Effect Of Immune Suppression Of Treg Cells In Burn Sepsis And Intervention Activity With Drug

Objective:To develop and evaluate an animal model of sepsis that established by swabbed pseudomonas aeruginosa onto scald wound in rats for the research applications of sepsis that caused by burn wound infection.Methods:Forty-eight SD rats were randomly divided into scald group (B, n=16), scald sepsis A group (BS-A, n=16), scald sepsis B group (BS-B, n=16) according to the random number table.Then,each group were randomly divided into2sub-groups,there were8mice in each sub-group. Immersing rat back into boiling water which was (92±2)℃for18s to duplicating the full-thickness scald wound after anesthesia. After scald, lml of normal saline containing103CFU of pseudomonas aeruginosa (PA, ATCC27953) was respectively injected into the wound tissue for the rats of group BS-A. Meanwhile, lml of normal saline containing1×105CFU of PA was respectively swabbed onto the surface of scald wound for the rats of group BS-B. The number of survival rats was recorded on every day after scald. At the post burn day (PBD)3and8, the survival ratio of PBD8subgroup was counted and the blood bacterial culture of each subgroup was tested. Results:(1) The survival ratio of PBD8subgroup BS-A (50.0%,37.5%) and PBD8sub group BS-B (62.5%,50%) were obviously lower than that of PBD8subgroup B (87.5%,75.0%). Compared with the sub-group BS-A, the survival ratio of both PBD3and PBD8sub-group of BS-B sub-groups was slight higher, but without statistical significance.(2) The positive ratios of the blood bacterial culture of PBD3sub-group BS-A and PBD3sub-group BS-A were75.0%and100%respectively, while60%,100%for PBD3sub-group BS-B and PBD3sub-group BS-B and0%,0%for PBD3sub-group B and PBD3sub-group B. Compared with group B, both sub-groups BS-A and BS-B were significantly higher (P<0.05). But there were no any statistical difference between the sub-groups of BS-A and BS-B.Conclusions:Swabbed pseudomonas aeruginosa (ATCC27953) onto full-thickness scald wound on rat to induce a sepsis that caused by wound infection is a useful animal model for relative researches in burn surgery. Compared with injection bacteria into wound tissue, this model is characterized by slower progressing and high mortality that more correspond to the feature of burn sepsis in clinic. Objective:To explore the effects of regulatory T (Treg) cells on the changes of immunity in the status of severer burns and sepsis, and to evaluate the protective effects of thymosin-1, Xuebijing injection, Jieduhuayu soup.Methods:Sixty-two SD rats were randomly divided into nine groups:1) Normal control group (N),2) Scaled group(S),3) Scaled sepsis group(SS),4) Scaled thymosin treatment group(S-T),5) Scaled Xuebijing treatment group(S-X),6) Scaled Jieduhuayu soup treatment group(S-J),7) Scaled sepsis thymosin treatment group(SS-T),8) Scaled sepsis Xuebijing treatment group(SS-X),and9) Scaled sepsis Jieduhuayu soup treatment group(SS-J),6-8rats in each group. Specimens of the rats peripheral blood were harvested for detections of the level of Treg cells and T lymphocyte subsets by FCM, and the level of serum TNF-α、IFN-γ and IL-4, IL-10, PCT and soluble CD25by the ELISA. Specimens of spleen tissues were harvested to determine the HLA-DR proteins and the Foxp3mRNA expression in the spleen tissue by real time PCR and immunohistochemistry respectively.Results:(1) The level of Treg cells in peripheral blood, the Foxp3mRNA and the HLA-DR expression in the spleen in Group S were significantly higher than that in Group N, but significantly lower than that in Group SS (P<0.05or<0.01). Compared with the Group N, the serous TNF-a, IFN-y and IL-4,IL-10levels and IFN-y/IL-4ratio in the Group S were significantly increased, but significantly lower than the burn sepsis group(P<0.05or<0.01).(2) Compared to the Group S, in the groups of S-T, S-X and S-J, the levels of Treg cells, Foxp3mRNA and HLA-DR expression in spleen and serous TNF-α, IFN-γ, and IL-4, IL-10reduced variously. But all these results were still significantly higher than that of Group N (P<0.05or<0.01). Among all treatment groups, in the Group S-T, the level of Treg cells in peripheral blood, Foxp3mRNA expression in spleen decreased and T lymphocyte subsets of CD3+, CD4+T cells were increased and the level of CD8+T cells decreased significantly, while the levels of serous TNF-a, IFN-yand IL-4, IL-10level and TFN-y/IL-4ratio of both Group S-X and S-J were decreased than that in Group S-T without significant difference.(3) Compared with the group SS,in the groups of S-T, S-X and S-J, the levels of Treg cells in blood, Foxp3mRNA and HLA-DR expression in spleen, TNF-a, IFN-yand IL-4, IL-10and the TFN-γ/IL-4ratio in serum were significantly lower. By compared with these three treatment groups each other, in Group SS-T, the degree of the decline of the levels of Treg cells in peripheral blood, Foxp3mRNA expression in the spleen, the increase of the level of CD3+, CD4+T cells and the decline on CD8+T cells were significantly higher than the other two groups (P<0.05或<0.01).And there were slightly decrease about the levels of serous TNF-a, IFN-yand IL-4, IL-10and the TFN-y/IL-4ratio, but no significant difference between them.Conclusion:In the status of severe burns and burn sepsis, the organism in the late progress of the diseases showed obvious changes on the Thl/Th2cell shift based on the changes of Treg cells, T lymphocyte subsets and cytokines, inflammatory mediators, and expressed varying degrees of immunosuppression. The medicines of thymosin-α1, Xuebijing injection and Jieduhuayu soup may improve immune suppression variously. But the effects of protective activities were slightly different.