| Objective:Gastric cancer is one of the most common malignant tumors of the digestive system,which is being a major security challenge to public health.At present,the development of nutritional function factors with anti-gastric cancer efficacy and safety has attracted more and more attention from researchers.A number of studies have been shown that polysaccharide are potential anti-gastric cancer substances.Lotus is widely cultivated in our country.The polysaccharide of the lotus plant has a rich nutritional function.For example,lotus root polysaccharide has been proved to have anti-gastric cancer effect.The lotus seedpod is the dry and mature receptacle of the lotus.In comparison to polysaccharide from other parts of the lotus,there are few reports on lotus seedpod polysaccharide（LSP）and whether it has anti-gastric cancer effect is still unclear.In this study,lotus seedpod polysaccharide was used as the research object.Using human gastric cancer AGS cells,the anti-gastric cancer effect of LSP was studied in vitro.At the same time,the differentially expressed genes in AGS cells treated with LSP were screened based on transcriptomics.N-methyl-N’-nitro-N-nitrosoguanidine（MNNG）induced gastric cancer rat model was used to further study the anti-gastric cancer effect of LSP in vivo.Simultaneously,the differentially expressed genes in MNNG gastric cancer rats treated with Lotus polysaccharides were screened using transcriptomes.The aim of this study is to provide data supporting the nutritional use of lotus seedpod polysaccharide in the treatment of gastric cancer.Methods:1.In vitro experiments:The effect of LSP on apoptosis of AGS cells was detected by Hoechst staining and flow cytometry.The effects of LSP on the viability,morphology and cell cycle of AGS cells were detected by CCK-8 kit and flow cytometry.Fluo-3AM,JC-1 and DCHF-DA fluorescent probes were used to detect the changes of Ca2+concentration,mitochondrial membrane potential（ΔΨm）and reactive oxygen species（ROS）levels in AGS cells.Wound healing assay and Transwell assay were used to detect the effect of LSP on the migration and invasion of AGS cells.The levels of transforming growth factor-β1（TGF-β1）and vascular endothelial growth factor（VEGF）in AGS cells were measured by ELISA method.The differentially expressed genes in AGS cells treated with LSP were screened based on transcriptome.2.In vivo experiments:Furthermore,MNNG induced rat gastric cancer model was used to evaluate the anti-gastric cancer effect of LSP in vivo.Hematoxylin-eosin staining（HE staining）was used to observe the gastric tissue pathological changes of rats.The levels of Immunoglobulin-A（Ig A）and glutathione（GSH）in gastric tissue of rats with gastric cancer were determined by ELISA and kit methods.The differentially expressed genes in MNNG gastric cancer rats treated with LSP were screened based on transcriptome.Results:1.In vitro experiments:（1）LSP decreased AGS cell viability,affected cell morphology and arrested cell cycle at G0/G1 phase.（2）LSP could induce apoptosis of AGS cells.（3）LSP increased Ca2+concentration,decreasedΔΨm,and promoted ROS generation.（4）LSP inhibited the migration and invasion of AGS cells.（5）LSP increased the level of TGF-β1 and decreased the level of VEGF in AGS cells.（6）Transcriptome screening revealed 9492 differentially expressed genes in AGS cells after LSP treatment,including 4413 up-regulated genes and 5079 down-regulated genes.KEGG analysis showed that 342 pathways were significantly enriched after AGS cells were treated with LSP,among which the cell cycle interaction pathway was the most significantly enriched.The key genes CDK1,CDC20,CDC6,CDC27,FZR1,MAD2L1,CCNA2,BUB1B and ESPL1 were screened by protein interaction network analysis of differentially expressed genes in cell cycle.2.In vivo experiments:（1）HE staining showed that LSP could alleviate MNNG induced gastric cancer tissue injury in rats.（2）LSP significantly increased the contents of Ig A and GSH in gastric tissue.（3）Transcriptome screening revealed 90significantly enriched pathways in MNNG gastric cancer compared with control rats Compared with MNNG gastric cancer rats,three pathways were significantly enriched after LSP treatment,which were acute myeloid leukemia,cancer transcriptional dysregulation and cancer pathway.Conclusion:1.Lotus seedpod induces apoptosis and inhibits the growth and proliferation of AGS cells,which may be related polysaccharide to the reduction of cell viability and arrest of cell cycle,the enrichment of cell cycle interaction pathways in AGS cells treated with lotus seedpod polysaccharide was the most significant.2.Lotus seedpod polysaccharide has a certain protective effect on MNNG gastric cancer rats,which may be related to the enhancement of immunity and anti-oxidation.Moreover,it was found that lotus seedpod polysaccharide may affected acute myeloid leukemia,transcriptional dysregulation in cancer and cancer pathway in MNNG rats. |
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