Abstract
Purpose The research aimed to identify the active component from Punica granatum L. to alleviate ischemia/reperfusion injury and clarify the underlying mechanism of the active component alleviating ischemia/reperfusion injury. Materials and Methods The SH-SY5Y cell model of oxygen-glucose deprivation/reoxygenation (OGD/R) was established to simulate the ischemia/reperfusion injury. According to the strategy of bioassay-guided isolation, the active component of punicalin from Punica granatum L. was identified. Flow cytometry and Western blotting were employed to evaluate the effects of OGD/R and/or punicalin on cell cycle arrest. Immunofluorescence assay was applied to assess the nucleus translocation. The relative content of ROS and GSH and the enzyme activities of CAT and SOD were examined using ELISA. Results The data of bioassay-guided isolation showed that punicalin from Punica granatum L. could alleviate OGD/R-induced cell injury in SH-SY5Y cells. Flow cytometry analysis and Western blotting for probing the expression of CDK1, p-CDK1, cyclin B1, and p21 revealed that punicalin could relieve OGD/R-induced cell cycle G0/G1 arrest. Additionally, immunofluorescence assay and Western blotting for probing the expression of TGF-β and p-Smad2/p-Smad3 showed that punicalin could relieve the OGD/R-induced TGF-β/Smad pathway. Furthermore, the TGF-β/Smad pathway inhibitor of LY2157299 was employed to confirm that the TGF-β/Smad pathway is crucial to the effect of punicalin. At last, it was indicated that punicalin could relieve OGD/R-induced oxidative stress. Conclusion Punicalin, an active component from Punica granatum L., was identified as a protective agent to alleviate the OGD/R-induced cell injury, which could exert the protective effect via TGF-β/Smad pathway-regulated oxidative stress and cell cycle arrest in SH-SY5Y cells.
Highlights
Cerebral ischemic stroke is considered as a brain injury disease with the symptoms of hemiplegia, cognitive impairment, and disability, which are caused by clogging blood vessel leading to insufficiency of cerebral blood supply [1]
E primary antibody of CDK1 (Cat No ab133327), p-CDK1 (Cat No ab201008), cyclin B1 (Cat No ab32053), p21 (Cat No ab109520), TGF-β (Cat No ab215715), and Smad 2/Smad3 (Cat No ab202445) and the secondary antibody were purchased from Abcam (USA). e dyes of DAPI (Cat No 268298) were purchased from Sigma (USA). e ELISA assay kits of ROS (Cat No E004-1-1), CAT (Cat No A007-1-1), GSH (Cat No A005-1-2), and SOD (Cat No A001-3-2) were obtained from Nanjing Jiancheng Bioengineering Institute (China). e LY2157299 (Cat No HY-13226), a TGF-β inhibitor, was purchased from MedChemExpress (MCE). e organic solvents ethanol, petroleum ether, chloroform, and ethyl acetate were purchased from Sinopharm Chemical Reagent Co., Ltd (China)
The ethanol extraction was extracted with petroleum ether, chloroform, and ethyl acetate, respectively, to obtain three fractions of PE, CF, and EA. e three fractions were dried to solid in rotary evaporation, and the active compound was identified according to bioactivity-guided isolation
Summary
Cerebral ischemic stroke is considered as a brain injury disease with the symptoms of hemiplegia, cognitive impairment, and disability, which are caused by clogging blood vessel leading to insufficiency of cerebral blood supply [1]. E TGF-β/Smad pathway is recognized as a process of signaling cascade response to the growth factor for maintaining the individual’s dynamic balance of physiology and pathology It plays many crucial roles in cell proliferation, apoptosis, oxidative stress, and inflammation, participating in a series of disease events such as cancer, diabetes, and cardiocerebrovascular disease [6, 7]. Recent shreds of evidence showed that TGF-β could serve as an injury-related cytokine involving brain injury and neurodegenerative diseases It is reported by Abdel et al that propolis could relieve cerebral injury via the regulation of the TGF-β/Smad pathway [8]. We established an oxygen-glucose deprivation/ reoxygenation (OGD/R) model in human neuroblastoma cell SH-SY5Y to simulate IRI, identify a TGF-β inhibitor of punicalin alleviating OGD/R-induced neuroblastoma injury from PGL extraction, and elucidate the mechanism of punicalin-suppressing cell injury via TGF-β-mediated oxidative stress and cell cycle arrest. We established an oxygen-glucose deprivation/ reoxygenation (OGD/R) model in human neuroblastoma cell SH-SY5Y to simulate IRI, identify a TGF-β inhibitor of punicalin alleviating OGD/R-induced neuroblastoma injury from PGL extraction, and elucidate the mechanism of punicalin-suppressing cell injury via TGF-β-mediated oxidative stress and cell cycle arrest. e present study revealed the role of TGF-β in IRI development and treatment and provided a novel therapeutic approach for IRI
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