The Study On The Signal Pathway Of Anti-inflammatory Of Mongolian Medicine Compound Terun Sodolol Petroleum Ether Extracts And Its Main Monomer

Inflammation, a common clinical symptom, happened generally in every part oforganism. Mongolian medicine is the ancient and traditional medicine. The previousresearch has already established the Mongolian medicine compound Terun sodololwhich has good function of anti-inflammation and increasing immune. It hassignificant treatment efficacy in animal model and the clinical cow mastitis. However,it still has excessive medication, unclear mechanism of action and other problems.Therefore, this research continues the issue to do more research of anti-inflammatorysignal transduction pathway of its petroleum ether extracts and its main monomer forthe base of the second development in cell molecular level, because its petroleumether extracts has significant function of anti-inflammation.Therefore, the effective active ingredients can be separated and identified bynature medicinal chemistry, organic chemistry and other knowledge and using liquidchromatography technology to establish the effective monomer components of thepetroleum ether extract. By LPS-induced RAW264.7macrophages, it can establishinflammation model in vitro to observe the function of Mongolian medicine compoundTerun sodolol mainly reflected on petroleum ether extract and monomer and monomercomplex on anti-inflammatory and LPS-TLR4-cytokines/NF-κB signal transductionpathway.The results of research about Mongolian medicine compound petroleum etherextracts on anti-inflammatory function as following: to interfere RAW264.7macrophages after24hours with different LPS concentration (the concentrations are0.5,1,2,5,10,25μg/mL), and then to collect cell supernatant to do the ELISAdetection. To detect the standard production and to draw the standard curve forcalculating the supernatant of tumor necrosis factor α(TNF-α)、Interleukin-6(IL-6),Interleukine1β(IL-1β)content. According to the results to establish the most effectiveLPS concentration is25μg/mL. Mongolia medicine compound Petroleum ether extract1,5,10,25,50mg/mL act on RAW264.7macrophages, to detect the proliferationability of cell (CCK-8method). The results showed that the obvious differencebetween the group of concentration50mg/mL and blank control group. This is thefunction of suppression to cell growth; other groups have no significant difference. This showed that there are no influences on cell growth, so the later stages ofexperiments choose the concentration steps5mg/mL、10mg/mL、25mg/mL. Theexperiment is divided into blank control group, the inflammation model group, theinflammation model and positive control group, inflammatory model and drugtreatment group. Tumor necrosis factor α (TNF-α）、Interleukin-6(IL-6), Interleukin1β(IL-1β) content were detected with ELISA method, tumor necrosis factor α(TNF-α),Interleukin-6(IL-6), Interleukin1β(IL-1β) mRNA expression were detected withReal-time PCR method. The results showed: LPS model group compare with the blankcontrol group on tumor necrosis factor α(TNF-α)、Interleukin-6(IL-6), Interleukin1β(IL-1β) protein secretion and mRNA expression showed all increase significantly（P<0.05）. When the concentrations of5mg/mL、10mg/mL、25mg/mL petroleumether extract exist, tumor necrosis factor α(TNF-α), Interleukin-6(IL-6), Interleukin1β(IL-1β)protein secretion, mRNA expression and LPS model group compared withLPS model group all decrease significantly（P<0.05）. There is obvious difference andappearing dose-dependent. This demonstrates that anti-inflammatory and cellprotection effect of Mongolian medicine Terun sodolol petroleum ether extract arerelated to the produce of inhibition of inflammatory cytokines.The establishment of effective concentration and time for Mongolian medicinecompound petroleum ether extract main monomer and its monomer complex act onmacrophages RAW264.7cell function: Mongolian medicine compound Terun sodololmain monomer and its monomer complex as the experiment medicine act onmacrophages RAW264.7, the ability of cell proliferation was detected. The result ofexperiment (CCK-8)of the ability of cell proliferation showed: Mongolian medicinecompound main monomer ingredients which are picroside II, curcumin, emodin, rheinin high, medium and low dose groups at different time points (0、6、12、24h) act onRAW264.7cell, there were no obviously influence for the cell exist vitality. But itshowed that at every monomer concentration groups acting time48h can decrease cellexist rate significantly. Additionally, the effect of monomer complex is a little betterthan monomer. Therefore, the later experiments, according to the rule of effectivedefence and medicine composition under different concentrations, to choose highconcentration monomer group and medium concentration monomer complex group,and the time point is24h.The influence of Mongolia medicine compound petroleum ether extract mainmonomer and monomer complex to LPS introduce RAW264.7on the ability of cellproliferation. The experiment was divided into normal cell control group, the inflammation model group, inflammation model add every monomer administrationgroup, inflammation model add monomer compound group, inflammation model addPyrrolidine dithiocarbamate group (PDTC;20microns), inflammation model addPDTC add the monomer complex group, inflammation model add dexamethasonegroup (Dex;10μg/mL). The results showed that it can more decrease the cell vitalityafter stimulating with LPS comparing with normal cell control groups. Monomer andmonomer complex for testing drugs and PDTC、Dex for positive drug inhibit thedeath of cell, and restore the survive of cells on the different level compared with LPSmodel groups.The influence of Mongolian medicine compound petroleum ether extract mainmonomer and monomer complex to RAW264.7producing cell factors introduced byLPS: The experiment was divided into normal cell control group, the inflammationmodel group, inflammation model add monomer administration group, inflammationmodel add monomer compound group, inflammation model add PDTC group,inflammation model add PDTC add the monomer complex group, inflammation modeladd dexamethasone group. The result of ELISA showed that TNF-α, IL-6, IL-1βsecretion of LPS model groups increased obviously comparing with normal cellcontrol groups；The secretion of TNF-α, IL-6, IL-1β comparing in each monomer andthe monomer complex test drugs, positive control drug groups with model groupswere decrease significantly.The study on LPS introduce RAW264.7to cell TLR-4/NF-κB signal pathway wasacted by Mongolian medicine compound petroleum ether extract main monomer andmonomer complex. The experiment was divided into normal cell control group, theinflammation model group, inflammation model add monomer administration group,inflammation model add monomer compound group, inflammation model add PDTCgroup，inflammation model add PDTC add the monomer complex group, inflammationmodel add dexamethasone group. At the protein level, by using Western blot toanalyze the influence of each monomer and the monomer complex test drugs toTLR-4/NF-κB signal pathway. To detect phosphorylation IκBα(P-IκBα)、P65、P-P65(phosphorylation P56), TLR-4protein. The results showed that LPS model groupscompared with normal control groups TLR-4、 P-IκB、 NF-κBP-P65the proteinexpression is obvious which demonstrates that LPS stimulates and activatesTLR-4/NF-κB signal pathway. Mongolia medicine compound petroleum ether extractmain monomer and monomer complex test drugs against the function of endotoxinLPS and decrease the protein expression of TLR-4、P-IKB、NF-κBP-P65. The results of real-time detected TLR-4、NF-κBP65showed that at the transcriptional level, LPSmodel groups compared with normal control groups TLR-4、 NF-κBP65mRNAexpressed significantly. However, Mongolia medicine compound petroleum etherextracts main monomer and monomer complex test drugs against the function ofendotoxin LPS and obviously decrease TLR-4、 NF-κBP65mRNA of proteinexpression. The result is the same as Western blotting. Using electrophoretic mobilityshift assays (EMSA) to detect the combine ability of DNA, to detect NF-κBP65(Nucleoprotein) activity. The results showed that LPS model groups compared withnormal control groups express significantly, the activity and content of NF-κBP65(Nucleoprotein) increase. However, Mongolia medicine compound petroleum etherextracts main monomer and monomer complex test drμgs weaken the function ofendotoxin LPS, decrease significantly the activity and content of NF-κBP65(Nucleoprotein).All results above demonstrate that Mongolia medicine compound petroleumether extracts and main monomer and monomer complex test drugs weaken thefunction of endotoxin LPS by TLR4and cytokines/NF-κB signal pathway to realize.