Study on the Immune Activity of RAW264.7 Macrophages Affected by Crude Polysaccharides from Manjing
Exploring the immune activity of RAW264.7 Macrophages affected by extracellular polysaccharides from Manjing through in vitro cell experiments.Took logarithmic growth stage macrophages and adjust the cell concentration to 1.2 × 105 cells/mL,inoculate on a 96 well plate for 24 h,and set up a negative control group(only complete culture medium added)and a positive control group(LPS,1 μg/mL),the crude polysaccharide group of Manjing(50,100,200,400 μg/mL),after 24 h of cultivation,MTT assay was used to detect cell proliferation activity,phagocytic ability,and NO secretion ability;Mouse cervical dislocation was euthanized,and the spleen was taken in a sterile environment.The cell concentration was adjusted to 1.0 × 106 mL and inoculated onto a 96 well plate,set up a control group and a crude polysaccharide group(50,100,200,400 μg/mL)and Con A(10 μg/mL)positive control group or LPS(5 μg/mL)positive control group,cultivate for 24,48,72 h.4 h before the end of cultivation,add MTT(5 mg/mL)10 μL to each well.Centrifuge the supernatant and sufficient shake after adding 100 μL DMSO,measure the absorbance at 570 nm using an enzyme-linked immunosorbent assay(ELISA)reader and calculate the proliferation index.Compared with the negative control group,the group treated with crude polysaccharides from Manjing(50 μg/mL),the survival rate of macrophages significantly increased(p=0.001＜0.01),LPS group,Manjing crude polysaccharide group(200,400 μg/mL)the survival rate of macrophages increased(p=0.011,0.021,0.027＜0.05);Compared with the negative control group,the phagocytic index of macrophages in the LPS group and the various dose groups of crude polysaccharides from Primulaceae significantly increased(p＜0.01),compared with the LPS group,the crude polysaccharide group of Manjing crude polysacchande group(50,100,200μg/mL),the phagocytic index of macrophages significantly increased(p＜0.01),Manjing crude polysaccharide group(400μg/mL),the phagocytic index of macrophages increased(p＜0.05);Compared with the negative control group,the LPS group and the crude polysaccharide group of Manjing crude polysacchande group(50 μg/mL),the expression level of NO significantly increased(p＜0.01),compared with the LPS group,the crude polysaccharide group of Manjing(200 μg/mL)the expression level of NO significantly decreased(p＜0.01),Manjing crude polysaccharide group(100,400 μg/mL)the expression level of NO significantly decreased(p＜0.05);Compared with the negative control group,the group treated with crude polysaccharides from Manjing(50,100,200 μg/mL),the proliferation index of spleen cells(T cells)significantly increased(p＜0.01),the proliferation index of splenocytes(T cells)in the conA group increased(p＜0.05),compared with the conA group,the crude polysaccharide group of Manjing(200 μg/mL),the proliferation index of spleen cells(T cells)significantly increased(p＜0.01),while the group treated with crude polysaccharides from Primulaceae showed a significant increase(50 μg/mL),the proliferation index of spleen cells(T cells)increased(p＜0.05);Compared with the negative control group,the crude polysaccharide group of Manjing(100,200,400 μg/mL),the proliferation index of spleen cells(B cells)significantly increased(p＜0.01),LPS group and crude polysaccharides group of Manjing crude polysacchande group(50％ μg/mL),the proliferation index of spleen cells(B cells)increased(p＜0.05),compared with the LPS group,the crude polysaccharide group of Manjing(200,400 μg/mL),the proliferation index of spleen cells(B cells)increased(p＜0.05).Manjing crude polysaccharide had good immunomodulatory activity on RAW264.7 cells;Manjing crude polysaccharide had a strong promoting effect on both cellular and humoral immunity in mice.
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