Prevention Effect Of Total Ginsenosides And Ginseng Extract From Panax Ginseng On Immunosuppressed Mice Induced By Cyclophosphamide

Objective:Based on the chemical composition and pharmacological activity of ginseng extract（GE）and total ginsenoside（TGS）from ginseng root,this study compared the prevention effects of GE and TGS on immunocompromised mice induced by cyclophosphamide（CP）.Method:1.Thermo Fisher Scientific Ulti Mate 3000 UHPLC+focus×2 dual gradient system and CORTECS UPLC C18 column（2.1 mm×100 mm,1.6μm）were used to detect the saponins in TGS and GE.The monosaccharide content was determined by HPLC pre-column derivatization.Amino acid content was analyzed by automatic amino acid analyzer.2.40 mice were randomly divided into four groups:control group,model group,TGS group and GE group.The mice were administered orally for 28 days and modeled for 4days（days 25,26,27,and 28）.After modeling,blood routine,hematopoiesis-related cytokines,serum biochemical markers,and inflammatory cytokines were detected using mouse venous plexus blood.The spleen and thymus were taken for organ index and histopathological examination.The proliferation of T lymphocytes,B lymphocytes,CD₄⁺T lymphocytes and CD₈⁺T lymphocytes were detected from spleen cells.The cell apoptosis,cell cycle and histopathological changes of bone marrow（BM）were analyzed.The protein changes of Bcl-xl,Blc-2,P53,Bax,Cyclin A and Cyclin D1 were detected by Western blot.Cardiac and liver mitochondrial function,liver biochemical indexes were detected and pathological sections were prepared 3.Hematopoietic stem cells（HSCs）were isolated from mouse bone marrow and their purity was determined.The sorted HSCs samples were incubated with FITC labeled Gr-1,PE labeled TER-119 and CD33,and APC labeled CD11b and CD41.Flow cytometry was used to detect the expression of each antibody in HSCs.The apoptosis and cell cycle of HSC were analyzed.The morphology of colonies was observed under microscope.4.The intestinal flora of mice was analyzed by 16S r DNA sequencing and bioinformatics to evaluate the moderator effect of TGS and GE on the imbalance of intestinal microflora in CP-induced immunocompromised mice.RESULTS:The results of component analysis showed that the total contents of 12kinds of ginsenosides of TGS and GE were 67.21%and 2.04%,respectively.The total content of 17 amino acids in TGS（1.41%）was lower than that in GE（5.36%）.The total contents of 10 monosaccharides were similar to that of TGS（74.12%）and GE（76.36%）.The results of animal experiments showed that both TGS and GE could protect the histopathology of BM by improving the histopathology of BM,increasing the parameters of blood cells,serum biochemical parameters,hematopoiesis-related cytokines and anti-inflammatory factors,inhibiting the apoptosis of BM cells,and improving BM-S arrest.TGS and GE could promote the proliferation of spleen lymphocytes(including T and B lymphocytes,CD₄⁺and CD₈⁺T lymphocytes,CD₁₉⁺B lymphocytes),inhibit the decrease of myocardial mitochondrial membrane potential and reactive oxygen species,and increase body weight and organ index,liver mitochondrial complex I-V and liver redox-related enzyme activity in model mice.Flow cytometry showed that CP obviously inhibited the proportion of HSCs differentiated into CD11b⁺,CD41⁺,CD33⁺,Gr-1⁺,and TER-119⁺.TGS and GE significantly improved the differentiation of HSCs induced by CP,and the protection effect of GE was superior to that of TGS.According to the change in the number of colony cells,CP inhibits the proliferation and differentiation of BM.Both TGS and GE maintained the normal proliferation and differentiation of BM.Meanwhile,TGS and GE could improve the S phase arrest and apoptosis of HSCs in CP-induced immunosuppressive mice.GE showed stronger protective effect than TGS on HSCs.TGS and GE could protect the composition and relative abundance of intestinal bacteria,such as Lactic acid bacteria,Bifidobacterium,Lachnospiraceae,Eubacterium＿Costanoligenes and Clostridiales,and can effectively recover the disturbance of intestinal immune;specifically,the protective effect of GE on the lactobacillus was superior to TGS.Conclusion:1.There were some differences in composition and content of ginsenoside,monosaccharide and amino acid between TGS and GE.2.TGS and GE have protective effect on the immune function of CP-induced immunosuppressed mice,and the preventive effect of GE has better than TGS in some parameters.3.Both TGS and GE could prevent HSCs damage in model mice induced by CP,and GE had a better effect than TGS on cell cycle of HSCs.4.TGS and GE could effectively recover the disturbance of intestinal immune and alleviate the side effects of CP by regulating intestinal microflora.The synergistic effect of ginsenosides and polysaccharide in GE played an important role in the in immunosuppressed mice induced by CP.