Decline in the expression of IL-2 after trauma and changes in the nuclear transcription factors NFAT and AP-1

OBJECTIVE: To investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1). METHODS: Mice with closed impact injury with fracture in both hind limbs were adopted as the trauma model. Spleen lymphocytes were isolated from traumatized mice and stimulated with Con-A. Culture supernatants were assayed for IL-2 activity, and total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. DNA binding activity of NFAT and AP-1 were measured by electrophoretic mobility shift assay (EMSA). The expression of c-Fos, c-Jun and JunB proteins was determined by the Western blot analysis. RESULTS: DNA binding activity of NFAT and AP-1 gradually decreased to a minimum of 41% and 49%, respectively, of the control on the 4th day after injury, which was closely followed by the decline in IL-2 activity and IL-2 mRNA. A decrease in the expression of c-Fos on the 1st and 4th day after trauma had no significant effect on c-Jun expression; the increase in expression of JunB was only on the 1st day after injury. CONCLUSION: Decreased IL-2 expression is, at least in part, due to a decline in the activation of NFAT and AP-1 in traumatized mice. The decline in DNA binding activity of NFAT and AP-1 is partly due to a trauma-induced block in the expression of c-Fos.

Qibai Pingfei capsule medicated serum inhibits the proliferation of hypoxia-induced pulmonary arterial smooth muscle cells via the Ca^(2+)/calcineurin/nuclear factor of activated T-cells 3 pathway

OBJECTIVE: To observe the effect of Qibai Pingfei capsule(QBPF) medicated serum on the proliferation of rat pulmonary arterial smooth muscle cells(PASMCs) under hypoxia conditions and to investigate its key molecular effects on the Ca^(2+)/calcineurin/nuclear factor of activated T-cells 3(NFATc3) signaling pathway.METHODS: QBPF was provided to rats via continuous gavage for 10 days. Primary rat PASMCs were cultured using the direct adherent culture method.Methyl thiazolyl tetrazolium assay was used to eval-uate the effect of QBPF on PASMCs proliferation under hypoxia conditions. Laser scanning confocal microscopy was used to detect changes in intracellular free calcium([Ca^(2+)]_i) in PASMC-loaded Fluo-3-AM.Real-time quantitative polymerase chain reaction and western blot were used to detect the transcription and protein expression levels of calcineurin and NFATc3 genes in PASMCs.RESULTS: Compared with normoxia conditions,PASMCs proliferated at 12, 24, 48, and 72 h under hypoxia conditions. QBPF at concentrations of 5%,10%, and 20% could inhibit hypoxia-induced PASMC proliferation to different degrees. The inhibitory effect was most significant in the 20% QBPF group under 24 h hypoxia conditions. The concentration of [Ca^(2+)]_iin PASMCs under hypoxia was increased and [Ca^(2+)]_iwas significantly decreased when co-incubated with QBPF at 5%, 10%, and 20%. Compared with normoxia conditions, the m RNA and protein expression levels of calcineurin and NFATc3 in PASMCs induced by hypoxia were up-regulated.QBPF application significantly down-regulated m RNA and protein expression levels of calcineurin and NFATc3 in PASMCs.CONCLUSION: QBPF can effectively inhibit hypoxia-induced proliferation of PASMCs through down-regulation of key molecular expression via the Ca^(2+)/calcineurin/NFATc3 pathway.