Cynanchum Paniculatum Induced Experimental Study Of The Human Hepatoma Cell Hepg-2 Apoptosis

2. ObjectiveTo search an effective therapy of hepatocellular carcinoma, we detect the cellarapotosis of the Hep-G 2 cells induced by Radix Cynanchi Panicullati (Xuchangqing), andstudy the molecular mechanism of Radix Cynanchi Panicullati for the basic experimentaldata.2. Methods:We study the responses of the relative cellar decease genes affected by the RadixCynanchi Panicullati with pharmacological methods. The parameters and methods asfollowing:2.1 the toxic effects of Radix Cynanchi Panicullati on Hep-G2:Detach the cells with 0.06% trypsin solution, suspend the cells and adjust theconcentration to 3×10⁵/ml with completed growth medium. Inocultate the cells with0.1ml/well on the 96-well plastic culster, culture in 37℃,saturated humidity,5% CO₂. 48hours later, remove medium and add fresh medium contained with Radix Cynanchi Panicullatiwith 0.1ml/well, 3 wells per diluted concentration. The aqueous abstract of Radix CynanchiPanicullati is diluted with twofold serial dilutions from 50 to 0.39 g /L; and dilutethe Radix Cynanchi Panicullati abstracted by ethanol same as above, culture in 37℃,saturated humidity,5% CO₂ for 12 days, set the normal contrast simutaneously. We evaluatethe toxic effects of Radix Cynanchi Panicullati on Hep-G2 by MTT method.2.2 the effects on the growth curve of HepG-2 affected by Radix CynanchiPanicullati:When the cells are in the logarithmic growth phase, suspend the cells and adjustthe cells to 10⁴/ml with completed growth medium. Inoculate the cells with 1ml per wellon 24-well plastic cluster. 24 hours later, remove the medium and add fresh maintain mediumcontained with 22g/L and 11g/L of Radix Cynanchi Panicullati abstracted by water, and11g/L and 5.5g/L of Radix Cynanchi Panicullati abstracted by ethanol respectively, Fourwells per diluted concentration. Set the normal contrast simutaneously. Then collect thesupernatant on the 0,1,2,3,4,5,6,7th day, 4 wells per sample, stained with typanblue and caculate the live cells ratio. Acoording to the culture time, draw the cellargrowth curve.2.3 the effects on the cellar cycle of Hep-G2 affceted by aqueous abstractof Radix Cynanchi PanicullatiAfter detaching the cells in the logarithmic growth phase, stain with typan blue. Aftercaculating the live cells ratio(more than 95%) then take the cells into experiment.â‘ suspend the cells and adujst to 1×10⁵/ml with completed growth medium. Inoculate the cellsto 50 cm² flask, culture in 37℃, 5%C02,saturated hunidity for 24 hours.â‘¡experimental groups: 2% DMEM medium contained with 22g/L,11g/L of Radix Cynanchi Panicullatiabstracted by water, set normal contrast group sinmutaneously. After 48hours, terminutesate the assay. DNA staining: remove medium and rinse with Hanks twice.Then detach the cells with 0.06% trypsin, 300g centrifuge for 5 minutes. After removalaqueous phase, suspend the cell with 1 ml of cold ethanol and fixed in 4℃for more than24 hours. Then centrifuge the mixture at 300rpm for 5 minutes, remove liquid phase andadd 1ml RNA enzyme(50μg/ml), aspirate cells in room temperature in dark for 30 minutes.After 300 rpm centrifugation for 5 minutes, remove liquid phase and add 1ml of PI(60μg/ml), aspirate cells in room temperature for 30 minutes. FACS detection: detect thesample with FACS, and analyse the results with ModFit software.2.4 the apotosis effects on Hep-G2 induced by Radix Cynanchi Panicullai2. 4. 1 observe the degradation segment of cellar DNA with electrophoresis:DNA abstract: after 48 hours, abstract the cellar DNA as the protocol applied.Detection method: agarose gel elcctrophoresis.Estimation standar: the strips of normal cells are near the sample well, the DNAof necrotic cells degrade to a serial segments since the anormal DNA decomposed, andthe apotosis cells decompose to 180bp in size or the strips of ladder.2.4.2 detection the apotosis of Hep-G2 induced by Radix Cynanchi Panicullaiwith FACS method:Preparation of HepG-2 cells: suspend the cells in the logarithmic growth phasewith0.06% trypin and stain with 0.4% typan blue. Caculate the live cells ratio (97.5%),then inoculate the ceils into the 50cm² flask, about 10⁶ cells per flask, culture in in37℃, 5%C02,saturated hunidity for 24 hours. Remove the medium and add fresh mediumcontained with Radix Cynanchi Panicullai abstract. Experimental group: the mediumcontained with 22g/L and 11g/L of the Radix Cynanchi Panicullai respective; the normalcontrast group: the medium contained with 2% fetal bovine serum. Take 3 flask at 24th and48th hours each time for apotosis detection.The apotosis detection of cells: remove the medium, detach cellswith 0.06% trypsin and aspirate gentlty. Centrifuge at 1000r/minutes for 10minutes. Remove aqueous phase, suspend cells with Binding Buffer adjusted to1×10⁶/ml. Take 100ul cellar suspension into FACS tube, add 5μl Annexin V-FITCand 10μl F into the mixture, incubate in room temperature for 15 minutes.Add 400 ul BindirBuffer per tube, analyse in one hour. Stain the contrast tube, the supplement tube (non-stained cells), the tube stained with Annexin V-FITonly and the tube stained with PI onlly simutaneously.3. Results3.1 the propagation effects on HepG-2 afftected by Radix Cynanchi Panicullai The toxic assay shows that the TC50 of the Radix Cynanchi Panicullaiabstracted by water is 22.07g/L and the 11.32g/L for ethanol abstract, the detaildata shows as table 3. On the concentration of 50g/L, the cells reveals toxin effects,such as vacuole, shrink and shelling. The destructive rate of Radix Cynanchi Panicullaabstracted by water is 64% for aqueous abstract and 57% for ethanol abstract. On theconcentration of 12.5g/L of aqueous abstract and 6.25g/L for ethanol abstract, the cellsreveal normally as contrast group.3.2 the effects on the growth curve of HepG-2 affected by Radix CynanchiPanicullati:In the experiment, the amount of total cells in the normal contrast group is 1,1.8,3.5,6.9,11.8,20.3,31.7,42.4 x 10⁴/ml during the 7 days. Compared with the normalcontrast, the amount of total cells of Radix Cynanchi Panicullati is mush lower onthe concentration of 22g/L and 11g/L, which shows the aqueous abstract of Radix CynanchiPanicullati has ability of inhibition the propagation of Hep-G2. Whereas the enthanolabstract of Radix Cynanchi Panicullati has no significant difference compared withthe normal contrast on experimental concentration, which shows that the enthanol abstracthas no effects on the propagaton of hepatocellular carcinoma.3.3 the effects on the cellar cycle affceted by Radix Cynanchi Panicullati:The experimental group treated with high dose of Radix Cynanchi Panicullatiabstracted by water for 48 hours, the cycle of HepG-2 changes apparently. The resultsshows that the most of HepG-2 stay on the phase of G0-G1, about 66.22% of the total cells.The figure of normal contrast is 58. 43%, P＜0.05. Compared with the normal control(26.41%), the figure of experimental group is 24.1% on the S phase, which is much lowerthan the normal control, P＜0.05. The total amount of cells in experimental group is 9.68%on the G2-M phase, wheras the figure of normal contrast is 15.1%. The analysis of cellarcycle shows that the abstract of Radix Cynanchi Panicullatii inhibit the propagationof cells on G0/G1 phases. But the low dose of the Radix Cynanchi Panicullati has noeffects on the celler propagation. The results reveals that the dose of Radix CynanchiPanicullati has linear relation with the inhibition effect.3.4 the apotosis effects on Hep-G2 induced by Radix Cynanchi PanicullaiAccording to the DNA ladder elecctrophoresis, the cellar DNA degrade to serialsegments as ladder on the concentration of 22g/L and 11g/L; the analysis of FACS: inthe treatment with high dose of Radix Cynanchi Panicullati for 24 hours, the apotosisrate is 5.37%, and the apotosis rate with the treatment for 48 hours is 25.34%; wherasthe apotosis rate in the treatment with low dose of aqueous abstract for 24 hours is 4.01%, and 18.31% for 48 hours, the blank control is 3.54%, which shows significantly difference. 4. Conclusion:4.1 The aqueous abstract of Radix Cynanchi Panicullai have the ability of inhibitionpropagation on the Hep-G2 cells in vivo, and the toxic effect of the aqueous abstractis much lower than that of ethanol abstract.4.2 Compared with the aqueous abstract of Radix Cynanchi Panicullai, the ethanolabstract has no significant effect on inhibition of cellar propagation.4.3 The abstract of Radix Cynanchi Panicullati can inhibit the cells on the S phasein vivo, prohibite the ceils entering G2-M phase;4. 4 Radix Cynanchi Panicullati can induce the Hep-G2 cell to apotosis.