| Objective1. Clinical study:To include advanced Hepatocellular Carcinoma patients, and divided into two groups. In the treatment group, Xin JianPi LiQi decoction (250ml, po, bid) combined with extracorporeal High Frequency Thermotherapy was given, while Xin JianPi LiQi decoction (250ml, po, bid) was given only in the control group. The purpose is to investigate the efficacy of the combination of these two treatments, to provide direction for clinical treatment, and providing ground and guideline for the use of the two treatments.2. Experimental study:To use Human hepatocellular carcinoma HepG-2cell line to further investigate the effect and mechanism of XJPLQF combined with hyperthermia and whether it has synergic effect when they are used together in vitro.Methods1.Clinical study:Forty-one patients of advanced-stage hepatocellular carcinoma were recruited according to the inclusion criteria, and were divided into treatment and control group randomly. Patients were given Xin JianPi LiQi decoction (250ml, po, bid) combined with extracorporeal High Frequency Thermotherapy in the treatment group. Patients were given Xin JianPi LiQi decoction (250ml, po, bid) only in the control group.30days was a treatment period. After3cycles of treatment, the disease control rate (DCR), time to progression (TTP), overall survival (OS), and safety were assessed between two groups. SPSS13.0was used for statistical analysis. Descriptive analysis was used for baseline characteristics, short-term efficacy and safty. Kaplan-Meier analysis was performed for the comparison of TTP and OS. Variance analysis was conducted for the clinical characteristics between groups. All reported P values were two sided.2. Experimental study:Hepatocellular carcinoma HepG-2cell line was selected in this study. It was cultivated in DMEM-H. The HepG2cells were divided into four groups:control group(normal cultured HepG2cells), JPLQF group(concentration of0.25mg/ml,0.5mg/ml,1.0mg/ml,1.5mg/ml,2. Omg/ml), hyperthermia group (cultured in the water bath43℃for1h and XJPLQF combined with hyperthermia group. The inhibition of cellular proliferation was detected by MTT method. The morphological alteration was observed by Hoechst33258staining. The apoptosis rates were analyzed by flow cytometry. The levels of Bax and Bcl-2proteins were detected by Western blotting analysis.Results1. Clinical study(1) Baseline characteristics41cases were recruited from the In-patient Oncology Department of Guangdong Provincial Hospital of TCM from September2010to December2012. The baseline characteristics, such as age, sex, staging, Eastern Cooperative Oncology Group (ECOG) performance status score, Child-Pugh liver function class and Chronic hepatitis virus infection, had no significant difference and was balanced between groups. One patient withdrew in control group because of change in self-decision. There were40evaluable cases,31cases deceased, while follow-up was conducting.(2) Time to Progression (TTP)By the cut-off date, patients in the treatment group had median time to progression of4.5months, as compared with3.1months in the control group. The TTP was prolonged for1.4months, indicating significant difference between two groups(P<0.01).(3) Overall Survival (OS)By the cut-off date, patients in the treatment group had median overall survival of6.6months, as compared with5.5months in the control group. The OS was prolonged for1.1months, indicating significant difference between two groups(P<0.05).(4) Short-term EfficacyShort-term efficacy was evaluated after3cycles of treatment. Disease control rate (DCR) was25%and15%in the treatment and control group. There was no significant difference between two groups.(5) SafetySafety evaluation indicated that there were no significant differences before and after treatment on blood routine, liver and Kidney function and ECG examination. There were no hepatotoxicity, gastrointestinal symptoms, alopecia or severe cardio, renal or hepatotoxicity.2. Experimental study(1) Effect of Xin JianPi LiQi decoction combined with hyperthmia on cell proliferation by MTT method.Xin JianPi LiQi decoction administrated alone and combined with hyperthmia can inhibit the proliferation of human hepatocellular carcinoma HepG-2cells. The inhibition ratio was gradually enhanced with the rise of medicine concentration and time, that is they present time and dose dependent relation. Hyperthmia can also suppress the proliferation of human hepatocellular carcinoma HepG-2cells. The effect is in a time-dependent way and the72-hour’s anti-proliferation rate is the highest one. The inhibitory effect on proliferation was promoted obviously when they were used together. The synergetic anti-proliferation rate was highest when they used together for24h.(2) Effect of Xin JianPi LiQi decoction combined with on morphological alteration by Hoechst33258staining, and apoptosis rates by flow cytometry method.Cells in each group been drug or hyperthmia administrated for24h without exception appeared a change on apoptosis morphology. The change of apoptosis morphology in electron microscope was most obvious in the group of Xin JianPi LiQi decoction combined with hyperthmia. The apoptotic rate was higher in each group compared with control group. The effect was in a dose dependent way. The apoptotic rate of was higher in the group of Xin JianPi LiQi decoction combined with hyperthmia than that of single XJPLQF or hyperthermia.(3) Effect of Xin JianPi LiQi decoction combined with hyperthmia on Bax and Bcl-2proteins levels by Western blotting analysisXin JianPi LiQi decoction and hyperthmia administrated alone and together for24h can raise the expression of Bax proteins and the level of Bax/Bcl-2, but degrade the expression of Bcl-2proteins. The level of Bax/Bcl-2was up regulated most obviously after cells treated for24h by Xin JianPi LiQi decoction with concentration of0.5mg/ml, combined with hyperthmia. When Xin JianPi LiQi decoction and hyperthmia was administrated alone and together for different time by the concentration of Xin JianPi LiQi decoction of0.5mg/ml, the protein level of Bax and level of Bax/Bcl-2were also up regulated, the protein level of Bcl-2was also down regulated.When Xin JianPi LiQi decoction and hyperthmia used together, the24-hour’s expression of Bax proteins and the48-hour’s level of Bax/Bcl-2was the highest, while the48-hour’s expression of Bcl-2proteins was the lowest.Conclusions1.Clinical studyThe time to progression and overall survival were siganificantly longer in Xin JianPi LiQi decoction combined with hyperthmia group than in the control group. Therefore, Xin JianPi LiQi decoction combined with hyperthmia can improve the survival and effectively prolong the TTP and OS.2. Experimental study(1) XJPLQF combined with hyperthermia has anti-proliferation effect on Human hepatocellular carcinoma HepG-2cells, of which anti-proliferation was higher than that of single XJPLQF or hyperthermia.(2) XJPLQF combined with hyperthermia has apoptotic effect on Human hepatocellular carcinoma HepG-2cells, of which the apoptotic rate was higher than that of single XJPLQF or hyperthermia.(3) XJPLQF combined with hyperthermia can promote the expression of Bax proteins and reduce the expression of Bcl-2proteins. |
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