The Extraction Of Polysaccharide From Anoectochilus Roxburghii And Its Immunomodulatory Activity On Immunosuppressed Mice In Vivo

Objective:The optimum extraction condition of polysaccharide from Anoectochilus roxburghii(ARP)and effects of its immunomodulation on immunosuppressed mice induced by cyclophosphamide(Cy)in vivo were reported in this paper.Methods:1.The extraction technology was optimized by orthogonal tests with extraction temperature,extraction time and solid to liquid ratio as factors and the extraction rate of polysaccharides was used as index.2.150 Kunming male mice with 18?22g were randomly divided into five groups,each consisting of 30 mice and allowed 1 week to adapt to their environment before the treatments,one of which used for control group,one of which used for model group,and the rest for treatments.Cy was given to each mouse both Cy group and three treatment groups by intraperitoneal injection with 80mg/kg body weight at Dayl to Day 3.ARP was given at the dose of 100 mg/kg'·BW,200 mg/kg·BW and 400mg/kg·BW to each mouse of three treatment groups respectively by gastrogavage at Day4 to Day 10.24h after the last administered,the animals were weighed and then killed by decapitation,the spleen and thymus were excised from the animal and weighed;carbon clearance test was used to assay mononuclear phagocytic system(MPS)function;the splenocytes proliferation was assessed by MTT;The WBC was detected by automatic biochemical analyzer;Content of cytokines in mouse serum was detected by ELISA;The expression of cytokines and regulatory factors in murine spleens were detected by qRT-PCR;The percentages of CD4+ and CD8+ T lymphocytes,the ratio of CD4+/CD8+ in peripheral blood of mice were detected by Flow Cytometry(FCM).Results:1.The optimum extraction conditions were as follows:extraction temperature was 70?,extraction time was 4h,solid to liquid ratio was 1:20,and the extraction rate of polysaccharides was 28.69%;2.Compared with model group,body weight gain in ARP high dosage group was increased(P<0.05);the spleen index in three ARP groups were decreased and the thymus index were increased(P<0.05);the phagocytic index in ARP low and medium dosage groups were increased(P<0.05);the stimulating index of T lymphocyte proliferation in ARP high dosage group was increased(P<0.05);the total number of white blood cells in ARP high dosage group was decreased(P<0.05);the content of cytokine IL-2 in serum of ARP low and high dosage groups were increased(P<0.05),and IL-6 in serum of ARP high dosage group was increased(P<0.05);the IL-4 mRNA expression level in ARP high dosage group was decreased(P<0.05),the IL-6 and IFN-? mRNA expression level in ARP medium and high dosage groups were decreased(P<0.05);the T-bet mRNA expression level in ARP groups were decreased(P<0.05),the GATA3 mRNA expression level in ARP high dosage group was increased(P<0.05);the percentage of CD4+,CD8+ T lymphocytes in ARP groups were increased(P<0.05),the ratio of CD4+/CD8+ were decreased(P<0.05).Conclusion:The above results indicated that ARP could promote the growth of immunosuppressed mice induced by Cy,mitigate the enlargement of spleen and the atrophy of thymus,enhance the phagocytosis of macrophage,stimulate T lymphocyte proliferation,regulate the expression and secretion of cytokines.It was concluded that ARP coould modulate the immune function of immunosuppressed mice in some degree,which related to regulate the T lymphocyte differentiation.