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Aim To analyze the anti-A549 and HI299 lung ade-nocarcinoma activities via using examples of baicalin, astragalo-side, hesperidin and cisplatin based on real time cellular analysis (RTCA) technology, and to build a new strategy for EC50 e-valuation reflecting the time-dimensional characteristic. Methods Using RTCA Software Pro for data analysis and GraphPad Prism and Origin Pro plotting, the in vitro anti-A549 and H1299 lung adenocarcinoma activities of baicalin, astragaloside, hesperidin, and cisplatin were characterized using the endpoint method and time dimension, respectively. Results (X) There were significant differences in EC50 values of A549 and H1299 cells at 24 h and 48 h endpoint methods. (2) The correlation coefficient of the curve fitted with the four-parameter equation was > 0. 9, and the dynamic change of EC50 remained relatively stable (the linear fitting of EC50 at adjacent 4 points I slope 1
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Realtime xCELLigence analysis (RTCA) is a new cell detection technology to continuously monitor, record and analyze a variety of information generated by cell activity. In drug research, it plays an important role in assessing myocardial toxicity and cell biological activity. Here, we first introduce the underlying mechanisms and characteristics of RTCA. Then we review the applications of RTCA in the research of myocardial toxicity and cell biological activity, to provides the fundamental baseline for understanding and exploiting RTCA. With the real-time, unlabeled, non-invasive, high throughput, and high accuracy features, RTCA not only promotes drug research and development, but also has a broad and good application prospect in other fields.
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Preparaciones FarmacéuticasRESUMEN
The chemical constituents of Rehmanniae Radix Preparatawere prepared according to the traditional method of "jiu zheng jiu shai" and investigated using multiple chromatographic methods. Six alkaloids were isolated and their structures were elucidated from spectral data and physicochemical properties, as follows: rehmanniae alkaloid A (4-{[(5-O-á-D-galactopyranosyloxy)methyl]-1H-pyrrole-2-carbaldehyde-1-yl}butyric acidmethyl ester) (1), baimantuoluoamide B (2), capparisine C (3), harman-3-carboxylic acid (4), (2S)-1-[2-(furan-2-yl)-2-oxoethyl]-5-oxopyrrolidine-2-carboxylate (5), and 1-[2-(furan-2-yl)-2-oxoethyl]pyrrolidin-2-one (6). Among them, compound 1 is a new alkaloid. Compounds 2-6 were newly isolated from Rehmannia glutinosa Libosch.The effect of compounds 1-6 on NRK-52e cell injury induced by LPS was investigated. The results show that compounds 1-3 exhibit protective effects against LPS-induced damage to NRK-52e cells.
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Aim To establish an in vitro arrhythmia detection technique based on real-time cell analysis (RTCA) Cardio sys-tem with aconitine as a tool drug, and to provide a reliable method for the development of antiarrhythmic drugs. Methods The effects of aconitine on rat cardiac rhythm were detected by eight-channel physiological recorder at the level of whole animal. In vitro cultured cardiac myocytes, the inoculation density of cardiac myocytes was investigated by HTCA method, the effect of aconitine on cardiac beating was monitored by RTCA Cardio system, and the CI value, beating rate, amplitude and irregular rhythm of cardiac myocytes were analyzed. Results Eight-channel physiological recorder was used to detect the effects of aconitine on whole animals. The results showed that aconitine(50 mg • g"1) could induce arrhythmias such as ventricular tachycardia, ventricular fibrillation, shortened RR interval and increased heart rate. RTCA Cardio system showed that aconitine (2-8 p,M) could induce arrhythmias such as increased cardiac cell beating frequency, decreased beating amplitude and abnormal beating state in a dose-dependent manner. Conclusions RTCA Cardio system can rapidly, sensitively and accurately detect the arrhythmia induced by aconitine in cardiac myocytes, which provides methodological reference for the development of antiarrhythmic drugs.
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Objective: To compare the bio-activities of different parts of Tripterygium wilfordii from different regions based on RTCA system with HPLC. Methods: Real-time xCELLigence analysis system was established to detect dynamically cellular activity at real-time: The seeding density was 2 × 105 cells/mL, the administration time was about 24 h, and the activity of RBL-2H3 cells from T. wilfordii was detected; The fingerprints of different parts of T. wilfordii were established by HPLC and the common peaks were analyzed. Results: The activity of T. wilfordii from Zhejiang was similar at the same site; The root barks and leaves of T. wilfordii had similar biological activity, and the roots and stems had similar activity; The biological activity for RBL-2H3 cells in root barks and buds was significantly higher than that in roots and stems, especially in the root barks, biological activity was the strongest; The fingerprints showed that the components of different parts were similar but the contents were largely different; The inhibitory rate of chloroform extract on RBL-2H3 cells was significantly higher than that of water extract. Conclusion: At the level of cell biology, the velamen of T. wilfordii and other aerial parts possess value of research and medication, which is deserved to further developed and utilized.
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Objective To analyze inhibitory effect of taspine derivative TasD1-6 on human liver cancer cells of Hep3B, HepG2, and BEL7402, and to study their preliminary mechanism of anticancer action. Methods Effect of TasD1-6 on cell growth of Hep3B, HepG2, and BEL7402 were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and real-time cellular analysis (RTCA) assay. Effect of TasD1-6 on Hep3B cell morphology was investigated with crystal violet staining. Next, Hep3B cell apoptosis was analyzed by Annexin V FITC/PI and Hoechst 33258 staining. Flow cytometry was used to determine Hep3B cell cycle. Effect of TasD1-6 on cell apoptosis protein expression in Hep3B cell was determined by Western blotting. Results MTT and RTCA assay demonstrated that TasD1-6 significantly inhibited the growth of Hep3B cell and the IC50 was (11.3 ± 1.6) μmol/L. Hep3B cell morphology indicated the shrinkage and obvious morphology changes. And TasD1-6 induced the Hep3B cell apoptosis in dose-dependent manner. Cell was stopped as G1 phase by TasD1-6. Western blotting analysis showed TasD1-6 could upregulate the protein expression of p53 and Bax and downregulate Mcl-1 protein expression (P < 0.05). Conclusion Taspine derivative TasD1-6 shows better inhibitory action on the growth of human liver cancer Hep3B cell than HepG2 and BEL7402 cells. At the same time, TasD1-6 can change cell cycle and induce cell apoptosis, which probably related to upregulation of p53 and Bax protein expression and downregulation of Mcl-1 protein expression.
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This study was aimed to investigate the protective effect and mechanism of β-asarone on PC12 cells injury induced byAβ₁₋₄₂ activated astrocytes, and provide experimental basis for β-asarone application in the prevention and control of Alzheimer's disease (AD). Firstly, RA-h and PC12 cells were co-cultured in the special transwell chamber, and the Real time cell analysis (RTCA) system was used to real-time observe its effect on PC12 cells survival rate in the co-culture system after astrocytes injury induced by Aβ₁₋₄₂. The best intervention time of β-asarone was selected according to the survival curve and parameters generated automatically. β-asarone with different concentrations was used for intervention on astrocytes, then the changes of PC12 cells survival rate in the co-culture system were observed. Secondly, MTT assay was used to detect the effect of Aβ₁₋₄₂ on PC12 cells survival rate as well as the intervention effect of β-asarone, and verify the testing results of RTCA. The levels of IL-1β, TNF-α and BDNF in culture media of the lower chamber were detected by ELISA. The NF-κB activity and phosphorylation levels of ERK, p38 and JNK were detected by Western blot. Results showed that β-asarone (55.5 mg•L⁻¹) could significantly slowdown the decline of PC12 cells survival rate caused by Aβ₁₋₄₂-induced RA-h activation (P<0.01), significantly reduce the levels of IL-1β, TNF-α and the phosphorylation levels of ERK, p38 and JNK in culture media of the lower chamber (P<0.01). β-asarone(166.7 mg•L⁻¹) could promote the release of BDNF in culture media of the lower chamber(P<0.05). These results indicated that Aβ₁₋₄₂ could induce RA-h activation and its release of IL-1β, TNF-α and other inflammatory factors to aggravate the PC12 cells injury; β-asarone could reduce the levels of IL-1β, TNF-α, promote the release of BDNF, and inhibit the NF-κB activity as well as phosphorylation levels of ERK, p38 and JNK protein in PC12 cells.
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Aim To establish an in vitro early drug cardiac tox-icity evaluation method by human embryonic stem cells derived cardiomyocytes ( hESC-CM) and real-time cell analysis Cardio (RTCA Cardio) system. Method The hESC-CM were cultured at RTCA Cardio E-Plate 96. Impedance signals from hESC-CM were analyzed for beating rate, contraction amplitude and beating rhythm irregularity to determine the optimum inoculation density and detection duration. Based on this, we used 0. 1 % DMSO to be the solvent and quinidine (0. 2, 0. 78, 3. 13, 12. 5, 50 and 100 μmol·L - 1 ) known as affecting cardiac activity to validate this method. Result The results revealed no significant changes in the cell index (CI), transient pulse patterns, beating rate and amplitude of hESC-CM. Quinidine will affect the CI and transi-ent pulse patterns of hESC-CM and decrease the beating rate and amplitude of hESC-CM when its concentration ≥3. 13 μmol · L - 1 . And this effect is concentration-dependent, the higher the concentration,the more time they need to recover beating and the more significant the beating rate and amplitude inhibition of quinidine on hESC-CM. Conclusion The method established by hESC-CM and RTCA Cardio system can detect the effect of quinidine on the contraction of hESC-CM, and this indicates that this method has the potential to be an attractive high-throughput tool for screening potential drugs in early evaluation of drug car-diotoxicity.
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OBJECTIVE: To provide reference for further application of RTCA in the field of TCM(traditional Chinese medicine) development, and summarize the RTCA technology application progress in recent years. METHODS: The RTCA technology principles, characteristics and applications are summarized based on referring to the related literature in recent years. RESULTS AND CONCLUSION: RTCA is a kind of unmarked, non-invasive real-time cell electronic analysis method. It is recently used in drug screening, toxicology evaluation, cell growth, division and death mechanism research. The dynamic real-time analysis of cell is more and more concerned. The RTCA technology application prospects in the field of traditional Chinese medicine and compound are demonstrated. It is hoped to provide reference for further application of RTCA in the field of pharmaceutical development.