| ObjectiveThis study aims to investigate the changes of high mobility group box-1 protein (HMGB1) expression and activity of cholinergic nerve in brain tissuues in sepsis, and then explores the effect of central HMGB1 on cellular immune response in sepsis and its relationship with the cholinergic anti-inflammatory pathway.Methods1. Forty SD rats were divided into four groups (according to random number): Control, Sham, CLP 24h, CLP 48h. Rats were sacrificed at 24 h and 48 h after sepsis. Then,the entire brain was extracted and cortex, hippocampus, striatum and thalamus tissues were isolated immediately. The expression of HMGB 1 in brain tissues was evaluated by Western blotting, PCR and tissue immunofluorescence. Indicators of cholinergic activity were measured by enzyme-linked immunosorbent assay (ELISA).2. Eighty SD rats were divided into four groups (according to random number): Control, Sham, HMGB1 (1μg), HMGB1 (10μg), setting 24 h and 48 h two time points (10 rats/group). Rats were sacrificed at 24 h and 48 h after central HMGB 1 stimulation, and then spleens were separated to isolate Tregs, DCs and CD4+T cells. Changes of cell surface molecules on Tregs and DCs were measured by flow cytometry analysis. The proliferation of T cells were measured by CCK8. cytokine secretion of Tregs and T cells were measured by ELISA.3. Sixty SD rats were divided into three groups (according to random number):Sham+NS, CLP+NS, CLP+BoxA, setting24 h and 48 h two time points (10 rats/ group). Rats were sacrificed at 24 h and 48 h after CLP and central BoxA stimulation, and then spleens were separated to isolate Tregs. DCs and CD4+T cells. Changes of cell surface molecules on Tregs and DCs were measured by flow cytometry analysis. The proliferation of T cells were measured by CCK8. cytokine secretion of Tregs and T cells were measured by ELISA.4. Experiment was divided into two parts:central HMGB1 stimulation and central BoxA intervention. In experiment of central HMGB1 stimulation, sixteen wild C57BL/6 mice and sixteen a7 nAChR knock-out C57BL/6 mice were randomly (random number) divided into Control group and HMGB1 (1μg) group. In experiment of central BoxA intervention, twenty-four wild C57BL/6 mice and twenty-four a7 nAChR knock-out C57BL/6 mice were randomly (random number) divided into three groups:Sham+NS, CLP+NS, CLP+BoxA. The processing of mice was same to the same experiment mentioned above. Mice were sacrificed at 24 h after stimulation, and then spleens were separated to isolate Tregs and CD4+T cells. Changes of cell surface molecules on Tregs were measured by flow cytometry analysis. The proliferation of T cells were measured by CCK8. Cytokine secretion of Tregs was measured by ELISA.Results1.Compared to sham groups, the expressions of HMGB1 in cortex, hippocampus, striatum and thalamus were significantly enhanced in the septic groups (cortex: P<0.05; hippocampus:P<0.05; thalamus:P<0.01; striatum:P<0.01), there were no remarkable differences between septic 24 and 48 h groups in four brain regions. Compared to sham groups, the level of cholinergic activity in four brain region were significantly enhanced in the septic groups:AchE (cortex:P<0.05; hippocampus: P<0.05; thalamus:P<0.05; striatum:P<0.05), Ach (cortex:P<0.01; hippocampus: P<0.01; thalamus:P<0.05; striatum:P<0.01), ChAT (cortex:P<0.01; hippocampus: P<0.01; thalamus:P<0.01; striatum:P<0.01), there were no remarkable differences between septic 24 and 48 h groups.2. Effect of central high mobility group box-1 protein on immune function of Treg, DC and CD4+T:(1) Compared to sham group, the expressions of Foxp3 and IL-10 secretion in Treg were significantly up-regulated in HMGB1 (1μg) group at 48h (P<0.01-0.05); Compared to sham group, the expressions of Foxp3 and CTLA-4 in Treg were up-regulated, the inhibition of Treg to T cell proliferation was enhanced, the secretion of TGF-P and IL-10 in Treg were significantly up-regulated in HMGB1(10μg) group at 24 and 48h (all P<0.01). (2) Compared to sham group, the expressions of CD80 and MHCⅡ were decreased in HMGB1 (1μg) group at 24 and 48h (all P<0.05); Compared to sham group, the expressions of CD80, CD86 and MHCⅡ were decreased, enhancement of DC to T cell proliferation was decreased in HMGB1 (10μg) group at 24 and 48h (all P<0.01). (3) Compared to sham group, the secretion of IFN-y in T cells was decreased, the secretion of IL-4 in T cells was enhanced in HMGB1 (1μg) group at 24h(all P<0.05); Compared to sham group, T cell proliferation was decreased, the secretion of IL-2 and IFN-y in T cells was decreased, the secretion of IL-4 in T cells was enhanced in HMGB1(10μg) group at 24 and 48h(all P<0.01).3. Effect of central BoxA on immune function of Treg, DC and CD4+T in CLP rats:(1) Compared to sham group, the expressions of Foxp3 and CTLA-4 in Treg were up-regulated, the inhibition of Treg to T cell proliferation was enhanced, the secretion of TGF-P and IL-10 in Treg were significantly up-regulated in CLP group at 24 and 48h (all P<0.01); Compared to CLP group, the expressions of Foxp3 and CTLA-4 in Treg were down-regulated, the inhibition of Treg to T cell proliferation was decreased, the secretion of TGF-β and IL-10 in Treg were significantly down-regulated in CLP+BoxA group at 24 and 48h (all P<0.01). (2) Compared to sham group, the expressions of CD80, CD86 and MHCⅡ were decreased, enhancement of DC to T cell proliferation was decreased in CLP group at 24 and 48h (all P<0.01); Compared to CLP group, the expressions of CD80, CD86 andMHCⅡ were up-regulated, enhancement of DC to T cell proliferation was up-regulated in CLP+BoxA group at 24 and 48h (all P<0.01-0.05). (3) Compared to sham group, T cell proliferation was decreased, the secretion of IL-2 and IFN-y in T cells was decreased, the secretion of IL-4 in T cells was enhanced in CLP group at 24 and 48h (all P<0.01); Compared to CLP group, T cell proliferation was enhanced, the secretion of IL-2 and IFN-y in T cells was up-regulated, the secretion of IL-4 in T cells was declined in CLP+BoxA group at 24 and 48h (all P<0.01).2..4 The role of α7 nAChR in central high mobility group box-1 protein regulate immune function of Treg:(1) In wild C57BL/6 mice, compared to sham group, the expressions of Foxp3 and CTLA-4 in Treg were up-regulated, the inhibition of Treg to T cell proliferation was enhanced, the secretion of TGF-β and IL-10 in Treg were significantly up-regulated in HMGB1 (1μg) group (all P<0.01); In a7 nAChR knock-out C57BL/6 mice, compared to sham group, the expressions of Foxp3 in Treg were up-regulated (P<0.05), the expressions of CTLA-4 in Treg, the inhibition of Treg to T cell proliferation, the secretion of TGF-P and IL-10 in Treg were no remarkable differences in HMGB1 (1μg) group. (2) In wild C57BL/6 mice, compared to CLP group, the expressions of Foxp3 and CTLA-4 in Treg were down-regulated, the inhibition of Treg to T cell proliferation was decreased, the secretion of TGF-β and IL-10 in Treg were down-regulated in CLP+BoxA group (all P<0.01-0.05); In a7 nAChR knock-out C57BL/6 mice, all indicators of Treg mentioned above were no remarkable differences between CLP and CLP+BoxA groups.Conclusions1. The expressions of HMGB1 and cholinergic activity in cortex, hippocampus, striatum and thalamus were significantly enhanced in the septic rats.2. Elevated central HMGB1 can enhance the immune inhibiting effect of Tregs, which appears to be closely related to the pathogenesis of immune dysfunction of T cells and DCs following severe sepsis.3. Antagonism of central HMGB1 can obviously attenuate the immune inhibiting effect of Tregs, and improve immune function of T cells and DCs in sepsis.4. Central high mobility group box-1 protein regulate immune function of Treg through the cholinergic anti-inflammatory pathway. |
PDF Full Text Request