Protein removal process and anti-inflammation effect of polysaccharides from Polygonum cuspidatum
Objective To improve the extraction of polysaccharides from Polygonum cuspidatum and determine their effect on RAW264.7 cell activity and mechanism in lipopolysaccharide-induced inflammation.Methods The free protein in the crude extract of Polygonum cuspidatum polysaccharide was treated by Sevage method,and then the single factor experiment was conducted.Finally,the response surface method was used to screen the extraction of Polygonum cuspidatum polysaccharide and protein removal scheme.RAW264.7 cells were divided into a normal group and different concentrations of Polygonum cuspidatum polysaccharide groups(5,10,20,40,80 and 120 μg·mL-1).CCK-8 kit was used to observe how Polygonum cuspidatum polysaccharide affected the cell viability.RAW264.7 cells were divided into a blank group,a model group(100μg·L-1 lipopolysaccharide),and different concentration groups(20,40,and 80 μg·mL-1 Polygonum cuspidatum polysaccharide solution).The secretion levels of inflammatory factors TNF-α and IL-6 were detected by ELISA.The protein expression levels of TLR4,MyD88 and NF-κBp65 were detected by Western blot.Results The optimal extraction scheme of polysaccharides from Polygonum cuspidatum was as follows:the volume ratio of Sevage reagent to the test solution was 1∶4,the shaking intensity was 8,shaking for 12 min,and the protein was removed 4 times.The cell viability was greatly increased at the concentration range of 20～40 μg·mL-1,and the growth of RAW264.7 cells was much promoted.Compared with the blank group,the cell viability of the model group was increased,the secretion levels of TNF-α and IL-6,and the expression levels of TLR4,NF-κBp65 and MyD88 proteins were obviously increased,indicating that the macrophage inflammation model was successfully constructed.Compared with the model group,the viability of RAW264.7 cells in Polygonum cuspidatum polysaccharides 20,40 and 80 μg·mL-1 groups was greatly decreased.In addition,the secretion levels of TNF-α and IL-6 were reduced in the cell supernatant,and the expression levels of TLR4,MyD88 and NF-κBp65 proteins were also reduced.Conclusion Polygonum cuspidatum polysaccharides can reduce the secretion of TNF-α and IL-6 in RAW264.7 cells induced by lipopolysaccharide,and the inhibition of NF-KB signaling pathway may be a mechanism for its anti-inflammatory effect.
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