| 1. Objective:ShuGanQingDuTang is a self-mastery medicinal herb which has a relatively objective therapeutic effect on cancer. This research project used its extract to treat the HepG-2 hepatic carcinoma cell line and investigated in vitro its effective mechanism in fighting hepatic cancer.2. Methodology:2.1 Cell culture:The HepG-2 cell line was separated into a single cell suspension with 0.06% parenzyme. It was concentrated to 3×105/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 after which the cells were cultivated in a 37℃, humidity saturated,5%CO2 incubator.2.2 Observing the effect of ShuGanQingDuTang to the HepG-2 cell line from the toxic experiment:Multiple proportions of diluted ShuGanQingDuTang. aqueous extract stock solution were divided into eight concentrations (100g/L,50g/L,25g/L,12.5g/L,6.25g/L, 3.13g/L,1.56g/L, and 0.78g/L). After separating the HepG-2 cells into a single cell suspension using 0.06% parenzyme, its density was prepared into a 3×105/ml cell suspension using a 10%fetal calf serum DMEM culture solution. The cells were inoculated in a 96 pore plate at 0.1ml/pore and cultivated in a 37℃, humidity saturated,5%CO2 incubator. Cell adherence occurred after 24 hours. After 2 days the culture medium was changed to a medium containing the Chinese herbal extract at 0.1ml/pore with 2 pores for each concentration. A control group also was set. The cells were cultivated in a 37℃, humidity saturated,5% CO2 incubator. The supernatant was abscised after 12 days. Cytotoxity on the remnant cells were detected using MTT.2.3 The effect of ShuGanQingDuTang extract on the growth curve of the HepG-2 cell line:Logarithmic phase cells were acquired and prepared to 1×104/ml density single cell suspension using a DMEM culture solution containing 10% fetal calf serum. The cells were inoculated in a 24 pore plate at 1ml/pore. Cells adherence occurred after 24 hours at which time the ShuGanQingDuTang. aqueous extract was diluted to 60g/L and 30g/L and the alcohol extract was diluted to 9g/L and 4.5g/L by complete medium. A control group used 2% DMEM to replace the aqueous and alcohol herbal extracts. The cells then were cultivated in a 37℃, humidity saturated,5%CO2 incubator. After incubation, samples of four pores each were acquired instantly and after the 1st,2nd,3rd,4th,5th,6th, and 7th day. After cellula digestivum samples of 40μl per pore were obtained and 10μl of 0.4% trypanblue staining solution was added. They were placed in a hemocytometer and viable cells which resisted staining were counted. This then was plotted graphically against culture time.2.4 The effect of ShuGanQingDuTang extract on HepG-2 cell AFP secretion:Multiple proportions of diluted ShuGanQingDuTang aqueous extract stock solution was divided into eight concentrations (60g/L,30g/L,15g/L,7.5g/L,3.751g/L,1.875g/L,0.938g/L, and 0.469g/L). After separating the HepG-2 cells into a single cell suspension using 0.06% parenzyme, a DMEM culture solution containing 10% fetal calf serum was used to prepare a cell suspension with a density of 3×105/ml. The cells then were inoculated in a 96 pore plate at 0.1ml/pore and cultivated in a 37℃, humidity saturated,5% CO2 incubator. Cells adherence occurred after 24 hours. After 2 days, the common culture medium was changed to a culture medium containing the herbal extract at O.lml/pore with 2 pores per concentration. A control group was established. Cell cultivation continued in a 37℃, humidity saturated,5%CO2 incubator. Supernatant was collected after 12 days and AFP secretion was detected using an AFP diagnostic kit.2.5 The effect of ShuGanQingDuTang extract on HepG-2 cell ALT and LDH secretions:The HepG-2 cells were separated into a single cell suspension using 0.06% parenzyme. DMEM culture solution containing 10% fetal calf serum was used to prepare the cell suspension to a density of 3×105/ml. The cells were inoculated into a 96 pore plate at 0.2ml/pore and cultivated in a 37℃, humidity saturated,5%CO2 incubator. Cell adherence occurred after 24 hours. The culture solution was abscised and 60g/L and 30g/L of the ShuGanQingDuTang aqueous extract was added with 2 pores per concentration. A control group was established using 2% DMEM to replace the ShuGanQingDuTang. aqueous extract. The cells were cultivated for 24 hours and 48 hours after which supernatant was collected by centrifuge and detected for ALT and LDH by ELISA.2.6 The apoptotic effect of ShuGanQingDuTang aqueous extract on HepG-2 cells: Logarithmic phase cells were acquired and prepared into a single cell suspension by 0.06% parenzyme and stained with 0.4% trypanblue. The viable cell count which resisted staining was 97.5%. The cells were inoculated in 50cm2 culture flasks with 106 cells per bottle and incubated at 37℃, with saturated humidity and 5% CO2 for 24 hours. The culture was a DMEM solution containing 10% fetal calf solution and 100μ/ml mycillin. The culture solution was changed to contain 5 ml of ShuGanQingDuTang aqueous extract per bottle at concentrations of 60g/L and 30g/L. A control group was established which used 2% DMEM to replace the herbal aqueous extract. Add the herbal extracts 24 and 48 hours later,each time point from each of 3 bottles of cell were detected for apoptosis. Apoptosis detection methodology:The culture solution was abscised carefully, trypsinized for 3 minutes by 0.06% parenzyme and lightly tapped to create a single cell suspension from the HepG-2 divided cell line and centrifuged for 10 minutes at 1000rpm. The cells were resuspended to 1×106/ml with Binding Buffer. 100μl of cell suspension solution was obtained and placed into 5ml FACS pipes to which was added 5μl of Annexin V-FITC, 10μl F and misce bene. After 15 minutes of incubation away from light.400μl of Binding Buffer was added to each pipe, whereupon flow cytometry was performed within one hour. Simultaneously, a control pipe with adelomorphous cells and a compensation installation pipe with unstained cells, simple staining Annexin V-FIT pipe and a simple staining PI pipe were set. FCM flow cytometry analysis:A FACS Caliber (Beckman Coulte, USA) ALTRA-type flow cytometer was used with a 488nm excitation light source. The control pipe was first used as a sample to obtain a distinct and localized cell colony in the FSC/SSC by calibrating the FSC and SSC parameters. A gate was set for the cell colony after which the continued fluorescence signal detection commenced for the cells in the gate. Fluorescence intensity was set for the control pipe (null cells) for non-specificity background fluorescence. This established the basis for determining the expression ratio of positive fluorescence. The upflow cytometer was calibrated to make the.fluorescence. scatter of the null cell in the graph to be concentrated in the left inferior quadrant. Without altering FL1, FL2, and FL3, the simple staining AV-FITC pipe and the simple staining PI pipe were placed as.samples. Fluorescence compensation was adjusted and analysis commenced after calibrating the optical spectrum overlay.10,000 cells from each sample were obtained and analyzed with CellQuest software.2.7 Determining the ability of ShuGanQingDuTang aqueous extract to induce HepG-2 cell apoptosis using a AO/EB fluorescence staining method:1×106/ml HepG-2 cells were inoculated in 10% fetal calf serum DMEM after which ShuGanQingDuTang aqueous extract was added at densities of 60g/L and 30g/L. The suspension was cultured in a 37℃, humidity saturated,5%CO2 incubator. Results were observed 24 and 48 hours after incubation. Results of AO/EB staining:a 100μl cell suspension was obtained to which was added 4μl of AO/EB colorant and misce bene. One drop was placed on a glass slide and covered with a coverslip.200 cells were counted under a fluorescent microscope.2.8 The effect of ShuGanQingDuTang aqueous extract on the apoptosis control gene p53:Cells slices were divided into groups. Sterile glass slides were inserted in each pore of the 24 pore cultivation plate. HepG-2 logarithmic phase cells were acquired and then digested and prepared into a cell suspension. Cell density was established at 1×105/ml and then inoculated in the 24 pore cultivation plates with 1ml/pore and incubated for 24 hours: ShuGanQingDuTang aqueous extract was added to the herbal group at concentrations of. 60g/L and 30g/L.2% DMEM was added instead to the control group. Each group had three pores set in parallel and cultivation resumed for 48 hours after which the slides which were full of cells were dislodged and rinsed twice with PBS, and fixed for 30 minutes with 4% paraformaldehyde. They were then air dried after being rinsed with PBS and preserved for later use in -20℃refrigerator. Detection was preformed according to the instructions provided with the kit. The main steps were as follows:50μl peroxydase blocking agent was dropped in and left at 37℃for 10 minutes. They were then rinsed with PBS 3 times after which 50μl of normal non-immune goat blood serum was dropped in and left at 37℃for 10 minutes. They were directly dried and the first p53 antibody as separately added and left at 37℃for 2 hours. They were rinsed with PBS 3 times after which the second biological tag antibody was added and left at 37℃for 10 minutes. After rinsing with PBS 3 times, streptomycete antibiotic albumen-peroxydase solution was dropped in and left at 37℃for 10 minutes. They were rinsed with PBS 3 times and 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 a neutral resin photographed.2.9The effect of ShuGanQingDuTang on the Fas/FasL expression on HepG-2:Cells preparation:Logarithmic phase cells were acquired and prepared as a 1×104/ml concentration of single cell suspension with 10% fetal calf serum DMEM culture solution. The cells were inoculated in 24-pore plates at 1ml/pore. Cell adherence occurred after 24 hours. ShuGanQingDuTang aqueous extract was diluted to concentrations of 60g/L and 30g/L using complete culture medium. The control group used 2% DMEM to replace the ShuGanQingDuTang aqueous extract. Cells were cultured in a 37℃, 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 60g/L and 30g/L groups with ShuGanQingDuTang aqueous extract) were separately extracted and reverse transcripted into cDNA. 1μl of the reverse transcription reaction product was taken as a mold and the Fas gene fragment and intra-referenceβ-actin were coamplified. PCR reactive condition:35 cycles were performed of 94℃force-denature for 5 minutes,94℃denature for 45 seconds,64℃anealing for 30 seconds, and 72℃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-referenceβ-actin gene acted as a comparative contrast among the groups. The same experiment was repeated 3 times. 1μl of reverse transcription reaction product was obtained as a mold and the FasL gene fragment and the intra-referenceβ-actin were coamplified. The PCR reactive condition:38 cycles were performed of 94℃denature for 5minutes,94℃denature for 45 seconds,58℃of anealing for 30 seconds,and 72℃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-referenceβ-actin gene acted as a comparative contrast among the three groups. The same experiment was repeated 3 times. 3. Results:3.1 Experiment on the cytotoxic effect of ShuGanQingDuTang extract on HepG-2:(1) The TC50 of HepG-2 cells was 62.65g/L with concentrations of ShuGanQingDuTang aqueous extract at 100g/L,50g/L,25g/L,12.5g/L,6.25g/L,3.13g/L,1.56g/L, and 0.78g/L. The rate of HepG-2 cell line destruction was 107.66,37.83%,5.82%,-0.97%,-3.88%,-15.52%,4.85%, and 21.34%. (2) Cells manifested vacuoles, atrophy, and abscission, with destruction rates of 107.66% and 88.90%, respectively, with ShuGanQingDuTang aqueous extract and alcohol extracts at 100g/L. With an aqueous extract density of 50g/L and an alcohol extract density of 6.25 g/L the cell structure appeared roughly normal under the microscope.3.2 The effect of ShuGanQingDuTang extract on the growth curve of HepG-2 cells: With an ShuGanQingDuTang aqueous extract density of 60g/L, cell populations from days 0-7 were 1,1.7,1.8,1.2,0.9,0.7,0.4, and 0.3 105/ml. At a density of 30g/L cell populations from days 0-7 were 1,1.7,1.9,1.6,1.4,0.9,0.6, and 0.4 105/ml. Cell proliferation was. slow. Cell proliferation was fast. This shows that ShuGanQingDuTang aqueous extract (60g/L and 30g/L) could inhibit the growth of HepG-2 cells while transparent inhibiting effect on cell multiplication.3.3 The effect of ShuGanQingDuTang extract HepG-2 cell AFP secretion:With densities of ShuGanQingDuTang. aqueous extract at 60g/L and 30g/L, the volume of AFP secretion of HepG-2 cells were 13.22ηg/ml and 18.08ηg/ml. Comparatively the volume of AFP secretions of the control group was 214.52ηg/ml. The standard reference values is less than 20ηg/ml showing that ShuGanQingDuTang aqueous extract at concentrations of 60g/L and 30g/L could inhibit AFP secretions of the HepG-2 cells in vitro.3.4 The effect of ShuGanQingDuTang extracts on ALT and LDH secretions of HepG-2 cells:Different concentrations of ShuGanQingDuTang aqueous extract were added and measured after 24 hours. At a concentration of 60g/L there was 12.34 U/L of ALT and 182.42 U/L of LDH. At a concentration of 30g/L, the ALT was 10.89 U/L and LDH was 165.33 U/L which was significantly difference than the control group.48 hours after adding a 60g/L concentration of the aqueous extract the ALT was 18.60 U/L and LDH was 284.40 U/L whereas the 30g/L group measured ALT at 15.18 U/L and LDH at 18.30 U/L which also was significantly difference than the control group.3.5 The effect of ShuGanQingDuTang aqueous extract on HepG-2 apoptosis:HepG-2 cell apoptosis increased proportionately with the increase in the concentrations of ShuGanQingDuTang aqueous extract showing a typical apoptotic peak. This was significantly different than the control group (P<0.05). However, the apoptotic rate was small which might be because the entire apoptotic process was temporary, the apoptotic times were uneven, or portions of apoptotic cell fragments failed to be detected. Nevertheless the experiment results still demonstrated that Euphorbia Antiquorum Linn. could directly induce HepG-2 cell apoptosis which likely could be one of the important mechanisms by which ShuGanQingDuTang inhibits hepatic carcinoma. 3.6 Determining whether ShuGanQingDuTang aqueous extract induces HepG-2 apoptosis using the AO/EB fluorescence staining method:The apoptotic rate of the cells in the control group at 24 hours was 2.10% and at 48 hours was 3.21%. Comparatively, the rate at which the ShuGanQingDuTang aqueous extract (60g/L concentration) induced apoptosis at 24 hours was 8.90% and at 48 hours was 18.32%. The apoptotic rate of the aqueous extract at 30g/L concentration at 24 hours was 6.40% and at 48 hours was 12.54%.3.7 The effect of ShuGanQingDuTang aqueous extract on the apoptosis control gene-p53:The coloration of p53 expression of the ShuGanQingDuTang group was obviously deeper than that of the control group. It had stronger staining and increased proliferation in the positive cell population. At an herbal extract density of 60g/L, the p53 positive cell rate was 33.56%. At a 30g/L concentration, the p53 positive cell rate was 29.45%. This was significantly different than the control group (P<0.05) and showed dose dependency.3.8 The effect of ShuGanQingDuTang on Fas/FasL expression in HepG-2: (1)Relative expression variation of Fas mRNA:The Fas gene andβ-actin coamplified by RT-PCR showed a significantly higher relative value of Fas gene expression in the HepG-2 cells treated by ShuGanQingDuTang at a 60g/L concentration in comparison to the control group.The amplified product was electrophoresis in 1.8% agarose. Its brightness was scanned using a gel imaging system. The relative value of the brightness of Fas gene compared to the intra-referenceβ-actin was used to differentiate the groups. The same experiment was repeated 3 times.(2)Relative expression variation of FasL mRNA:The FasL gene and B-actin coamplified by RT-PCR showed a significantly higher relative value of the Fas gene expression in the HepG-2 cells treated by ShuGanQingDuTang at a concentration of 60g/L compared to the control group and negative control group.4. Conclusion:4.1 ShuGanQingDuTang aqueous extract can inhibit the growth of the HepG-2 cell line while its alcohol extract has no apparent inhibition on cell multiplication. The aqueous extract has an obvious cytotoxic effect on HepG-2 cells. Further, the effect increases obviously with the prolongation over time of the drug action.4.2 The inhibitory effect in vitro of the ShuGanQingDuTang. aqueous extract on HepG-2 AFP secretions is much better than that of its alcohol extract which has no inhibition on AFP secretion.The anticarcinogenic mechanism of ShuGanQingDuTang is apoptosis. With induction time extended,the effect of induction was increasing, the cell apoptosis rate is higher. 4.3 The anticarcinogenic mechanism of ShuGanQingDuTang is apoptosis. The rate at which it induces apoptosis increases gradually over time.4.4 ShuGanQingDuTang aqueous extract can up-regulate the expression of the apoptosis related gene, p53, in HepG-2 cells in a dose dependent manner.4.5 ShuGanQingDuTang aqueous extract can up-regulate Fas/FasL expression thereby inducing apoptosis of hepatic carcinoma cells.
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