Research Of Anti-tumor Effects Of The Extract Of Prunella Vulgaris L. On Lymphoid And Hematopoietic Malignancies In Vivo

Background and ObjectiveThe incidence of lymphoid and hematopoietic malignancies is gradually increasing by 4% each year. Combined therapy is a common way to treat lymphoid and hematopoietic malignancies, but chemotherapeutics have some serious side-effects, low healing rate, invasion, and primary or secondary drug resistances which will make the treat fail. It is time to seek new anti-tumor drugs which have high therapeutic effect and mild side-effect. Professor Zhang mingzhi has found that Prunella Vulgaris L has the effect of anti-tumor, and referred to many relative articles. There are little articles about lymphoid and hematopoietic malignancies. Our research team has the relative explores in order to prove the effect of Prunella Vulgaris L on lymphoid and hematopoietic malignancies, explore the mechanism, extract effective component which can provide the theory evidences for using Prunella Vulgaris L on clinic by large dosages. Prunella Vulgaris L. is a traditional Chinese herb and has the "QingGanXieHuo" and "RuanJianSanJie" effects. It is often used to cure mastalgia, thyromegaly, acute tonsillitis, swelling of throat and high blood pressure. In the earlier time, people had used Prunella Vulgaris L. to treat the carcinoma of thyroid gland, lymphosarcoma, parotidean carcinoma, tonsillar cancer, nasopharyngeal carcinoma, breast cancer, cervix cancer, liver cancer, et al. The modern pharmacology has proved Prunella Vulgaris L. has many pharmacologic activities. The experiments in vivo and in vitro showed that it had certain anti-tumor effects. However, nowadays the anti-tumor study on Prunella Vulgaris L. still concentrates on the level of mixture, screening effective anti-tumor monomer is rare. Moreover, up to now, it has little relative experimental reports about its anti-tumor components , so it has most significance.This experiment offered experimental foundations for exploiting Prunella Vulgaris L. to be monomer and applying to the clinic by observing the anti-tumor effects of the extract of Prunella Vulgaris L. (EPVL) in vivo and primarily approach its mechanism of action by molecular biology technology.Methods1. EL-4 cells were growing in DMEM with high glucose containing 10% fetal horse serum, at 37℃in a humidified 5% CO₂ incubator. When the cell-growth was in exponential phase of growth, tumor cells were collected under sterile conditions and were made a single cell suspension by sterile N. S in the concentration of 1×10⁷/ml. Each C57BL/6 mouse was subcutaneously injected in the right flank with 0.2ml cell suspension. When tumor cells were passaged stable in vivo , tumor-bearing mice were killed, tumor mass was dissected and made a single cell suspension by sterile N-S in the concentration of 4×10⁵个/ml under sterile conditions. Each C57BL/6 mouse was subcutaneously injected in the right flank with 0.2ml cell suspension.2. On the next day, tumor-bearing mice were randomly divided into five groups and each group was consisted of 10 mice: large,moderate,small three dosage groups of EPVL. 500mg/kg/d,300mg/kg/d,100mg/kg/d of EPVL injection were respectively injected subcutaneously every day for 10 days; positive control group, 20mg/kg/d of CTX injection was injected subcutaneously for 3 days; negative control group, the same volume of N-S injection was injected subcutaneously every day for 10 days. Kinetics of tumor growth and survival time of tumor-bearing mice were measured, tumor growth inhibition rate (IR) was calculated.3. Tumor tissue samples were taken and examined by HE staining to observe pathomorphologic difference between experiment group and negative control group and assess the inhibitory effects of EPVL on tumor-bearing mice.4. Tumor tissue samples were examined by immunohistochemistry method to observe expressing of bcl-2 protein and bax protein between experimental group and negative control group.Statistical analysisSPSS 10.0 software was used to carry out statistical analysis. T-test and x²-test were employed. The size of test was "α= 0.05".Results1. The ratio of inhibition(IR) of tumor growth of large,moderate,small three dosage groups of EPVL were 39. 54%, 65. 26%, 22. 30% , respectively. The net tumor weights were 1.4054g, 0.8075g, 1.8055g respectively and obviously less than that of negative control group, 2.3246g. The difference had statistical significance (P<0.05) . IR of moderate dosage group was obviously more than that of small and large dosage groups, the difference had statistical significance(P<0.05). However, the difference of IR between small dosage group and large dosage group had no statistical significance ( P>0.05 ) . The difference of CTX group' s weight had statistical significance(P<0.05) between pre-therapy and post-treatment, the weight of post-treatment is less than pre-therapy obviously. The weight-changing among three groups of EPVL is not evident and has no statistical significance (P>0. 05). Activity and reactivity has no change.2. The survival time of the tumor-bearing mice of large,moderate,small three dosage groups of EPVL (25.50d, 28. 50d, 24. 86d) were longer than that of negative control group (22.67d) , the difference had statistical significance (P<0. 05). The survival time of the tumor-bearing mice of moderate dosage group was longer than that of small and large dosage groups, the difference had statistical significance (P<0.05) . However, the difference of the survival time of the tumor-bearing mice between small dosage group and large dosage group had no statistical significance (P>0. 05) .3. More widespread necrosis domain of EL-4 cells in the moderate dosage group of EPVL than in the negative control group was revealed by histopathological examination.4. It was compared with moderate dosage group of EPVL and negative control group that the expressing of bcl-2 protein was down-regulation and bax protein was up-regulation examined by immunohistochemistry method.Conclusion1. The extract of Prunella Vulgaris L. (EPVL) can remarkably inhibit the transplanted Mouse T-lymphoma cells EL-4 in vivo.2. It was proved that inducing apoptosis of tumor cells was one of important anti-tumor mechanisms.3. The extract of Prunella Vulgaris L. (EPVL) can prolong the survival time of the tumor-bearing mice remarkably.