Effects Of Training On Immunological Functions Of Elite Athletes And The Potential Immunomodulatory Effect Of Cucurbitacin E

Aim: Firstly, we sought to establish the reference ranges of white blood cell differential counts and serum immunoglobulin levels for elite athletes from Guangdong Province through a large scale screening, to provide reasonable assessment for training and recovery. Secondly, we wanted to explore the effects of 4-week high intensity training on immunological functions of elite athletes and to investigate the inhibitory effect of prolong intense training on immunological functions of elite athletes. Thirdly, we sought to explore the immunomodulatory effect of cucurbitacin E(Cuc E) and the related mechanism, and to evaluate its potential application for athletes as a promising agent in modulating the adaptive immune respone.Methods:(1) 603 samples from baseball team, softball team, basketball team, hockey team, handball team, and football team were collected. White blood cell differential counts and serum immunoglobulin levels were measure. Data were analyzed using SPSS software. Reference ranges of white blood cell differential counts and serum immunoglobulin levels of elite athletes were established by calculating 95% confidence interval.(2) 11 elite rowers from Guangdong canoe-kayak team were chose as a study model. Venous blood samples were obtained before and 24 h after the 4-week high intensity training period. White blood cell differential counts were measure by automatic blood cell analyzer. Serum immunoglobulin levels were measure by automatic biochemical analyzer. Serum IL-6、IL-8、TNF-α and IL-10 were detected by ELISA kits. Lymphocyte subsets and regulatory T cells were detected by flow cytometry. Data were analyzed by SPSS software to compare the pamemeters before and after the training period.(3) Human PBMC and Jurkat T cells were stimulated with phorbol 12,13-dibutyrate(PDB) and ionomycin(Ion). The expression levels of human PBMCs activation antigens CD69 and CD25 were evaluated by immunofluorescent staining together with flow cytometry. The cytokines interleukin-2(IL-2), tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ) expressed by activated human PBMCs and Jurkat cells were measured by cytometric bead array. The NF-κBand MAPKs signaling were detected by western blot analysis. NF-κB/p65 translocation was examined by immunofluorescence microscopy.Results:(1) The white blood cell differential counts and serum immunoglobulin levels reference ranges of elite athletes were as follows: WBC count:(3.7-8.8) ×109 cell/L; Neutrophils count:(1.6-5.2) ×109 cell/L; Lymphocytes count:(1.4-3.4) ×109 cell/L; Monocytes count:(0.16-0.58)×109 cell/L; Eosinophils count:(0-0.5)×109 cell/L; Basophils count:(0-0.1)×109 cell/L; Neutrophils percentage:(37-66) %; Lymphocytes percentage:(25-52) %; Monocytes percentage:(2.5-8.4) %; Eosinophils percentage:(1-7) %; Basophils percentage:(0-1.5) %; Ig G:(7.58-16.91) g/L; Ig A:(0.80-3.49) g/L; Ig M:(0.53-3.41) g/L.(2) After a 4-week intensity training period, white blood count was significantly decreased(P < 0.01). There were no statistical differences in the numbers ofneutrophils, lymphocytes, monocytes, eosinophils, and basophils. The percentages of neutrophils, lymphocytes, monocytes, eosinophils, and basophils had also no significant changes. Serum Ig G levels weremarkedly decreased(P < 0.01) after training. There were no statistical differences in serum Ig A or Ig M levels(P > 0.05). Serum IL-6 levels were significantly increased(P < 0.05), serum IL-8 and TNF-α levels were significantly increased(P < 0.01) while IL-10 levels were significantly decreased(P < 0.01). The percentages of B cells and CD3+CD4+ cells as well as the numbers of B cells and CD3+CD4+ cells were markedly decreased(P < 0.05), while the CD3+CD4+/CD3+CD8+ ratio was also significantly decreased(P < 0.01). There were no statistical differences in the percentages of NK cells, T cells, and CD3+CD8+ cells as well as in the numbers of T cells, NK cells, and CD3+CD8+ cells(P > 0.05). No statistical differences were observed in CD4+CD25+Fox P3+ Treg/CD4+ ratio or CD4+CD25+Fox P3+ Treg counts(P > 0.05)。(3) Cuc E significantly suppressed the expression of CD69 and CD25 on the surfaces of CD3+ T cells in PBMC stimulated with PDB plus Ion. PDB+Ion-induced expression of IL-2, TNF-α and IFN-γ were dose-dependently inhibited by Cuc E. The m RNA levels of these cytokines in activated Jurkat T cells were also decreased upon Cuc E treatment. Cuc E decreased the phosphorylation levels of inhibitor of κB(IκB) and NF-κB/p65 in PDB+Ion-stimulated cells. The nuclear translocation of NF-κB/p65 was also significantly suppressed in the presence of Cuc E. The phosphorylation of p38 MAPK, JNK and Erk1/2 was not decreased by Cuc E treatment.Conclusion:(1) White blood cell differential counts and serum immunoglobulin reference ranges of elite athletes are not all the same with that of normal population.(2) 4-week intensity training could suppress the production of white blood cells, especially Tcells, thereby inhibitingthe humoral immunity mediated by B cells, accompanied with increased levels of several proinflammatory cytokines.(3) Cuc E could significantly decreased IL-2, TNF-α and IFN-γ expression in both protein and m RNA levels via downregulating the NF-κB signaling, highlighting its application as a promising agent in dampening the adaptive immune and treating inflammatory diseases.