RESUMO
ObjectiveTo explore the protective effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix （SP） extract on oxygen-glucose deprivation/reoxygenation （OGD/R）-injured SH-SY5Y cells based on oxidative stress and apoptosis. MethodThe extracts of the two medicinal materials mixed in different ratios were prepared. Human neuroblastoma SH-SY5Y cells were cultured in vitro and the injury was induced by OGD/R. Cell counting kit-8 （CCK-8） assay was used to screen the optimal ratio of the two medicinals and then the extract was used for further experiment. SH-SY5Y cells were classified into normal control group， OGD/R group， and low-， medium-， and high-dose SP （2∶1） extract groups （10， 30， 100 mg·L-1， respectively）. Cells in the groups， except the normal control group， were rapidly reoxygenated for 12 h after 4 h OGD for modeling. Then cell viability was detected by CCK-8 and cell morphology was observed under the microscope. The release rate of lactate dehydrogenase （LDH）， superoxide dismutase （SOD） activity， and content of glutathione （GSH） and malondialdehyde （MDA） were determined by spectrophotometry. The level of reactive oxygen species （ROS） was detected with 2，7-dichlorodihydrofluorescein diacetate （DCFH-DA） and mitochondrial membrane potential with JC-1 assay. The nuclear morphology was observed based on Hoechst 33342 staining， and apoptosis was examined by flow cytometry combined with Annexin V-FITC/PI staining. ResultThe viability of the cells was highest in the presence of the extract of the two medicinals mixed at the ratio of 2∶1. Compared with normal control group， OGD/R group showed damaged cell morphology， high release rate of LDH and levels of ROS and MDA （P<0.01）， low SOD activity and GSH level （P<0.01）， low mitochondrial membrane potential， and high apoptosis rate （P<0.01）. Compared with OGD/R group， SP extract improved cell viability and cell morphology and reduce cell LDH release rate in a concentration-dependent manner （P<0.01）. In addition， SP extract at 30， 100 mg·L-1 reduced the level of intracellular ROS and increased SOD activity and GSH level （P<0.05， P<0.01）， and SP extract at 100 mg·L-1 decreased the content of MDA （P <0.05）. Moreover， SP extract increased mitochondrial membrane potential， and SP extract at 30， 100 mg·L-1 lowered the apoptosis rate （P<0.01）. ConclusionThe extract of Salvia miltiorrhiza Bunge and Radix Puerariae mixed at 2∶1 shows better protective effect on OGD/R-injured SH-SY5Y cells. The mechanism is the likelihood that it alleviates oxidative damage of cells and inhibits cell apoptosis.
RESUMO
ObjectiveTo explore the protective effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix （SP） extract on oxygen-glucose deprivation/reoxygenation （OGD/R）-injured SH-SY5Y cells based on oxidative stress and apoptosis. MethodThe extracts of the two medicinal materials mixed in different ratios were prepared. Human neuroblastoma SH-SY5Y cells were cultured in vitro and the injury was induced by OGD/R. Cell counting kit-8 （CCK-8） assay was used to screen the optimal ratio of the two medicinals and then the extract was used for further experiment. SH-SY5Y cells were classified into normal control group， OGD/R group， and low-， medium-， and high-dose SP （2∶1） extract groups （10， 30， 100 mg·L-1， respectively）. Cells in the groups， except the normal control group， were rapidly reoxygenated for 12 h after 4 h OGD for modeling. Then cell viability was detected by CCK-8 and cell morphology was observed under the microscope. The release rate of lactate dehydrogenase （LDH）， superoxide dismutase （SOD） activity， and content of glutathione （GSH） and malondialdehyde （MDA） were determined by spectrophotometry. The level of reactive oxygen species （ROS） was detected with 2，7-dichlorodihydrofluorescein diacetate （DCFH-DA） and mitochondrial membrane potential with JC-1 assay. The nuclear morphology was observed based on Hoechst 33342 staining， and apoptosis was examined by flow cytometry combined with Annexin V-FITC/PI staining. ResultThe viability of the cells was highest in the presence of the extract of the two medicinals mixed at the ratio of 2∶1. Compared with normal control group， OGD/R group showed damaged cell morphology， high release rate of LDH and levels of ROS and MDA （P<0.01）， low SOD activity and GSH level （P<0.01）， low mitochondrial membrane potential， and high apoptosis rate （P<0.01）. Compared with OGD/R group， SP extract improved cell viability and cell morphology and reduce cell LDH release rate in a concentration-dependent manner （P<0.01）. In addition， SP extract at 30， 100 mg·L-1 reduced the level of intracellular ROS and increased SOD activity and GSH level （P<0.05， P<0.01）， and SP extract at 100 mg·L-1 decreased the content of MDA （P <0.05）. Moreover， SP extract increased mitochondrial membrane potential， and SP extract at 30， 100 mg·L-1 lowered the apoptosis rate （P<0.01）. ConclusionThe extract of Salvia miltiorrhiza Bunge and Radix Puerariae mixed at 2∶1 shows better protective effect on OGD/R-injured SH-SY5Y cells. The mechanism is the likelihood that it alleviates oxidative damage of cells and inhibits cell apoptosis.
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The aim of this study was to investigate the mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion(OGD/R) injury in H9 c2 cardiomyocytes. First, Discovery Studio 2019 was used for the molecular docking of luteolin with three key enzymes including lipoxygenase 5(ALOX5), lipoxygenase 12(ALOX12), and lipoxygenase 15(ALOX15) in lipoxygenase pathway. The docking results showed that luteolin had high docking score and similar functional groups with the original ligand. From this, H9 c2 cardiomyocytes were cultured in vitro, and then the injury model of H9 c2 cardiomyocytes was induced by deprivation of oxygen-glucose for 8 h, and rehabilitation of oxygen-glucose for 12 h. Cell viability was detected by tetrazolium(MTT) colorimetry. H9 c2 cardiomyocytes were observed with a fluorescence inverted microscope, and colorimetry was used to detect the level of lactate dehydrogenase(LDH) in cell supernatant. The results showed that luteolin could significantly protect the morphology of H9 c2 cells, significantly improve the survival rate of H9 c2 cardiomyocytes in OGD/R injury model, reduce the level of LDH in cell supernatant, inhibit cytotoxicity, and maintain the integrity of cell membrane. The inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with the model group, luteolin can significantly reduce the release of IL-6 and TNF-α. Western blot was employed to detect the protein levels of ALOX5, ALOX12, and ALOX15 in lipoxygenase pathway. After luteolin intervention, the protein levels of ALOX5, ALOX12, and ALOX15 were significantly down-regulated compared with those in model group. These results indicate that luteolin can inhibit the release of IL-6 and TNF-α by restraining the activation of lipoxygenase pathway, thereby playing a protective role in the cardiomyocyte injury model induced by OGD/R.
Assuntos
Humanos , Apoptose , Glucose , Lipoxigenases , Luteolina/farmacologia , Simulação de Acoplamento Molecular , Miócitos Cardíacos , Oxigênio , Traumatismo por Reperfusão , Transdução de SinaisRESUMO
Aim To investigate the effect of ginsenoside Rgl on PC 12 cell hypoxia-reoxygenation injury and its possible mechanism. Methods PC 12 cells were randomly divided into six groups. Except for the blank control group, all the other groups were hypoxia and hypoglycemia for 6 hours, and then reoxygenated and glycosylated for 24 hours to make OGD/R models. Each drug group was given corresponding drugs 2 hours before modeling pretreatment. DCFH-DA method was used to detect the ROS production in cells, Annexin V- FITC/PI double staining method was performed to detect cell apoptosis rate, ELISA method was used to detect LDH activity and IL-1 (3 content in cell supernatant, and Western blot was applied to detect the ex- pression of proteins of N0X2, p22phox, p47phox, NLRPl, ASC, Caspase-1, PSD95, Tau, p-Tau and observe the intervention effect of ginsenoside Rgl. Re sults Tempol, Apocynin and Rgl (5, 10 jjLmol • L"1) groups could significantly inhibit ROS production and apoptosis, reduce LDH release and IL-1 (3 content in cell supernatant; Apocynin and Rgl (5, 10 |xmol • L"1) groups could significantly down-regulate the expression of N0X2, p22phox and p47phox in cells. The Tempol, Apocynin and Rgl (5, 10 jxmol • L"1 ) groups could significantly decrease the protein expression of NLRP1, Caspase-1, ASC, IL-1 (3 and p-Tau, and markedly down-regulate the expression of PSD95 protein. Conclusion Rgl is likely to reduce the is- chemia-reperfusion injury of PC 12 cells by inhibiting the NOX2-NLRP1 pathway.