| Previous study have confirmed that severe trauma or infection can cause glucocorticoid resistance (down-regulation of the expression or the function of glucocorticoid receptor), hyperactivated mediators of inflammation (nuclear factor-kappa B, activator protein-1) as well as hypersecretion of the pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). This kind of situation will continue and the interaction of the three factors will lead to the amplification of inflammation reaction and the initiation of systemic inflammatory response syndrome (SIRS). SIRS is the result of hyperactivity of innate immunity and the main cause of subsequent whole body damage, too. The excessive release of endogenous mediators of anti-inflammatory in concomitant with the development of SIRS is an important causative factor of subsequent immunosuppression, mainly of the cell immune. Therefore, investigating the molecular mechanism of the development of SIRS and the subsequent immunosuppression is of theoretical and clinical significance.The family of steroid receptor coactivator (SRC) is composed of SRC-1, SRC-2 and SRC-3. These cofactors interact with nuclear receptors in a ligand-dependent manner and enhance transcriptional activation of the receptor via histone acetylation/methylation and recruitment of additional cofactors. A great deal of information about SRCs has already been accumulated in regulating growth and development, energy metabolism as well as formation of some tumors. But the regulatory effects of SRCs on inflammatory/immune response still remain unclear.The SRC-3 knockout mice were introduced from Baylor College of Medicine (Huston, USA) to our laboratory and colonized. On the bases of successful reproduction and identification, we carried out two parts of experiments to explore the regulatory effects of SRCs on inflammatory/immune response. In the first part of experiments, the function of SRC-3 during the development of SIRS was studied in vivo and in vitro: 1) on the model of an intraperitoneal injection of LPS (5mg/kg body weight), the effects of SRC-3 knock-out on the secretion of pro-inflammatory cytokines as well as the expression and nuclear translocation of glucocorticoid receptor, nuclear factor-kappa B, activator protein-1 in liver and spleen were observed; 2) after 10μg/ml LPS challenging, the effects of SRC-3 protein depletion on the activation of primary cultured peritoneal macrophages were studied, including the transcription and bioactivity of pro-inflammatory cytokines. In another part of experiment, the role of SRC-3 in innate immunity was studied on bacteria loading model. The relation of SRC-3 and immunosuppression was studied on animal models of burn- and LPS injection-induced inflammatory response.Main results:1. Based on successful reproduction and colonial expanding, we used PCR and Western blot to characterize the off-springs on both gene and protein level. A number of SRC-3 knock-out mice were identified, which provided a good condition for the following experiments.2. SRC-3 was expressed in the hepatic and splenic tissue from SRC-3+/+ mice, with much higher expression level (net excess 55.8%) in spleen than in liver tissue.3. After an intraperitoneal injection of LPS with a dose of 5mg/kg body weight, general physiological status in SRC-3-/- group was much easier than that of SRC-3+/+ group, but no obvious pathological changes were found in liver, spleen, kidney, myocardium and thymus in mice of the two groups.4. After an intraperitoneal injection of LPS, the two groups exhibited a higher expression of serum TNF-α, IL-1β, IL-6 and IL-10 than the normal level. But in contrast to the SRC-3+/+ group, the serum level of TNF-α, IL-1βand IL-6 in SRC-3-/- group was significantly lower, however, the IL-10 significantly higher than that of wildtype mice.5. Under normal condition, no statistic differences in the level of TNF-α, IL-1β, IL-6, IL-10 mRNA of peritoneal macrophages were observed between the SRC-3+/+ group and SRC-3-/- group. After LPS stimulation in vitro, the levels of above cytokines in both groups were markedly elevated at 4h, while the increased extent of TNF-α, IL-1β, IL-6 mRNA were obviously less but IL-10 mRNA more in SRC-3-/- group than that of SRC-3+/+ group.6. The levels of GR expression and its nuclear translocation in liver were significantly reduced in both SRC-3+/+ group and SRC-3-/- group after LPS challenging. However, the reduction extent of SRC-3-/- group is significant less than that of SRC-3+/+ group. The reduction extent of GR expression in spleen from SRC-3-/- group is significant less than that from SRC-3+/+ group. The nuclear translocation of GR in SRC-3+/+ mice was significantly reduced, but no significant changes were observed in SRC-3-/- group.7. After LPS stimulation, the IκB-αin liver and spleen was markedly reduced and reached the nadir at 1h in both groups, and the reduction extent of SRC-3-/- group was obviously lower than that of SRC-3+/+ group. The levels of NF-κB p65/p50 expression and its nuclear translocation in liver and spleen in both groups was markedly elevated, and the elevated extent of its expression in SRC-3-/- group was obviously larger compared with that of SRC-3+/+ group, but the level of its nuclear translocation in SRC-3-/- group was obviously lower than that of SRC-3+/+ group. The interferon regulatory factor-1 (IRF-1) expression in liver and spleen in both groups was markedly elevated, and it was obviously less elevation in SRC-3-/- group than that of SRC-3+/+ group at matched time points.8. After LPS stimulation, the levels of c-Jun/c-Fos expression and its nuclear translocation in liver and spleen in both groups were markedly elevated and reached the peak at 1h, but the increased levels in SRC-3-/- group were significantly lower than those in SRC-3+/+ group.9. After LPS stimulation, the hepatic SRC-1 in both groups were significantly reduced, but the changes in SRC-3-/- group were less than those in SRC-3+/+ group. And the level of intranuclear in the liver was significantly reduced in SRC-3+/+ group, but increased in SRC-3-/- group. No SRC-1 in spleen of two groups was detectable10. At matched time points, the expression and intranuclear levels of SRC-2 were obviously higher in SRC-3-/- group than those of SRC-3+/+ group. They were both significantly increased in the liver of two groups, but in the spleen, they were reduced in SRC-3+/+ group and increased in SRC-3-/- group.11. Under normal condition, the expression level of GR on peritoneal macrophage in SRC-3+/+ group did not differ from that of SRC-3-/- group. The GR expression on peritoneal macrophage in both groups at 4h after LPS stimulation were markedly reduced, but the reduction extent of SRC-3-/- group was obviously lower than that of SRC-3+/+ group.12. Under normal condition, the expression level of peritoneal macrophage NF-κB p65 in SRC-3+/+ group was obviously higher than that of SRC-3-/- group. The expression level of peritoneal macrophage NF-κB p65 in both groups at 4h after LPS stimulation were markedly elevated, but the increased extent of SRC-3-/- group was obviously larger than that of SRC-3+/+ group.13. Under normal condition, the expression level of peritoneal macrophage c-Jun/c-Fos in SRC-3+/+ group was not different from that of SRC-3-/- group. After LPS stimulation, the expression level of peritoneal macrophage c-Jun/c-Fos in both groups were markedly elevated at 4h, but the increased extent of SRC-3-/- group was obviously lower than that of SRC-3+/+ group.14. The results acquired through bacteria loading experiment showed that the viable organism content in peripheral blood in SRC-3-/- group was obviously more than that of SRC-3+/+ group. And at 24h and 48h after bacteria injection, the bacterial content in liver, spleen as well as thymus in SRC-3-/- group was markedly more than those of SRC-3+/+ group, but the bacterial content in nephridial tissue was low and no difference between two groups.15. Under normal condition, no significant differences in the serum level of IL-2 and sIL-2R were observed between two groups. After TBSA 15%-20%Ⅲ°burn injury, the level of serum IL-2 and sIL-2R significantly reduced at 24h in both SRC-3+/+ group and SRC-3-/- group, but serum IL-2 was was markedly lower, serum sIL-2R was obviously higher in SRC-3-/- group than those of SRC-3+/+ group, respectively. The levels of serum IL-2 after 5mg/kg body weight of LPS intraperitoneal injection in both groups were no significant difference from normal levels, but sIL-2R was markedly elevated. And the increased extent of serum sIL-2R in SRC-3-/- group was obviously larger than that of SRC-3+/+ group.16. Under normal condition, the fraction of CD3+, CD4+ cell of T cell subgroup decreased with distinct extent in peripheral blood, spleen and thymus in SRC-3-/- group, in concomitant with the increase of the ratio of CD8+ cells and the decrease of the ratio of CD4+/CD8+. After burn injury or LPS injection, the proportion of CD3+, CD4+ as well as the ratio of CD4+/CD8+ in peripheral blood, spleen and thymus were significantly reduced at 24h in both SRC-3+/+ group and SRC-3-/- group. However, the reduction of CD3+, CD4+ in SRC-3-/- group was significant less than that of SRC-3+/+ group, and the ratio of CD4+/CD8+ of two groups were no difference.Conclusion:1. SRC-3 is related with the synthesis and release of pro-inflammatory cytokines. The depletion of SRC-3 protein could suppress the gene transcription and release of TNF-α, IL-1β,IL-6 induced by LPS, improve system effect during inflammatory reaction, relieve the activation of peritoneal macrophage.2. SRC-3 is relavent to the expression and nuclear translocation of GR. The depletion of SRC-3 protein could relieve the LPS-induced response of down-regulation of the expression and nuclear translocation of GR as well as glucocorticoid resistance.3. The expression and activity of NF-κB may be concerned with the SRC-3 protein which involves in regulation of NF-κB signal transduction pathway. The absence of SRC-3 protein could result in suppression of gene transcription activity through reversing the down-regulation of IκB-αand subsequently inhibiting nuclear translocation of NF-κB during the early stage of inflammatory response.4. SRC-3 is concerned with the activation of AP-1 signal transduction pathway. The absence of SRC-3 protein could result in partial inhibition of LPS-induced the expression and activity of AP-1 owing to relative deficient of the synthesis and release of pro-inflammatory cytokines, such as TNF-α,IL-1β.5. There is compensation effect among the members of SRC family. The SRC-1 and SRC-2 could compensate consequence, to certain extent, induced by the absence of SRC-3 protein, but it is limited.6. SRC-3 plays an important role in mantaining normal immune response. The absence of SRC-3 protein could result in the attenuation of innate immunity function including suppressing the LPS-induced transcription activity and release of TNF-α,IL-1β,IL-6 and cutting down the function of phagocytotic and eliminating bacterium as well as its production and the suppression of cellular immune function through anomaly depression of CD3+ and CD4+ T cells as well as the CD4+/CD8+ ratio.7. SRC-3 may work as a fine tuner to adjust the cell immune in a"bi-direction"manner. The absence of SRC-3 protein could aggravate immunosuppression through promoting the expression of IL-10 on one side, and on the other side extenuate the degression of CD3+ and CD4+ T cells as well as the CD4+/CD8+ ratio, which indicate SRC-3 involving in the occurance of immunosuppression accompanied with SIRS.
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