Abstract
Objective: To explore the effects and possible mechanism of rapamycin (RAPA) on apoptosis of CD4(+)CD25(+) Tregs from the mouse severe aplastic anemia (SAA) model. Methods: The BALB/c female SAA model mice were induced by interferon-gamma in combination with busulphan. The SAA model mice were intraperitoneal injection with RAPA at daily dose of 0.5 mg/kg for 5 days (the RAPA-treated group, n=15) in the SAA group (n=15) and the un-treated group (n=15) were control. Bone marrow hematopoiesis changes were observed by the patho-morphological examination of femurs. The mononuclear cells of the peripheral blood and spleen were subjected to assess the intracellular Foxp3 expression in CD4(+)CD25(+) Tregs by flow cytometry (FCM). In addition, after being pured by immunomagnetic beads, the splenic CD4(+)CD25(+) Tregs was subjected to assess apoptosis by FCM and the Akt and Stat3 phosphorylation by using of western blot. Results: The patho-morphological examination of femurs showed normal marrow cell proliferation in un-treated group and hypocellularity in both SAA group and RAPA-treat group, with an increase in the number of fat cells. The bone marrow hematopoietic tissue ratio in RAPA-treat group was higher than SAA group [(9.75±1.83)% vs (7.00±2.00)%, Δx=2.15% (95%CI 0.15%-5.35%), P=0.037]. In the SAA group, FCM analysis showed down-expression of Foxp3 in CD4(+)CD25(+) Tregs compared with the un-treated group. However, after treatment with RAPA, the expression of Foxp3 in CD4(+)CD25(+) Tregs was increased (P<0.017). Compared with the un-treated group, increased CD4(+)CD25(+) Tregs apoptosis [(19.84±1.39)% vs (29.85±2.72)%] with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group (P<0.05, respectively). On the contrary, RAPA-treated group exhibited CD4(+) CD25(+) Tregs with a reduction in apoptosis rate [(22.39±3.71)%], Akt phosphorylation and Stat3 phosphorylation compared with the SAA group (P<0.05, respectively). Conclusion: These results indicate that RAPA may increase the expression of Foxp3 by down-regulation the levels of Akt and Stat3 phosphorylation and reduce apoptosis in splenic CD4(+)CD25(+) Tregs from the mice model of SAA.