Proteomics Analysis Of Immunomodulatory And Anti-tumour Mechanism Of Hedysari Radix Polysaccharide

BackgroundHedysari radix. known as "Hongqi'in traditional Chinese medicine, which is the dried root of Hedysarum polybotrys Hand.-Mazz., belongs to the the Fabaceae family but not belongs to the same genus as a species of Astragali radix and was believed to possess the effects of invigorating qi for consolidating superficies, replenishing blood, diuresis for detoxification, drainage, astringing wound for regenerating tissue. Hedysari radix polysaccharide (HPS), which is one of main chemical constituents of Hedysari radix, is composed of rhamnose, xylose, arabinose. galactose and glucose. Pharmacological research indicate that HPS possess the effects of immunomodulatory, antitumor and antioxidant. Furthermore. HPS may be considered as a new drug of traditional Chinese medicine with high value for development.Proteomics, known as an important systems biology tool in the post-genomics era, provided a novel approach for multicomponent, multitargets and multilinks dynamic analysis of traditional Chinese medicine as a powerful biomarker detection tool. Furthermore, Previous research showed that HPS possess the immunomodulatory and antitumor effects and the mechanism remains unclear. In this study, proteomics approach was applied to identify differentially expressed proteins to investigate the complex molecular mechanisms at a global qualitative and quantitative level in various model systems treated by HPS and provided further insights into theoretical progress for clinical application of RH and RHP in traditional Chinese medicine research.ObjectiveTo investigate the differentially expressed proteins in various model systems after treated by HPS through a proteomics approach and provide preliminary results for further study to explore the complex molecular mechanism of immunomodulat ory and antitumor effects of HPS. Furthermore, to explore the immunity in immunosuppressed mice after HPS treatment.MethodsThe target cells or tissues collected from various model systems treated by HPS were lysed by lysis buffer for protein extraction. The protein concentration were measured by Bradford method after acetone precipitation respectively. The proteins extracted from cells or tissues were separated by means of two dimentional gel electrophoresis (2-DE) based on17cm immobilized pH gradient. After Coomassie brilliant blue G250staining or silver staining and image scanning,2-DE image comparison was performed to analyze the differentially expressed proteins including theoretical Mr. theoretical pI value and3D analysis using PDQuest8.0software. Furthermore, mass spectrometry was applied to identify the differentially expressed proteins and the data were searched against NCBInr database by an in-house Mascot search engine. Western blot was employed to assess the expression of heat shock protein beta-1in S180sarcoma. Immunosuppression model was established by administrating cyclophosphamide to normal mice. The thymus gland index and ultrastructure, spleen index, ultrastructure of thymocyte, phagocytosis of the macrophage were determined after HPS treatment.Results1. A total of69protein spots in2-DE images of thymus cells from mice were found to be differentially expressed between control group and RPS-treated group, of which33spots were up-regulated,26spots down-regulated,3spots disappeared,7spots expressed only in HPS-treated group. Finally,4spots were successfully identified to be protein synthesis initiation factor4, carbonic anhydrase, peroxiredoxins6and proteasome by MALDI-TOF/TOF and Mascot software.2. Compared with cyclophsphamide (Cy) group, the HPS combined with Cy group showed obviously inhibitory effect on growth of S180sarcoma (P<0.05). A total of28protein spots in2-DE images of S180sarcoma were found to be differentially expressed between control group and RPS-treated group, of which8spots were up-regulated,19spots down-regulated,1spots expressed only in Cy group. Finally,5spots were successfully identified to be heat shock protein hsp84, unnamed protein product, apolipoprotein A-I, albumin, heat shock protein beta-1by MALDI-TOF/TOF and Mascot software. Results from western blot manifested the same trend as from proteomics analysis.3. A total of21protein spots in2-DE images of HeLa cells were found to be differentially expressed between control group and HRP-treated group, of which14spots were up-regulated,4spots down-regulated,3spots expressed only in control group. Finally,4spots were successfully identified to be nucleophosmin, alpha-enolase, triosephosphate isomerase, acetoacetyl-coenzyme A reductase by MALDI-TOF/TOF and Mascot software.4. A total of35protein spots in2-DE images of A549cells were found to be differentially expressed between control group and RPS-treated group, of which24spots were up-regulated,8spots down-regulated,3spots expressed only in control group and theoretical Mr, theoretical pI value and3D structure of each spot were calculated with PDQuest software.5. HPS treatment significantly raised thymus gland index, spleen index and phagocytosis of the macrophage, and decreased damage of thymocyte.Conclusion1. The protein expression profile of mice thymus was altered by HPS treatment and the successfully identified proteins including synthesis initiation factor4, carbonic anhydrase, peroxiredoxins6and proteasome were associated with immunomodulatory effect.2. HPS treatment strengthened chemotherapy effect of Cy in tumor-bearing mice. The successfully identified proteins including heat shock protein hsp84, unnamed protein product, apolipoprotein A-I, albumin, heat shock protein beta-1were associated with synergistic effect with Cy and immunomodulatory in tumor-bearing mice.3. The protein expression profile of human cervical carcinoma HeLa cells was altered by HPS treatment and the successfully identified proteins including nucleophosmin. alpha-enolase, triosephosphate isomerase, acetoacetyl-coenzyme A reductase were associated with anti-proliferation effec of HPS on HeLa cells.4. The protein expression profile of human lung adenocarcinoma A549cells was altered by HPS treatment and the35differentially expressed spots were associated with anti-proliferation effect of HPS on A549cells.5. HPS treatment significantly increased immunity effect in immunosuppressed mice.