Analysis on chemical constituents from Glycyrrhizae Radix et Rhizoma by HPLC-Q-TOF-MS / 中草药

Objective: To analyze the main chemical constituents of Glycyrrhizae Radix et Rhizoma by HPLC-Q-TOF-MS in the positive and negative ions mode. Methods: The chromatographic fingerprint was obtained with Diamonsil II C18 column (250 mm×4.6 mm, 5 μm) and gradient elution with 0.05% H2O-formic acid (A)-acetonitrile (B), and the flow rate was 1 mL/min. The column temperature was maintained at 35℃. The detection wavelength was of 200-600 nm. Positive and negative ions MS information of Bruker Daltonics 1200 HPLC-Q-TOF was coupled with elemental analysis and compared with the literature data to analyze the compounds information. Results: Combined with the accurate relative molecular mass of compounds provided by HPLC-Q-TOF-MS, 40 compounds were identificated in the ethanolic extract from Glycyrrhizae Radix et Rhizoma, which include 28 flavonoids, 11 triterpenoids saponins, and one coumarin. Conclusion: The method provides the technical support for the quality control of Glycyrrhizae Radix et Rhizoma, and contributes the reference data to elucidating the potential basis of Glycyrrhizae Radix et Rhizoma

Study on chitosan-modified tripterygium glycoside nanoparticles and its renal targeting property / 中国中药杂志

<p><b>OBJECTIVE</b>To prepare chitosan-modified tripterygium glycoside nanoparticles (LMWC-TG-PLA-NPs), and assess its renal targeting property in rats.</p><p><b>METHOD</b>Chitosan-modified tripterygium glycoside nanoparticles (LMWC-TG-PLA-NPs) were prepared by modified spontaneous emulsification solvent evaporation method, and modified with 50% deacetylated low molecular weight chitosan (LMWC). The shape of nanoparticles was observed under a transmission electron microscope. The mean diameter of nanoparticles was measured by particle size analyzer. The drug encapsulation efficiency and drug loading were measured by centrifuge method. The in vitro release behavior was studied with dialysis bags. Renal microdialysis technique and renal artery administration technique were combined to study the renal targeting property of nanopartcles. LMWC-TG-PLA-NPs were administrated in rats by tail vein injection (TVI) and renal artery administration (RAA), respectively, with TG-PLA-NPs as the control group. Renal dialysis fluid was regularly collected to determine the drug concentration in the dialysis fluid, map drug concentration-time curves, and calculate AUC ratio in kidneys through the two injection approaches as the renal targeting parameter (RTP), in order to assess the renal targeting property of LMWC-TG-PLA-NPs.</p><p><b>RESULTS</b>The prepared LMWC-TG-PLA-NPs looked smooth and round. Their average diameter, polydispersity index, encapsulation efficiency and drug loading were (207.6 +/- 3.4) nm, (0.078 +/- 0.009)%, (61.83 +/- 2.43)%, and (10.70 +/- 0.37)%, respectively. The pH 7.4 PBS buffer solution containing 20% ethanol showed obvious sustained release behavior. LMWC-TG-PLA-NPs showed a RTP of 71.97%, which was 3.6 times of TG-PLA-NPs of the control group.</p><p><b>CONCLUSION</b>The prepared LMWC-TG-PLA-NPs showed high drug encapsulation efficiency and drug loading, with obvious sustained release characteristics and renal targeting property. LMWC-TG-PLA-NPs are expected to become a new type vector for reducing toxic and side effects of tripterygium glycoside. Meanwhile, a new method is established for assessing renal targeting property with AUC ratio in kidneys after administrated through caudal veins and renal arteries as the renal targeting parameter.</p>

Effects of blocking inhibitory KIR receptors on cytotoxic activity of human NK cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of blocking the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 with monoclonal antibody on cytotoxic activity of human NK cells.</p><p><b>METHODS</b>Human peripheral blood NK cells were isolated by Rosettesep NK sorting kit. The cytotoxic activity of NK cells against human leukemia NB4, K-562, Raji cells and allogeneic mature or dendritic cells (DCs) was detected before or after KIR2DL1 and KIR2DL2/2DL3 were blocked. The effect of NK cells on T lymphocyte proliferation was detected by mixed lymphocyte reaction and TGF-β1 concentration in culture supernatant was measured.</p><p><b>RESULTS</b>The cytotoxicity of NK cells to NB4 cells was augmented with increasing concentration of the antibody. Combination of both antibodies enhanced killing activity of NK cells. NK cells had strong cytotoxicity to K-562 cells, but were not enhanced by the blockade of inhibitory receptors. The cytotoxicity to Raji cells was not evidently augmented. The cytotoxicity of NK cells to mature DC was enhanced remarkably with the increase of concentration of the antibodies (2.20% ±1.10% compared with 37.59% ±5.06%, P<0.05). In mixed lymphocyte reaction, the blockade of two antibodies enhanced the inhibition effect of NK cells on T cell proliferation (77.85% ± 8.31% compared with 43.05% ± 5.95%, P<0.05) and the content of TGF-β1 in the supernatant was increased.</p><p><b>CONCLUSION</b>The cytotoxic effects of human NK cells against target cells were significantly enhanced with the blockade of inhibitory KIR receptor; and the cytokine TGF-β1 secreted by NK cells further inhibits T cells proliferation.</p>

Synergistic cytotoxic effects of rapamycin and idarubicin on human acute T-cell lymphoblastic leukemia Jurkat cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the cytotoxic effects of mTOR inhibitor rapamycin (Rapa) and idarubicin (IDA) on human T-cell acute lymphoblastic leukemia Jurkat cell line.</p><p><b>METHODS</b>The proliferation of Jurkat cells was observed by CCK-8 assay. The combined index was analyzed by Isobologram method. Apoptosis was detected by electron microscopy and flow cytometry with Annexin V/PI staining. Protein expressions of Caspase 3, PARP, Caspase 8, Caspase 9, Akt, p-Akt, P85S6K, p-P85S6K, P70S6K, p-P70S6K, ERK1/2 and p-ERK1/2 were determined by Western blotting.</p><p><b>RESULTS</b>The IC(50) of IDA for Jurkat cells was significantly reduced when combined with 10 nmol/L rapamycin. The combined index was <1. Both electron microscopy and Annexin V/PI staining flow cytometry revealed that rapamycin significantly increased apoptotic sensitivity to IDA. The combination of IDA with rapamycin enhanced the expressions of Caspase 3, PARP, Caspase 8 and Caspase 9. Rapamycin significantly inhibited mTOR signaling upstream Akt and downstream S6K activation triggered by IDA, and the combination of the two agents led to synergistic inhibition of ERK phosphorylation.</p><p><b>CONCLUSION</b>Combination of IDA with rapamycin exerted a synergistic anti-proliferative effect and promoted apoptosis by both extrinsic and intrinsic apoptotic pathways in Jurkat cells. Inhibition of ERK phosphorylation and mTOR signaling by rapamycin may play a certain role in this synergistic effect.</p>

Polysorbate-80 modified neurotoxin nanoparticle with its transport and cytotoxicity against blood-brain barrier / 药学学报

This study was aimed at the transport across blood-brain barrier (BBB) of polysorbate-80 modified neurotoxin loaded polybutylcyanoacrylate nanoparticle (P-80-NT-NP) and its cytotoxicity. An in vitro model of BBB using rat brain microvascular endothelial cells (rBMECs) was established. The cytotoxicity of P-80-NT-NP was measured by the MTT assays, where neurotoxin (NT), nanoparticle (NP), neurotoxin nanoparticle (NT-NP) as control, and the permeability of P-80-NT-NP was determined by using of Millicell insert coculture with rBMECs and fluorescence spectrophotometry. MTT results showed that NT, NP, NT-NP and P-80-NT-NP were avirulent to rBMECs when the concentration of NT was lower than 200 ng x mL(-1). But the cytotoxicity of NP, NT-NP and P-80-NT-NP would be augmented accordingly as concentration increased (P < 0.01), causing obvious reductions of cell survival rate, with no significant difference between them (P > 0.05). When the concentration of NT was 150 ng x mL(-1), the permeability on rBMECs of P-80-NT-NP and NT-NP were both significantly higher than that of NT (P < 0.01), and the permeability of P-80-NT-NP was greater than that of NT-NP (P < 0.05). In conclusion, polysorbate-80 modified neurotoxin nanoparticles can transport across the BBB, while concentration of NT is greater than 200 ng x mL(-1), P-80-NT-NP has a little cytotoxicity against rBMECs.

Celecoxib-induced apoptosis in acute promyelocytic leukemia cell line MR2 and its mechanism / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate celecoxib-induced apoptosis of acute promyelocytic leukemia cell line MR2 and related mechanism.</p><p><b>METHODS</b>MR2 cells were treated with celecoxib at different concentrations (0, 20, 40, 80, 120 and 160 micromol/L). The proliferation of MR2 cells was observed by MTT assay and apoptosis was detected by DNA fragmentation analysis and flow cytometry with Annexin V-FITCïPI staining. The expression of survivin and PML/RARalpha mRNA was examined by RT-PCR and nested-PCR, and the protein expression of caspase-3, 9 and PARP was analyzed by Western-blot.</p><p><b>RESULTS</b>After treatment with celecoxib the viability of MR2 cells decreased markedly in a dose- and time-dependent manner, and a DNA ladder pattern of internucleosomal fragmentation was observed. The translocation of phosphatidylserine at the outer surface of the cell plasma membrane was induced by celecoxib and its level increased following the augmentation of the drug concentration. The expression of survivin mRNA decreased dramatically while no significant change with PML/RARalpha. Treatment with celecoxib for 24 h resulted in the activation of caspase-3 and 9, cleavage of PARP.</p><p><b>CONCLUSION</b>Celecoxib could inhibit MR2 cell proliferation by inducing apoptosis, which might be mediated by the caspase-3 and 9 activation and PARP cleavage. Moreover, the down-regulation of survivin may play a certain role in apoptosis of MR2 cells induced by celecoxib.</p>