The Effects Of Chikusetsusaponin ?a On Inflammatory Response In RAW264.7 Cells Induced By LPS

Livestock and poultry inflammation seriously threatens animal health and the economy of breading industry.It is of great significance to explore an effective,low toxic and environmental friendly medicine for treating livestock and poultry inflammation.Chikusetsusaponin ?a(CHS-?a),a glycosidic compound with three nucleoids,is a main active ingredient in Panax japonicus and has hypoglycemic,hypolipidemic,antiviral and other pharmacological activities.At the same time,CHS-?a also has anti-inflammatory effect,but its anti-inflammatory molecular mechanism still needs further exploration.This experiment was to establish mouse macrophage RAW264.7 cells inflammatory model induced by lipopolysaccharide(LPS)to investigate the anti-inflammatory effect and molecular mechanism of CHS-?a.The methods and results were as follows:1.The action time and concentrations of LPS to establish the inflammatory model were determined by MTT assays combined with morphological observation and the expression level of IL-6 in the cells.And the results showed that the cell viability of RAW264.7 cells induced by 0.1,1,5,10 ?g/mL LPS for 6,12,24 h had no obvious change compared with the control group.However,after LPS stimulation for 24 h,the cell membrane extended outward,showing a shuttle shape or polygon.The higher concentration of LPS,the more obvious the change was.At the same time,the relative mRNA expression of IL-6 in cells treated with different concentrations of LPS for 24 h were higher than the control group with a dose dependent.Comprehensive consideration,we used 1 ?g/mL LPS stimulate cells for 24 h to established an inflammation model.2.MTT assays were used to determine the safe concentration of CHS-?a on RAW264.7 cells.We found 25 ?g/mL CHS-?a with obvious cytotoxicity significantly reduced the relative viability of cells in 12 and 24 h.There was no obvious effects on the viability of RAW264.7 cells treated with 3.125,6.25,12.5 ?g/mL CHS-?a,followed by treatment with LPS or vehicle.Therefore,3.125,6.25,12.5 ?g/mL were safe concentrations.3.The production(in cell supernatant)and the relative mRNA expressionof IL-6,IL-10,IL-1?,TNF-? in RAW264.7 cells were detected by ELISA and Q-PCR.The results showed that CHS-?a can inhibited the secretion and relative mRNA expression of IL-1?,IL-10,TNF-?,and IL-6 in LPS-stimulated RAW264.7 cells.4.Western blot,Q-PCR,ELISA were used to detect the effect of CHS-?a on the expression of COX-2 protein,COX-2 mRNA,iNOS mRNA,PGE2 and NO secretion in cells.We found CHS-?a can inhibit the expression of COX-2 and iNOS,as well as the production of PGE2 and NO in RAW264.7 cells induced by LPS.5.The effects of CHS-?a on relative protein expression of I?B-?,p65 in NF-?B pathway,p-p38,p-JNK,p-ERK in MAPK pathway,p-STAT1 in JAK-STAT1 pathway in RAW264.7 cells induced by LPS were detected by Western blot.The results indicated CHS-?a can increase the expression of I?B-?,p65,and inhibited the expression of p-p38,p-JNK,p-ERK and p-STAT1.In conclusion,CHS-?a possesses anti-inflammatory activity may reduce the expression of inflammatory genes and the secretion of inflammatory factors by inhibiting NF-?B,MAPK and STAT1 activation.