Pathological Significance Of CD4~+CD25~+Tregs Cells In The Immune Tolerance Of Microenvironment Among Patients With Middle To Late Staged NPC And The Reversing Effect Of Qi-Boosting Toxin-Resolving Formula

OBJECTIVETo detect the percentage of CD4+CD25+Tregs cells,the levels of Foxp3mRNA, the content of its relative cytokines (IFN-γ、IL-2、TGF-β、 IL-10、IL-6、IL-17), intervention reaction of QBTRF in the patients with middle to late staged NPC and QBTRF’s influence on BALB/c animals, the pathological significance of CD4+CD25+Tregs cells and the reversing effect of Qi-Boosting Toxin-Resolving Formula(QBTRF) were investigated in the immune tolerance of tumor microenvironment among patients with middle to late staged nasopharyngeal carcinoma (NPC), which it is provided a new clue for establishing immune regulation therapy of traditional Chinese medicine in NPC.METHODS1.The peripheral blood samples were taken form healthy population, patients with middle to late staged NPC. Flow-cytometry was performed to detect the percentage of CD4+CD25+Tregs cells, CD4+CD25+Foxp3Tregs cells and Th17cells of peripheral blood cells, which the differences was compared between these groups.2.The percentage of CD4+CD25+Tregs cells, CD4+CD25+Foxp3Tregs cells and Th17cells were detected by Flow-cytometry in the peripheral blood among middle to late staged NPC patients treated by conventional therapy and conventional therapy add to QBTRF, which the differences was compared between these groups.3.The percentage of CD4+CD25+Tregs cells, CD4+CD25+Foxp3Tregs cells and Th17cells were detected by Flow-cytometry in peripheral blood of the blank group, saline group and QBTRF group of BALB/c mice, which the differences was compared between these groups.4.The serum levels of IFN-γ, IL-2, TGF-p, IL-10, EL-6and IL-17were determined by ELISA in the healthy population and patients with middle to late staged NPC, which the differences of every cytokine was compared between these groups.5.The serum levels of IFN-γ, IL-2, TGF-β, IL-10, IL-6and IL-17were determined by ELISA in the peripheral blood among middle to late staged NPC patients treated by conventional therapy and conventional therapy add to QBTRF, which the differences of every cytokine was compared between these groups.6.To further confirm the regulatory effect of QBTRF on immune system, the serum levels of IFN-γ, IL-2, TGF-β, IL-10, IL-6and IL-17were determined by ELISA in peripheral blood of the blank group, saline group and QBTRF group of BALB/c mice, which the differences of every cytokine was compared between these groups.7.The transcriptional levels of Foxp3mRNA and ROR-yt mRNA were detected by real-time PCR in the peripheral blood among the healthy population and patients with middle to late staged NPC, which the differences the relation of CD4+CD25+Tregs cells, Foxp3mRNA and ROR-yt mRNA was compared between these groups.8.The transcriptional levels of Foxp3mRNA and ROR-yt mRNA were detected by real-time PCR in the peripheral blood among middle to late staged NPC patients treated by conventional therapy and conventional therapy add to QBTRF, which the differences was compared between these groups.RESULTS1. The results of flow cytometry show that the percentage of CD4+CD25+Tregs cells were respectively (2.65±0.31)%、(4.23±0.53)%, the percentage of CD4+CD25+Foxp3Tregs cells were respectively (0.48±0.05)%、(0.98±0.15)%, and the percentage of Th17cell were respectively (1.80±0.30)%、(0.50±0.21)%in peripheral blood among healthy control and middle to late staged NPC patients. Compared with the healthy control, the percentage of CD4+CD25+Tregs cells and CD4+CD25TFoxp3Tregs cells was significantly higher in peripheral blood among middle to late staged NPC patients (P<0.05), while the percentage of Th17cell is significantly decreased (P<0.05).2. The results of clinic treatment show that the percentage of CD4+CD25+Tregs cells were respectively (4.75±0.39)%、(3.09±0.49)%, the percentage of CD4+CD25+Foxp3Tregs cells were respectively (0.93±0.15)%、(0.53±0.04)%, and the percentage of Th17cell were respectively (0.63±0.20)%、(2.09±0.34)%in peripheral blood among NPC patients treated by conventional therapy and conventional therapy add to QBTRF. Compared with NPC patients treated by conventional therapy, the percentage of CD4+CD25+Tregs cells and CD4+CD25+Foxp3Tregs cells was significantly lower in peripheral blood among NPC patients treated by conventional therapy add to QBTRF (P<0.05), while the percentage of Th17cell is significantly increased (P<0.05).3. The results of animal experiment show that the percentage of CD4+CD25+Tregs cells were respectively (6.53±1.09)%、(6.79±1.22)%、(4.52±0.61)%, and the percentage of CD4+CD25+Foxp3Tregs cells were respectively (12.16±2.51)%、(11.01±2.00)%、(8.25±1.43)%in peripheral blood among the blank group, the NS group and QBTRF of BALB/c mouse. Compared with the blank group and the NS group of BALB/c mouse, the percentage of CD4+CD25+Tregs cells and CD4+CD25+Foxp3Tregs cells was significantly lower in peripheral blood among BABL/c mice intervened by QBTRF (P<0.05), while the percentage of CD4+CD25+Tregs cells and CD4+CD25+Foxp3Tregs cells had no significant difference between the blank group and the NS group (P>0.05).4. The detection of cytokines show that the serum levels of IFN-γ、IL-2、 IL-17、TGF-β、IL-10、IL-6were respectively (220.36±18.76) pg/ml、(174.79±7.55) pg/ml、(7.71±0.34) pg/ml、(488.82±36.91) pg/ml、(0.68±0.08)pg/ml、(1.37±0.03)pg/ml in peripheral blood among among the healthy control, the serum levels of IFN-γ、IL-2、IL-17、TGF-β、 IL-10、IL-6were respectively (124.95±4.22) pg/ml、(81.88±10.84) pg/ml、(4.61±0.09) pg/ml、(645.56±39.61) pg/ml、(1.27±0.21) pg/ml、(1.88±0.13) pg/ml in peripheral blood among middle to late staged NPC patients. Compared with the healthy control, the serum levels of TGF-β、IL-10、IL-6was significantly higher in peripheral blood among middle to late staged NPC patients (P<0.05), while the serum levels of IFN-γ、IL-2、IL-17is significantly decreased (P<0.05).5.The experimental result of clinical treatment in middle to late staged NPC show that the serum levels of IFN-γ、IL-2、IL-17、TGF-β、IL-10、 IL-6were respectively (155.26±4.99) pg/ml、(128.89±9.27) pg/ml、(2.97±0.14) pg/ml、(544.47±21.96) pg/ml、(0.99±0.04) pg/ml、(2.86±0.19) pg/ml in the NPC group treated by conventional therapy. The serum levels of IFN-γ、IL-2、IL-17、TGF-β、IL-10、IL-6were respectively (321.60±41.19)pg/ml、(231.91±24.24)pg/ml、(5.54±0.14) pg/ml、(421.62±25.42) pg/ml、(0.67±0.11) pg/ml、(1.47±0.05) pg/ml in the NPC group treated by conventional therapy add to QBTRF. Compared with the NPC group treated by conventional therapy, the serum levels of IFN-γ、IL-2、IL-17was significantly higher in the NPC group treated by conventional therapy add to QBTRF (P<0.05), while the serum levels of TGF-P, IL-10, IL-6is significantly decreased (P<0.05).6. The serum levels of IFN-γ、IL-2、IL-17、TGF-β、IL-10、IL-6were respectively (229.72±5.72) pg/ml.(70.24±3.34) pg/ml、(11.22±0.35) pg/ml、(126.31±16.50)pg/ml、(128.43±9.86)pg/ml、(20.55±0.92) pg/ml in peripheral blood among BABL/c mice intervened by QBTRF. Compared with the blank group and NS group of BALB/c mouse, the serum levels of TGF-β、IL-10、IL-6was significantly decreased (P<0.05), but IFN-γ、IL-2、IL-17was significantly higher in peripheral blood among BABL/c mice intervened by QBTRF (P<0.05), while there was no significant difference between the blank group and the NS group (P>0.05).7.Compared with the healthy controls, the transcriptional levels(3.699±0.309) of Foxp3mRNA was significantly higher in peripheral blood among patients with middle to late staged NPC (P<0.05), while ROR-yt mRNA(O.303±0.115) was significantly decreased (P <0.05). The ratio of CD4+CD25+Tregs cells was positively related with CD4+CD25+Foxp3Tregs cells in peripheral blood among patients with middle to late staged NPC (r2=0.163, P<0.05),but there was no relation between the transcriptional levels of Foxp3mRNA and ROR-yt mRNA(r2=-0.127, P>0.05). The transcriptional levels of ROR-yt mRNA is significantly higher in peripheral blood among the NPC treated by QBTRF (P<0.05), while the transcriptional levels of Foxp3mRNA is significantly decreased (P<0.05).CONCLUSION1.The advantaged percentage and functional activity of CD4+CD25+Tregs cells is one of important reasons produced the phenomenon of immune tolerance in the tumor microenvironment in patients with middle to late staged NPC, which was maintained by influencing the differentiatial factors of CD4+CD25+Tregs cells and Th17cells and its immune activity.2.The activity regulation and effect factors of CD4+CD25+Tregs cells is negatively related with the cytokines of IFN-y, IL-2, IL-17and transcriptional factor ROR-yt mRNA in patients with middle to late staged NPC, but positively with the level of TGF-β、IL-6、IL-10and transcriptional factor Foxp3mRNA3. Intervention effect of compound Chinese medicine QBTRF can reduce the percentage and functional activity of suppressor cell CD4+CD25+Tregs cells in patients with middle to late staged NPC, improve the percentage and functional activity of effect cell Th17cells, which be help to reverse the phenomenon of immune tolerance in tumor microenvironment and enhance the resistance of tumor in patients with NPC.4. Inhibition effect of QBTRF for CD4+CD25+Tregs cells may be realized on the extensively intervention of negative and positive regulating factors, which embodies pharmacological pattern characteristics of more target micro effect in traditional Chinese medicine compound.5. The percentage and functional change of CD4+CD25+Tregs cells, the expression levels of negative regulation factors(TGF-β、IL-10) and the secretion levels of positive regulation factors (IFN-γ、IL-2、IL-17、IL-6) in BALB/c mice intervened by QBTRF further supported the role characteristics of QBTRF for CD4+CD25+Tregs cells in patients with middle to late staged NPC.6.The reversal of immune tolerance dominated by CD4+CD25+Tregs cells in the tumor microenvironment should be the important way of antitumor therapy of traditional Chinese medicine, which embody and give full play to the unique advantages of traditional Chinese medicine anticancer.