Effects Of Sophora Saponins On The Invasion And Metastasis Of Non - Small Cell Lung Cancer

ObjectiveZhuang-medicine "Gocaekmbaw", namely Gynostemma pentaphyllum (Thunb.) Makino, a member of the Cucurbitaceae family, is considered to be one of the main ingredients in the clinical treatment of lung cancer drug and shows obvious curative effect. But its active components and mechanism of anti-tumor are unclear, which affected further development and application of the national drug. Therefore, under the guidance of theory of zhuang medicine, combining with modern technology, this paper mainly studies on exploring the anti-cancer efficacy and pharmacological mechanism of "Gocaekmbaw" and defining its real pharmacological composition, in order to lay the foundation for the preparation of anti-tumor drugs with high efficiency and low toxicity.Methods1. The extracting technology was optimized by single factor experiment and orthogonal design. The absorption and elution conditions of total saponins purificatin by various macroporous adsorption resins were investigated with the content of total saponins as index.2. A549 cell line was cultured and treated with gypenoside LVI and drug-containing serum of total saponins to evaluate the anti-cell mobility effect for 48 hours, respectively. The invasive ability of A549 cells was examined in transwell chamber with gypenoside LVI and drug-containing serum of total saponins respectively for 48 hours. The number of migrate cells were observerd and the inhibitive ratio was calculated. The blank and drug-containing serum were comparatively analyzed by liquid chromatography-ion trap time of flight tandem mass spectrometry (LCMS-IT-TOF) method to explore the real compositions absorbed into body.3. C57BL/6J mice lewis lung carcinoma subcutaneous transplantation tumor model was established.80 successful tumor-bearing mice were selected and then randomly divided into eight groups:model group, "Gocaekmbaw" high-dose group, medium-dose group, low-dose group, cis-platinum group, combination group, Shenyi Capsule group and gypenoside LVI group. The ninth group was 10 normal mice. Mental state, activity of freedom movement, hair, diet and wears were observed. Changes in body weight and tumor volume were monitored every two days during the administration. On the 21st day, after weighting, executing animal and taking material. Pick the eyeball method for blood, use conventional methods to fetch tumor tissues, spleen, thymus and weight them, calculate inhibition rate. Utilize conventional method to take out the whole lung tissue in mice and use Bouin’s liquid dye to fix the tissue.4. By each group of mice spleen and thymus weight, calculate spleen index and thymus index. Using enzyme-linked immunosorbent assay (Elisa) to detect interleukin-2 (IL-2) and interleukin-10 (IL-10) levels in mice serum.5. HE staining method was used to observe pathological changes of mice tumor tissue.6. After lung tissue was fixed 24 hours, the number of pulmonary metastasis was observed under a biological microscope, and then the metastasis ratio was calculated. The expression changes of microvessel density (MVD), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were detected by the method of immunohistochemistry (IHC-P). VEGF, HIF-1α, Matrix metallo-proteinases-2 (MMP-2) and Matrix metallo-proteinases-9 (MMP-9) protein expression were detected by the technique of Western blot (WB). VEGF mRNA, HIF-1α mRNA, MMP-2 mRNA, MMP-9 mRNA transcript levels were detected by Real-time Polymerase Chain Reaction (Q-PCR) technique.7. Six rats were halved into two groups at random. Then the two groups were administrated orally and intravenously with 20(S)-gypenoside LVI, respectively. Urine samples were collected from 0-48 h after administration. After pretreated by SPE-C18 cartridges, each urine sample was comparatively analyzed by LCMS-IT-TOF.8. Molecular docking was used to investigate the interaction of six gypenosides with VEGFR-2. Results1. The optimal technology was that gynostemma pentaphylla was extracted with 12 fold of 70% ethanol for 3 times,1 hour for each time. HP-20 type macroporous adsorption resin was the best choice for the purification of total saponins. The optimized parameters were as follows:the content of total saponins at 6.23-12.47 mg/ml, eluting solvent of 70% aqueous ethanol, and elution volume at 20 BV.2. Drug-containing serum of "Gocaekmbaw" was shown to have inhibitive effects on mobility and invasion ability of A549 cells, while gypenoside LVI can hardly restrain that of A549 cells. Analysis result of blank and drug-containing serum showed that gypenoside LⅥ, gypenoside XLⅥ, gypenoside L, gypenoside LI, damulin B and damulin A might be the effective compositions absorbed into body.3. Each treated group could significantly inhibit the growth of tumor (P <0.05). Spleen index and thymus index of cisplatin group were significantly lower than model group and other treated group (P<0.05). Compared with model group, spleen index and thymus index of each "Gocaekmbaw" group, gypenoside LⅥ group, ShenYi capsule group increased significantly (P<0.05). Compared with model group, IL-2 content of "Gocaekmbaw" groups increased significantly (P<0.05) and IL-10 content of "Gocaekmbaw" groups decreased significantly (P<0.05).4. Result of IHC-P showed that expression of MVD of "Gocaekmbaw" high-dose group, medium-dose group, combination group, Shenyi Capsule group and gypenoside LⅥ group decreased significantly (P<0.05). Expression of VEGF and HIF-1α of "Gocaekmbaw" high-dose group, medium-dose group, combination group and gypenoside LVI group decreased significantly (P<0.05). MMP-2 protein expression levels of all treated groups were lower, but the difference is insignificant. MMP-9 protein expression levels of each treated group excepted for "Gocaekmbaw" medium-dose group was lower, but the difference is insignificant. Q-PCR results show that, compared with model group, VEGF mRNA relative expression levels of each treated group decreased significantly (P<0.05). MMP-2 mRNA relative expression levels of each treated group decreased, but only the difference of gypenoside LVI group and combination group is significant (P<0.05). MMP-9 mRNA relative expression levels of each treated group decreased, but only "Gocaekmbaw" high-dose group and combination group decreased significantly (P<0.05). HIF-la mRNA relative expression levels of each treated group decreased, but only gypenoside LVI group decreased significantly (P<0.05).5. After administration, a total of eight metabolites of gypenoside LVI were plausibly assigned, six from the rat urine of orally administered group and eight from the rat urine of intravenously administrated group. They were speculated as gypenoside LⅥ, gypenoside XLⅥ, gypenoside L, gypenoside LI, damulin A, damulin B,2α-OH-20(S)-ginsenoside Rh2 and 2a-OH-20(R)-ginsenoside Rh2.6. Five residues (Asn923, Asp1046, Leu840, Cys 919, Glu917) of VEGFR-2 ground gypenosides could form H-bonds with gypenosides, which can produce significant contribution to biological activities.Conclusion1. The optimal extracting techniques were refluxing extraction for 3 times with 12 fold 70% ethanol,1 hour for each time. HP-20 type macroporous adsorption resin was the best choice for the purification of total saponins. The optimized parameters were as follows:the content of total saponins at 6.23-12.47 mg/ml, eluting solvent of 70% aqueous ethanol, and elution volume at 20 BV.2. Drug-containing serum of "Gocaekmbaw" was shown to have inhibitive effects on mobility and invasion ability of A549 cells. Gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin B and damulin A were the effective compositions absorbed into serum.3. Gypenosides could inhibit the growth of Lewis lung cancer cells in mice, reduce tumor volume and the number of lung metastases, improve the general condition of tumor-bearing mice, and also increase its body weight.4. The mechanism of anti-lung cancer of Gypenosides might be related to the following pathways:enhancing immunity by regulating the immune cytokines IL-2 and IL-10 expression in tumor-bearing mice; reducing VEGF, HIF-1α, MMP-2 and MMP-9 mRNA transcript levels and protein expression in tumor-bearing mice.