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AIM: To investigate the effects of isonlosinine on proliferation, invasion, migration and autophagy of PC9 cells in non-small cell lung cancer (NSCLC), and to explore its possible molecular mechanism. METHODS: The effect of Isoliensinine on the proliferation of PC9 cells were measured by CCK-8 assay, and the IC50 value of PC9 cells was calculated. Wound healing and transwell experiments were used to study the effect of Isoliensinine on migration and invasion of PC9 cells in vitro, respectively. The formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The expression levels of LC3, pERK and ERK in the PC9 cells were determined by western blot. RESULTS: Isonlosinine significantly inhibited the proliferation of PC9 cells. IC50 of isonlosinine (24 h) for the PC9 cells was 34.11 µmol / L. Isonlosinine significantly inhibited cell migration and invasion of PC9 cells. The results of acridine orange fluorescent staining showed that the number of the intracellular acid dye follicular bright red fluorescence in PC9 cells was significantly increased after isonlosinine treatment, while the autophagic lysosomes were rarely observed in control group. The expression of LC3-II in PC9 cells was significantly enhanced after isonlosinine treatment. Furthermore, molecular mechanism study showed that isonlosinine could activate the expression level of p-ERK. CONCLUSION: Isoliensinine significantly inhibits the proliferation, migration and invasion, and induces autophagy of PC9 cells, which may be correlated with the activation of ERK signaling pathway.
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Objective To establish a high-throughput evaluation model for anaphylactic reactions; To screen and identify potential anaphylactogens from TCM monomeric compounds.MethodsCell model of stably expressed MrgX2 was established. Recombinate plasmid pmCherry-C1-MrgX2 was transfected to HEK293 to establish cell line for screening model. MrgX2 agonist and antagonist were used to identify the validation and stability of the cell line. A small library consisting of 180 compounds was profiled by using a cell-based calcium mobilization assay to find novel compounds targeting the MrgX2 receptor. EC50 test, IC50 test, specificity validation and cytotoxicity evaluation were carried out to detect the function of the positive agonist.ResultsThe EC50 of C48/80 to MrgX2 model was 2.7 μg/mL and the IC50 of 2-APB (evoked by 10 μg/mL C48/80) was 46.29 μmol/L. The first generation cell model of MrgX2 was similar to the 20th generation, and the Z factor of MrgX2 cell model was 0.78. In the primary screening for agonist, isoliensinine was identified as a novel agonist targeting receptor MrgX2 with an EC50 of 4.5 μmol/L and IC50 of39.47 μmol/L. Moreover, isoliensinine was validated to activate MrgX2 receptor specifically without cytotoxicity. Conclusion A high-throughput evaluation method for anaphylactic reactions can be established in vitro through calcium mobilization assay. A potential anaphylactogen isoliensinine is identified and validated.
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Objective:To compare the inhibitory effect of neferine and isoliensinine on 5α-reductase to provide reference for the development of 5α-reductase inhibitors. Methods: Different reaction tubes and control tubes were prepared, liver was homogenated, and reducing coenzyme II ( NADPH) , testosterone, pending test sample, the positive drug and buffer was respectively added into 96-well plates. The change in the absorbance of NADPH at 340nm in 1h was determined by a microplate reader. Compared the experimen-tal group with the blank control group, the inhibition rate ( I%) of the test drugs against 5α-reductase was calculated. Results:As for the six concentration gradients (2-40 mg·ml-1 ) in the experiments, the best inhibitory concentration of neferine and isoliensinine was 10 mg·ml-1(I% =25.00 ±1.030% and 29.90 ±2.410%, respectively). Compared with the control group, neferine and isoliensi-nine showed significant inhibition against 5α-reductase (P<0. 05). Compared with the positive group at the same concentration (10 mg ·ml-1), the inhibition of neferine and isoliensinine was significantly lower (P<0. 05). The inhibition effect of isoliensinine was rel-atively better than that of neferine (P<0. 05). Conclusion:Neferine and isoliensininein have notable inhibitory effect on 5α-reduc-tase, which show certain application prospect in the treatment of prostatic hyperplasia in clinics.
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Objective To observe protective effects of four active liposoluble alkaloids of a Chinese herb, lotusine (Lot), liensinine (Lien), isoliensinine (Iso) and neferine (Nef) of embryo loti (the green embryo), against H2 O2-induced oxidative damage on human umbilical vascular endothelial cell ECV-304.Methods The protective effects of Lot, Lien, Iso and Nef on the survival of normal and oxidatively damaged ECV 304 cells were studied by cell morphology observation and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) assay.The levels of nitric oxide (NO) and nitric oxide synthase ( NOS) were measured using colorimetric assay.Results Lot, Lien, Iso and Nef did not affect cell morphology and cell viability of normal ECV 304 cells.The survival of oxidative damaged vascular endothelial cells was rescued by incubating with Lot at 100μmol/L, and Lien, Iso and Nef at 0.1 μmol/L.The proliferative activity of medicated groups increased to 112.8%, 129.3%, 125.6 and 118.2%, respectively (P<0.01 or 0.05), relative to that of the group with H2O2 induced oxidative damage.The four alkaloids restrained oxidative injury of endothelial cells induced by H2 O2 and the protective influences were similar with captopril, which served as a positive control.Each alkaloid except Lot reduced intercellular space and increased the connections of oxidative damaged cells, concomitant with more recognizable cell borders.Lien, Iso and Nef also increased the concentration of NO ( P<0.05 ) .Besides, all of the four alkaloids activated NOS in damaged vascular endothelial cells ( P<0.05 ) . Conclusion The four alkaloids of embryo loti, especially Lien, Iso and Nef, have certain protective effects against H2 O2-induced oxidative damage on vascular endothelial cells.The protective mechanism may be promotion of NO release through the increase of NOS production.
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AIM To evaluate the beneficial effects of isoliensinine on paraquat(PQ)-induced acute lung injury and pulmonary fibrosis and explore the mechanism of its action. METHODS PQ (45 mg·kg-1, ip)-induced acute lung injury and PQ (100 mg·kg-1, ig)-induced pulmonary fibrosis were prepared. At 8, 24 and 48 h after PQ administration, the effects of isoliensinine (20 mg·kg-1, ig, 3 times a day, from 24 h before PQ administration to the end of experiment) on activity of superoxide dismutase (SOD), alkaline phosphatase (ALP) and the content of malondialdehyde (MDA) in plasma and bronchoalveolar lavage fluid (BALF) of acute lung injury groups were evaluated respectively. On the 14 d following PQ ingestion, the effects of isoliensinine (10, 20 and 40 mg·kg-1, ig, twice a day, from 24 h before PQ administration to the end of experiment) on hydroxyproline content, transforming growth factor β1 (TGF-β1) and matrix metalloproteinase-2 (MMP-2) expressions and the histopathological changes in lung tissues of pulmonary fibrosis groups were observed. RESULTS In the acute lung injury model, isoliensinine (20 mg·kg-1) significantly increased SOD activity, and decreased MDA content and ALP activity, as well as ameliorated the histopathological damage of lung tissue compared with PQ group. However, the indexes mentioned above in isoliensinine alone group did not change obviously compared with normal saline group. In the pulmonary fibrosis model, isoliensinine (10, 20 and 40 mg·kg-1) resulted in a dose-dependent decrease of hydroxyproline content compared with PQ group [(2.11±0.21), (1.94±0.24) and (1.89±0.26), respectively, vs (2.44±0.33) mg·g-1 wet tissue]. The expressions of TGF-β1 and MMP-2 in the lung tissue of the isoliensinine 40 mg·kg-1+PQ group were significantly less than those of the PQ group. Furthermore, isoliensinine could improve the histopathological changes of fibrosis as comparison with PQ group. CONCLUSION Isoliensinine has protective effects on PQ-induced acute lung injury and pulmonary fibrosis.
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Background Isoliensinine(IL) is a kind monomer alkaloid of double benzyl group quinoline separated from Plumula nelumbinis,which has been speculated to have antiarrhythmic effect,Ca~(2+) and?1-receptor blockade, and reversal the left ventricular hypertrophy(LVH).It has been reported the activity of Sarco/Endoplasmic reticulum Ca~(2+)-ATP_(ase)(SERCA) in essential hypertention(EH)/LVH patients was lower than that of healthy people. Objective To investigate the effects of Isoliensinine on left ventricular hypertrophy and activity of SERCA in 2 kidney 1 clamp(2K1C) hypertensive rats.Methods 2K1C hypertensive rats were randomly received Isoliensinine (RHR-IL,5 mg/kg per day p.o,n=16) or placebo.Blood pressure,left ventricle mass index(LVMI) and the activity of myocardial SERCA were measured after 10 weeks treatment.Six untreated SD rats were served as control. Results Isoliensinine decreased BP and LV/BM ratio(IL:136.4?14.6 vs control:189.8?4.4 mm Hg,P
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Aim To investigate the inhibitory effects and mechanism of action of isoliensinine (IL) on the proliferation of porcine coronary arterial smooth muscle cells (CASMCs) induced by phenylephrine (Phen) and its mechanisms of action. Methods MTT assay, immunohistochemical method and Western blotting were adopted. Results IL (0.03-3 μmol·L-1) could inhibit the CASMCs proliferation induced by Phen (0.1 μmol·L-1) in a concentration-dependent manner. IL (0.1 μmol·L-1) antagonized Phen-induced overexpression of PDGF-β and bFGF from 0.545±0.026 and 0.47±0.03 to 0.458±0.019 and 0.376±0.017 (P<0.01, P<0.01). IL (0.1 μmol·L-1) also decreased c-fos, c-myc and hsp70 overexpression induced by Phen from 0.57±0.04, 0.44±0.04 and (173±36)% to 0.46±0.05, 0.372±0.021 and (115±35)% respectively (P<0.01, P<0.01, P<0.01). Conclusion IL exerted antiproliferative effect on CASMCs induced by phenylephrine, and its mechanisms were related to decrease the overexpression of growth factors (PDGF-β, bFGF), protooncogene (c-fos, c-myc) and hsp70.
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Antiarrhythmic effect of isoliensinine (IL) was studied on experimental arrhythmic mo-dels induced by coronary artery ligation and 4 different arrhythmogenic drugs in comparison with quinidine(Qu). Results of the study showed that the antiarrhythmic potency of IL was stronger than that of Qu atthe same dosage. The effects of IL on fast and slow response action potentials of myocardium were ob-served in guinea pig papillary muscles by standard microelectrode technique, which showed that IL couldreduce APA and Vmax and shorten the APD50. The results suggested that the antiarrhythmic mechanismof IL is related to its non-specific inhibition of the currents of Na+ and Ca2+.
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Objective To investigate the adsorption function of macroporous adsorption resins for the separation and purification of the effective components in Plumula Nelumbinis. Methods AKS-W, AB-8, and H214 resins were systematically studied for their adsorption capability, and the contents of the liensinine, isoliensinine, and neferine were determined by HPLC. Results AKS-W Resin had a preferable adsorptive and separateive effect for the effective components in Plumula Nelumbinis. The products containing 10.2% liensinine, 6.7% isoliensinine, and 11.9% neferine could be obtained. Conclusion Macro-porous-type and non-polar adsorption resin AKS-W is an ideal adsorbent for the extraction of the effective components in Plumula Nelumbinis.