| Objective: We aimed to study the effect of Dendrobium officinale polysaccharides and Rhizoma Coptidis extract on the gene expression profiling of HUVEC and Hep G2,respectively.Methods: 1.Extraction of Dendrobium officinale polysaccharides and Rhizoma Coptidis by a spin evaporator.2.Cell proliferation activity was detected by MTS assay.3.Extraction and detection of total RNA.4.c DNA synthesized and amplified.5.Probe labeling and chip hybridization.6.Image acquisition and data analysis.7.Bioinformatics analysis.8.Validation the results of gene chip by q RT-PCR.9.SPSS software was used to analyze the data.Values were expressed as the mean ± standard deviation,and T test was used for comparison between the two groups.P < 0.05 was statistically significant.Results: 1.Cell proliferation activity assay Dendrobium officinale polysaccharides can inhibit the proliferation of HUVEC cells.Compared with the control group,the higher the concentration of polysaccharide of Dendrobium officinale above 100 ?g/m L,the lower the survival rate of HUVEC cells(P < 0.05 or P < 0.01).The high concentration of crude extract of Rhizoma Coptidis has a certain inhibitory effect on the proliferation of Hep G2 cells.Compared with the control group,the higher the concentration of Rhizoma Coptidis above 30 ?g/m L,the lower the survival rate of Hep G2 cells(P < 0.05).2.Results of gene expression profile microarray hybridization Compared with the control group,after 400 ?g/m L Dendrobium officinale polysaccharides for 24 hours,there were 91 genes with expression differences in HUVEC cells,among which 84 up-regulated genes(Ratio > 2)and 7 down-regulated genes(Ratio < 0.5).After 75 ?g/m L Rhizoma Coptidis treated Hep G2 cells for 24 hours,there were 1621 differentially expressed genes,including 869 up-regulated genes(Ratio > 2)and 752 down-regulated genes(Ratio < 0.5).3.Bioinformatics analysis of differentially expressed genes GO enrichment analysis of differentially expressed genes showed that the up-regulation of differentially expressed genes was mainly concentrated in the biological processes of cytokine production,immune response and chemokine,while the down-regulation of differentially expressed genes was mainly concentrated in the biological processes of cytokine activity,chemokine,immune response and stress response.KEGG pathway analysis showed that the up-regulated genes of Dendrobium officinale polysaccharides were mainly involved in TNF signaling pathway,rheumatoid arthritis,herpes simplex virus,measles,malaria,Toll-like receptor signaling pathway and Nod-like receptor signaling pathway,while the down-regulated genes of Dendrobium officinale polysaccharides were mainly concentrated in chemokine signaling pathway.After Rhizoma Coptidis treated Hep G2 cells,GO enrichment analysis of differentially expressed genes showed that the up-regulation of differentially expressed genes was mainly concentrated in biological processes such as apoptosis,protein activity and so on,while the down-regulation of differentially expressed genes was mainly concentrated in biological processes such as cell mitosis and oxidoreductase activity.KEGG pathway analysis showed that up-regulated genes of Rhizoma Coptidis were mainly involved in p53 signaling pathway,TNF signaling pathway,MAPK signaling pathway and cancer-related signaling pathway,while down-regulated genes were mainly involved in lipid metabolism-related signaling pathway.4.Verification the results of gene chip by q RT-PCR In the experiment of Dendrobium officinale polysaccharides on HUVEC cells,in order to verify the results of microarray,we selected representative genes related to immunity from up-regulated and down-regulated genes: IL6,ICAM1,CCL14 genes for q RT-PCR verification according to the results of KEGG gene function enrichment.In the experiment of Rhizoma Coptidis on Hep G2 cells,the representative genes ACTG1 and ITGB1 related to cancer were selected from up-regulated genes,and the representative genes ACAT2 and HMGCS1 related to lipid metabolism were selected from down-regulated genes for q RT-PCR verification.The results of verification are consistent with those of the chip.Conclusion:Compared with the control group,the gene expression of HUVEC cells treated by Dendrobium officinale polysaccharides was significantly different,and the differentially expressed genes were significantly enriched in the signaling pathways of immune response,production of cytokines and chemokines.After Rhizoma Coptidis treated Hep G2 cells,differentially expressed genes were significantly enriched in cancer and lipid metabolism signaling pathways.This study provides a more comprehensive understanding of the action targets of Dendrobium officinale polysaccharides and Rhizoma Coptidis,providing a basis for further study of the pharmacological mechanism of Dendrobium officinale polysaccharides and Rhizoma Coptidis. |
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