The aim of this study was to investigate the underlying mechanism of chrysophanol(Chr) in reducing inflammation and foam cell formation induced by oxidized low-density lipoprotein(ox-LDL) and to investigate the targets and pathways related to effects of Chr on coronary atherosclerosis, providing a theoretical basis for the development of new clinical drugs. RAW264.7 macrophages were cultured in vitro, and after determining the appropriate concentrations of Chr and ox-LDL for treating RAW264.7 macrophages using a cell counting kit-8(CCK-8), the macrophages were treated with different concentrations of Chr(10, 15 μmol·L~(-1)) and ox-LDL(with or without 80 mg·mL~(-1)) for 24 h. RAW264.7 macrophages were divided into four groups: control group, model group(80 mg·mL~(-1) ox-LDL), treatment group(80 mg·mL~(-1) ox-LDL+10 μmol·L~(-1) Chr), and treatment group(80 mg·mL~(-1) ox-LDL+15 μmol·L~(-1) Chr). Lipid accumulation in each group was detected by oil red O staining. CD36 expression was analyzed by flow cytometry. Western blot was used to detect the expression of scavenger receptor class A1(SR-A1), scavenger receptor class B type Ⅰ(SR-B1), autophagy-related protein 5(Atg5), Beclin-1, autophagy adaptor protein p62(P62), the ratio of microtubule-associated protein light chain 3(LC3)Ⅱ to LC3Ⅰ(LC3Ⅱ/LC3Ⅰ), nuclear factor kappa B P65(NF-κB P65), inhibitor of κB kinase β(IKKβ), nuclear factor of κB inhibitor(IκB), high mobility group box protein 1(HMGB1), phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), and phosphorylated mammalian target of rapamycin(mTOR). Real-time quantitative polymerase chain reaction(RT-qPCR) was used to detect the mRNA expression levels of ATP-binding cassette transporter A1(ABCA1), ATP-binding cassette transporter G1(ABCG1), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), HMGB1, inducible nitric oxide synthase(iNOS), arginase 1(Arg1), macrophage galactose-type lectin-1(Mgl-1), and NF-κB P65. Immunofluorescence analysis was performed to determine the localization of HMGB1 in RAW264.7 cells in each group. The autophagy inhibitor 3-methyladenine(3-MA) was added as a control for reverse validation, and the RAW264.7 macrophages were divided into four groups again: control group, model group(80 mg·mL~(-1) ox-LDL), treatment group(80 mg·mL~(-1) ox-LDL + 15 μmol·L~(-1) Chr), and inhibitor group(80 mg·mL~(-1) ox-LDL+15 μmol·L~(-1) Chr+3-MA). The results showed that Chr effectively reduced foam cell formation by regulating the expression levels of SR-A1, ABCA1, ABCG1, the LC3Ⅱ/LC3Ⅰ ratio, Atg5, Beclin-1, and p62, and inhibited the NF-κB/HMGB1-PI3K/Akt/mTOR signaling pathway. Moreover, the inhibitory effects of Chr on autophagy and the NF-κB/HMGB1-PI3K/Akt/mTOR pathway were reversed by the autophagy inhibitor 3-MA. In conclusion, Chr exhibits therapeutic potential for the treatment of atherosclerosis by inducing autophagy and modulating the NF-κB/HMGB1 and PI3K/Akt/mTOR pathways to inhibit the formation of macrophage inflammatory foam cells.
Animals ; Lipoproteins, LDL/metabolism* ; Mice ; TOR Serine-Threonine Kinases/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Macrophages/cytology* ; RAW 264.7 Cells ; Proto-Oncogene Proteins c-akt/genetics* ; Signal Transduction/drug effects* ; NF-kappa B/genetics* ; Anthraquinones/pharmacology* ; Foam Cells/cytology* ; HMGB1 Protein/genetics* ; Humans

OBJECTIVE: To assess the effect of tumor cell lysate (TCL) with low high-mobility group B1 (HMGB1) content for enhancing immune responses of dendritic cells (DCs) against lung cancer. METHODS: TCLs with low HMGB1 content (LH-TCL) and normal HMGB1 content (NH-TCL) were prepared using Lewis lung cancer (LLC) cells in which HMGB1 was inhibited with 30 nmol/L glycyrrhizic acid (GA) and using LLC cells without GA treatment, respectively. Cultured mouse DCs were exposed to different doses of NH-TCL and LH-TCL, using PBS as the control. Flow cytometry was used to detect the expressions of CD11b, CD11c and CD86 and apoptosis of the stimulated DCs, and IL-12 levels in the cell cultures were detected by ELISA. Mouse spleen cells were co-cultured with the stimulated DCs, and the activation of the spleen cells was assessed by detecting CD69 expression using flow cytometry; TNF-β production in the spleen cells was detected with ELISA. The spleen cells were then co-cultured with LLC cells at the effector: target ratios of 5:1, 10:1 and 20:1 to observe the tumor cell killing. In the animal experiment, C57/BL6 mouse models bearing subcutaneous LLC xenograft received multiple injections with the stimulated DCs, and the tumor growth was observed. RESULTS: The content of HMGB1 in the TCL prepared using GA-treated LLC cells was significantly reduced (P < 0.01). Compared with NH-TCL, LH-TCL showed a stronger ability to reduce apoptosis (P < 0.001) and promote activation and IL- 12 production in the DCs. Compared with those with NH-TCL stimulation, the DCs stimulated with LH-TCL more effectively induced activation of splenic lymphocytes and enhanced their anti-tumor immunity (P < 0.05). In the cell co-cultures, the spleen lymphocytes activated by LH-TCL-stimulated DCs showed significantly enhanced LLC cell killing activity (P < 0.01). In the tumor-bearing mice, injections of LH-TCL-stimulated DCs effectively activated host anti-tumor immunity and inhibited the growth of the tumor xenografts (P < 0.05). CONCLUSION Stimulation of the DCs with LH-TCL enhances the anti-tumor immune activity of the DCs and improve the efficacy of DCbased immunotherapy for LLC in mice.
Animals ; Humans ; Mice ; Apoptosis ; Dendritic Cells/immunology* ; Glycyrrhizic Acid/pharmacology* ; HMGB1 Protein ; Lung Neoplasms/immunology*

OBJECTIVE: To investigate the protective effect against intestinal mucosal injury in rats following traumatic brain injury (TBI) and explore the underlying mechanism. METHODS: SD rat models of TBI were established by fluid percussion injury (FPI), and the specimens were collected at 12, 24, 48, and 72 h after TBI. Another 15 rats were randomly divided into shamoperated group (n=5), TBI with saline treatment (TBI+NS) group (n=5), and TBI with PD treatment (TBI+PD) group (treated with 30 mg/kg PD after TBI; n=5). Body weight gain and fecal water content of the rats were recorded, and after the treatments, the histopathology of the jejunum was observed, and the levels of D-lactic acid (D-LAC), diamine oxidase (DAO), ZO-1, claudin-5, and reactive oxygen species (ROS) were detected. Lipid peroxide (LPO) and superoxide dismutase (SOD) 2 content, jejunal pro-inflammatory factors (IL-6, IL-1β, and TNF- α), Sirt1 activity, SOD2 and HMGB1 acetylation level were also determined after the treatments. RESULTS: The rats showed significantly decreased body weight and fecal water content and progressively increased serum levels of D-LAC and DAO after TBI (P < 0.05) with obvious jejunal injury, significantly decreased expression levels of ZO-1 and claudin-5, lowered SOD2 and Sirt1 activity (P < 0.05), increased expression levels of LPO, ROS, and pro-inflammatory cytokines, and enhanced SOD2 and HMGB1 acetylation levels (P < 0.05). Compared with TBI+NS group, the rats in TBI+PD group showed obvious body weight regain, increased fecal water content, reduced jejunal pathologies, decreased D-LAC and DAO levels (P < 0.05), increased ZO-1, claudin-5, SOD2 expression levels and Sirt1 activity, and significantly decreased ROS, LPO, pro-inflammatory cytokines, and acetylation levels of SOD2 and HMGB1 (P < 0.05). CONCLUSION PD alleviates oxidative stress and inflammatory response by activating Sirt1-mediated deacetylation of SOD2 and HMGB1 to improve intestinal mucosal injury in TBI rats.
Animals ; Brain Injuries, Traumatic ; Glucosides/pharmacology* ; HMGB1 Protein/metabolism* ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Stilbenes/pharmacology* ; Superoxide Dismutase/metabolism*

Objective To investigate the effect and mechanism of isorhapontigenin (ISO) in the protection of mice from the lipopolysaccharide (LPS)-induced acute lung injury (ALI). Methods RAW264.7 cells were cultured in vitro with different concentrations of ISO and the viability of the cells was measured by CCK-8 assay.Further,RAW264.7 cells were induced with 200 ng/ml LPS and then treated with ISO and the autophagy inhibitor 3-methyladenine (3-MA).Western blotting was employed to determine the expression of inflammatory cytokines [interleukin (IL)-1β,IL-6,tumor necrosis factor-α (TNF-α),P65,phospho-P56 (p-P65),IκB,phospho-IκB (p-IκB),inducible nitric oxide synthase (iNOS),cyclooxygenase-2 (COX-2),and high mobility group box-1 (HMGB1)] and autophagy markers (LC3Ⅱ/Ⅰ,Beclin1,and P62).The reactive oxygen species (ROS) production of the cells was measured with the DCFH-DA probe.The mouse model of ALI was established by intraperitoneal injection of LPS (15 mg/kg).The pathological changes of the lung tissue were observed via HE staining.The expression of inflammatory cytokines and autophagy markers in the lung tissue was determined by Western blotting and the content of ROS in bronchoalveolar lavage fluid (BALF) by flow cytometry. Results ISO down-regulated the expression of IL-1β,IL-6,TNF-α,iNOS,COX-2,and HMGB1 and inhibited the ROS production in the LPS-induced RAW264.7 cells (all P<0.05).Furthermore,it promoted the expression of LC3Ⅱ/Ⅰ and Beclin1 and inhibited the expression of P62,thereby activating autophagy (all P<0.05).However,the addition of 3-MA up-regulated the expression of p-P65/P65,p-IκB,iNOS,COX-2,and HMGB1,down-regulated that of IκB (all P<0.001),and promote the production of ROS.ISO mitigated the pathological changes in the lung tissue of ALI mice.It down-regulated the expression of p-P65/P65,p-IκB,iNOS,COX-2,and HMGB1 and up-regulated that of IκB in the lung tissue (all P<0.001) and decreased the ROS production in BALF.However,such protective effect was reversed by 3-MA. Conclusion ISO may induce autophagy of macrophages to protect mice from LPS-induced ALI.
Animals ; Mice ; Acute Lung Injury/pathology* ; Beclin-1/pharmacology* ; Cyclooxygenase 2/metabolism* ; Cytokines ; HMGB1 Protein/metabolism* ; Interleukin-6 ; Lipopolysaccharides ; Lung/pathology* ; NF-kappa B/metabolism* ; Reactive Oxygen Species/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

The study aimed to explore the mechanism and targets of Shenfu Injection in the regulation of inflammatory injury in chronic heart failure rats based on the high mobility group box-1/Toll like receptor 4/nuclear factor kappa-B(HMGB1/TLR4/NF-κB) signaling pathway. The rat model of chronic heart failure was established using isoproterenol. The modeled rats were divided into three groups by random number table: the model group, Shenfu group and glycopyrrolate group, and the normal group was also set. The rats were administrated for 15 consecutive days, and on the following day after the last administration, they were sacrificed for sample collection. The cardiac mass index and left ventricular mass index of the rats in each group were measured, and the echocardiogram was used to analyze the cardiac function indices, and ELISA to test the inflammatory indices in rat serum. The pathological morphology and fibrosis status of rat heart tissues were observed by HE staining and Masson staining, respectively. The content of HMGB1 was determined by immunofluorescence staining. The protein and mRNA expression of HMGB1/TLR4/TLR4 signaling pathway was detected by Western blot and RT-qPCR, respectively. The results showed that the chronic heart failure rat model was successfully prepared. The rats in the model group had reduced cardiac function, increased levels of HMGB1 and inflammatory factors(P<0.05), and elevated protein and mRNA expression of HMGB1, TLR4, MyD88, and NF-κB P65 in myocardial tissue(P<0.05), with fibrous connective tissue hyperplasia, inflammatory cell infiltration and severe fibrosis. Shenfu Injection improved cardiac function, decreased the levels of HMGB1 and inflammatory factors(P<0.05) and the protein and mRNA expression of HMGB1, TLR4, MyD88, and NF-κB P65 in myocardial tissue(P<0.05), ameliorated interstitial fibrous connective tissue hyperplasia and inflammatory cell infiltration, and reduced fibrosis. In conclusion, Shenfu Injection can reduce inflammatory damage and improve cardiac function in chronic heart failure rats by regulating the HMGB1/TLR4/NF-κB signaling pathway.
Rats ; Animals ; NF-kappa B/metabolism* ; HMGB1 Protein/pharmacology* ; Toll-Like Receptor 4/metabolism* ; Myeloid Differentiation Factor 88/metabolism* ; Hyperplasia ; Rats, Sprague-Dawley ; Signal Transduction ; RNA, Messenger ; Heart Failure/genetics* ; Fibrosis

The aim of this study was to investigate the effects of dexmedetomidine (Dex) on hepatic ischemia/reperfusion injury (HIRI) and the underlying mechanism. The in vitro HIRI was induced by culturing HL-7702 cells, a human hepatocyte cell line, under 24 h of hypoxia and 12 h of reoxygenation. Quantitative real time PCR (qRT-PCR) and Western blot were performed to detect the expression levels of long non-coding RNA MALAT1, microRNA-126-5p (miR-126-5p) and high mobility group box-1 (HMGB1). Bioinformatics prediction and double luciferase assay were used to verify the targeting relationship between miR-126-5p and MALAT1, HMGB1. Reactive oxygen species (ROS), malondialdehyde (MDA) and ATP levels in culture medium were detected by corresponding kits. The results showed that Dex significantly reduced the levels of ROS and MDA, but increased the level of ATP in HL-7702 cells with HIRI. HIRI up-regulated the expression levels of MALAT1 and HMGB1, and down-regulated the level of miR-126-5p. Dex reversed these effects of HIRI. Furthermore, Dex inhibited HIRI-induced cellular apoptosis, whereas MALAT1 reversed the effect of Dex. This inhibitory effect of Dex could be restored by up-regulation of miR-126-5p. The results suggest that Dex protects hepatocytes from HIRI via regulating MALAT1/miR-126-5p/HMGB1 axis.
Dexmedetomidine/pharmacology* ; HMGB1 Protein/genetics* ; Humans ; MicroRNAs/genetics* ; RNA, Long Noncoding/genetics* ; Reperfusion Injury/genetics*

Objective: To investigate the effect of silencing the high-mobility group box-1 protein (HMGB1) combined with docetaxel (DTX) on the proliferation and apoptosis of PCa cells and its possible action mechanism. METHODS: The expression of HMGB1 mRNA in different PCa cell lines and normal prostatic epithelial cells was detected by RT-qPCR. The PC-3 cells were transfected with different HMGB1 small interfering RNAs (si-HMGB1, si-HMGB1-2 and si-HMGB1-3), and the silencing effect was detected. The effects of different concentrations of DTX on the proliferation of the PC-3 cells was determined by MTT. Then the PC-3 cells were randomly divided into five groups: control (conventional culture), si-HMGB1-NC (si-HMGB1-NC transfection), si-HMGB1 (si-HMGB1-3 transfection), DTX (20 nmol/L DTX), and si-HMGB1+DTX (si-HMGB1-3+20 nmol/L DTX transfection), followed by measurement of the survival rate of the cells by MTT, their apoptosis rate by flow cytometry, and the expressions of HMGB1, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) proteins in different groups by Western blot. RESULTS: The expression of HMGB1 mRNA in the PC-3 cells was the highest and the lowest after transfection with si-HMGB1-3. DTX inhibited the proliferation of the PC-3 cells at various concentrations. Compared with the control group, the si-HMGB1 and DTX groups showed significantly decreased A values, cell survival rates and HMGB1 and Bcl-2 expressions, but increased cell apoptosis rates and Bax expressions (P < 0.05). In comparison with the si-HMGB1 and DTX groups, the si-HMGB1+DTX group exhibited a remarkably decreased A value, cell survival rate and Bcl-2 expression, but increased cell apoptosis and Bax expression. The expression of the HMGB1 protein was markedly lower in the si-HMGB1+DTX than in the DTX group (P < 0.05). CONCLUSIONS Silencing HMGB1 combined with DTX chemotherapy can inhibit the proliferation and promote the apoptosis of PCa cells, which may be attributed to its regulatory effect on the expressions of the Bcl-2 family-related proteins.、.
Apoptosis ; Cell Proliferation ; Docetaxel/pharmacology* ; HMGB1 Protein/genetics* ; Humans ; Male ; Prostatic Neoplasms/genetics*

Carbon monoxide (CO), as a vital small molecule in signaling pathways, is found to be involved in ischemia-reperfusion injury (IRI) in renal transplantation. CO-releasing molecule-2 (CORM-2), a CO-releasing molecule, is a type of metal carbonyl complexes which can quickly release CO in vivo. In this study, an in vitro oxidative stress injury model was established to examine the effect of CORM-2 pretreatment on the nuclear-cytoplasmic translocation of high mobility group box 1 protein (HMGB1) in mouse primary renal proximal tubular epithelial cells (RPTECs). Immunofluorescence staining showed that HMGB1 in the medium- and CORM-2-treated groups was predominantly localized in the nucleus of the cells, whereas higher amounts of HMGB1 translocated to the cytoplasm in the HO- and inactive CORM-2 (iCORM-2)-treated groups. Western blotting of HMGB1 showed that the total amounts of cytoplasmic HMGB1 in the HO-treated (0.59±0.27) and iCORM-2-treated (0.57±0.22) groups were markedly higher than those in the medium-treated (0.19±0.05) and CORM-2-treated (0.21±0.10) groups (P<0.05). Co-immunoprecipitation showed that the levels of acetylated HMGB1 in the HO-treated (642.98±57.25) and iCORM-2-treated (342.11±131.25) groups were markedly increased as compared with the medium-treated (78.72±74.17) and CORM-2-treated (71.42±53.35) groups (P<0.05), and no significant difference was observed between the medium-treated and CORM-2-treated groups (P>0.05). In conclusion, our study demonstrated that in the in vitro oxidative stress injury model of primary RPTECs, CORM-2 can significantly inhibit the nuclear-cytoplasmic translocation of HMGB1, which is probably associated with the prevention of HMGB1 acetylation.
Active Transport, Cell Nucleus ; drug effects ; Animals ; Carbon Monoxide ; pharmacology ; Cell Nucleus ; metabolism ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; metabolism ; Kidney Tubules ; cytology ; Mice ; Organometallic Compounds ; pharmacology ; Oxidative Stress

OBJECTIVETo investigate the effect of Liuwei Wuling tablets on the cytoplasmic translocation and release of high-mobility group box-1 (HMGB1) in hepatocytes in mice with acute live injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS).

METHODSA Balb/c mouse model of acute liver injury was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (5 ug/kg). A total of 24 healthy mice were randomly and equally divided into acute liver injury control group and Liuwei Wuling tablet treatment group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in both groups at each time point within one week. Liver tissues were collected at 36 hours to perform pathological examination. The serum levels of HMGB1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), complement 3a (C3a), and complement 5a (C5a) were measured. Immunohistochemistry was used to determine the expression and cytoplasmic translocation of HMGB1 in hepatocytes.

RESULTSAt 6, 12, and 24 hours, the Liuwei Wuling tablet treatment group had significantly lower serum levels of ALT than the control group (225.33±181.64 U/L vs 471.17±174.72 U/L, t = 3.38, P < 0.01; 1509.53±182.51 U/L vs 7149.52±734.25 U/L, t = 25.82, P < 0.01; 162.89±86.51 U/L vs 1318.16±557.71 U/L, t = 7.09, P < 0.01), as well as significantly lower serum levels of AST than the control group (179.22±94.57 U/L vs 561.91±209.6 U/L, t = 5.76, P < 0.01; 590.92±190.92 U/L vs 2266.48±705.64 U/L, t = 7.94, P < 0.01; 231.24±87.7 U/L vs 444.32±117.01 U/L, t = 5.05, P < 0.01). The treatment group had significantly lower levels of HMGB1 than the control group at 6 and 12 hours (54.21±11.89 ng/ml vs 72.07±13.65 ng/ml, t = 3.41, P < 0.01; 49.23±5.97 ng/ml vs 68.71±13.07 ng/ml, t = 4.70, P < 0.01). The treatment group had significantly lower levels of TNF-α, IL-1β, and IL-6 than the control group at 12 hours (163.62±9.12 pg/ml vs 237.09±51.47 pg/ml, t = 4.86, P < 0.01; 15.66±13.13 pg/ml vs 37.43±18.68 pg/ml, t = 3.30, P < 0.01; 7.10±3.06 pg/ml vs 21.42±8.23 pg/ml, t = 5.65, P < 0.01). The treatment group had significantly lower levels of C3a and C5a than the control group at 12 hours (2.52±1.27 pg/ml vs 9.83±2.96 ng/ml, t = 7.86, P < 0.01; 2.16±1.03 ng/ml vs 7.23±1.55 ng/ml, t = 9.67, P < 0.01). Compared with the control group, the treatment group had significantly reduced liver inflammation and necrosis, and a significantly lower cytoplasmic translocation rate of HMGB1 in hepatocytes (38.76%±7.37% vs 8.15%±2.11%, P < 0.01).

CONCLUSIONLiuwei Wuling tablets can reduce the cytoplasmic translocation of HMGB1 in hepatocytes and relieve liver inflammation in mice with acute liver injury.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Complement C3a ; analysis ; Complement C5a ; analysis ; Cytoplasm ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Galactosamine ; HMGB1 Protein ; metabolism ; Hepatocytes ; drug effects ; Interleukin-1beta ; blood ; Interleukin-6 ; blood ; Lipopolysaccharides ; Liver Failure, Acute ; drug therapy ; Mice ; Mice, Inbred BALB C ; Protein Transport ; Tablets ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo 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.

METHODSNinety-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.

RESULTS(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).

CONCLUSIONSUlinastatin 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.

Animals ; Burns ; drug therapy ; CTLA-4 Antigen ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Glycoproteins ; pharmacology ; HMGB1 Protein ; blood ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spleen ; drug effects ; T-Lymphocytes, Regulatory ; cytology ; drug effects