虎杖多糖中蛋白去除工艺及多糖的抗炎作用研究

Protein removal process and anti-inflammation effect of polysaccharides from Polygonum cuspidatum

目的优化虎杖多糖提取工艺,研究其对RAW264.7细胞活性的影响以及在脂多糖诱导的炎症过程中的作用机制。方法用Sevage法对虎杖多糖粗提取物中游离蛋白进行处理,然后进行单因素实验,最后利用响应面法筛选虎杖多糖提取及除蛋白方案。将RAW264.7细胞分为正常组和不同浓度虎杖多糖组(5、10、20、40、80、120μg·mL^(-1)),采用CCK-8试剂盒检测虎杖多糖对细胞活力的影响。将RAW264.7细胞分为空白组,模型组(100μg·L^(-1)脂多糖),不同浓度给药组(20、40、80μg·mL^(-1)虎杖多糖溶液),采用ELISA试剂盒检测肿瘤坏死因子-α(TNF-α)、白细胞介素-(IL-6)分泌水平;采用Western blot法检测TLR4、MyD88、NF-κBp65蛋白表达水平。结果筛选得虎杖多糖最优提取方案为Sevage试剂与供试液体积比1∶4、振摇强度8档、振摇时间12 min、除蛋白4次。虎杖多糖质量浓度在20~40μg·mL^(-1)内使细胞活力显著提升,对RAW264.7细胞生长有明显促进作用。与空白组相比,模型组细胞活力升高,TNF-α和IL-6分泌水平及TLR4、NF-κBp65和MyD88蛋白表达水平显著升高,表明巨噬细胞炎症模型构建成功。与模型组相比,虎杖多糖20、40、80μg·mL^(-1)组RAW264.7细胞活力显著降低。此外,在细胞上清液中发现TNF-α和IL-6分泌水平明显减少,并且TLR4、MyD88和NF-κBp65蛋白表达水平也显著降低。结论虎杖多糖可以减少脂多糖诱导的RAW264.7细胞中TNF-α和IL-6分泌,抑制NF-κB信号通路可能是其发挥抗炎作用的一种机制。

Objective To improve the extraction of polysaccharides from Polygonum cuspidatum and determine their effect on RAW264.7 cell activity and mechanism in lipopolysaccharideinduced 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 120μg·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 80μg·mL^(-1)Polygonum cuspidatum polysaccharide solution).The secretion levels of inflammatory factors TNF-αand 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 20~40μg·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-αand 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 80μg·mL^(-1)groups was greatly decreased.In addition,the secretion levels of TNF-αand 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-αand IL-6 in RAW264.7 cells induced by lipopolysaccharide,and the inhibition of NF-κB signaling pathway may be a mechanism for its anti-inflammatory effect.