The Research On Mechanisms Of Rhubarb In Treatment Of Endotoxemia Based On TLR4-MAPK-NFκB Anti-inflammatory Signal Pathway

Object:To explore the dynamic changes in expression level of relative proteins within the TLR4-MAPK-NFκB inflammatory pathways in endotoxemia rat model, then to find the appropriate time points for index measurement. Based on this, the regulation effect on TLR4-MAPK-NFκB pathway of rhubarb was observed, with the purpose of explorating molecular mechanism on the "purging fire detoxification" of rhubarb on the treatment of endotoxemia disease initially. To provide better guide for clinical practice of rhubarb.Methods:1. LPS was administrated by intravenous injection at the dose of 5mg/kg to establish rat endotoxemia model, then the general situation of rats were observed,and rectal temperature was measured at each time point. Water extract of rhubarb were continuously administrated by ig for 7 times to observe the effects of rhubarb "purging fire detoxification" on the treatment of endotoxemia disease initially.2. The content of inflammatory cytokines, such as IL-6, IL-1β, TNF-a and NO, was measured at each time point using enzyme-linked immunosorbent assay (Elisa); the expression levels of proteins, sush as proteins in TLR4-MAPK-NFκB pathway, IL-6, EL-1β, TNF-a and iNOs in rats, was tested using western blotting; the Relative expression of IL-6, IL-1β, TNF-α and iNOs mRNAs was detected with QRT-PCR assay.3. The multivariate analysis of variance and principal component analysis were used for analyzing the expression level of all tested proteins in TLR4-MAPK-NFκB pathway of model rat, hoping to find the appropriate time points for index measurement and target proteins of rhubarb.Results:1. LPS after injection via the tail vein into the body, can stimulate the body to produce large amounts of endogenous pyrogenic cytokines, causing fever, and the emergence of a bimodal heat, the biggest changes in body temperature appear at 4h and 6h. LPS can cause immune organs coefficient changes, such as spleen and thymus, the performance was much obvious at 4h and 6h. The mRNAs and proteins expression levels of NO, IL-1β, IL-6 and TNF-α at 4h and 6h after modeling was increased. All of these deminstrate that the endotoxemia rat model was established successfully.2. The mRNA and protein expression levels of TLR4 in rat model was increased, the phosphorylation levels of Erk, JNK, p38 in MAPK pathway and IκBα, NFκB p65 in NFκB pathway was modulated. At 4h and 6h after modeling the increase was apprent. After doing Multivariate analysis of variance repeated sets, The 4h,6h and 8h were chosed primaryly as a follow-up experiment observation time window.3. Take the result of primary test of rhubarb into congratulation, the two time points of 4h and 6h were chosen for the follow-up experiments for observation of the treatment on endotoxemia rat model. At the dose of 3.0g/kg and 1.5g/kg, water extract of rhubarb show the ability to decrease the inflammatary cytokines content, the expression level of correspondent mRNA and proteins.4. The regulation of rhubarb on TLR4-MAPK-NFκB pathway:4.1 At the dose of 3.0g/kg and 1.5g/kg, water extract of rhubarb show the ability to decline the expression level of TLR4 mRNA and protein in model rats of 4h and 6h.4.2 At the dose of 3.0g/kg and 1.5g/kg, water extract of rhubarb can reduce the phosphorylation levels of Erk, JNK, p38 in MAPK pathway in model rats of 4h and 6h.4.3 At the dose of 3.0g/kg and 1.5g/kg, water extract of rhubarb can reduce the phosphorylation levels of IκBα, NFκB p65 in NFκB pathway in model rats of 4h and 6h.5. Do single factor variance analysis and principal component analysis for the dates above, aqueous extract of rhubarb can regulated the TLR4-MAPK-NFκB pathway by lowering the phosphorylation level of Erk, JNK, IκBα and NF-κB p65.Conclusions:1. There was over expression of inflammatory cytokines in endotoxemia rat, the expression of relevant proteins in TLR4-MAPK-NFκB pathway shown a certain dynamic changes. After doing analysis, the three time points,4h,6h and 8h after modeling, were selected primarily as a optimal sample time for follow-up experiment.2. Rhubarb can decrease rectal temperature, decline the content of inflammatory cytokines, and then improve the body’s inflammatory injury.3. Rhubarb can play a role in anti-inflammatory effect, by comprehensive regulating the TLR4-MAPK-NFκB pathway thoroughly by targeting at much proteins in this pathway, especially phosphorylation levels of Erk, JNK, IκBα and p65 NFκB.