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Objective:To investigate the effects and mechanism of sciadopitysin combined with CK2 inhibitor CX-4945 on proliferation and apoptosis of glioblastoma U87 cells.Methods:Glioblastoma U87 cells were cultured in vitro, and treated with 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin respectively. U87 cells were treated with 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945. U87 cells were divided into control group (without any treatment), sciadopitysin group (100.00 μmol/L of sciadopitysin), CX-4945 group (5.00 μmol/L of CX-4945), sciadopitysin combined with CX-4945 group (100.00 μmol/L of sciadopitysin plus 5.00 μmol/L of CX-4945). MTT method was used to detect cell viability, Caspase3/7 activity assay and Annexin Ⅴ/ PI double staining were used to detect cell apoptosis, and Western blotting was used to detect the expressions of Notch1 pathway related proteins ICN1, HES1 and DLL3. Results:The cell viabilities of U87 cells treated with 0, 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin were (100.00±6.30) %, (112.02±7.63) %, (140.84±6.73) %, (113.92±7.92) %, (102.60±7.12) % and (73.16±2.74) % respectively, and there was a statistically significant difference ( F=55.21, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 0.01, 0.10, 1.00, 100.00 μmol/L of sciadopitysin treatment ( P=0.009; P<0.001; P=0.003; P<0.001). The cell viability of U87 cells was inhibited by 100.00 μmol/L of sciadopitysin, while sciadopitysin at other low concentrations manifested as enhancement or no obvious effect. The cell viabilities of U87 cells treated with 0, 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945 were (100.00±5.53) %, (108.70±10.24) %, (93.14±2.82) %, (81.46±4.92) %, (56.92±3.99) % and (31.24±2.67) % respectively, and there was a statistically significant difference ( F=135.18, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 1.25, 5.00, 10.00, 20.00 μmol/L of CX-4945 treatment ( P=0.022; P<0.001; P<0.001; P<0.001). Low concentration (1.25 μmol/L) of CX-4945 enhanced the cell viability of U87 cells, however higher concentrations (5.00, 10.00, 20.00 μmol/L) of CX-4945 shown inhibitory effect. The cell viabilities of U87 cells in the control group, sciadopitysin group, CX-4945 group and sciadopitysin combined with CX-4945 group were (100.00±5.53) %, (71.96±2.10) %, (77.66±4.12) % and (42.56±4.22) % respectively, and there was a statistically significant difference ( F=160.56, P<0.001). There were statistically significant differences between the control group and each treatment groups (all P<0.001). There were statistically significant differences between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (both P<0.001). The Caspase3/7 activities of U87 cells in the above four groups were 2.34±0.47, 4.02±0.22, 3.67±0.32 and 5.85±0.28 respectively, and there was a statistically significant difference ( F=55.80, P<0.001). The apoptosis rates of each groups were (0.40±0.10) %, (17.37±0.57) %, (3.00±0.66) % and (33.47±0.87) % respectively, and there was a statistically significant difference ( F=1 822.18, P<0.001). Further pairwise comparison showed that there were statistically significant differences in Caspase3/7 activities and apoptosis rates between the control group and each treatment groups ( P<0.001, P=0.001, P<0.001; P<0.001, P=0.001, P<0.001). There were statistically significant differences in Caspase3/7 activities and apoptosis rates between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (all P<0.001). The protein expression levels of Notch 1 pathway related proteins ICN1 (0.55±0.07 vs. 1.01±0.09), HES1 (0.66±0.08 vs. 1.00±0.06) and DLL3 (0.74±0.04 vs. 1.01±0.09) in U87 cells decreased significantly after treatment with 100.00 μmol/L of sciadopitysin ( t=5.94, P=0.004; t=5.15, P=0.007; t=4.00, P=0.016) . Conclusion:Sciadopitysin can synergize with CK2 inhibitor CX-4945 to inhibit the proliferation and promote apoptosis of glioblastoma U87 cells by inhibiting Notch1 signaling pathway.
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Background In addition to the typical signs of skin damage, long-term arsenic exposure is often accompanied by signs and symptoms of neurobehavioral abnormalities. Objective To investigate potential intervention effect of sciadopitysin on senescence of neurons induced by sodium arsenite in rats and possible underlying mediating effect of cell cycle-related transcription factor E2F1. Methods SH-SY5Y cells were treated with 4 μmol·L−1 sodium arsenite for 24 h and intervened with 50 μg·mL−1 Ginkgo biloba extract (EGb761) or four major biflavonoids in Ginkgo biloba leaves (isoginkgetin, bilobetin, sciadopitysin, and ginkgetin) for 24 h respectively. Then, cell viability was measured by CCK-8 assay. Thirty-two 180-200 g SPF rats were randomly divided into a control group, an arsenic treatment group (10 mg·L−1), a Ginkgo biloba extract intervention group (10 mg·kg−1), and a sciadopitysin intervention group (10 mg·kg−1), 8 rats in each group, half male and half female. The rats were treated with sodium arsenite by free drinking water for 3 consecutive months, and the intervention treatment was conducted after 2 months of poisoning with drug intake by gavage for 1 month. HE staining was used to detect structural changes in the hippocampus, while Nissl's staining was used to detect changes in hippocampal morphology and neuron numbers. Moreover, senescence-associated β galactosidase (SA-β-gal) staining and Western blotting were used to detect senescence of hippocampal neurons and the expression level of E2F1, respectively. Results Compared to the arsenic treatment group, EGb761 and the four biflavonoids in Ginkgo biloba leaves effectively antagonized the inhibitory effect of sodium arsenite on cell viability (all Ps＜0.05), and sciadopitysin showed better restoration of cellular viability than Ginkgo biloba extract (P＜0.05). The results of HE staining and Nissl's staining showed that the hippocampal neurons in the arsenic treatment group were reduced in cell count and the synaptic structure was abnormal, with swelling, nuclear shrinkage, and vacuole, compared with the control group. The results of SA-β-gal staining showed that the number of senescent cells in the arsenic treatment group (15.75±3.01) was significantly increased compared with the control group (2.88±0.84) (P＜0.05); the numbers of senescent cells in the Ginkgo biloba extract group (9.38±1.92) and the sciadopitysin treatment group (7.75±2.38) were significantly decreased compared with the arsenic treatment group (all Ps＜0.05). The results of Western blotting showed that compared with the control group, the expression of E2F1 protein in hippocampus of the arsenic treatment group was significantly decreased (1.00±0.17 vs. 0.65±0.19, P<0.05); compared with the arsenic treatment group, the protein expression level of E2F1 in hippocampus of the sciadopitysin treatment group (0.89±0.18) was significantly recovered (P<0.05); compared with Ginkgo biloba extract (0.68±0.19), sciadopitysin had a better recovery effect on E2F1 expression level (0.89±0.18) (P＜0.05). The results of correlation analysis showed that the E2F1 protein expression level was negatively correlated with the positive rate of SA-β-gal staining in hippocampal neurons (r=−0.518, P＜0.05). Conclusion Sciadopitysin is an effective component of Ginkgo biloba extract. It can effectively inhibit the senescence of hippocampal neurons induced by sodium arsenite, and E2F1 may play an important mediating role.
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This study was designed to investigate the inhibitory effects of sciadopitysin on the catalytic activities of human 12 kinds of UDP-glucuronosyltransferases (UGTs) in vitro. The risk of drug-drug interactions (DDI) is predicted by in vitro-in vivo extrapolation (IV-IVE). Methods A panel of recombinant human UGT isoforms and human liver microsome (HLM) as well as a series substrates including 4-methyl umbelliferone (4-MU), trifluoperazine (TFP) and N-3-carboxypropyl-4-hydroxy-1, 8-naphthalimide (NCHN) (UGT1A1 specific fluorescent probe substrates) were used to characterize the inhibitory effects of sciadopitysin on human UGTs in vitro. The half maximum inhibitory concentration (IC50) and the constant of inhibition kinetics (Ki) were obtained by nonlinear regression using GraphPad Prism 6.0 software. The potential risk of DDI induced by UGT1A1 was predicted based on in vitro parameters. The results demonstrated that sciadopitysin had strong inhibitory effects on UGT1A1, UGT1A3, UGT1A8 and UGT1A10, with the remaining activity being below 30% at a final concentration of 10 μmol·L-1. For UGT1A1, UGT1A3, UGT1A8 and UGT1A10, the IC50 was 0.20 μmol·L-1 to 1.34 μmol·L-1, the inhibition kinetic constant Ki was 0.07 μmol·L-1 to 2.12 μmol·L-1. The AUC ratio of UGT1A1 can be increased by 19% to 147% at the oral dose of 240 mg·d-1. The sciadopitysin competitively inhibited the formation of 4-MU-O-glucuronide by UGT1A1, UGT1A3, UGT1A8, and UGT1A10. At the same time, the inhibition of NCHN-O-glucuronidation by UGT1A1 was consistent with the competitive inhibition. The strong inhibition of sciadopitysin on UGT1A1 led to reduction of the metabolism of UGT1A1 substrates, and increased the risk of DDI. When co-administrated with other drugs, special attentions should be given to the DDI from inhibition of drug metabolism enzymes to prevent serious clinical consequences.
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Objective: To purify the biflavonoids from Ginkgo biloba leaves and determine their contents in the leaves collected in different periods. Methods: Dichloromethane fraction of ethanol extract was used to purify biflavonoids through silica gel column and semi-preparative HPLC. The contents of four biflavonoids were simultaneously determined using RP-HPLC. Results: According to the reported 1H-NMR data, four compounds were identified as bilobetin (LB-1), ginkgetin (LB-2), isoginkgetin (LB-3), and sciadopitysin (LB-4). Content determination demonstrated that the biflavonoids accumulation decreased as the leaves collected from the beginning to the end of September, and the content difference can be as large as 30%. Conclusion: RP-HPLC method can be used in the quantitative determination of biflavonoids in G. biloba leaves. The semi-preparative condition can be used in the purification of biflavonoids from G. biloba leaves. The contents of four biflavonoids could be decreased significantly when the leaves turned yellow, which provides the useful information for the collection of G. biloba leaves. However, the mechanism and the accumulation of other kinds of compounds need further study.