increased protein levels of SHH,PTCH1 and GLI1 in brain tissue of CIRI mice.3.The effect of different concentrations of GXNI pretreatment for 24 hours on neuronal cell viability was examined via using CCK-8 assay.It was found that GXNI at 0.03 μL/m L,0.1 μL/m L,0.3 μL/m L and 1.0 μL/m L had no remarkable effect on the cell viability of neurons,and when the GXNI dose was 3.0 μL/m L,10.0 μL/m L and 30.0 μL/m L,cell viability was significantly inhibited.ICC results showed that OGD/R injury can significantly decrease the number of neurons,the fluorescence intensity of Tuj1 protein of neurons,and increase the fluorescence intensity of mitochondria,while GXNI can markedly improve the reduction of cell numbers and fluorescence intensity of Tuj1 protein caused by OGD/R,and decreased mitochondrial fluorescence intensity.Cyclopamine can significantly inhibit the protective effect of GXNI on primary neurons.Conclusion: 1.In this experiment,GXNI exerted protective effects on the brain of stroke mice,and effectively alleviate cerebral ischemia/reperfusion injury.The protective effect of GXNI at a dose of 5 m L/kg was most significant.2.We revealed and verified for the first time that SHH-mediated axonal guidance signaling was the most critical signaling pathway for GXNI to protect stroke mice from cerebral ischemia/reperfusion injury. |
