After the culture period, BMDCs were harvested and further cultured in the presence of p-OVA (50 g/mL) at 37 C. production; and the cell killing activity of cytotoxic T lymphocytes. In summary; oral administration of MAP ameliorated chronic EFS stress-induced immunosuppression. = 20. ** 0.01, compared to control levels. 2.2. Alleviative Effect of MAP on Chronic EFS Stress-Induced Disturbances in Lymphocyte Subsets As shown in Figure 2, both the total cell numbers and the number of each lymphocyte subset were dramatically decreased in the thymus (Figure 2A) and spleen (Figure 2B) by chronic EFS stress, and this effect was considered to be related to the atrophy of the lymphoid organs. The thymocyte subsetsincluding CD4+, CD8+, and CD4+CD8+ lymphocyteswere all significantly protected by the high dose of MAP (160 mg/kg), whereas the splenocyte subsetsincluding CD4+, CD11b+, and CD11c+ lymphocyteswere significantly increased by administration of both doses of MAP (80 and 160 mg/kg). Open in a separate window Figure 2 Protective effect of MAP against chronic EFS stress-induced disturbance in thymocyte and splenocyte cellularity. Mice are grouped as described in Figure 1. Lymphocyte subsets of the thymus (A) and spleen (B) were analyzed using flow cytometry, in which 10,000 cells were scored. Values are means SEM of three experiments, = 7. * 0.05, ** 0.01 compared with control. 2.3. Protective Effects of MAP against Chronic EFS Stress-Induced Suppression of Lymphocyte Proliferation Oral administration of MAP enhanced mitogen (Con A or LPS)-stimulated lymphocyte proliferation (Figure 3), which was impaired by chronic EFS stress. Furthermore, both doses of MAP (80 and 160 mg/kg) protected the proliferative function of lymphocytes. Open in a separate window Figure 3 MK-0773 Protective effects of MAP on chronic EFS stress-induced suppression of lymphocyte proliferation. Mice are grouped as described in Figure 1. Isolated mice splenocytes were co-cultured with (A) Con A or (B) LPS. Proliferation of splenocytes was measured using 3(H)-thymidine incorporation assay. Values are means SEM of three experiments, = 7. ** 0.01 compared with control. 2.4. Protective Effect of MAP against Chronic EFS Stress-Induced Disturbance in Lymphoid Organ Subsets of Ovalbumin (OVA)-Immunized Mice The number of each lymphocyte subset in both spleen and thymus was decreased by chronic EFS stress and significantly restored by MAP treatment, regardless of immunization (Figure 2 and Figure MK-0773 4). When immunized, the CD4+CD8+ T lymphocytes were significantly protected by the high dose of MAP (160 mg/kg; Figure 4A), and the number of splenocyte subsetsincluding the CD4+ and HDAC10 CD11b+ lymphocyteswas significantly restored by the administration of high-dose MAP (Figure 4B). The absolute numbers of CD11b+ and CD11c+ cells, which are responsible for antigen processing, were considerably increased MK-0773 overall in all groups (Figure 4B) compared to those in non-immunized groups (Figure 2B), and the degree of decrease caused by EFS stress lessened in all subtypes after the immunization. Open in a separate window Figure 4 Protective effect of MAP against chronic EFS stress-induced disturbance of lymphocyte cellularity in OVA-immunized mice. Mice are grouped as described in Figure 1. Immunization with OVA was previously achieved in mice. Cellularity of lymphocytes in the (A) thymus and (B) spleen was analyzed using flow cytometry in which 10,000 cells were scored. MK-0773 Values are means SEM of three experiments, = 5. * 0.05, ** 0.01 compared with control. 2.5. Immune Enhancing Effect of MAP on Immunoglobulin G (IgG) Production and Generation of OVA-Specific T Cells in Chronically Stressed Mice T cells isolated from the OVA-immunized spleen were co-cultured with p-OVA-pulsed bone marrow-derived cells (BMDCs), and the proliferative activity of T cells was examined on the last day of incubation. T cell proliferation was markedly weak in the chronic EFS stress-induced samples MK-0773 compared to that in those not subjected to stress. It is interesting to note that oral MAP administration protected cells against the effects of chronic EFS stress in a dose-dependent manner (Figure 5A). Open in a separate window Figure 5 Immune enhancing effect of MAP on OVA-specific IgG production and OVA-specific T cell generation in chronically stressed mice. Mice are grouped as described in Figure 1. Immunization of OVA peptide was achieved in mice. Serum lgG levels of collected blood were monitored using ELISA analysis (A); Isolated T cells.