Study On Immune Regulation Of COX Signal Pathway Mediated By Guizhi Shaoyao Zhimu Decoction In AA Rats

Purpose:To study pharmacodynamic action of Gui shao zhimu Decoction on the adjuvant arthritis rats, and explore the molecular biology mechanism of Gui shao zhimu Decoction on rheumatoid arthritis, by means of impact on the expression of cyclooxygenase signaling pathway and relative proteins.Material and method:Female SPF Wistar rats were randomly divided into normal group, the controlled group, the ibuprofen group, Gui shao zhimu(GSZM) high-dose group,GSZM middle-dose group, GSZM low-dose group. All rats except the normal group were injected complete Freund’s adjuvant into the paws to establish adjuvant arthritis model. Eight rats were left in each guoup. Gavage administration of treatment were made at one week after modeling: saline was administrated in controlled group, ibuprofen suspension(63mg/kg) was administrated in ibuprofen group, Gui shao zhimu Decoction(34.65, 17.325, 8.6625g/kg)were given in GSZM high, middle, low-dose group. Drug administration lasted for 3 weeks。In general pharmacodynamic research, general state and weight changes were observed, paw swelling ratio were calculated to study the edema situation. Reflect time to thermal pain by hot plate was measured to observe thermal hyperalgesia. Level of Tumor necrosis factor α,interleukin 1β, interleukin 10 in peripheral blood and plantar soft tissue of rats were observed by ELISA method, to research peripheral and local concentration of pro-inflammatory and inhibiting inflammatory cytokines.Articular cartilage damage was estimated by pathological slices in rat ankle. Radiographic changes in ankle anteroposterior were detected by means of digital radiographgic X-ray collimator. Peripheral and local interferon γ and interleukin 4were checked by ELISA method. T-bet and Gata-3 m RNA expression in paw tissue were detected by real-time quantitative reverse transcription PCR method. Helper T lymphocyte drift were reflected by the ratios of INF-γ/IL-4 and T-bet/Gata-3, Th1 subpopulation represented by INF-γ and T-bet, Th2 subpopulation represented by IL-4 and Gata-3.Correlation analysis between the two ratios were made. Western blot method was taken to detect the protein expression of cyclooxygenase 1(COX-1), cyclooxygenase 2(COX-2),inducible nitric oxide synthase(i NOS), TNF-α in paw soft tissue, to observe the effect on the expression of COX isozymes and the related proteins in each group. Expression of COX-2and its host protein nuclear factor-κB(NF-κB), lower protein vascular endotheelial growth factor(VEGF) in COX signaling pathway were checked by immunohistochemistry(IHC),and pathological image analysis were made to compare the impact of the treatment groups on COX signaling pathway.Results:1. General pharmacodynamic results showed that: Poor generally state were showed after modeling. Paw swelling ratio were significantly raised up. Thermal hyperalgesia was significantly reduced. Inflammatory cell infiltration, pannus formation and synovial hyperplasia could be seen in HE staining slices of the ankle joint cartilage, pathological scores were significantly enhanced. Significantly ankle joint and soft tissue swelling, rough articular surface, narrowing joint space were reflected in ankle radiologic findings. After three weeks of treatment, all indexes compared with the model group in each treatment group were improved(P<0.05): general state was improved, paw swelling were significantly reduced(P<0.05), thermal hyperalgesia was significantly promoteded(P<0.05). ELISA outcomes shows that peripheral and local concentration of TNF-α and IL-1βwere significantly raised up(P<0.01), IL-10 level was significantly descended(P<0.01) in controlled group. After administration TNF-α, IL-1β levels were depressed compared with the controlled group(P<0.01), IL-10 levels were significantly increased(P<0.01). Pharmacodynamic action of GSZM high-dose group, middle-dose group were similar to that of the ibuprofen group except that IL-10 levels were significantly lower(P<0.01), indexes of GSZM low-dose group were significantly different with the other three groups(P<0.01). Situation of synovial structures and inflammatory infiltration were improved in slices of treatment groups, pathological score was significantly reduced(P<0.05). X-ray displayed reduced ankle swelling. Among the treatment groups, GSZM high-dose group got better efficacy, close to ibuprofen group,efficacy of GSZM low-dose group was significantly lower than the other three treatment groups.2. Th drift results: IL-4 level of the controlled group were significantly descended(P<0.01),INF-γ level were significantly raised up(P<0.01), INF-γ / IL-4 ratio was significantly enhanced(P<0.01). In plantar soft tissues T-bet expression was significantly increased(P<0.01), with significantly reduced GATA-3 expression(P<0.01), T-bet / Gata-3 ratio was significantly enhanced(P<0.01). The result reflected Th1 direction of Th1 / Th2 drift. The ratio of INF-γ / IL-4 and T-bet / Gata-3 were both reduced significantly in four treatment group(P<0.01), suggesting a role in promoting the recovery of Th drift. The recovery action of Th drift in GSZM high-dose group and middle-dose group was similar to that of the ibuprofen group.3. Results of COX isozymes and related protein expression checking: The expression of COX-2, i NOS and TNF-α showed a significant increase in controlled group(P<0.01). After treatment an significant decrease of COX-2, i NOS and TNF-α expression could be seen in buluofen group, GSZM high-dose group and middld-dose group(P <0.01). The COX-1expression in ibuprofen group and GSZM low-dose group was significantly lower than that of other groups(P<0.01).4. Protein expression of COX signaling pathway: expression of NF-κBp65, COX-2, VEGF were significantly increased in controlled group(P<0.01), significantly descended in treatment groups(P<0.01). Comparison of the three protein expression among the four groups showed that there was no significant differences between GSZM middle-dose group and ibuprofen group, three protein expression in GSZM low-dose group were significantly higher than ibuprofen group(P<0.01), but VEGF expression of GSZM high-dose group was significantly lower than that of ibuprofen group(P<0.01).Conclusion:1. Gui shao zhimu Decoction has improving action on articular soft tissue inflammation in AA rat.2. Gui shao zhimu Decoction can significantly reduce peripheral and local concentration of pro-inflammatory cytokines in and AA rat, elevate levels of anti-inflammatory cytokines, the effect and dose levels were positively correlated.3. Gui shao zhimu Decoction can significantly descend the ratio of INF-γ / IL-4 and T-bet /Gata-3, its therapeutic mechanism may be related to the adjustment of Th1/Th2 drift.4. Gui Shao Zhimu Decoction can significantly inhibit COX-2 expression, maintaining or mildly suppressing the expression of COX-1, which partly shows the reason of its exact therapeutic effect with less side-effect to RA.5. The pharmacodynamic action of Gui shao zhimu Decoction on AA rats may be closely related to reduction of NF-κBp65, COX-2, VEGF expression in COX signaling pathway.