Study On The Saponins From Gocaekmbaw(Gynostemma Pentaphyllum) And Its Inhibition On The Proliferation Of Clear Renal Cell Carcinoma

Renal cell carcinoma(RCC) encompasses a heterogeneous group of cancers derived from renal tubular epithelial cells and is among the 10 most common cancers worldwide.About 35%of ancer patients had regional lymph node or distant metastasis at the time of diagnosis,and the prognosis is poor.Therefore,early diagnosis and treatment of RCC are extremely important.Clear cell renal cell carcinoma(ccRCC)accounts for about 80%of RCC cases.The histological appearance shows the accumulation of intracellular lipid droplets and is closely related to abnormal lipid metabolism.Lipid accumulation is currently considered to be an important marker of the aggressiveness of RCC,and obesity is an independent risk factor recognized by RCC.Therefore,basic research targeting lipid metabolism provides new ideas for the treatment of RCC.Gynostemma pentaphyllum(Thunb.)Makino belongs to family Cucurbitaceae.It is widely used in Zhuang medicine.G.pentaphyllum has the functions of clearing away heat and detoxification,regulating Qi Flow and San-Dao&Liang-Lu.Clinically,it can be used to treat various chronic bronchitis,pyelonephritis,as well as hypertension,carbuncle swelling and toxin.Gypenosides were the main active ingredients of G.pentaphyllum.The anticancer activity of gypenosides in vivo and in vitro had been widely reported,such as prostate cancer,gastric cancer,hepatocellular carcinoma,lung cancer,colon cancer and breast cancer.However,the effects and mechanism of gypenosides in RCC remain unclear.Based on the theory of Zhuang medicine,it is speculated that the Zhuang medicine Gynostemma pentaphyllum and its saponins may have anti-kidney cancer effects.ObjectiveBased on the theory of Zhuang medicine and research progress,from the perspective of lipid metabolism network and its related upstream and downstream pathways,based on technologies such as network pharmacology,transcriptomics,and lipid metabolomics at the cells level and nude mouse xenograft tumor models,To clarify the effect of gypenosides on renal cancer cells inhibiting proliferation and inducing apoptosis.The active compounds in Gynostemma pentaphylla were separated and prepared.Clarify the effect of Gyp L and Gyp LI regulating the metabolism of arachidonic acid mediated by MAPK pathway and affecting the proliferation of RCC.Strive to make innovative discoveries to provide scientific basis for the development of Zhuang medicine preparations and the treatment of RCC.Methods1.To explore the antitumor effect of gypenosides by vitro experiments.CCK8、colony-formation assay and Annexin V/Propidium iodide(PI)were performed to evaluate cell death treated with gypenosides.Construct a subcutaneous xenograft tumor model in BALB/c nude mice.Divide into normal saline group and gypenosides treatment group.When the tumor volume reached 100mm3,the nude mice will be treated with normal saline and drugs daily.Weigh the nude mice and measure the long diameter of the transplanted tumor every 3 days,and calculate the volume.Perform HE staining and immunohistochemistry on the tumor to detect the expression of apoptotic proteins and related enzymes of the arachidonic acid pathway.Extract lipids from nude mice tissues and plasma and analyze them by UPLC/MS/MS.2.The location of the active compounds in Gynostemma pentaphyllum extract was confirmed by using mass spectrometry.The active compounds were isolated by synthetically using HP20 macroporous resin,silica gel,medium pressure preparative chromatography and semi-preparative chromatography.The anti-tumor activity of active compounds on ccRCC cells proliferation was proved by CCK8 proliferation assay.3.The effects of Gyp L and Gyp LI on the proliferation of RCC cells were detected by colony-formation assay,CCK8,Hoechst and Annexin V/PI experiments.The effects of Gyp L and Gyp LI on cell cycle was demonstrated by Rnase/PI flow cytometry.Western Blot was used to detect the changes of apoptosis and cell cycle related proteins.Based on network pharmacology and RNA sequencing data analysis,pathway enrichment analysis and other bioinformatics methods,to find the different key genes and metabolic pathways of two drugs,and analyze the underlying mechanism of drug action.Verify differential genes and key proteins of RNAseq related results of by RT-qPCR,Western Blot and immunofluorescence technology.The content of arachidonic acid metabolites were detected by using UPLC/MS/MS.Results1.Gypenosides extracted from G.pentaphyllum showed strong activity against 786-O and Caki-1 cells,and cell apoptosis were detected through Annexin V/PI dual staining assay.Gypenosides inhibits the growth of transplanted tumors in BALB/c nude mice.In vivo experiments,the content of arachidonic acid in the cells was reduced to inhibit tumors.Gypenosides does not affect on liver founction and bodyweight of nude mice.2.A total of 4 saponins monomers were isolated and prepared from the original medicinal materials of Gynostemma pentaphyllum,namely Gyp L,Gyp LI,damulin A,and damulin B.The four components were screened for their anti-tumor activity by the CCK8 assays.The results showed that Gyp L and Gyp LI can effectively reduce the viability of cancer cells in the normal concentration.Therefore,we will study the mechanism of this pair of isomers in inhibiting the proliferation of RCC.3.We conducted CCK8,colony-formation assay,Hoechst 33258 staining and Annexin V/PI flow cytometry to ascertain that Gyp L and Gyp LI have a significant inhibitory effect on renal cancer cell lines.Western Blot experiment showed that GypL and GypLI can induce apoptosis of renal cancer cells by up-regulating Bax and down-regulating Bcl2.Next,flow cytometry was used to detect the cell cycle distribution,and Western Blot was used to detect the cycle-related proteins CDK1,Cyclin B1,CDK2,and Cyclin A.The results showed that after GypL and GypLI treatments,769-P and ACHN cells were blocked in G2/M phase and G1/S phase.In order to further explore the mechanism of GypL and GypLI affecting the proliferation of RCC,we used network pharmacology and RNAseq to analyze different genes,and found that the enzymes related to arachidonic acid metabolism in renal cancer cell lines after intervention by Gyp L and Gyp LI,the mRNAs of PLA2G4,COX7A,ALOX12 and CYP2E1 are significantly down-regulated,while Gyp L and Gyp LI can significantly up-regulate the related genes Fos,JUN and DUSP1 in the MAPK pathway and verified by RT-qPCR.The results of Western Blot and immunofluorescence experiments show that Gyp L and Gyp LI down-regulate cPLA2,P-P38,P-ERK and P-MEK,Increase the expression of P-JUN,P-JNK.At the same time,UPLC/MS/MS was used to detect the decrease in the content of arachidonic acid.Conclusion1.Gypenosides inhibit the proliferation of renal cancer cells and induce apoptosis.2.Gypenosides can inhibit the growth of RCC in nude mice by reducing intracellular arachidonic acid content.3.Isolation and preparation of the active compounds Gyp L,Gyp LI,damulin A,and damulin B in Gynostemma pentaphyllum,and Gyp L and Gyp LI can effectively reduce the viability of cancer cells in the normal concentration.4.Gyp L and Gyp LI induce renal cancer cell apoptosis by down-regulating genes related to arachidonic acid metabolism and up-regulating genes related to MAPK pathway and the content changes of arachidonic acid in cells.