Effects of ulinastatin on immune function of spleen in severely burned rats and its mechanism / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effects of ulinastatin on immune function of splenic CD4(+) T lymphocytes and CD4(+) CD25(+) regulatory T lymphocytes (Tregs) and content of high mobility group box 1 (HMGB1) in peripheral blood of severely burned rats, and to analyze the possible mechanisms.</p><p><b>METHODS</b>Ninety-six male SD rats were divided into sham injury group, burn group, and ulinastatin group according to the random number table, with 32 rats in each group. Rats in sham injury group were sham injured on the back by immersing in 37 ℃ warm water for 12 s. Rats in burn group and ulinastatin group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back by immersing in 94 ℃ hot water for 12 s. Immediately after injury, rats in each group were intraperitoneally injected with saline (40 mL/kg), meanwhile rats in ulinastatin group were intraperitoneally injected with ulinastatin (4×10(4) U/kg), once per 12 h, till post injury hour 72. Eight rats of each group were respectively selected on post injury day (PID) 1, 3, 5, and 7 to collect abdominal aortic blood samples. Serum content of HMGB1 was detected by enzyme-linked immunosorbent assay (ELISA). And then, rats of the 3 groups were sacrificed immediately to collect spleens and separate CD4(+) CD25(+) Tregs and CD4(+) T lymphocytes. Flow cytometer was used to detect positive expression rates of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead-winged helix transcription factor p3 (Foxp3) in CD4(+) CD25(+) Tregs. Content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs, and content of interleukin 2 (IL-2), IL-4, and γ interferon (IFN-γ) in culture supernatant of CD4(+) T lymphocytes was detected by ELISA. The proliferative activity of CD4(+) T lymphocytes was determined by microplate reader. The sample number of above-mentioned experiments was 8 at each time point in each group. Data were processed with analysis of variance of factorial design and LSD test.</p><p><b>RESULTS</b>(1) Compared with that in sham injury group, serum content of HMGB1 of rats in burn group was significantly increased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, serum content of HMGB1 of rats in ulinastatin group was significantly decreased from PID 1 to 7 (with P values below 0.01). (2) Compared with those in sham injury group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in burn group were significantly increased from PID 1 to 7 (with P values below 0.01), peaking on PID 3 [(65±10)%, (76±10)%, and (28.2±4.4) pg/mL respectively]. These 3 indexes of rats in sham injury group on PID 3 were (45±7)%, (46±7)%, and (11.2±2.3) pg/mL respectively. Compared with those in burn group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in ulinastatin group were significantly decreased from PID 1 to 7 (P<0.05 or P<0.01), reaching the nadir on PID 7 [(43±6)%], PID 1 [(50±8)%], and PID 7 [(12.4±3.4) pg/mL] respectively. These 3 indexes of rats in burn group on PID 7, 1, and 7 were (58±8)%, (71±9)%, and (19.7±2.8) pg/mL respectively. (3) Compared with those in sham injury group, the content of IL-2 and IFN-γ in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased in burn group from PID 1 to 7, with P values below 0.01. Compared with that in burn group, the content of IL-2 and IFN-γ in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased in ulinastatin group from PID 1 to 7, P<0.05 or P<0.01. (4) Compared with that in sham injury group, the proliferative activity of CD4(+) T lymphocytes of rats in burn group was significantly decreased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, the proliferative activity of CD4(+) T lymphocytes of rats in ulinastatin group was significantly increased from PID 1 to 7 (with P values below 0.01).</p><p><b>CONCLUSIONS</b>Ulinastatin can weaken the immunosuppressive function mediated by splenic CD4(+) CD25(+) Tregs in severely burned rats, and improve proliferative function and secretory function of splenic CD4(+) T lymphocytes, which may be attributed to the inhibiting effect of ulinastatin on the release of HMGB1 in large amount.</p>

Effects of ulinastatin on immune function of patients with severe burn injury / 中华烧伤杂志

<p><b>OBJECTIVE</b>To primarily evaluate the effects of ulinastatin on immune function of patients with severe burn injury.</p><p><b>METHODS</b>Forty patients with severe burn admitted to our ward from March 2013 to October 2015, conforming to the study criteria, were divided into conventional treatment group (CT, n=20) and ulinastatin treatment group (UT, n=20) according to the random number table and patient's consent. After admission, patients in group CT received antishock treatment, antibiotic treatment, debridement, skin grafting, and nutrition support, etc. On the basis of the above-mentioned treatment, patients in group UT received intravenous drip of ulinastatin from first day after admission twice a day, with a dosage of 8×10(5) U every time, for 7 days in addition. Peripheral venous blood samples were collected from patients in groups CT and UT on post treatment day (PTD) 1, 3, 5 and 7, respectively. Twenty healthy volunteer were selected as health control group (HC), and peripheral venous blood samples were collected on the first day of the study. Percentage of CD4(+) CD25(+) regulatory T lymphocytes (Tregs) was determined by flow cytometer. The proliferative activity of T lymphocytes was detected by microplate reader (denoted as absorbance value). Content of interleukin 2 (IL-2) in culture supernatant of T lymphocytes, and content of IL-4 and γ interferon (IFN-γ) in serum were detected by enzyme-linked immunosorbent assay. Expression of human leukocyte antigen-DR (HLA-DR) on CD14(+) monocytes was determined by flow cytometer. Data were processed with analysis of variance for repeated measurement, chi-square test, and LSD-t test.</p><p><b>RESULTS</b>(1) Compared with that of volunteer in group HC, the percentage of CD4(+) CD25(+) Tregs of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 13.303 to 26.043, P values below 0.01). Compared with that in group CT, the percentage of CD4(+) CD25(+) Tregs of patients in group UT was significantly decreased on PTD 5 and 7 (with t values respectively 8.317 and 15.071, P values below 0.01). (2) The proliferative activity of T lymphocytes of patients in group CT on PTD 1, 3, 5, and 7 was respectively 0.71±0.11, 0.61±0.15, 0.54±0.12, and 0.67±0.17, which was significantly lower than that in group HC (1.21±0.22, with t values from 8.686 to 11.957, P values below 0.01). The proliferative activity of T lymphocytes of patients in group UT on PTD 3, 5, and 7 were respectively 0.81±0.11, 0.85±0.14, and 1.08±0.13, which was significantly higher than that in group CT (with t values from 4.808 to 8.568, P values below 0.01). (3) Compared with those of volunteer in group HC, content of IL-2 in culture supernatant of T lymphocytes of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 8.073 to 9.288, P values below 0.01), content of IL-4 in serum of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 18.926 to 41.451, P values below 0.01), and content of IFN-γ in serum of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 4.543 to 27.659, P values below 0.01). Compared with those in group CT, content of IL-2 in culture supernatant of T lymphocytes of patients in group UT was significantly increased from PTD 3 to 7 (with t values from 6.507 to 8.869, P values below 0.01), content of IL-4 in serum of patients in group UT was significantly decreased from PTD 3 to 7 (with t values from 6.922 to 8.843, P values below 0.01), and content of IFN-γ in serum of patients in group UT was significantly increased on PTD 5 and 7 (with t values respectively 5.369 and 13.521, P values below 0.01). (4) The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group CT on PTD 1, 3, 5, and 7 were respectively (28±6)%, (25±7)%, (25±7)%, and (39±10)%, which were significantly lower than the percentage of volunteer in group HC [(87±8)%, with t values from 16.323 to 25.645, P values below 0.01]. The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group UT on PTD 3, 5, and 7 were respectively (40±6)%, (42±9)%, and (49±10)%, which were significantly higher than those in group CT (with t values from 3.071 to 7.324, P values below 0.01).</p><p><b>CONCLUSIONS</b>On the basis of CT, additional ulinastatin intervention can decrease CD4(+) CD25(+) Tregs percentage, improve the immune function of T lymphocytes and T helper cells, and increase expression of HLA-DR on CD14(+) monocytes of patients with severe burn injury, thus improve the immune function of patients.</p>

The effect of high mobility group box-1 protein in central nerve system on septic brain injury / 中华急诊医学杂志

Objective To investigate changes in high mobility group box-1 protein ( HMGB1 ) level in brain tissues with severe sepsis, and the relationship between HMGB1 and septic brain injury.Methods Forty wild C57BL/6 mice were randomly ( random number) divided into 4 groups: sham group, sepsis group, cerebroventricular injection control group, and sepsis with BoxA ( HMGB1 inhibitor) cerebroventricular injection group.Septic model was reproduced by cecal ligation and puncture, and the cerebroventricular catheterization model was established by motorized mice brain stereotaxic instruments.After septic challenge, 1 μg BoxA was injected into the ventricle of brain via cerebroventricular catheter immediately.Mice were sacrificed and brains were harvested at 24 h after sepsis, and hippocampus tissue was separated immediately.Expressions of brain HMGB1 and caspase-3 changed in apoptotic neurons and brain injury were determined by brain tissue immunofluorescence, Western blotting, TUNEL and HE staining respectively.One-way analysis of variance ( ANOVA) for analyzing inter-group differences, student t test for comparing difference between two groups . Results (1) HMGB1 expression in hippocampus was significantly enhanced in the septic group compared to the sham group [ (22.74 ±9.29) vs.4.57 ±2.18, P<0.01].(2) Compared to the sham group, neuronal apoptosis [ (35 ±9.17) vs.(1.67 ±1.53) , P<0.01) and caspase-3 expressions [ (16.79 ±8.17) vs.( 3.39 ±2.09), P<0.05] were significantly increased in hippocampus with aggravated brain injury in the septic group.(3) Cerebroventricular injection of BoxA significantly inhibited HMGB1 in hippocampus [ (2.66 ± 2.06) vs.( 22.74 ±9.29), P<0.01];(4) Cerebroventricular injection of BoxA obviously alleviated acute brain injury, and decreased neuronal apoptosis [ ( 12 ±4.36 ) vs.( 35 ±9.17 ) , P <0.01 ] as well as caspase-3 activity [ (4.10 ±2.11) vs.(16.80 ±8.17), P<0.05].Conclusions The elevated expression of brain HMGB1 is closely related to pathogenesis and development of septic brain injury, and treatment with antagonist towards brain HMGB1 can markedly attenuate acute brain injury following severe sepsis.