Research In Decreasing Toxicity And Increasing Efficacy And Mechanism Of Treating RA On The Compatibility Of Nux Vomica And Sappan Wood,Nux Vomica And Radix Dipsci

Objective: Under the guide of TCM theory, on the basis of clinical application, thepaper systematic reviewed the ancient and modern application of Nux vomicacompatibility, which in decreasing toxicity and increasing efficacy. With the compatibilityof Nux vomica and Sappan Wood,Nux vomica and Radix Dipsci (hereinafter referred toas N-S, N-R) which of different ratio, through the experiment on toxicity, analgesic,anti-inflammatory effects, the article observed the reduction poison effect, tried to seek thebest proportion, and searched after the mechanism of radical damage resistance, cellfactors and apoptosis three aspects separately. Using the theory of odour compatibility, thesignificance of N-S, N-R compatibility was interpreted, which in decreasing toxicity andincreasing efficacy, and the treatment of rheumatoid arthritis (RA) either. At the sametime,the paper will provide the scientific basis of clinical application for treatment of RA withNux vomica.Methods:1. The content of nux vomica alkali were determinated before and aftercompatibility on the N-S, N-R, which of different ratio decoction, by high performanceliquid chromatography(HPLC); Through the test of acute toxicity, to observe the status ofthe mice died, and record the number of deaths, and then calculate the median lethal dose(LD50) by Bliss. Finally, to analyze the toxic strength changes of different proportions, andseek the best ratio.2. Adopting the mice hot plate and writhing, the licking latency were measured andthe total number of writhing in15min were recorded after the drug administration. Thenthe inhibition rate of the different ratio on writhing was calculated. The experiments wereto observe the analgesic impact in mice, and seek the best analgesic ratio.3. Establishing the rat model of adjuvants arthritis(AA), the degree of rat paw edema were measured by micrometer; the coefficient of the immune organ was calculated; and thecontent of NO, SOD, MDA in serum were determinated. The expression levels of IL-1β,IL-6, IL-10, and TNF-α, CRP, CIC in serum, and PGE2, Cor, Fas, Bcl-2in synovium weredetected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression ofcytokines such as IL-1β, IL-10, TNF-α in synovium were semi-quantitative analyzed byreverse transcription-polymerase chain reaction (RT-PCR). Which in the aspect ofanalgesic and immune, these experiments were to clarify the mechanism and find the bestratio. The expression of Fas in the synovium was detected by immunohistochemistry (IHC)and the anti-apoptosis mechanism was observed initially.4. On the long-term toxic effects, histological study of liver, kidney, brain, wasobserved by haematoxylin and eosin (HE) stains. The pathological changes of synoviumwere observed by HE, and then analyze anti-inflammatory efficacy after drugadministration.Results:1. The results of HPLC showed that, comparing with the single decoction ofnux vomica group, the contents of brucine were separately reduced by68.8%,76.8%,99.3%, and the strychnine were reduced by56.8%,66.9%,93.6%in N-S1:2,1:4,1:6decoction. The contents of brucine were separately increased by17.6%, reduced by23.5%29.0%, and the strychnine were respectively increased by60.7%, reduced by8.51%,16.6%in N-R1:2,1:4,1:6decoction.2. Experiment on the acute toxicity of N-S, N-R By calculating, the value of LD50were605.92,827.21,1084.7,1479.4,401.64,664.1,953.79mg/kg, respectively in thesingle decoction of nux vomica group, N-S1:2,1:4,1:6and N-R1:2,1:4,1:6decoctiongroup.3. Experiment on the analgesic effect of N-S, N-R Hot plate test: After theadministration, the pain threshold were increased and the pain reaction time were extended(P <0.01or P <0.05) in N-S1:4,1:6and N-R1:6decoction group, which N-S and N-S1:6decoction group were better. Writhing: The inhibition of writhing in N-R1:6decoctiongroup was significantly stronger than the nux vomica and blank group (P <0.05), and thewrithing inhibitory rate was26.75%. The other groups had no significant changes.4. Experiment on anti-inflammatory effects of AA rats The primary paw swelling:During treatment, the rat paw swelling degree were significantly reduced in N-S1:4,1:6and N-R1:6decoction group, the anti-inflammatory effect was more significant (P <0.01or P <0.05). The secondary paw swelling: Paw swelling were significantly reduced after administration of the compatibility groups, the N-S1:4,1:6and N-R1:6decoction group ofanti-inflammatory effect were more prominent (P <0.01or P <0.05), the best ratio wasN-S1:6.5. Experiment on the immune organ coefficient of AA rats The thymus coefficientswere decreased in nux vomica group, N-S of1:4,1:6and N-R1:2,1:4,1:6decoction group(P <0.01or P <0.05). The spleen coefficients were decreased significantly (P <0.05or P<0.01) in nux vomica group, N-S1:2,1:4and N-R1:6decoction group.6. Experiment on the NO, SOD, MDA in serum of AA rats The content of NO inserum could be reduced in N-S1:6decoction group (P<0.05); the single decoction of nuxvomica group, N-S1:2,1:4,1:6and N-R1:2,1:4,1:6decoction group could increase thecontent of SOD (P <0.05or P <0.01); N-S1:4,1:6and N-R1:2,1:4,1:6decoction couldsignificantly decrease the content of MDA (P <0.01).7. Experiment on the inflammatory cytokines of AA rat After the administration, thelevels of IL-1β, CRP were decreased in N-S1:2decoction (P <0.01or P <0.05); thecontents of IL-1β and TNF-α, CRP in N-S1:4decoction were reduced significantly (P<0.01or P <0.05); and the levels of IL-1β, TNF-α, IL-6, CIC, CRP N-S1:6decoction werereduced more obviously (P <0.01or P <0.05). After the administration, the levels of IL-6,IL-10, CRP were decreased in N-R1:2decoction (P <0.01or P <0.05), and the contents ofIL-1β, IL-6, IL-10, CRP and CIC, Cor in N-R1:4decoction were significantly lowed; thelevels of IL-1β, TNF-α, IL-6, IL-10, CRP and CIC, Cor in N-R1:6decoction weresignificantly reduced (P <0.01or P <0.05).8. Experiment on the mRNA expression of cytokines in AA rat synovium ThemRNA expression of cytokines as IL-1β were significantly decreased in Nux vomica groupand N-S1:2,1:4,1:6decoction group (P <0.05or P <0.01). Compared with the modelgroup, the mRNA expression of cytokines as IL-10were significantly up-regulated in N-S1:6decoction group (P <0.05). The mRNA expression of cytokines as TNF-α had nosignificant changes.9. Experiment on the cells apoptotic factors of AA rat The contents of Fas weresignificantly reduced in Nux vomica and N-S1:6decoction group (P <0.01). At thesametime, the result of IHC showed that, comparing with the model group, the positiveexpression of Fas was decreased in N-S1:6decoction, and mainly located in the cytoplasm.N-S1:6decoction could significantly improve the content of Bcl-2(P <0.05).10. Experiment on the pathomorphology of liver, kidney, brain and synovium The inflammatory cell infiltration, fibrous tissue proliferation of the liver could be significantlyimproved by the compatibility group. At the sametime, the kidney glomerular atrophy andmesangial proliferation were reduced, the stenosis of kidney tubules and luminal protein,brain tissue cortex edema and neuronal solid contraction could be improved. Thethickening of the synovial tissue, inflammatory cell infiltration, fibrous tissue proliferationand necrosis were improved as well. The most obvious groups are N-S, N-R1:6decoction.Conclusion: After the compatibility of N-S, N-R, the toxicity of nux vomica can bereduced, meanwhile the analgesic and anti-inflammatory effect can be enhanced. Theappropriate ratio has effects of decreasing toxicity and increasing efficacy. Theanti-inflammatory immune mechanism of N-S is decreasing NO, MDA, IL-1β, IL-1βmRNA and TNF-α, reducing the secretion of IL-6, CIC and CRP, decreasing thepro-apoptotic factors Fas and increasing the content of SOD, the Suppression of apoptosisfactor as Bcl-2, increasing the mRNA expression of IL-10. The anti-inflammatory immunemechanism of N-R is reducing MDA, increasing the content of SOD IL-1β, TNF-α, thesecretion of IL-6, reducing the generation of the CIC, CRP and Cor,. N-S, N-R havemultiple levels and way, many of the targets anti-inflammatory and immune adjustmenteffects. In this research, the best ratio, which have the effects of decreasing toxicity andincreasing efficacy, are N-S and N-R1:6.