High mobility group box-1 protein inhibits regulatory T cell immune activity in liver failure in patients with chronic hepatitis B

BACKGROUND: Liver failure in chronic hepatitis B (CHB) patients is a severe, life-threatening condition. Intestinal endotoxemia plays a significant role in the progress to liver failure. High mobility group box-1 (HMGB1) protein is involved in the process of endotoxemia. Regulatory T (Treg) cells maintain immune tolerance and contribute to the immunological hyporesponsiveness against HBV infection. However, the roles of HMGB1 and Treg cells in the pathogenesis of liver failure in CHB patients, and whether HMGB1 affects the immune activity of Treg cells are poorly known at present, and so were explored in this study. METHODS: The levels of HMGB1 expression were detected by ELISA, real-time RT-PCR, and Western blotting, and the percentage of CD4(+)CD25(+)CD127(low) Treg cells among CD4(+) cells was detected by flow cytometry in liver failure patients with chronic HBV infection, CHB patients, and healthy controls. Then, CD4(+)CD25(+)CD127(low) Treg cells isolated from the peripheral blood mononuclear cells from CHB patients were stimulated with HMGB1 at different concentrations or at various intervals. The effect of HMGB1 on the immune activity of Treg cells was assessed by a suppression assay of the allogeneic mixed lymphocyte response. The levels of forkhead box P3 (Foxp3) expression in Treg cells treated with HMGB1 were detected by RT-PCR and Western blotting. RESULTS: A higher level of HMGB1 expression and a lower percentage of Treg cells within the population of CIA(+) cells were found in liver failure patients than in CHB patients (82.6+/-20.1 mu g/L vs. 34.2+/-13.7 mu g/L; 4.55+/-1.34% vs. 9.52+/-3.89%, respectively). The immune activity of Treg cells was significantly weakened and the levels of Foxp3 expression were reduced in a dose- or time-dependent manner when Treg cells were stimulated with HMGB1 in vitro. CONCLUSIONS: The high level of HMGB1 and the low percentage of Treg cells play an important role in the pathogenesis of liver failure in patients with chronic HBV infection. Moreover, HMGB1 can weaken the immune activity of Treg cells. It is suggested that effectively inhibiting HMGB1 expression could be a feasible way to treat liver failure by suppressing endotoxemia and enhancing Treg cell activity.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

High mobility group box 1 protein(HMGB1)as an immune-modulating factor for polarization of human T lymphocytes

Objective This study was performed to investigate the effect of high mobility group box-1 protein(HMGB1)on immune function of human T lymphocytes in vitro and explore its potential role in cell-mediated immune dysfunction.Methods Fresh blood was obtained from healthy adult volunteers and peripheral blood mononuclear cells(PBMCs)were isolated,then rhHMGB1 was added to PBMCs.Four-color flow cytometric(FCM)analysis was used for the measurement of intracellular cytokine including interleukin IL-4 and interferon IFN-?ELISA kits were employed for the determination of IL-2 and sIL-2R protein levels in cell culture supernatants.Results(1)Different stimulating time and dosage of rhHMGB1 did not alter the number of IFN-αpositive cells(Th1).rhHMGB1 stimulation provoked a dose-dependent and time-dependent increase in Th2 subset and decrease in ratio of Th1 to Th2.(2)Compared with the untreated cells,when the cells were coincubated with rhHMGB1(10-100ng/ml)for 12 hrs,protein release of IL-2 and sIL-2R were significantly up-regulated.At 48 hrs,in contrast,protein production was relatively lower in cells after exposure to 100-1000 ng/ml rhHMGB1.Conclusions These findings demonstrated that HMGB1 has a dual influence on immune functions of human T lymphocytes.

血必净注射液对创伤性急性肺损伤患者HMGB1水平的影响及其治疗价值

目的探讨血必净注射液对创伤性急性肺损伤患者血清高迁移率族蛋白B1水平的影响。方法将50例创伤性急性肺损伤患者随机分成两组,常规组给予常规治疗,血必净组在常规治疗基础上加用血必净注射液100 mL,Q12 h;治疗前及治疗后7 d,利用酶联免疫吸附法检测外周血HMGB1浓度,同时监测血气分析,记录治疗前后急性生理学与慢性健康状况Ⅱ(acute physiology and chronic health evaluation,APACHE-Ⅱ)评分、pH值及氧合指数。结果两组患者24 h内血清HMGB1浓度较正常健康组显著升高;治疗7 d后,与常规组相比,血必净组血清HMGB1下降更为明显(P<0.05);血必净组较常规组pH值、氧合指数改善更为显著(P<0.05);两组死亡率及ARDS发生率无明显差异(P>0.05)。结论创伤性急性肺损伤患者早期HMGB1即开始明显升高,而血必净注射液治疗后可显著降低其外周血HMGB1水平及改善缺氧状态,具有一定治疗价值。

黏蛋白1、细胞周期调节蛋白p16和高迁移率族蛋白B1在原发性喉癌临床诊断效能分析

目的评估黏蛋白MUCI(Mucin-1)、p16和高迁移率族蛋白BI(HMGB1)在原发性喉癌中诊断.疾病进展及预后的相关性。方法通过免疫组化方法分析原发性喉癌患者临床肿瘤样本中p16和MUCI表达水平,ELISA法检测原发性喉癌患者血清样本中HMGB1的表达水平。Spearman相关分析MUCI,p16和HMGBI表达水平与喉癌分期、分级的相关性。并通过Kaplan-Meier法分析MUCI,p16和HMGBI表达水平与原发性喉癌患者预后的相关性。结果免疫组化结果示MUCI表达水平与原发性喉癌的分期具有显著相关性(r=0.513,P<0.001),p16与原发性喉癌分期有显著相关性(r=00.437,P<0.001)。HMGBI与喉癌患者的临床分期和病理分级有相关性(r=0.523,r=0.671,P均<0.001)。结论MGBI、p16和MUCI的表达水平状态可做为原发性喉癌患者疾病进展的诊断指标,有望成为原发性喉癌诊断与治疗的新生物标志物。

2型糖尿病合并冠心病患者HMGB1与VEGF的相关性研究

目的探讨2型糖尿病合并冠心病患者高迁移率族蛋白B1(HMGB1)与血管内皮细胞生长因子(VEGF)及冠状动脉病变严重程度的相关性,为临床诊断提供参考。方法对该院2014年10月至2015年10月收治的83例糖尿病合并冠心病患者(病变组)和28例糖尿病非合并冠心病患者(对照组)测定HMGB1和VEGF水平,同时进行冠状动脉造影(CAG)检查,以Gensini积分进行统计,比较分析HMGB1、VEGF水平与冠状动脉病变程度的相关性。结果病变组HMGB1水平[(7.61±1.14)ng/mL]明显高于对照组[(3.96±0.67)ng/mL],病变组VEGF水平[(43.16±1.64)pg/mL]明显高于对照组[(23.30±3.19)pg/mL],差异均有统计学意义(P<0.01)。HMGB1在轻度病变组[(5.28±1.62)ng/mL]vs中度[(7.34±1.53)ng/mL]vs重度[(10.68±1.79)ng/mL],VEGF在轻度病变组[(27.96±0.67))pg/mL]vs中度[(38.84±1.21))pg/mL]vs重度[(66.50±1.61))pg/mL],两两比较,差异均有统计学意义(P<0.01)。相关性分析显示,HMGB1及VEGF水平与Gensini积分呈正相关。结论 HMGB1、VEGF水平与冠状动脉粥样病变的程度及范围有关,HMGB1、VEGF水平可以作为预测糖尿病合并冠心病患者病情发展的指标,是病变严重程度的一个预测因素。在无法进行CAG检查时,可提高诊断率,减少漏诊和误诊,值得临床应用。

San-Cao Granule (三草颗粒) Ameliorates Hepatic Fibrosis through High Mobility Group Box-1 Protein/Smad Signaling Pathway

Objective： To investigate the possible mechanism of San-Cao Granule（SCG, 三草颗粒） mediating antiliver fibrosis. Methods： A total of 60 male Sprague-Dawley rats were randomly divided into the normal control group, porcine serum-treated group, ursodesoxycholic acid（UDCA, 60 mg/kg）, SCG（3.6 g/kg） group, SCG（1.8 g/kg） group and SCG（0.9 g/kg） group, with 10 rats in each group. Liver fibrosis was induced with porcine serum by intraperitoneal injection for 8 weeks, except for the normal control group. Then, the rats in the three SCG-treated groups and UDCA group were administered SCG and UDCA respectively for 4 weeks. The serum levels of alanine transaminase（ALT）, aspartate transaminase（AST）, albumin（ALB）, total bilirubin（TBIL）, hyaluronic acid（HA）, laminin（LN）, and type Ⅳcollagen（ⅣC） were examined using commercial kits and hepatic histopathology was examined with hematoxylin and eosin and Masson staining. Moreover, the protein expression levels of high mobility group box-1 protein（HMGB1）, transforming growth factor β1（TGF-β1）, phosphorylated mothers against decapentaplegic homolog 3（p-Smad3）, Smad7, toll-like receptor 4（TLR4）, myeloid differentiation factor 88（My D88）, nuclear factor-kappa B（NF-κB） and α-smooth muscle actin（α-SMA） were determined by western blot, immunohistochemistry and real time quantitativereverse transcription polymerase. Results： Both SCG（3.6 and 1.8 g/kg） and UDCA significantly ameliorated the liver fibrosis induced by porcine serum as indicated by retarding the serum levels increasing of ALT, AST, TBIL, HA, LN and ⅣC and preventing the serum level reducing of ALB compared with the model group（all P〈0.01）. Meanwhile, the collagen deposition was attenuated by SCG and UDCA treatment. Furthermore, SCG markedly reduced the expressions of HMGB1, TGF-β1, p-Smad3, TLR4, My D88, NF-κB and α-SMA, and enhanced the expression of the Smad7 compared with the model group（all P〈0.01）. Conclusion： SCG ameliorates hepatic fibrosis possibly through inhibiting HMGB1, TLR4/NF-κB and TGF-β1/Smad signaling pathway.

去整合素金属蛋白酶10和高迁移率族蛋白B1在声门型喉癌患者中的表达及预后分析

目的探讨去整合素金属蛋白酶10(a disintegrin and metalloprotease 10,ADAM10)和高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1)与声门型喉癌患者病理特征及预后关系分析。方法回顾性收集2017年3月~2020年12月于南京医科大学附属南京医院确诊及治疗的声门型喉癌患者50例(观察组),另取相对喉癌组织切缘0.5cm以上部位标本作为对照组。观察并比较ADAM10和HMGB1在两组中的阳性表达率,分析其阳性表达与声门型喉癌患者的病理特征关系。单因素分析影响声门型喉癌预后的危险因素,Cox多因素回归分析声门型喉癌患者不良预后的独立危险因素。结果ADAM10和HMGB1在观察组的阳性表达率均高于对照组,差异均有统计学意义(P<0.05)。声门型喉癌组织中的ADAM10与淋巴结转移和T分级差异比较有统计学意义,而与年龄、性别、饮酒史、吸烟史、分化程度差异比较无统计学意义(P>0.05);HMGB1与分化程度差异比较有统计学意义(P<0.05),而与年龄、性别、饮酒史、吸烟史、淋巴结转移、T分级差异比较无统计学意义(P>0.05)。单因素分析结果表明,淋巴结转移、T分级、分化程度、ADAM10、HMGB1是患者预后的影响因素。Cox多因素回归分析结果表明,淋巴结转移、T3+T4分级、低分化程度、ADAM10阳性、HMGB1阳性为声门型喉癌患者预后不良的独立影响因素(P<0.05)。结论ADAM10和HMGB1可作为声门型喉癌不良预后的风险评估指标。

Upregulated inflammatory associated factors and blood-retinal barrier changes in the retina of type 2diabetes mellitus model

AIM： To examine the expression of high mobility group box-1（HMGB-1） and intercellular adhesion molecule-1（ICAM-1） in the retina and the hippocampal tissues; and further to evaluate the association of these two molecules with the alterations of blood-retinal barrier（BRB） and blood-brain barrier（BBB） in a rat model of type 2 diabetes.METHODS： The type-2 diabetes mellitus（DM） model was established with a high-fat and high-glucose diet combined with streptozotocin（STZ）. Sixteen weeks after DM induction, morphological changes of retina and hippocampus were observed with hematoxylin-eosin staining, and alternations of BRB and BBB permeability were measured using Evans blue method. Levels of HMGB-1 and ICAM-1 in retina and hippocampus were detected by Western blot. Serum HMGB-1 levels were determined by enzyme-linked immunosorbent assay（ELISA）.RESULTS： A significantly higher serum fasting blood glucose level in DM rats was observed 2wk after STZ injection（P 〈0.01）. The serum levels of fasting insulin,Insulin resistance homeostatic model assessment（IRHOMA）,total cholesterol（TC）, total triglycerides（TG） and low density lipoprotein cholesterol（LDL-C） in the DM rats significantly higher than those in the controls（all P 〈0.01）.HMGB-1（0.96±0.03, P 〈0.01） and ICAM-1（0.76±0.12, P 〈0.05） levels in the retina in the DM rats were significantly higher than those in the controls. HMGB-1（0.83±0.13, P 〈0.01） and ICAM-1（1.15 ±0.08, P 〈0.01） levels in the hippocampal tissues in the DM rats were alsosignificantly higher than those in the controls. Sixteen weeks after induction of DM, the BRB permeability to albumin-bound Evans blue dye in the DM rats was significantly higher than that in the controls（P 〈0.01）.However, there was no difference of BBB permeability between the DM rats and controls. When compared to the controls, hematoxylin and eosin staining showed obvious irregularities in the DM rats.CONCLUSION： BRB permeability increases significantly in rats with type-2 DM, which may be associated with the up-regulated retinal expression of HMGB-1 and ICAM-1.