[Cell metabolomics study of ginkgo flavone aglycone combined with doxorubicin against liver cancer in synergy].

Ultra-high-performance liquid chromatography-Q exactive orbitrap tandem mass spectrometry(UHPLC-QEOrbitrap-MS/MS) was used to explore the inhibitory effect and mechanism of ginkgo flavone aglycone(GA) combined with doxorubicin(DOX) on H22 cells. The effects of different concentrations of GA and DOX on the viability of H22 cells were investigated, and combination index(CI) was used to evaluate the effects. In the experiments, control(CON) group, DOX group, GA group, and combined GA and DOX(GDOX) group were constructed. Then the metabolomics strategy was employed to explore the metabolic markers that were significantly changed after combination therapy on the basis of single medication treatment, and by analyzing their biological significance, the effect and mechanism of the anti-tumor effect of GA combined with DOX were explained. The results revealed that when 30 µg·mL~(-1) GA and 0.5 µmol·L~(-1) DOX was determined as the co-administration concentration, the CI value was 0.808, indicating that the combination of GA and DOX had a synergistic anti-tumor effect. Metabolomics analysis identified 23 metabolic markers, including L-arginine, L-tyrosine and L-valine, mostly amino acids. Compared with the CON group, 22 and 17 metabolic markers were significantly down-regulated after DOX treatment and GA treatment, respectively. Compared with the DOX and GA groups, the treatment of GA combined with DOX further down-regulated the levels of these metabolic markers in liver cancer, which might contribute to the synergistic effect of the two. Five key metabolic pathways were found in pathway enrichment analysis, including glutathione metabolism, phenylalanine metabolism, arginine and proline metabolism, ß-alanine metabolism, and valine, leucine and isoleucine degradation. These findings demonstrated that the combination of GA and DOX remarkably inhibited the viability of H22 cells and exerted a synergistic anti-tumor effect. The mechanism might be related to the influence of the energy supply of tumor cells by interfering with the metabolism of various amino acids.

Effects of Senegenin against hypoxia/reoxygenation-induced injury in PC12 cells.

OBJECTIVE: To investigate the effect and the potential mechanism of Senegenin (Sen) against injury induced by hypoxia/reoxygenation (H/R) in highly differentiated PC12 cells. METHODS: The cultured PC12 cells were treated with H/R in the presence or absence of Sen (60 µmol/L). Four groups were included in the experiment: control group, H/R group, H/R+Sen group and Sen group. Cell viability of each group and the level of lactate dehydrogenase (LDH) in culture medium were detected for the pharmacological effect of Sen. Hoechst 33258 staining and annexin V/propidium iodide double staining were used to analyze the apoptosis rate. Moreover, mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) and intracellular free calcium ([Ca(2+)]i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 and activity of NADPH oxidase (NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay, respectively. RESULTS: Sen significantly elevated cell viability (P<0.05), decreased the leakage of LDH (P<0.05) and apoptosis rate (P<0.05) in H/R-injured PC12 cells. Sen maintained the value of △Ψm (P<0.05) and suppressed the activity of caspase-3 (P<0.05). Moreover, Sen reduced ROS accumulation P<0.05) and [Ca(2+)]i increment (P<0.05) by inhibiting the activity of NOX (P<0.05). CONCLUSION: Sen may exert cytoprotection against H/R injury by decreasing the levels of intracellular ROS and [Ca(2+)]i, thereby suppressing the mitochondrial pathway of cellular apoptosis.

Enhancing ergosterol production in Pichia pastoris GS115 by overexpressing squalene synthase gene from Glycyrrhiza uralensis.

The present study was designed to determine the effects of copy number variations (CNVs) of squalene synthase 1(SQS1) gene on the mevalonate (MVA) pathway. SQS1 gene from G. uralensis (GuSQS1) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy number of GuSQS1 were constructed. HPLC was used to assay the level of ergosterol in all transgenic P. pastoris strains containing GuSQS1. HPLC analysis showed that the contents of ergosterol in all of the transgenic P. pastoris containing GuSQS1 were higher than that in the negative control. And with the increase of copy number of GuSQS1, the content of ergosterol showed an increasing-decreasing-increasing pattern. The contents of ergosterol in 10-copy-GuSQS1 P. pastoris and 47-copy-GuSQS1 P. pastoris were significantly higher than that in the rest recombinant P. pastoris strains. In conclusion, the CNVs of GuSQS1 influence the content of secondary metabolites in the MVA pathway. The present study provides a basis for over-expressing GuSQS1 and increasing the content of glycyrrhizin in G. uralensis cultivars.

[The effect of retrobulbar injection with anisodamine on ocular blood velocity in anterior ischemic optic neuropathy].

OBJECTIVE: To study the effect of retrobulbar injection with anisodamine on ocular blood velocity in anterior ischemic optic neuropathy (AION). METHODS: 15 minutes before injection, 15 minutes and 1 hour after injection, the blood flow velocity in 39 cases (39 eyes) diagnosed as AION was measured and analyzed by CDI, timed average maximum velocity, peak systolic velocity, end diastolic velocity, resistance index and pulsatility index of OA, CRA and PCA were recorded. RESULTS: Compared with the normal eyes, each parameter of SPCA was statistically significant. 15 minutes and 1 hour after post ocular injection of anisodamine, every parameter examined in the experiment was increased except resistance index compared with pre-injection (P < 0.01). CONCLUSIONS: retrobulbar injection with anisodamine can effectively improve flow velocity of nasal and tempo short posterior ciliary artery and central retinal artery.