| Object:Use ultra-high performance liquid chromatography-Photo diode array (UPLC-PDA) to determine the the content of the active ingredients in Radix Paeoniae Alba decoction and the absorption of the active ingredients in rat plasma after administration of Radix Paeoniae Alba decoction; explor the protein expression changes of CIA synovial tissue, and draws its signal transduction network map, revealing the mechanism of the development of RA; Clarify the effects of the active ingredients on the protein expression of CIA synovial tissue and its regulation on signal transduction networks, explain its role in the treatment of RA,and to lay the foundation of Bizhongxiao Decoction to treat RA.Methods:(1) The quantitative analysis on Paeoniflorin in Radix Paeoniae Alba,the analysis was perform on UPLC system with BEH (R) C18column (50mm x2.1mm,1.7μm).The mobile phase is consist of acetonitrile (A) and1%acetic acid (B),Flow rate and detection wavelength was set at0.25ml/min and190-480nm.Gragh the standard line、Precision、sample stability and reproducibility was examined in this study.(2) Use the UPLC-PDA method to analyze the active ingredient in rats plasma after oral administration of Radix Paeoniae Alba decoction.(3) Use low, medium and high doses of Paeoniflorin standard solution to gavage intervention the CIA rats, then using enzyme-linked immunosorbent assay (ELISA) to determine the levels of serum inflammatory cytokines IL-1β, TNF-α to screen the the optimal dose of paeoniflorin in the treatment of CIA rats.(4)The Collagen-induced arthritis(CIA) rats model was established by injected subcutaneous of bovine Ⅱ collagen(B Ⅱ C)and complete Freunds adjuvant(CFA). Use the optimal dose of Paeoniflorin to intervent the CIA rats, dynamic observation of the general situation in rats, body weight increased,arthritis integral, joint swelling, joint X-ray and synovial histopathology,and detect the serum IL-1β, TNF-α levels by ELISA.(5) Quantitative proteomics detection:42d synovial tissue were extracted for each group, and the extracted total protein was enzymatic digestion;each group were labeled by different iTRAQ reagents,determin the labeled results and mixed them, then separated by SCX/RPLC and identification of differentially expressed proteins in each group by LC-MS/MS. Ingenuity Pathway Analysis is used to analyze the biological function of DEPs, the canonical pathway and networks were also performed by it.Results:(1) Paeoniflorin was separated successfully in four minutes.Determined the content of paeoniflorin in Radix Paeoniae Alba is0.74mg/g. The calibration curve showed good linear regression within test ranges of Paeoniflorin,R2>0.9994.The relative standard deviations (RSD) of intra-day and inter-day Precision were from0.99%to3.64%,indicating good precision. The recoveries of Paeoniflorin ranged from98.6%to103.6%,with RSD value<2.8%,showing good recoveries.The sample of Radix Paeoniae Alba decoction has good stability in24h,with RSD value<2.7%.(2) The Paeoniflorin was detected successfully in plasma of rat after oral administration of Radix Paeoniae Alba decoction in30minutes.(3) The medium doses of Paeoniflorin(1mg/kg.d) is the optimal dose to treat of CIA rats,it can significantly reduce the levels of serum inflammatory cytokines IL-1β,TNF-α.(4) The Collagen-induced arthritis(CIA) rats model was successfully established by injected subcutaneous of bovine Ⅱ collagen(B Ⅱ C)and complete Freunds adjuvant(CFA),valued the models on the42nd, the success rate is90%. Use the medium doses of Paeoniflorin to intervent the CIA rats,we found that:During the disease process, rats presented low spirits, somnolence, slow action, diet reduction and dry hairs in the later stage. On the42nd day, some rats had difficulty in weight bearing and movements., but the treatment group and normal group’s rats lively, eating more, coat shiny and soft,and the weight growth of model group rats is reduce comparing to normal group. In the first week of immunization, the CIA rat’s foot swelling was significant. Arthritis index integral of model group increased comparing to normal group in the first week and got to peak on the21st day, the AI of treatment group in21th、28th35thand42rd is significantly lower than the model group (P<0.05). Histopathology observation show that model rat’s synovial tissue had inflammatory cell infiltration., large amount of pannus generated,vascular proliferation and synovial thickening, but there was a few new vessels and inflammatory cell infiltration in synovial tissue and synovial thickened a little in treatment group. The model group rats had swelling tissue around the ankle joint in x-ray, the foot joint presented obscure space, narrow and fusion.some joint soft tissue still swelled a little and the bone destruction was obvious. the swelling tissue around the joint of arthritis rat in the treatment group reduced and the bone destruction degree was greatly weakened. Using ELISA to detect serum IL-1β and TNF-α of rats in each group, we found that the level of IL-1β and TNF-α in model group and treatment group have increased, after the treatment of paeoniflorin, serum IL-1β, TNF-α level decreased significantly.(5) After the analysis of IPA, we found that there are128DEPs were identified in the model group and the normal group, Among these proteins,44protein are down-regulated, and84are up-regulated. After treatment by Paeoniflorin,there are9proteins in the44down-regulated are up-regulated,and14proteins in the84up-regulated are down-regulated,19proteins have no significant difference from the normal group. Bioinformatics analysis found69significant canonical pathways, include IL-1signaling, IL-6signaling, Wnt/β-catenin signaling, G-protein-coupled receptors signaling and so on. We also drawed network diagrams.Conclusions:(1) UPLC-PDA can determine the paeoniflorin in a effective way.(2) Paeoniflorin in Radix Paeoniae Alba decoction was found to be the bioactive component which rat plasma can absorb. This components may be an effective ingredients to treatment of RA by Radix Paeoniae Alba,and it may be the efficacy material basis of BZXD to treat of RA.(3) Paeoniflorin can reduce the level of inflammatory cytokines IL-1β and TNF-α, inhibit the inflammation and bone erosion of CIA rats.(5) The pathogenesis of RA is related to multiple abnormal expression of proteins,these proteins involving several signal transduction pathways and constitute a complex signaling network. Most of the proteins expression are reversed after the intervention of Paeoniflorin,and some pathways are changed. This may be one of the mechanism of Paeoniflorin for the treatment of RA,and lay a solid foundation for further research of BZXD on the mechanism of RA. |
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