| Paeoniae radix alba is the dried root of the Paeoniae lactiflora Pall..Paeoniae radix alba is sour and bitter in flavor,slightly cold in nature and acting on the liver and spleen channels,with the effects of tonifying blood and regulating menstruation,astringing yin to arrest sweating,regulating liver and alleviating pain and clearing the liver-yang and checking exuberance of Yang.The constituents isolated from Paeoniae radix alba include monoterpenes,tannins and polysaccharides,etc.In clinical,Paeoniae radix alba can be used to treat liver disease,rheumatoid arthritis,intestinal diseases,etc.Pharmacology studies showed that the extracts from Paeoniae radix alba possessed the activities of liver tissue protection,anti-inflammation,anti-oxidation,etc.Most previous studies concluded that the total glucosides with the activity of antioxidant was the therapeutic mechanism of Paeoniae radix alba for treating liver diseases.As we all know,the water decoction of Paeoniae radix alba was mainly used in traditional Chinese medicine?TCM?clinic.In fact,a large number of polysaccharides existed in the water decoction.According to the previous researches,polysaccharides from TCMs possess important medicinal value,especially the effect of immunoregulation about immune inhibition.Therefore,Paeoniae radix alba polysaccharides?PRAP?is likely to be another important pharmacodynamic substance basis for Paeoniae radix alba in the treatment of liver diseases,and the immunosuppressive regulation may be its main mechanism.The following research is carried out in this paper:1.Study on extraction technology of PRAPResponse surface methodology?RSM?was used to study the effects of the liquid-solid ratio,the extraction time and the extraction times on the yield of PRAP.The optimal extraction conditions were predicted as follows:the liquid-solid ratio is 10.65 mL/g,the extraction time is 2.10 h,the extraction times is2.44 and the predicted yield of PRAP is 10.46%.To validate the predicted extraction conditions,5 repeated experiments were conducted on the extraction of PRAP under these conditions.The actual average yield of PRAP is 10.17%,which is closed to the predicted value?10.46%?.Therefore,the optimal extraction conditions can be used to optimize the extraction technology of PRAP.And the contents of total carbohydrate,alduronic acid and protein in PRAP are 81.39%,3.17%and 3.88%,respectively.2.Separation,purification and characterization of PRAPThree uniform polysaccharide PRAP.A-1?snow white floccule?,PRAP.B-1?light yellow floccule?and PRAP.B-2?faint yellow floccule?were obtained after the separation of PRAP by anion-cation resins and cellulose,which were soluble in water but insoluble in organic solvents.The single symmetrical peak was detected by HPLC.UV showed that the three uniform polysaccharides were free of nucleotide and proteins.IR was measured to characterize PRAP and three uniform polysaccharides of PRAP.The characteristic absorption peaks of polysaccharides were detected at about 3000-2800 cm-1,1400-1200 cm-1 and3400 cm-1.The characteristic absorption of furanose ring in PRAP can be observed at 856.07 cm-1.The absorption peak in 1000-1200 cm-1 shows the pyranose ring in the three uniform polysaccharides.3.Study on the mechanism of treating autoimmune hepatitis with PRAPThe therapeutic effect of PRAP was studied in mice model of autoimmune hepatitis?AIH?induced by the same gene liver antigen.AIH mice model was well established and the pathological features on histomorphology and histopathology of liver and spleen,liver function and oxidative stress were evaluated and elucidated in detail.After treated with PRAP,the liver function of mice could be recovered by repairing the relevant histopathological damages of liver and spleen and relieving oxidative stress of liver.Liver function LDH,ALT,AST activities and TBIL content decreased.Indices of oxidative stress MDA content was decreased,GSH content and SOD activity were increased.Immunohistochemical detection showed that the expressions of CD4,CD8,IL-2,IL-6 and IL-10 were up-regulated and no obviously positive expression of TGF-?was found in the model group.After treatment,the expressions of CD4,CD8,IL-2,IL-6 and IL-10 were down-regulated,while the expression of TGF-?was up-regulated,indicating that PRAP can suppress the immune inflammatory response of AIH.High-throughput transcriptome studies were conducted on experimental mice,total RNA was extracted for sequencing.Through differential gene expression analysis,gene set analysis,functional enrichment analysis and protein interaction network analysis,the key nodal protein genes in this study were A2m?Cd14?Cdr1?Cxcl1?Cxcl13?Cxcl5?Cyba?Cybb?Elane?Fcer1g?Fcgr4?Fga?Fpr2?Hp?Ltf?Mmp8?Mmp9?Orm2?,etc.The signaling pathways related to the pathogenesis of AIH were screened out from the mechanism pathways:chemokine signaling pathway,cytokine-cytokine receptor interaction,osteoclast differentiation,node-like receptor signaling pathway,etc.,which laid a preliminary foundation for the further study and verification of the mechanism pathways.In this paper,the RSM is used to extraction technology of PRAP for the first time,and the separation,purification and preliminary characterization of PRAP were carried out.The mechanism of PRAP in the treatment of AIH,the pathological features on histomorphology and histopathology of liver and spleen,liver function and oxidative stress were evaluated and elucidated in detail for the first time.This study can provide scientific and clinical basis for PRAP to become a drug for treating AIH in clinical. |
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