| Objective:Isolation and identification of saponins from“Gocaekmbaw"(Gynostemma pentaphyllum(Thunb.)Makino).The inhibitory effect of G.pentaphyllum saponins on tumorwas investigated in vitro and in vivo.The relationship between G.pentaphyllum saponin and cholesterol was explored by constructing overexpressed cell lines to further elaborate the anti-tumor mechanism of G.pentaphyllum saponin.Method:1.Lewis mouse lung cancer model was established by subcutaneous inoculation of LLC cells in the axilla.The G.pentaphyllum saponin and cisplatin control drugs were given by intraperitoneal injection.After administration,the mice were dissected to obtain experimental materials.The similarities and differences between different groups were analyzed for experimental materials and data processing.2.G.pentaphyllum was treated with high temperature and high pressure,and the effective components were extracted with 80%ethanol reflux.The G.pentaphyllum saponin monomer was obtained by using macroporous resin for sugar removal,silica gel column separation,C18 reversed phase column medium pressure preparation and separation,and high performance liquid chromatography(HPLC)purification.The chemical composition of the compounds was analyzed by UV,IR,NMR,MS,TLC silica gel plate and high performance anion exchange chromatography-pulsed method.3.The monomer efficacy of the compounds obtained by screening for human lung cancer cell A549,H1299,human gastric cancer cell SGC7901,human renal cancer cell T24,human neuroblastoma cell SH-SY5Y and human leukemia cell K562.The in vitro activity of the isolated compounds was detected in human lung cancer A549 cells as model cells.The inhibition rate of tumor cells in vitro was detected by CCK8 assay.The apoptotic activity was detected by TUNEL staining.After staining with JC-1 reagent,mitochondrial membrane permeability was detected.The cell colony formation test and propidium iodide staining were followed by flow cytometry.The cell proliferation of G.pentaphyllum saponin was analyzed.Scratch assay and transwell chamber assay were used to analyze the effects of new compounds on invasion and metastasis.4.After the combination of G.pentaphyllum saponin and cholesterol in vitro,the cholesterol residue in the supernatant after the combination was detected by HPLC to verify the relationship between G.pentaphyllum saponin and cholesterol.5.Overexpression plasmids of cholesterol related genes HMGCR and LPCAT3 were constructed and transfected into A549 cells using adenovirus to construct overexpression cell lines.The effects of G.pentaphyllum saponin on overexpressed cell lines and wild type were investigated.The CRISPR knockout plasmid was constructed,and the plasmid was introduced into cells through electrical transformation.Result:1.Anatomical tumor weighing and measurement results of Lewis lung cancer mice showed that G.pentaphyllum alone group had a certain inhibitory effect compared with model group.The positive drug cisplatin group had better efficacy,and the low dose G,pentaphyllum saponin combined with cisplatin group had the best efficacy.The results showed that the tumor inhibition effect of Lewis lung cancer from strong to weak was as follows:PL group(90.55%)>P group(68.84%)>L group(21.67%)and H group(20.00%).HE staining showed that compared with the M group,the apoptotic cells in group H were increased,while the normal cells in group P and PL were significantly reduced.The results of four tests of blood lipid showed that G.pentaphyllum saponin had a certain effect on the reduction of triglyceride and an effect on the promotion of HDL-C.Gene level qPCR results showed that compared with the model group,cholesterol synthesis related genes were increased in the administration group.Apoptosis related genes were up-regulated;In the detection of transfer-related genes,there was a significant decrease in group H(p<0.01),and an increase in other groups;Tumor necrosis factor related genes were significantly increased(p<0.01).2.Two new saponins were isolated from Zhangzhou G.pentaphyllum after heat treatment:G.pentaphyllum damulin E(2?,3?,12?-trihydroxydammar-20(22),24-diene-3-O-?-D-gl-ucopyranoside);G.pentaphyllum damulin F(2,3 beta,12 beta trihydroxydammar-20(21),24-diene-3-O-beta-D-glucopyranoside).3.The IC50 values of damulin E and damulin F on A549,H1 299,SGC-7901,T24,SH-SY5Y and K562 cells were 19.8± 0.4 ?M and 38.9 ± 0.6 ?M,respectively.18.5 ± 0.5?m,33.4 ± 0.5?m;16.1±0.9 ?m,37.3±0.7 ?m;18.5 ± 0.6?m,64.7±0.5?m;31.8 ± 0.5?m,63.1± 0.7 ?m;20.6± 0.4 ?m,57.7± 0.5 ?m.TUNEL and JC-1 staining results showed that the two compounds induced apoptosis and mitochondrial membrane permeability changes.Western blot and qPCR results showed that damulin F up-regulated the expression of pro-apoptotic proteins and genes.In addition,cell scratch and transwell chamber experiments also showed that damulin E and damulin F had inhibitory effects on the invasion and metastasis of A549 cells,and damulin F reduced the expression of metalloprotein matrix enzyme genes MMP-2 and MMP-9.The results of cell cloning assay and cell cycle detection showed that both damulin E and damulin F affected the formation of cell colonies and inhibited the cell cycle mainly in the G0/G1 phase.4.HPLC was used to quantitatively analyze the cholesterol reference substance at 210 nm wavelength,with good linearity in the range of 100-2000 ng(R2=0.9999).In the same dose of cholesterol,different concentrations of G.pentaphyllum saponin were added,and it was found that G.pentaphyllum saponin added 50 ng had a lowering effect on cholesterol.5.OV-HMGCR and OV-LPCAT3 overexpressing cell lines were successfully constructed in A549 cells.Cell death occurred after long-term culture of OV-LPCAT3.When gp-saponin was added to the wild-type and OV-HMGCR,the IC50 of OV-HMGCR increased by 5.7%,and OV-HMGCR was more resistant than the wild-type when G.pentaphyllum saponin was added 60 ?M.The difference of gene expression between the two overexpressed cell lines and the wild type showed that the expression of genes related to cholesterol synthesis and cholesterol excretion were up-regulated in the overexpressed cell lines.OV-LPCAT3 transfer-related gene expression was significantly down-regulated;Tumor necrosis factor-related genes were significantly up-regulated in the two overexpressed cell lines.Gene expression test showed the difference between OV-HMGCR and wild-type after the addition of G.pentaphyllum saponin.The results showed that the pro-apoptotic gene Bax was down-regulated in OV-HMGCR compared with wild-type,while the transfer related gene MMP2 was relatively down-regulated in OV-HMGCR.Tumor necrosis factor-related genes were significantly increased in both overexpressed cell lines and wild type after addition(p<0.01).Cholesterol biosynthesis related genes were up-regulated in both OV-HMGCR and wild-type after addition.However,the expression of cholesterol excretion-related gene ABCA1 was down-regulated after the addition of OV-HMGCR,and the expression of ABCG2 gene was down-regulated after the administration of wild-type and OV-HMGCR.HMGCR was successfully constructed,and the plasmid pKO-HMGCR and pKO-LPCAT3 were knocked out and transformed into A549 cells.Conclusion:In vivo experiments,Lewis lung cancer mouse model was used to detect the inhibitory effect of thin saponins on lung cancer,and the modeling was relatively successful with good homogenization.Good effect was obtained by intraperitoneal injection.G.pentaphyllum had a certain inhibitory effect on lung cancer in mice with Lewis lung cancer,and the combined administration group had a better effect,indicating that G.pentaphyllum had a certain synergistic promoting effect on the inhibition of lung cancer with cisplatin,which provided a reference for clinical treatment of lung cancer in the future.The effective active parts were extracted and separated by conventional methods,and the new monomers were obtained by purification,detection and identification.It has good inhibitory effect on many kinds of tumor cells in vitro.The library of active monomer saponins was further enriched,which provided more materials for clinical drug development in the future.Further analysis of the mechanism of the compound inhibiting the action of lung cancer cell A549 showed that it had a great effect on the cholesterol-related genes and the genes related to cellular inflammatory factors.The combination of cholesterol in vitro and saponin GP-LI showed that saponin GP-LI could reduce cholesterol to a certain extent but not in a dose-dependent manner.Furthermore,the over-expressed mutant and knockout mutant models of A549 cells were constructed to analyze the mechanism of inhibition of A549 cells by G.pentaphyllum.Preliminary analysis results showed that G.pentaphyllum was overexpressed in A549 cells. |
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