The Beneficial Effects Of Splenic Active Polypeptide And Carnosine On Cyclophosphamide-induced Immunosuppression

Objective: Bioactive peptide possess characteristic of little side-effect.It has drawn public attention in the clinical area where bioactive peptides act as immunoregulator to reduce toxicity and heighten impact of chemotherapeutic drugs.However,the application of such bioactive peptide is limited bcause of absence of experimental basis.Therefore,in this study,we established immunosuppression mouse model by cyclophosphamide(CTX)to investigate the immunoregulatory activity of calf splenic bioactive polypeptide and dipeptide,in order to provide theoretical and experimental basis for further improving clinical application of splenic active peptide.Methods:(1)The effects of(Lienal Polypeptides Injection,LPI)on immunosuppression provoked by CTX in mice.Mice were randomly divided into five groups,including normal control,CTX,CTX+LPI-L(8 mg/kg;i.p.),CTX+LPI-H(16 mg/kg;i.p.)and CTX+TP5(2 mg/kg;i.p.)groups.LPI and TP5 were injected intraperitoneally 7 days prior to the injection of CTX(200 mg/kg;i.p.).Spleen,thymus and peritoneal fluid were collected to measure spleen index,thymus index,spleen lymphocyte counts and lymphocyte subsets at the eighth day.(2)The effects of LPI on immunosuppression provoked by CTX in Lewis lung carcinoma(LLC)xenograft mouse model.LLC cells were implanted subcutaneously in C57BL/6 mice.After inoculation of LLC for 6 days,mice were randomly divided into five groups,including normal control,CTX(20 mg/kg;i.p.),CTX+LPI-L(8 mg/kg;i.p.),CTX+LPI-H(16 mg/kg;i.p.),CTX+TP5(2 mg/kg;i.p.),LPI-L,LPI-H and TP5 groups.Then,mice were treated for 15 dsys and measured tumor volume.Spleen,thymus,peritoneal fluid and tumor were collected to measure tumor weight,spleen index,thymus index,spleen lymphocyte counts,lymphocyte subsets and phagocytosis of peritoneal macrophage at the 20 th day.Bioinformatics analysis was applied to invetigate the function of LPI.(3)The effects and underlying mechanisms of carnosine on immunosuppression induced by CTX.Kunming mice were divided into five groups,including normal control,CTX(20 mg/kg;i.v.),CTX+carnosine(100 and 200 mg/kg;i.p.),CTX+NAC(200 mg/kg;i.p.)groups.The levels of ROS and oxidative DNA adduct were measured after treatment for 24 h.Chromosome abnormality,cell cycle arrest and apoptosis have been futher detected in bone marrow cells of mice.Results:(1)In normal mice,LPI was able to markedly enhance immune organ index and increase spleen lymphocyte counts.Moreover,LPI was capable of increasing CD3+CD4+ and CD3+CD8+ T cells.(2)In LLC-bearing mouse model,LPI sigficantly attenuated inhibitory effect of immune organ index and spleen lymphocyte counts induced by CTX.At the same time,LPI evidently increased CD3+CD4+and CD3+CD8+ T cells.Phagocytosis of peritoneal macrophage was significantly promoted by LPI.Compared with CTX group,mouse tumor volum and tumor weight significantly decreased in LPI combined with CTX group,which indicated that LPI promotes anti-tumor effect of CTX.Bioinformatics analysis results indicated that LPI affects the immune function and phagocytosis of macrophage related signaling pathway may play a major role in the effect of LPI.(3)Splenic bioactive dipeptide carnosine significantly decreased levels of ROS and DNA adduct 8-oxo-d G and SCE frequency induced by CTX in bone marrow cells.Moreover,Carnosine significantly inhibited G2/M cell cycle arrest and decreasd p-Chk1/Chk1,p-p53/p53 ratio and reduced expression of p21.Besides,carnosine significantly decreased apoptosis proportion of bone marrow cells and reduced Cleaved-Caspase 3/Caspase 3 ratio and expression of Bax and Cyt c.Conclusions: Splenic bioactive polypeptide and dipeptide can significantly improve CTX-induced immunosuppression of mice.On one hand,LPI can effectively inhibit CTX-induced reduction on peripheral mature immune cells of normal and LLC-xenograft mice.Besides,LPI is beneficial for maintaining the stability of T lymphocyte subsets and enhancing phagocytosis of peritoneal macrophage.LPI can promote anti-tumor effect of CTX through regulation effect on immune.On the other hand,Carnosine can significantly inhibite CTX-induced SCE,G2/M cell cycle arrest and apoptosis in bone marrow cells probably by decreasing oxidative DNA damage.