Pharmacodynamic Evaluation And Mechanism Study Of CZK, An Alkaloid Derivative Derived From Phellodendron Chinense, In The Treatment Of Ischemic Strok

Objective:Network pharmacology was used to explore the molecular mechanism of Clausena lansium and its compounds for treating ischemic stroke.Pharmacodynamic experiments were conducted to verify the neuroprotective effect and mechanism of a novel carbazole alkaloid derivative from Clausena lansium called CZK on cerebral ischemia-reperfusion,which could provide more evidence for the developments of Clausena lansium against ischemic stroke.Methods:(1)The compounds of Clausena lansium were acquired by PubMed and CNKI databases,and their corresponding targets were screened by SEA database.The genes associated with ischemic stroke were obtained from GeneCards database.The protein-protein interaction was found by STRING database.(2)Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichments were set up by Metascape database.(3)Discovery Studio software was used for molecular docking of CZK and NF-E2-related factor 2(Nrf2)/Kelch-like ECHassociated protein 1(Keapl)complex.(4)SH-SY5Y cells were used to detect the cytotoxicity of CZK.(5)Rat middle cerebral artery occlusion(MCAO)model was established to verify the protective effect of CZK on ischemic stroke.Triphenyltetrazolium chloride(TTC)staining was used to show the infarct,edema ratio and neurologic deficit score.(6)The electron paramagnetic resonance(EPR)technique was used to detect the hydroxyl radical scavenging ability of CZK.(7)Reactive oxygen species(ROS)levels were revealed by dihydroethidium(DHE)staining.Superoxide dismutase(SOD),malonaldehyde(MDA),and glutathione(GSH)assay kits were employed for quantifying the antioxidative indexes.(8)TdT-mediated dUTP Nick-End Labeling(TUNEL)and Nissl staining were applied to observe neuronal damage in the brain.(9)Western Blot was acquired to measure the protein levels of Nrf2,Keap1,NADPH:Quinone Oxidoreductase 1(NQO1),and Heme oxygenase-1(HO-1)in the ischemic penumbra.(10)Immunofluorescence staining was used to observe the fluorescence expression of Nrf2 and 8-hydroxy-2deoxyguanosine(8-OHdG).Results:(1)Compounds and target screening indicated that 256 gene targets from Clausena lansium were related to ischemic stroke,GO and KEGG pathway enrichment results indicated that the mechanism of Clausena lansium in treating ischemic stroke was significantly associated with oxidative stress.(2)Molecular docking showed that CZK bound to Nrf2 protein complex.(3)Compared with Claulansine F,CZK had lower cytotoxicity when incubated with SH-SY5Y cells for 72 h and at a concentration of 6.25 μM(P<0.001).(4)CZK alleviated cerebral ischemic injury at the dose of 50 mg/kg,and the cerebral infarction rate(P<0.01),edema rate(P<0.01),and neurobehavioral score(P<0.01)were significantly decreased.(5)CZK had a strong scavenging ability of hydroxyl radical,and the IC50 value was 77.08 nM.(6)CZK significantly reduced ROS levels(P<0.01)and increased SOD activity(P<0.01)and GSH content(P<0.05)in the brain.CZK also reduced the number of 8-OHdG-positive cells(P<0.01).(7)CZK alleviated neuronal damage,which could be shown as the increase of Nissl bodies(P<0.05)and the decrease of apoptotic cells(P<0.01).(8)Immunofluorescent staining showed that CZK promoted the Nrf2 nucleus translocation.And western Blot data demonstrated that CZK treatment upregulated the expression of Nrf2(P<0.05),HO-1(P<0.01),and NQO1(P<0.01).Conclusion:Through network pharmacological analysis,it was suggested that Clausena lansium might treat ischemic stroke with anti-oxidation effect.(2)CZK could alleviate cerebral ischemiareperfusion injury by directly scavenging free radicals and activating Nrf2 pathway to reduce oxidative stress.