| 1.ObjectiveAs one of the major component of radix ginseng, a kind of Chinese strengthening body resistance herbs,20(R)-ginsenoside Rg3can inhibit the tumor angiogenesis, prevent the recurrence, diffusion and metastasis of tumor. Its clinical therapeutic effects have been proved by several tumor research center including Guang’anmen Hospital affiliated to China Academy of Chinese Medical Sciences. Otherwise, its therapeutic effects and clinical application are limited by its some defect such as backward drug preparation and low bioavailability. Therefore, it is meaningful for the improvement of Rg3’s therapeutic effects to prepare the PEG-PLGA-Rg3nanoparticles and give ginsenoside Rg3the sustained release character. Moreover, it can also provide some new ideas for designing and developing the new type high-performance anti-tumor Chinese herbs. Our study is to analyse the difference between Rg3and PEG-PLGA-Rg3nanoparticles on tumor angiogenesis and verify the feasibility and superiority of Rg3nanoparticles through in vitro an in vivo experiments.2.Methods2.1experiments in vitroThe effects of Rg3and PEG-PLGA-Rg3nanoparticles on the proliferation of EA.hy926cell and PG cell were observed by MTT assay with the aim to evaluate whether the cytotoxicity of them was distinctive.The effects of Rg3and PEG-PLGA-Rg3nanoparticles on the cell cycle of EA.hy926cell were detected by flow cytometry PI single-staining assay to examine the difference of their effect between Rg3and Rg3nanoparticles.The effects of Rg3and PEG-PLGA-Rg3nanoparticles on the apoptosis of EA.hy926cell were detected by flow cytometry Annexin V-FITC/PI double-staining assay to examine the difference of their effect between Rg3and Rg3nanoparticles.The effects of Rg3and PEG-PLGA-Rg3nanoparticles on the invasion and tube formation of EA.hy926cell were observed by transwell and tube formation assay to examine whether they could affect the function of endothelial cell while not killing them.At the same time, the difference of their effect between Rg3and Rg3nanoparticles was also observed.Expression of VEGF mRNA, the protein expression of E-cad,MMP-9,HIF-la,VEGF were examined by RT-PCR and western blot to explore the internal molecular mechanisms.2.2experiments in vivoLewis lung cancer mice model was established and60mice were randomly divided into5groups with twelve in each group:PEG-PLGA-Rg3nanoparticles group(Rg3-N), PEG-PLGA group(PEG), Rg3group(Rg3), normal control group(C), saline control group(NS), intragastric administration for14days.The weights of mice were measured every2days and weight curves were obtained. At the same time, color pattern, activity and mental status were observed.The mice were sacrificed when the administration was over, weighing the transplanted tumors and calculating tumor inhibitory rate and tumor:weight ratio. The microvessel densities(MVD) of the tumors were measured by immunohistochemical staining with anti-CD31antibody. The effects of Rg3and PEG-PLGA-Rg3nanoparticles on the tumor angiogenesis in vivo were observed based on these indexes.Expression of VEGF mRNA, the protein expression of TSP-1,E-cad,Ki-67,MMP-9,HIF-la,VEGF of transplanted tumors were examined by RT-PCR, immunohistochemistry and western blot to explore the internal molecular mechanisms of anti-tumor effects in vivo.3.Results3.1experiments in vitroCompared with control group, OD values were not significantly different(P>0.05) in five different concentration(0.004ug/ml,0.04ug/ml,0.4ug/ml,4ug/ml,40ug/ml) Rg3and Rg3nanoparticles groups after EA.hy926cells and PG cells were treated for24h and48h. There was not dose-effect relationship and significant difference(P>0.05) between Rg3and Rg3nanoparticles.After treated by three different concentrations(0.4ug/ml,4ug/ml,40ug/ml) Rg3and Rg3nanoparticles for24h and48h, the cell cycle of EA.hy926cells were not significantly different(P>0.05) compared with control group. It was the same between Rg3and Rg3nanoparticles as well as between different concentrations.After treated by three different concentrations(0.4ug/ml,4ug/ml,40ug/ml) Rg3and Rg3nanoparticles for48h, the apoptosis of EA.hy926cells were not significantly different(P>0.05) compared with control group. It was the same between Rg3and Rg3nanoparticles as well as between different concentrations.After treated by three different concentrations (1.6ug/ml,8ug/ml,40ug/ml) Rg3and Rg3nanoparticles for48h, the invasion abilities of EA.hy926cells decreased significantly (P<0.05) compared with control group and this trend was dose dependent. Otherwise, there was no significant difference(P>0.05) between Rg3and Rg3nanoparticles.After treated by three different concentrations Rg3and Rg3nanoparticles for48h, the numbers of tubes in1.6ug/ml group were of no significant difference(P>0.05) compared with control group. In contrast, the numbers of tubes in both groups(8ug/ml,40ug/ml) significantly decreased(P<0.05) but there was no significant difference(P>0.05) between the two concentrations. It was the same between Rg3and Rg3nanoparticles.In comparison with control group, the expression of VEGF and VEGF mRNA were not affected significantly (P>0.05) after treated by Rg3and Rg3nanoparticles at40ug/ml for48h. Otherwise, the expression of MMP-9and E-cad decreased significantly(P<0.05) even if there was no significant difference(P>0.05) between Rg3and Rg3nanoparticles. In addition, the expression of HIF-1a was not founded in any group.3.2experiments in vivoThe trends of variation of mice weight in NS group and PEG group were rising early but declining later. In contrast, the trends of the other three groups were rising early and stable later. In comparison with NS group, the mice of Rg3group and Rg3nanoparticles group had better general status:brighter color, more active and better spirit.The tumor weight in PEG group, Rg3group and Rg3-N group showed no significant difference(P>0.05) compared to NS group but the tumor:weight ratio and MVD in Rg3group and Rg3-N group declined significantly(P<0.01). Besides, there was no significant difference(P>0.05) between Rg3group and Rg3-N group.Compared to NS group, the expression of VEGF mRNA, MMP-9, HIF-1a and VEGF in Rg3group and Rg3-N group decreased. Furthermore, the expression of Rg3-N group was lower than that of Rg3group. At last, the expression of E-cad in Rg3-N group showed significant difference(P<0.01) compared to NS group and Rg3group.The expression of TSP-1, and Ki-67in Rg3group and Rg3-N group showed no significant difference(P>0.05) compared to saline control group. It was the same between Rg3and Rg3nanoparticles 4conclusion(1)Rg3and PEG-PLGA-Rg3nanoparticles showed no direct cytotoxicity and couldn’t affect the apoptosis, cell cycle and proliferation of EA.hy926endothelial cells. They couldn’t directly inhibit the proliferation of tumor cells in vivo, either.(2)Rg3and PEG-PLGA-Rg3nanoparticles could restrain the invasion ability and tube formation of EA.hy926endothelial cells and the effects maybe result from the inhibition of expression in E-Cad and MMP-9.(3)Rg3and PEG-PLGA-Rg3nanoparticles could supress the tumor angiogenesis in vivo, which was perhaps related to the inhibition of expression in MMP-9, HIF-1α and VEGF.(4)Rg3and PEG-PLGA-Rg3nanoparticles could improve the general status of animals.(5)The effects of PEG-PLGA-Rg3nanoparticles in vivo were superior to Rg3on several indexes, which was perhaps resulted by the sustained release character of PEG-PLGA nano-carriers and longer blood concentration.
|
PDF Full Text Request