Research On The Mediatorome Regulating Mechanism Of Treating Systemic Inflammatory Response Syndrome Rabbits With Decoction For Clearing Away Pestilent Factors And Detoxification

Objective:"Inflammatory Mediatorome",usually highly expressed during inflammation, were all mediators of inflammation in a cell or tissue in one time-phase.As an organic whole,mediators of inflammation,closely linked in function,were called "mediatorome" In this research,several kinds of protein were selected to study from a great deal kinds of protein.Because studying target was limited in a small circumscription,experimental detection could be finished by precise technique and data processing would be easy.This strategy was helpful to resolve problems both in experimental techniques and data processing and very important in TCM research by holistic cellular biology techniques.Uncontrolled inflammation in systemic inflammatory response syndrome(SIRS) was actually a mediator disease,mainly caused by chain reaction of cytokine.But up to now,no anti-mediator therapy had made sure effect in treating septic shock.There were two possible reasons.First,there were so many kinds of mediator of inflammation that pathogenetic condition could not be improved by treating only a few mediators.Second,mediators of inflammation could protect the body in septic shock and anti-mediator therapy could destroy the protection.For the first reason,many kinds of mediators of inflammation should be treated together。For the second reason,every mediator in every time-phase should be treated correctly and moderately instead of being simply cleaned up.Exactly treating many kinds of mediators together was a ideal strategy, which was called "mediatorome regulating".TCM could regulate the mediatorome because of its integrity characteristics.Decoction for clearing away pestilent factors and detoxification(DCPD) and Xingnaojing injection(XNJI) had sure therapeutic effect to exogenous febrile disease and could affect mediators.In this research,SIRS experimental animals were treated by DCPD and XNJI while the change of mediatorome was observed and analyzed.Method:This research,in which SIRS experimental animals were treated by DCPD and XNJI while many kinds of mediator of inflammation were detected,included 4 experiments.In "experiment 1",New Zealand rabbits received LPS injection of different times and different doses to indue different exogenous febrile disease model.In "experiment 2",New Zealand rabbits SIRS models were induced by twice(with a 24-hour interval) lipopolysaccharide(LPS) infusion through vein.SIRS rabbits received intragastric administration of DCPD containing different dose of gypsum.Symptoms and objective signs,such as rectal temperature,consciousness,skin and mucosa,were observed at 1,2.5 and 5.5 hour after dosage and blood samples were collected at 1 and 2.5 hour after dosage.Then,10 kinds of cytokines,including interleukin1(IL-1), interleukin4(IL-4),interleukin6(IL-6),interleukin10(IL-10), interleukin12(IL-12),interleukin13(IL-13),interleukin18(IL-18),tumor necrosis factor-α(TNF-α),interferon-γ(INF-γ) and transforming growth factor(TGF-β1),were tested by enzyme linked immunosorbent assay.Samples of lung and liver were got for pathological section at the end of experiment.In "experiment 3",SIRS models,indueed in the same way,received XNJI intravenous injection in ear vein.Symptoms and objective signs,such as rectal temperature,consciousness,skin and mucosa,were observed at 1,2.5 and 5.5 hour after dosage and blood samples were collected at 1 and 2.5 hour after dosage.Then,10 kinds of cytokines,including IL-1,IL-4,IL-6,IL-10,IL-12, IL-13,IL-18,TNF-α,INF-γand TGF-β1,were tested by enzyme linked immunosorbent assay.Samples of lung and liver were also got for pathological section.In "experiment 4",the level of inflammatory mediators and rectal temperature of 49 rabbits in other three experiments were analyzed through bivariate correlate,partial correlate,hierarchical cluster,factor date reduction,linear regression and Logistic regression to explore the variation rule of inflammatory mediatorome.Results:In the different-dose injecting experiment,the first LPS injection caused fever,less activity,lassitude and tachypnea in rabbits,then similar symptoms accompanied with prolonged venous hemorrhage time were found after the second injection.As the LPS dose increasing,pathogenetic condition intensified.Large-dose LPS injection caused endotoxin shock symptoms such as unconsciousness and ice cold ears,possibly causing death.In the multiple injecting experiment,injection with equal dose or progressively increasing dose could induce fever every time during 7 days.In the validity proving experiment,all the 38 rabbits had fever after first injection and 2 rabbits died in 24 hours,then all the remained 36 rabbits had fever again after second injection and no rabbit died in next 48 hours.The DCPD treated groups received intragastric administration of DCPD containing different dose of gypsum.Rectal temperature of both the little-dose and large-dose DCPD group was lower than the controlled group at 1h and 2.5h after dose and there was statistical significance in the difference. At 1h after dose,TNF-αlever of the large-dose DCPD group was lower than the controlled group while TGF-β1 lever was higher,the difference was statistically significant.Lever of inflammatory mediators in little-dose DCPD group was usually between the controlled group and large-dose DCPD group, while lever of inflammatory mediators in the controlled group was never between the little-dose and large-dose DCPD group.At 2.5h after dose,TNF-αlever of large-dose DCPD group was significantly lower than the controlled group, while IL-4 lever of both the little-dose and large-dose DCPD group was higher than the controlled group,the difference is nearly significant(P= 0.056).Lever of inflammatory mediators in little-dose DCPD group was usually between the other 2 groups.Rectal temperature of the XNJI treated group was significantly lower than the controlled group at 1h and 2.5h after dose.At 1h after dose,TNF-αlever of the XNJI treated group was lower than the controlled group and IL-18 lever was higher,the difference was statistically significant.At 2.5h after dose, no statistical significance was obtained in all the inflammatory mediators between 2 groups.Variation tendency of inflammatory mediators in all the treated groups was compared to the controlled group and variation of inflammatory mediatorome showed some regularity.For the 2 DCPD treated groups,the direction of variation of all the inflammatory mediators at different time-phase was almost same.According to Pearson coefficient correlation,Spearman coefficient correlation and partial coefficient correlation,positive correlation was found between pro-inflammatory mediators and between anti-inflammatory mediators,but correlation between pro-inflammatory mediators and antiinflammatory mediators was complicated.The coefficient correlation was high between several mediators.In a 9-step clustering analysis,IL-12 and IL-13 were clustered at the first step,IL-12,IL-13 and TGF-β1 were clustered at the second step,while TNF-αwas clustered with other inflammatory mediators at the last step.3 common factors were obtained in factor analysis.The first common factor included IL-12,IL-13,TGF-β1 and IL-4,the second common factor included IFN-γ,TNF-α,IL-18 and IL-10,while the last common factor consisted IL-1,IL-6 and IL-10.Regression equations obtained by linear regression and logistic regression were statistically significant,but coefficients of determination were poor and only a few variables had statistical significance.Conclusion:LPS injection could surely cause fever.LPS injection with different times and different doses could indue different exogenous febrile disease models of Weifen,qifen,yingfen and xuefen syndrome.Weifen and qifen syndrome could be endued by LPS infusion once,yingfen and xuefen syndrome could be endued by twice(with a 24-hour interval) LPS infusion,persistent qifen syndrome or deficiency of liver-yin and kidney-yin syndrome by multiple times infusion and unconsciousness and collapse syndrome could be caused by large-dose LPS injection.The results showed that DCPD containing different dose of gypsum gave antipyretic effect on SIRS rabbits and DCPD containing large dose of gypsum gave more obvious antipyretic effect.DCPD could antagonise inflammation possibly by restraining TNF-αand promoting TGF-β.All the data of inflammatory mediators showed regularity.DCPD with different dose of gypsum caused inflammatory mediators changing in the same direction and the effect of DCPD with large dose of gypsum was more obvious.The results showed that XNJZ also gave antipyretic effect on SIRS rabbits and antagonised inflammation possibly by restraining TNF-α,but the reason about promoting IL-18 was unknowed.Some variation characteristic of inflammatory mediatorome was revealed through several multivariate statistical analyzing.The regularity of variation of inflammatory mediators suggested that action mechanism of different treating drugs could be similar.Correlation analysis demonstrated that inflammatory mediators had close relationship.Clustering analysis showed that numerical data of IL-12,IL-13 and TGF-β1 was very similar and data characteristic of TNF-αwas quite different.3 common factors were obtained in factor analysis.The first common factor mainly reflected anti-inflammatory mediators,while the second and third common factors probably reflected different pro-inflammatory mediators.