The Effect And Mechanism Study Of Panax Notoginseng Compositions On LPS-induced Inflammatory Factors

Objective:To research the anti-inflammatory effect of Panax Notoginseng and its five mainmonomers,incluing ginseng saponin Rb1,Rg1,Rd,Re and notoginseng saponin R1,andcompare the anti-inflammatory effects to determine the main anti-inflammatorycompositions of Panax notoginseng and further study the molecular mechanisms.Methods:1. A stable cell inflammatory response model was constructed. LPS was used tostimulate the RAW264.7cells to produce the inflammatory response. then, weused the real-time PCR technology to detct the expression of inflammatoryfactors interleukins1-β(IL-1β), interleukins-6(IL-6), tumor Necrosis Factor-α(TNF-α) and inducible NO synthase(iNOS).2. MTT assay was used to detect the cytotoxicity of Panax notoginseng and Rb1,Rg1, Rd, Re and R1on RAW264.7cells in vitro and IC50was calculated.3. We used the cell inflammatory response model to screen which compositions hadthe anti-inflammatory effect from the PNG, Rb1, Rg1, Rd, Re and R1.Experimental groups: RAW264.7cell group, LPS and RAW264.7cell group, LPS,Panax notoginseng and RAW264.7cell group. Firstly, we detect the effects ofdifferent concentrations of PNG, Rb1, Rg1, Rd, Re and R1on the expression ofinflammatory factors.the concentrations were12.5μg/mL,25μg/mL,50μg/mLand100μg/mL; Seondly, we detect the effects of different time point of PNG, Rb1,Rg1, Rd, Re and R1on the expression of IL-1β, IL-6, TNF-α, iNOSinflammatory factors.4. By western blot assay, we detected the protein expression of NF-kappaB in theNF-kappaB signaling pathway and p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38in the MAPK signaling pathway.5. The effect of PNG,Rb1,Rg1,Rd,Re and R1on expression of accssory moleculesCD14and TLR4were ayalysed by Flow cytometry assay.Result:1. The expression of inflammatory factors IL-1β、IL-6、TNF-αand iNOS were raisedafter the100ng/mL LPS stimulated macrophages.2. We found that the PNG,Rb1,Rd,Rg1,Re and R1had no cytotoxicity toRAW264.7cells,the IC50was higher than400μg/mL.3. When we screened which compositions had the anti-inflammatory effect,wefound that PNG,Rb1,Rd reduced significantly the expression of IL-1β, IL-6,TNF-α, iNOS,especially Rd;R1reduced significantly the expression of TNF-α,iNOS. However, Rg1,Re only reduced significantly the expression of iNOS;Allof the inhibitory effect depended on the concentrations and time points.4. Then,we reserched the anti-inflammatory mechanisms of Panax Notoginseng andits five main monomers and found that PNG,Rb1,Rd reduced the proteinexpression of p-ERK1/2and NF-κB in a concentration-dependent mannersignificantly, blocked the protein expression of p-JNK and p-p38but had noeffect on the total ERK1/2, JNK, p38; Rg1, Re, R1didn’t affect the proteinexpression of phosphorylated and total ERK1/2, JNK, p38and NF-κB.5. CD14and TLR4are the LPS receptors on the macrophage,so we use flowcytometry to study Panax Notoginseng compositions have any impact on the LPSreceptors. We found that compared to the normal group, the LPS raised the CD14fluorescence intensity and reduced the TLR-4fluorescence intensity;Compared tothe LPS group, the panax notoginseng compositions reduced significantly the CD14fluorescence intensity with the degree of PNG>Rd>Rb1>Re>R1>Rg1,butdidn’t affect the TLR-4fluorescence intensity.Conclusion:We successfully constructed the stable cell inflammatory response model invitro,and found that PNG,Rb1,Rd,Rg1,Re and R1had some anti-inflammatoryeffect,but Rd had the best anti-inflammatory effect, followed by Rb1.Therefore, weconcluded that the main anti-inflammatory compositions of PNG were Rd and Rb1.Inaddition to this, we also research the anti-inflammatory signaling pathway and foundthat PNG, Rb1, Rd had good down-regulatory effect on the protein expression ofNF-kappaB and MAPK pathway, but Rg1, Re and R1had no effect on this twopathways, so we concluded the PNG, Rb1and Rd inhibited the expression ofinflammatory factors by inhibiting the NF-kappaB and MAPK pathway.