The Effect Of Anoectochilus Roxburghii Polysaccharides On Apoptosis Of Spleen Lymphocytes In Immunosuppressive Mice Induced By Cyclophosphamide

Objective:The effect of Anoectochilus roxburghii polysaccharide(ARP)on apoptosis of splenic lymphocytes in immunosuppressive mice induced by cyclophosphamide was reported in this paper.Methods:75 Kunming male mice with 18-22g were randomly divided into five groups,each consisting of 15 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.cyclophosphamide was given to each mouse both cyclophosphamide group and three treatment groups by intraperitoneal injection with 80mg/kg body weight at Day1 to Day 3.ARP was given at the dose of 100 mg/kg BW,200 mg/kg BW and 400 mg/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 killed by cervical dislocation,and were taken the spleen for following experiments.Morphological changes in the spleen tissues were assessed by HE staining method;DNA cycle of splenic lymphocyte were detected by Flow cytometry;BcL-2,Bax,Fas,FasL mRNA expression were tested by Real Time-PCR.Results:1.HE staining results showed as follows:In the blank control group,the spleen tissue structure was normal,and it was full of splenic nodule formed by tightly packed lymphocytes,red pulp arounded by plenty of red cells and fringe area filled with macrophages,lymphocytes,and vasculars;In model group,splenic nodules,germinal center was not obvious,lymphocytes decreased and structure was fuzzy;Compared with the model group,in ARP groups,cells increased in the fringe area of spleen significantly,splenic nodule was larger and lymphocytes increased,the boundary of red pulp and white pulp was obvious.2.TUNEL method results showed as follows:The apoptotic ratio of murine splenic lymphocytes in model group was 37.3%,which was significantly higher than that of the control group;The apoptotic ratio of murine splenic lymphocytes in three ARP groups was lower than that of the model group,which were 22.9%,15.7%,18.7%respectively.3.Annexin V-FITC/PI double-staining method results showed as follows:The ratio of early apoptotic cells in model group was higher than that of blank control group,Significantly the ratio of early apoptotic cells in three ARP groups were significantly lower than that of model group,the denser ARP was;The total percentage of apoptotic cells(the total number of FITC single positive cells and FITC/PI double positive cells)among the groups had significant difference,the total ratio in ARP groups were lower than that of model group and were higher than that of the control group,the medium group(ARP,200 mg/kg)had the lowest total ratio of apoptotic cells among three ARP groups.4.DNA cycle of splenic lymphocytes results showed as follows:The ratio of splenic lymphocytes in G1 period was higher than that of blank control group,and the ratio of splenic lymphocytes in S period was lower than that of blank control group.The ratio of splenic lymphocytes in sub-G,of mice treated by ARP decreased obviously,while that was on the contrary in the period of S and G2 significantly.5.qRT-PCR results showed as follows:The Bax mRNA expression levels in three ARP groups were lower than that of model group significantly,as with the rising density of ARP,the Bax mRNA expression levels reduced gradually;However,The BcL-2 mRNA expression levels in three ARP groups was higher than that of model group significantly.Polysaccharide concentration increased,the BcL-2 mRNA expression also increased gradually.When the density of APR was 400 mg/kg,the mRNA expression began to decrease,But it was higher than that of model group;The Fas mRNA expression levels in three ARP groups was lower than that of model group significantly,the higher density the APR was,the lower level Fas mRNA expression was,so was FasL.Conclusion:1.APR could improve splenic tissue lesions of mice induced by cyclophosphamide and could reduce the apoptotic ratio of spleen cells;2.APR could decrease the apoptotic ratio of splenic lymphocyte in cyclophosphamide-induced mice,promoted DNA replication and mitosis process;3.It was concluded that the inhibition of APR in splenic apoptotic lymphocytes of cyclophosphamide-induced mice related to upregulation of rate of BcL-2/Bax and downregulation of the expression of Fas mRNA.