Abstract:Objective To investigate the potential mechanisms of high-dose methylprednisolone treatment for immune thrombocytopenia (ITP) by assessing the levels of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the brain, colon, and serum.Methods A total of 18 BALB/c mice, SPF grade, weighing 18-22 g, with an equal distribution of males and females, were utilized. Blood platelet counts were measured using an automatic hematology analyzer. Based on platelet counts, the mice were randomly divided into three groups: a normal group, a model group, and a high-dose methylprednisolone group, each consisting of 6 mice. The normal group received injections of physiological saline, while the model and high-dose methylprednisolone groups were injected with anti-mouse platelet serum (APS) to replicate an ITP animal model. High-dose methylprednisolone treatment began in the high-dose methylprednisolone group on the 8th day after model induction, while the normal and model groups received physiological saline. Peripheral blood platelet counts were monitored at different time points: before APS injection, 4 days after injection, the 8th day of the experiment (day 1 of treatment), the 12th day of the experiment (day 5 of treatment), and the 15th day of the experiment (day 8 of treatment). Following the experiment, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VIP and PACAP in the brain, colon, and serum. Immunohistochemistry was employed to observe changes in P53 protein expression in lymphocytes of the mesenteric lymph nodes of mice, and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method was used to assess the apoptosis rate of lymphocytes in mesenteric lymph nodes. ELISA was also conducted to evaluate the levels of IFN-γ, IL-4, IL-10, and IL-17A in the spleen of mice.Results Compared to the normal group, both the model group and the high-dose methylprednisolone group exhibited reduced vitality and developed symptoms such as skin hemorrhage after APS injection. High-dose methylprednisolone treatment in the high-dose methylprednisolone group led to increased food intake, body weight, and improved coat condition, with reduced bleeding spots. Comparing platelet counts at various time points among the groups: (1) Platelet counts at different time points showed significant differences (F = 20.618, P = 0.000); (2) Platelet counts differed among the three groups (F = 59.621, P = 0.000); (3) Platelet counts displayed different trends over time among the three groups (F = 7.072, P = 0.000). Compared to the normal group, the model group exhibited elevated P53 protein expression in lymphocytes of the mesenteric lymph nodes (P < 0.05) and an increased rate of lymphocyte apoptosis (P < 0.05). When compared to the model group, the high-dose methylprednisolone group showed decreased P53 protein expression in lymphocytes of the mesenteric lymph nodes (P < 0.05) and reduced lymphocyte apoptosis (P < 0.05). Furthermore, compared to the normal group, the model group displayed decreased levels of VIP and PACAP in the brain, colon, and serum (P < 0.05). In comparison to the model group, the high-dose methylprednisolone group showed elevated VIP levels in the brain, colon, and serum (P < 0.05), as well as increased PACAP levels in the brain and colon (P < 0.05). Finally, when compared to the normal group, the model group had higher levels of IFN-γ and IL-17A in the spleen (P < 0.05) and lower levels of IL-4 and IL-10 (P < 0.05). In contrast, the high-dose methylprednisolone group had lower levels of IFN-γ and IL-17A (P < 0.05) and higher levels of IL-4 and IL-10 in the spleen (P < 0.05) compared to the model group.Conclusion The modulation of intestinal immune function via VIP/PACAP signaling pathways may represent one of the potential mechanisms underlying the effectiveness of high-dose methylprednisolone treatment for ITP.
