| Objective: To investigate the effect and mechanism of galangin on6-OHDA-induced model of Parkinson’s disease(PD),and whether the effect is relevant to the Keap1/Nrf2 pathway.Methods: The network pharmacology method was used to carry out the following research:(1)The targets of galangin and PD were obtained through database retrieval.(2)Screen the common targets of galangin and PD,import the common targets into Cytoscape 3.7.1 software,and construct a drug-target-disease network.(3)The protein-protein interaction(PPI)relationship of common targets was obtained with the help of database,and the PPI network was constructed and analyzed.(4)The DAVID 6.8 database was used to obtain the common targets gene ontology(GO)function and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.(5)Using Pub Chem database,Uni Prot website,Sybyl-X 2.0 software,and SYBYL’s Protocol generation technology to simulate the docking of galangin and core target molecules.According to the prediction results of network pharmacology,HT22 cells were cultured in vitro,and the following studies were carried out:(1)HT22cells were treated with a series of concentrations(3.13,6.25,12.5,25,50,100,200,400 μM)of 6-Hydroxydopamine hydrobromide(6-OHDA),and the effect at 24 h was detected.Therefore,the optimal modeling concentration was screened out.(2)HT22 cells were treated with a series of concentrations(1.56,3.13,6.25,12.5,25,50,100 and 200 μM)of galangin and cell activity was measured at 24 h using the MTT method.Thus,the safe concentration of galangin in HT22 cells was determined.(3)HT22 cells were treated with a series of concentrations(1.56,3.13,6.25,12.5,25,50 μM)of galangin for 24 h and then treated with 100 μM 6-OHDA for 24 h.Cell activity was assayed using the MTT method,thereby screening four concentrations of galangin,low,medium and high,for subsequent studies.(4)To investigate the effect of galangin on the PD cell model(in terms of cell morphology,apoptosis and cellular oxidative stress).We divided the cells into 6 groups: a blank control group,a model group(treated with 100 μM 6-OHDA for 24 h),and galangin low,medium and high dose groups(first treated with galangin 6.25,12.5,25,50 μM for 24 h,and then treated with 100 μM 6-OHDA for 24 h).The morphological changes of the cells in each group were observed by microscopy,apoptosis was observed by Hoechst33324/PI staining,and the apoptosis rate and reactive oxygen species(ROS)levels were detected by flow cytometry.(5)In order to explore the effect of galangin on apoptosis-related proteins of HT22 cells,Western Blot(WB)was used to detect the expression levels of Bcl-2 and Bax.(6)WB was performed to detect the expression levels of Keap1,Nrf2 and HO-1 in cells to explore the mechanism.(7)We took BZ555 worms expressing green fluorescent protein(GFP)in dopaminergic neurons as the research object.The BZ555 worms were divided into four groups: a blank control group,a model group(treated with 50 m M 6-OHDA),a galangin group(treated with 100 μM galangin)and a positive drug group(treated with 2 m M Levodopa(L-Dopa)).The GFP fluorescence intensity representing the number of dopaminergic neurons,was observed by fluorescence microscopy.And the function of dopaminergic neurons was measured by assessing the food-sensing behavior in BZ555 worms.Afterwards,the BZ555 worms were again divided into four groups,a blank control group,a model group(treated with 50 m M 6-OHDA),a galangin group(treated with 100 μM galangin),and a positive drug group(treated with 5 m M N-acetyl-L-cysteine(NAC)),and the fluorescence intensity representing the level of ROS was observed by fluorescence microscope.Results:(1)114 galangin targets,2224 PD-related targets,and 31 common targets were screened.Nine core targets were identified under the conditions of Degree>8,Betweenness Centrality>0.01815109,and Closeness Centrality>0.5,namely HMOX1,NFE2L2,SOD1,GSR,CASP9,KEAP1,MPO,GSTP1,and CREB1.(2)Common targets are mainly enriched in mitochondria,mitochondrial outer membrane,pore complex,nuclear membrane and other cellular components;cell aging,regulation of apoptosis process,cellular response to ROS,response to oxygen,etc.and enriched in apoptosis,PI3K/Akt,HIF-1,etc.signal path.(3)The direct relationship between galangin and the core target was simulated by molecular docking.The results showed that galangin and NFE2L2/Nrf2 protein showed strong affinity,suggesting that the mechanism maybe relevant to the Nrf2 signaling pathway.(4)The cell viability began to decrease significantly when 6-OHDA-treated for24 h(P ≤ 0.001).(5)HT22 cells were treated with a series of concentrations of galangin for 24 h.The cell viability was not significantly affected within the range of 200 μM,and 4 concentrations of 6.25,12.5,25,and 50 μM were selected within this range for subsequent studies.(6)MTT assay results showed that cell viability was greatly reduced in the model group after 6-OHDA induction compared to the blank control group(P ≤ 0.001),while galangin(6.25,12.5,25,50 μM)significantly increased cell activity in a dose-dependent manner(P ≤ 0.001).(7)Using microscope observation,compared with the model group,galangin dose-dependently improved the morphology of HT22 cells induced by 6-OHDA.(8)The results of Hoechst33324-PI staining assay showed that compared with the model group,galangin significantly inhibited the apoptosis induced by 6-OHDA.(9)The results of flow cytometry showed that galangin dose-dependently reduced apoptosis(P ≤ 0.001).(10)The results of flow cytometry showed that compared with the blank control group,the model group significantly stimulated the production of intracellular ROS levels(P ≤ 0.001),while galangin dose-dependently reduced the ROS production(P ≤0.01).(11)The results of WB showed that compared with the blank control group,galangin dose-dependently decreased the ratio of Bax to Bcl-2(P ≤0.001).(12)Galangin increased HO-1 expression(P ≤ 0.05)by decreasing Keap1 level(P ≤ 0.001)and increasing Nrf2 level(P ≤ 0.05).(13)Galangin reduced ROS levels in BZ555 worms(P ≤ 0.001),increased the number of dopaminergic neurons(P ≤ 0.001),and improved food-sensing behavior(P≤ 0.001).Conclusion: The current study reveals that galangin exhibits neuroprotective effects,as evidenced by the improvement of neuronal viability in 6-OHDA-treated HT22 cells and C.elegans,by activating the Nrf2/Keap1/HO-1 signaling pathway,which provides novel insight into the further development of galangin as a candidate for the treatment of PD in the future. |
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