| 1. Objective: This research project investigated the herb, Smilax Glabra Roxb. (tufuling), and its ability to induce apoptosis of the hepatic carcinoma HepG-2 cell line in vitro. Its effective molecular mechanism was observed in order to find experimental evidence for its curative effect in treating hepatic carcinoma.2. Methodology: This project employed a cytopharmacological approach to research the apoptotic effect of Smilax Glabra Roxb. on HepG-2 and the correlating control genes Bax, Bc 1-2, and Fas/FasL. We used the following major indicators and methodology:2.1 Investigating the toxological effect of Smilax Glabra Roxb. extract on the HepG-2 cell line: A 0.06% solution of parenzyme was used to prepare the HepG-2 cells as a single cell suspension. It was concentrated to 3x105/ml using a DMEM culture containing 10% fetal calf serum to attain a cell suspension. The cells were inoculated in a 96 pore plate at 0.1 ml/pore and cultivated in an incubator at 37℃, with saturated humidity and 5% CO2. Cell adherence occurred after 24 hrs. After 2 days the culture medium was changed to contain an herbal solution at 0.1 ml/pore with 3 pores per concentration. Multiple proportions of diluted Smilax Glabra Roxb. aqueous extract stock solution were divided into eight concentrations (50g/L, 25g/L, 12.5g/L, 6.25g/L, 3.13g/L, 1.56g/L, 0.78g/L, and 0.39g/L); likewise multiple proportions of diluted Smilax Glabra Roxb. alcohol extract stock solution were divided into to seven concentrations (50g/L, 25g/L, 12.5g/L, 6.25g/L, 3.13g/L, 1.56g/L, and 0.78g/L). The cells continued to be cultivated in a 37℃, humidity saturated, 5% CO2 incubator. A control group containing no herbal extract was also set. After 12 days the supernatant was abscised and the herbal cytotoxicity was detected by MTT.2.2 The effect of Smilax Glabra Roxb. extract on the growth curve of the HepG-2 cell line: Logarithmic phase cells were obtained and prepared at a density of 1x104/ml single cell suspension by DMEM culture solution containing 10% fetal calf serum. Cells were inoculated in 24 pore plates at 1ml per pore. Cell adherence occured after 24 hours at which time a complete culture medium was used to dilute a Smilax Glabra Roxb. aqueous extract to 22g/L and 11g/L and a Smilax Glabra Roxb. alcohol extract to 11g/L and 5.5g/L. A control group used 2% DMEM as a substitute for the aqueous and alcoholextracts. The cells were cultivated in an incubator at 37℃, with saturated humidity, and 5% CO2. Samples of four pores were taken instantly after incubation and after the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th day. After cellula digestivum we took 40μl samples per pore and added 10μl of 0.4% trypanblue staining solution, placed them on a hemocytometer and the counted viable cells which resisted staining. This then was plotted graphically against culture time.23 The influence of Smilax Glabra Roxb. aqueous extract on the cell cycle of HepG-2 cells: Logarithmic phase cells were acquired. These were prepared into a single cell suspension by using 0.06% parenzyme and light mixing. They then were stained with 0.4% trypanblue. Viable cells were counted by hemocytometer. The experiment proceeded when the viable cell ratio exceeded 95%. 1) The cell suspension was diluted to 105/ml with DMEM culture solution containing 10% fetal calf serum and was inoculated in 50cm2 culture flasks. Each flask contained 5ml and were incubated at 37℃ with saturated humidity, and 5% CO2 for 24 hours. At this point they were ready for treatment. 2) Herb group: Smilax Glabra Roxb. aqueous extract was added to the culture solution attaining final concentrations of 22g/L and 11 g/L. Control group: identical volumes of 2% DMEM were added into the control group solution. Action time was 48 hours with each group having 3 bottles. DNA staining: the culture solution was discarded and the cells were washed twice using Hanks detergent. They then underwent 0.06% trypsinization for 3 minutes whereupon they were lightly mixed to make a cell suspension and placed in a 300rpm centrifuge for 5 minutes. Supernatant was then removed. The cells were resuspended with lml of cold alcohol, lightly mixed and fixed at 4℃. Fixation time exceeded 24 hours. After fixing, they were centrifuged at a 300rpm for 5 minutes whereupon supernatant was removed. A lml RNA enzyme with a 50ug/ml density was added and lightly mixed. They were protected from hght for 30 minutes at room temperature. Cell RNA were removed. They then underwent another 5 minutes of centrifuge at 300 rpm after which supernatant was removed and lml of PI at a 60ug/ml concentration was added. They were lightly mixed and protected from hght for 30 minutes at room temperature. FACS detection: The samples finally were detected using FACS and analysed with ModFit software which calculated the percentage of cells at each phase.2.4 The influence of Smilax Glabra Roxb. extract on HepG-2 apoptosis: 2.4.1 Observing DNA degradation fragmentation by DNA Ladder: DNA extraction: HepG-2 logarithmic phase cells were co-cultured with 2% DMEM and Smilax Glabra Roxb. aqueous extract at concentrations of 22g/l and llg/L for 48 hours. They were each rinsed twice with PBS after which DNA was extracted using routine methods.Detection method: detection of the DNA Ladder was accomplished using agarose gel electrophoresis.Discussion of results: The genome straps of normal living cell DNA were located close to the well. Dead cells showed a single band continuous hymeno-form strap due to its irregular DNA degradation. Apoptosis corpuscular DNA showed a ladder strap on account of its oligonucleotide fragment created from DNA degradated to 180 bp or its multimer.2.4.2 Observing the influence of Smilax Glabra Roxb. aqueous extract on HepG-2 apoptosis by flow cytometry:Cultivation and treatment of HepG-2: Logarithmic phase cells were acquired and prepared into single cell suspension after digestion using 0.06% parenzyme and stained with 0.4% trypanblue. The viable count ratio of cells resisting stain was 97.5%. The cells were inoculated in 50cm2 culture flasks with 106 cells per flask and cultured in a 10% fetal calf serum DMEM solution with 10% FCS and lOOU/ml mycilhn. They were incubated at 37°C with saturated humidity at 5% CO2 for 24 hours after which the culture solution was changed. Herbal group: Smilax Glabra Roxb. aqueous extracts of 22g/L and llg/L were added to 5ml of cultured solution. The control group used equal amounts of 2% DMEM to replace the Smilax Glabra Roxb. aqueous extracts. 3 flasks of cells from each group were obtained 24 and 48 hours later and we proceeded to detect apoptosis.Detection of apoptosis (methodology): The culture solution was abscised carefully, trypsinized for 3 minutes using 0.06% parenzyme and lightly mixed creating a single cell suspension from the divided HepG-2 cell line. This was centrifuged for 10 minutes at lOOOrpm whereupon the cells were resuspended to lxlO6 per ml concentration using Binding Buffer. 100(4.1 of HepG-2 cell suspension was placed into 5ml FACS tubes into which was added 5ul Annexin V-FITC, lOul F and misce bene. 400ul Binding Buffer per tube was added after incubating for 15 minutes at room temperature away from light. Within one hour, flow cytometry was performed. Simultaneously, control pipes with adelomorphous cells and a compensation installation pipe with unstained cells, a simple stained Annexin V-FTT pipe and a simple staining PI pipe were set.FCM flow cytometry analysis: (Beckman Coulte, U.S.A.) ALTRA-type FACS Caliber flow cytometer was used with an excitation light source of 488nm. The control pipe was first placed as a sample to obtain a distinct, localized cell colony in the FSC/SSC by adjusting the FSC and SSC parameters. A gate was set for the cell colony after which a continuous fluorescent signal detection was applied to the cells in the gate. We set the fluorescence intensity of the control pipe (null cells) for non-specificity background fluorescence. This established the base for determining the expression ratio of the positive fluorescence. The upflow cytometer was calibrated to cause the fluorescence scatter of the null cells in the graph to concentrate in the left lower quadrant. Without altering, FL1, FL2, and FL3 the simple staining AV-FITC pipe and simple staining PI pipe were placed as samples. Fluorescence compensation was adjusted and the samples were analyzed after calibrating the optical spectrum overlay. 10,000 cells were obtained from each sample and analyzed using CellQuest software. 2.5 The influence of Smilax Glabra Roxb. aqueous extract on the apoptosis controlling genes, Bax and Bcl-2: Cells slices were divided into groups. Sterile glass slides were inserted in each pore of the 24-pored cultivation plate. HepG-2 logarithmic phase cells were acquired and then digested and prepared into a cell suspension. Cell density was adjusted to lxlO5 per ml and inoculated with 1 ml/pore in the 24 hole cultivation plate. They were then placed in the incubator for 24 hours after which Smilax Glabra Roxb. extract was added into the herbal group at final concentrations of 22g/L and 1 lg/L. Equal amounts of 2% DMEM were added into the control group. Each grouphad three pores set in parallel and cultivation resumed for 36 hours. The glass slides which were full of cells were dislodged and rinsed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes and air dried after rinsing with PBS. They were then preserved for later use in a -20°C refrigerator. Detection was performed according to instructions provided with the kit. The main steps were as follows: 50ul peroxydase blocking agent was dropped in and left at 37°C for 10 minutes. They were then rinsed 3 times with PBS after which 50ul of normal non-immune goat blood serum was dropped in and left for 10 minutes at 37 °C. They were dried directly and the first Bax and Be 1-2 antibodies were added separately and left at 37°C for 2 hours. They were then rinsed three times with PBS after which the second biological tag antibody was dropped in and left at 37°C for 10 minutes. After rinsing three times with PBS, streptomycete antibiotic albumen- peroxydase solution was dropped in and left at 37°C for 10 minutes. They were again rinsed with PBS 3 times after which freshly prepared DAB colorate coloration solution was dropped in and the coloration effect was observed under light microscope. When the coloration manifested they were rinsed with tap water and counterstained with hematoxylin. They were then fixed with neutral resin and photographed. 2.6 The effect of Smilax Glabra Roxb. on the Fas/FasL expression on HepG-2:Cells preparation: Logarithmic phase cells were acquired and prepared as a lxlO4/ml concentration of single cell suspension with 10% fetal calf serum DMEM culture solution. The cells were inoculated in 24-pore plates at 1 ml/pore. Cell adherence occurred after 24 hours. Smilax Glabra Roxb. aqueous extract was diluted to concentrations of 22g/L and llg/L using complete culture medium. The control group used 2% DMEM to replace the Smilax Glabra Roxb. aqueous extract. Cells were cultured in a 37°C, humidity saturated, 5% CO2 incubator.Relative expression variation of Fas/FasL mRNA: The cell's total RNA of the 3 groups (the control group and the 22g/L and llg/L groups with Smilax Glabra Roxb. aqueous extract) were separately extracted and reverse transcripted into cDNA. lul of the reverse transcription reaction product was takenas a mold and the Fas gene fragment and intra-reference P-actin were coamphfied. PCR reactive condition: 35 cycles were performed of 94oC force-denature for 5 minutes, 94oC denature for 45 seconds, 64oC anealing for 30 seconds, and 72oC extension for 1 minute. The amplified product was ionophoresised in 1.8% sepharose gel, and its brightness scanned in a gel imaging system. The relative value of the brightness of the Fas gene to the intra-reference P-actin gene acted as a comparative contrast among the groups. The same experiment was repeated 3 times, lul of reverse transcription reaction product was obtained as a mold and the FasL gene fragment and the intra-reference P-actin were coamplified. The PCR reactive condition: 38 cycles were performed of 94oC denature for 5 minutes, 94oC denature for 45 seconds, 58oC of anealing for 30 seconds, and 72oC extension for 1 minute. The amplified production was ionophoresised in 1.8% sepharose gel, and its brightness scanned in a gel imaging system. The relative value of the brightness of the FasL gene to the intra-reference P-actin gene acted as a comparative contrast among the three groups. The same experiment was repeated 3 times. 3. Results:3.1 The effect of Smilax Glabra Roxb. extract on the proliferation of the HepG-2 cell line: The TC50 of the eight set Smilax Glabra Roxb. aqueous extract densities of 50g/L, 25g/L, 12.5g/L, 6.25g/L, 3.13g/L, 1.56g/L, 0.78g/L, and 0.39g/L was 22.07g/L. The rates at which they destroyed the HepG-2 densities of 50g/L, 25g/L, 12.5g/L, 6.25g/L, 3.13g/L, 1.56g/L, 0.78g/L, and 0.39g/L were, respectively, 64%, 59%, 9%, 14%, -6%, 38%, 70%, and 70%; the TC50 of the Smilax Glabra Roxb. alcohol extract was 11.32 g/L for its seven setting densities of 50g/L, 25 g/L, 12.5 g/L, 6.25 g/L, 3.12 g/L, 1.56 g/L, and 0.78g/L. The rates at which they destroyed the HepG-2 cell line were 57%, 65%, 53%, 32%, 20%, 21% and 3%.Cells manifested vacuoles, atrophy, and abscission with a destruction rate of 64% and 57% with herbal aqueous and alcohol extract concentrations of 50g/L; At concentrations of 12.5% g/L (aqueous extract) and 6.25%g/L (alcohol extract), the cell structure generally appeared normal under the microscope.3.2 The effect of Smilax Glabra Roxb. extract on the growth curve of the HepG-2 cell line: There was rapid growth in the cells of the control group. During days 0-7 the cell populations were 1, 1.8, 3.5, 6.9, 11.8, 20.3, 31.7, and 42.4 105/ml. Comparatively, the cell populations at 0-7 days were 1, 1.8, 1.8, 1.2, 0.7, 0.4, 0.4, and 0.2 105/ml with the Smilax Glabra Roxb. aqueous extract of 22g/L and 1, 1.8, 1.9, 1.5, 0.9, 0.7, 0.5, and 0.4 105/ml at a density of llg/L. Cell proliferation was slow for these two extract concentrations. The cell populations from 0-7 days of the Smilax Glabra Roxb. alcohol extract at a density of llg/L were 1,1.8, 3.2,6.5, 9.7,16.3, 22.7, and 38.5 105/ml. At a density was 5.5g/L, the cell populations were 1, 1.9, 3.1, 6.3, 10.4, 17.8, 25.7, and 41.3 105/ml. These two alcohol extract concentrations displayed rapid cell proliferation. In sum, Smilax Glabra Roxb. aqueous extract (22g/L, llg/L) inhibited the growth of the HepG-2 cell line, while the alcohol extract (llg/L, 5.5g/L) had no obvious inhibition on cell proliferation.3.3 The effect of the Smilax Glabra Roxb. aqueous extract on the HepG-2 cell cycle: A high dose of Smilax Glabra Roxb. aqueous extract showed a marked change on the HepG-2 cell cycle after 48 hours. The majority of the cells (66.22% of the total) were in G0-G1 phase. This was more than the control group (58.43%, P<0.05). 24.1% of the total cells were in the S phase which was less than the control group (26.47%, P<0.05). Cells in the G2-M phase were 9.68% of total cells which was less than the control group (15.1%). Cell cycle analysis showed that Smilax Glabra Roxb. aqueous extract stopped the cells at the GO/Gl phase. Although there was no significant difference between the Smilax Glabra Roxb. low dose group and the control group, the low-dose group displayed an effect trend similar to the high dose.3.4 The effect of Smilax Glabra Roxb. extract on the apoptosis of HepG-2 cells: A DNA ladder electrophoretic analysis the HepG-2 cell's degraded condition of the DNA fragment. The result showed the obvious manifestation of a ladder molecule strap of the DNA in the HepG-2 treated with Smilax Glabra Roxb. aqueous extracts of 22g/L and llg/L concentrations. Flow cytometry analysis: a DNA histogram displayed a hypodiploid peak in front of the Gl peak (Sub-Gi peak), which is the apoptotic peak formed by the apoptotic cells. The effect of Smilax Glabra Roxb. aqueous extract at a high dose for 24 and 48 hours, had apoptotic rates of 5.37% and 25.34% while a small...
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