Immunomodulatory Effect Of Codonopsis Pilosula Polysaccharide

Objective:To explore the immunomodulatoryfunction and mechanism of Codonopsis pilosula polysaccharide soluble powder(CPPS),murine macrophage cell RAW 264.7 was applied in vitro and baby broilers were used in vivo.The overall goal is to obtain the required data for the clinical application of CPPS.Method:(1)Preparation and determination of CPPS:Briefly,8 volumesof 95%alcohol was added to perform the reflux extraction for 1 h and then extracted for 3 times using 10 volumes of the boiling water(1 h each time).The extracted liquids were combined,filtered,concentrated and ethanol-precipitated to obtain the dried CPPS for in vivo study.The proteins were removed via Sevage method.Briefly,the CPPS was mixed with Sevage reagent[chloroform:n-butanol=5:1(V/V)]byl:5(V/V)with shaking.The lower liquid portion was collected,concentrated and dried to obtain the refined CPPS for in vitro assay.The CPPS quantity was determined by phenol sulfuric acid assay.(2)In vitro assay:CPPS was added to RAW 264.7 cells by continual 2 time dilution from 4000 to 31.25 μg-ml-1.MTT was used to detect the effect on the cell proliferation by the different CPPS concentration.25、50、100、150 and 200 μg·mL-1 were selected based on the MTT analysis.ELISA kit was used to detect the TNF-a and IL-6.The TNF-a and IL-6 mRNA expression were determined by RT-PCR.Western blot analysis was used to assess the expression of NF-κB p65 and IκB-α.(3)In vivo study:300 broilersof 308 Ross aged 7 days were divided into three experimental groups with high(0.4 g/kg drinking water),medium(0.2 g/kg of the drinking water)and low dose(0.1 g/kg of the drinking water)of CPPS;the positive control group(0.2g Astragalus polysaccharide was added per Kg drinking water);In the immune control group and blank control group,no any drug was added.Except for the blank group,at the age of 7 days,the broilers were the firstly intranasally immunizated with Newcastle disease virus live vaccine(La Sota),and at 21 days of age were re-immunized as primary immunization.In the meanwhile,the drugs were simultaneously administrated with the immunization for three consecutive days.The Newcastle disease antibody titer was detected by β-micro method.The SIgG and IgG were detected by double antibody sandwich ELISA.The thymus,spleen,bursa index and body weight were calculated.Result:(1)The CPPS content was more than50%(for in vivo study).The content of CPPS was 83.97%after removing the proteins(for in vitro assay).(2)CPPS enhanced the cell proliferation of RAW 264.7 with the concentration of 31.25～125 μg·mL-1.When the concentration of CPPS was higher than 250μg-mL-1,the cell proliferation of RAW 264.7 cells was inhibited.The TNF-a(P<0.05)and IL-6(P<0.05)secretion and mRNA(P<0.05)expression were increased when CPPS at 100 and 150 μg·mL-1.NF-κB p65 expression in nuclear(P<0.05)was increased and IaB-α expression in cytoplasm(P<0.05)was decreased when CPPS at 100 μg mL-1.(3)The indexes of thymus,spleen,bursa of Fabricius,anti-ND antibody,IgG and sIgA were significantly increased(P<0.05)by adding CPPS to the drinking water,the weight gain were increased in the treatment group with CPPS(P<0.05),CPPS experiment group compared with positive control group was no significant difference in every indexes.Conclusion:(1)In vitro analysis demonstrated that the secretion of TNF-alpha and IL-6 of RAW 264.7were increased via activation of the cellproliferation and the NF-κB signaling pathway by CPPS.(2)In vivo study demonstrated that CPPS can increase the level of ND antibody,immune organ index and immune globulin content to play an role of increased immune function,and enhanced the body weight growth was promoted by CPPS.It was the best to add CPPS powder to the drinking water at 0.2g/kg.