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 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.

High mobility group box-1 release from H2O2-injured hepatocytes due to sirt1 functional inhibition

BACKGROUND High mobility group box-1 (HMGB1), recognized as a representative of damageassociated molecular patterns, is released during cell injury/death, triggering the inflammatory response and ultimately resulting in tissue damage. Dozens of studies have shown that HMGB1 is involved in certain diseases, but the details on how injured hepatocytes release HMGB1 need to be elicited. AIM To reveal HMGB1 release mechanism in hepatocytes undergoing oxidative stress. METHODS C57BL6/J male mice were fed a high-fat diet for 12 wk plus a single binge of ethanol to induce severe steatohepatitis. Hepatocytes treated with H2O2 were used to establish an in vitro model. Serum alanine aminotransferase, liver H2O2 content and catalase activity, lactate dehydrogenase and 8-hydroxy-2- deoxyguanosine content, nicotinamide adenine dinucleotide (NAD+) levels, and Sirtuin 1 (Sirt1) activity were detected by spectrophotometry. HMGB1 release was measured by enzyme linked immunosorbent assay. HMGB1 translocation was observed by immunohistochemistry/immunofluorescence or Western blot. Relative mRNA levels were assayed by qPCR and protein expression was detected by Western blot. Acetylated HMGB1 and poly(ADP-ribose)polymerase 1 (Parp1) were analyzed by Immunoprecipitation. RESULTS When hepatocytes were damaged, HMGB1 translocated from the nucleus to the cytoplasm because of its hyperacetylation and was passively released outside both in vivo and in vitro. After treatment with Sirt1-siRNA or Sirt1 inhibitor (EX527), the hyperacetylated HMGB1 in hepatocytes increased, and Sirt1 activity inhibited by H2O2 could be reversed by Parp1 inhibitor (DIQ). Parp1 and Sirt1 are two NAD+-dependent enzymes which play major roles in the decision of a cell to live or die in the context of stress . We showed that NAD+ depletion attributed to Parp1 activation after DNA damage was caused by oxidative stress in hepatocytes and resulted in Sirt1 activity inhibition. On the contrary, Sirt1 suppressed Parp1 by negatively regulating its gene expression and deacetylation. CONCLUSION The functional inhibition between Parp1 and Sirt1 leads to HMGB1 hyperacetylation, which leads to its translocation from the nucleus to the cytoplasm and finally outside the cell.

Predictive Value of High Mobility Group Box-1 and miR-146b in Septic Shock Patients

In the face of the elevated incidence and mortality rate of septic shock in the ICU,this retrospective study seeks to investigate the indicative and predictive value of high-mobility group box 1(HMGB1)and miR-146b in patients with septic shock.Quantitative RTPCR was employed in this study to quantify the HMGB1 and miR-146b levels in plasma samples obtained from the patient group and healthy controls.The investigation involved the comparison between the two groups and tracking changes in the patient group over time.The finding revealed that upon admission,the patient group exhibited markedly elevated relative expression levels of HMGB1,which subsequently decreased over time.Conversely,the patient group displayed significantly reduced relative expression levels of miR-146b upon admission,which subsequently increased over time compared to the control group.Receiver operating characteristic(ROC)curves showed good predictive value for HMGB1 and miR-146b.The experimental results suggest that HMGB1 and miR-146b serve as valuable and convenient biomarkers for evaluating the severity of septic shock and predicting mortality.Additionally,it is proposed that serum miR-146b may be inducible and potentially exerts a negative regulatory effect on the expression of HMGB1.

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.

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。

木犀草素对脑梗死动物模型大脑皮质HMGB1／TLR4／NF-κB通路的干预

目的观察脑缺血后HMGB1/TLR4/NF-κB的表达变化,探讨木犀草素的干预研究。方法用改良线栓法制作大鼠大脑中动脉闭塞(MCAO)模型。梗死后腹腔内立即注射木犀草素,在梗死后24h采用Western blot和RT-qPCR观察HMGB1/TLR4/NF-κB蛋白和基因表达变化。结果脑梗死后24 h HMGB1/TLR4/NF-κB的表达明显上调(P<0.05)。木犀草素低剂量组下调HMGB1/TLR4/NF-κB的表达不明显(P>0.05)。木犀草素高剂量组下调TLR4/NF-κB的表达明显(P<0.05)。木犀草素可能是通过抑制HMGB1/TLR4/NF-κB的表达起到脑保护作用。结论 HMGB1/TLR4/NF-κB可能是木犀草素的靶点之一,木犀草素可能是通过抑制HMGB1/TLR4/NF-κB的表达发挥其脑保护的作用。

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。

高迁移率族蛋白-1和白细胞介素-6在腰椎ModicⅡ型改变终板组织中的表达及意义

目的探讨高迁移率族蛋白-1(HMGB-1)和白细胞介素-6(IL-6)在腰椎ModicⅡ型改变软骨终板组织中的蛋白表达水平及其临床意义。方法收集因单节段退变性腰椎间盘疾病行椎间融合术的患者45例,其中男19例,女26例,年龄31~69岁,平均(47.22±11.49)岁。根据手术节段有无ModicⅡ型改变,将其分为ModicⅡ型改变组(A组)和无Modic改变组(B组)。对照组(C组)为同时期因腰椎爆裂性骨折行融合手术的4例年轻患者,其中男3例,女1例,年龄17~29岁,平均(23.25±5.32)岁。对收集到的软骨终板组织行HE染色以观察其组织形态学变化,免疫组化染色以检测HMGB-1和IL-6的蛋白表达水平。结果HE染色结果显示,A、B组软骨终板均出现了不同程度的退变,A组较严重,C组未见明显退变。免疫组化染色结果显示,A、B、C组HMGB-1阳性细胞指数分别为51.00±21.20、30.23±16.86、4.93±3.13,吸光度值分别为0.142±0.044、0.101±0.050、0.038±0.008。A、B、C组IL-6阳性细胞指数分别为43.19±14.46、25.83±12.49、5.08±3.22,吸光度值分别为0.177±0.078、0.116±0.081、0.032±0.016。A、B组HMGB-1、IL-6阳性细胞指数和吸光度值均明显高于C组,A组亦明显高于B组,差异有统计学意义(P<0.05),HMGB-1与IL-6的表达水平呈中度正相关关系(P<0.05)。结论软骨终板组织中HMGB-1和IL-6表达水平升高可能是ModicⅡ型改变引起下腰痛的原因之一。

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。

丙泊酚对急性肺损伤大鼠HMGB-1表达的影响

目的探讨丙泊酚的肺保护作用及其可能作用机制。方法采用尾静脉注射内毒素建立急性肺损伤模型,丙泊酚和锌原卟啉同样经过尾静脉给药。将Wistar大鼠按随机数字表法分为实验对照组(C组)、脂多糖组(L组)、脂多糖+丙泊酚组(LP组)。C组尾静脉注射生理盐水;L组尾静脉注射LPS 7.5mg·kg-1;LP组尾静脉注射LPS 7.5mg·kg-1,同时静脉注射丙泊酚30mg·kg-1。给药前及开始后第3、6和12h测定动脉血氧分压(PaO2),实验结束后观察肺湿/干重(W/D)比值、肺组织病理学检查并进行肺损伤轻重程度评分,同时观察各组大鼠肺组织高迁移率族蛋白1(HMGB-1)含量。结果实验前各组PaO2无明显差异,注射LPS后L组PaO2持续下降,和C组相比其PaO2显著降低(P<0.05)。实验结束后L组和C组相比其W/D比值、肺组织病理学检查评分、HO-1及HMGB-1含量显著增加(P<0.05)。而LP组和L组相比其PaO2显著改善,而W/D比值、肺组织病理学检查评分及HMGB-1含量显著降低(P<0.05)。结论丙泊酚具有肺保护作用,且该作用可能与其抑制HMGB-1过度表达作用相关。

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。

丙酮酸乙酯对脓毒症大鼠肠组织保护和HMGB1表达的影响

目的:通过观察丙酮酸乙酯对脓毒症大鼠肠组织HMGB1表达的影响,进一步揭示丙酮酸乙酯治疗脓毒症的分子作用机制。方法:将96只SD大鼠随机分为假手术组、脓毒症组、低剂量丙酮酸乙酯治疗组(LET组)、高剂量丙酮酸乙酯治疗组(ET组),以盲肠结扎穿刺法复制大鼠脓毒症模型。LET组(20 mg/kg)及ET组(40 mg/kg)即刻腹腔内注射EP注射液4 mL,每6 h重复注射一次,直至实验结束,假手术组及脓毒症组在相同时间用相同剂量的生理盐水腹腔内注射。各组大鼠分别于术后2 h、8 h、24 h、48 h 4个时间点随机处死6只大鼠,经腹主动脉取血,用ELISA方法检测血浆TNF-α、IL-6、HMGB1水平。术后24 h,取大鼠末端回肠,用RT-PCR法检测回肠组织HMGB1mRNA表达水平,用免疫组织化学两步方法观察肠组织HMGB1蛋白表达,在光镜下观察大鼠肠组织的病理变化。结果:与假手术组相比,脓毒症组血浆HMGB1在术后8 h、24 h、48 h显著增高,脓毒症组回肠组织HMGB1mRNA及蛋白表达在术后24 h显著增高(P<0.05)。与脓毒症组相比,ET组、LET组血浆HMGB1在术后8 h、24 h、48 h显著降低,ET组、LET组回肠组织HMGB1mRNA及蛋白表达在术后24h显著降低(P<0.05)。结论:脓毒症大鼠血浆HMGB1出现时间晚,作用时间长,提示HMGB1是脓毒症的重要晚期炎症介质。丙酮酸乙酯可抑制脓毒症大鼠血浆及肠组织HMGB1的表达,提示丙酮酸乙酯对脓毒症的治疗机制可能与其直接或间接抑制HMGB1的表达有关。

帕金森病患者血浆HMGB1、TLR4和NF-κB的检测及其临床意义

目的检测帕金森病(Parkinson's disease,PD)患者血浆高迁移率族蛋白1(high mobility group box 1,HMGBl)、Toll样受体4(toll-like receptors-4,TLR4)和核转录因子kappa B(nuclear transcription factor kappa B,NF-κB)表达水平及其与PD病情的相关性。方法选取本院收治并确诊为PD患者60例和健康志愿者40例,采用酶联免疫吸附法分别测定并比较两组患者外周血血浆HMGB1、TLR4及NF-κB表达水平。PD按Hoehn-Yahr分期标准进行临床分期、分型。结果与健康对照组比较,PD组血浆HMGB1、TLR4和NF-κB水平均明显升高,差异具有统计学意义(P <0. 001);Ⅰ期～Ⅳ期PD患者血浆HMGB1、TLR4和NF-κB水平均存在差异(P<0.001),与PD临床分期呈正相关(相关系数分别为0.889、0.785、0.831,P<0.001);上述因子在不同临床分型中未见明显差异(P>0.05)。结论 PD患者血浆HMGB1、TLR4和NF-kB水平高于健康人群,且与PD临床分期呈正相关,血浆HMGB1、TLR4和NF-κB高水平可以作为PD发病和分期的参考依据。

Interferon regulatory factor-1 mediates the release of high mobility group box-1 in endotoxemia in mice

Background The extracellular release of the danger signal high mobility group box-1 （HMGB1） has been implicated in the pathogenesis and outcomes of sepsis. Understanding the mechanisms responsible for HMGB1 release can lead to the identification of targets that may inhibit this process. The transcription factor interferon regulatory factor-1 （IRF-1） is an important mediator of innate immune responses and has been shown to participate in mortality associated with endotoxemia; however, its role in mediating the release of HMGB1 in these settings is unknown. Methods Male IRF-1 knockout （KO） and age matched C57BL/6 wild type （WT） mice were given intraperitoneal （IP） injections of lipopolysaccharide （LPS）. In some experiments, 96 hours survival rates were observed. In other experiments, mice were sacrificed 12 hours after LPS administration and sera were harvested for future analysis. In in vitro study, RAW 264.7 murine monocyte/macrophage-like cells or primary peritoneal macrophage obtained from IRF-1 KO and WT mice were cultured for LPS mediated HMGB1 release analysis. And the mechanism for HMGB1 release was analyzed by immune-precipitation. Results IRF-1 KO mice experienced less mortality, and released less systemic HMGB1 compared to their WT counterparts. Exogenous administration of recombinant HMGB1 to IRF-1 KO mice returned the mortality rate to that seen originally in IRF-1 WT mice. Using cultures of peritoneal macrophages or RAW264.7 cells, in vitro LPS stimulation induced the release of HMGB1 in an IRF-1 dependent manner. And the janus associated kinase （JAK）-IRF-1 signal pathway appeared to participate in the signaling mechanisms of LPS-induced HMGB1 release by mediating acetylation of HMGBI. Conclusion IRF-1 plays a role in LPS induced release of HMGB1 and therefore may serve as a novel target in sepsis~

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.

甘草甜素对脑缺血大鼠的脑保护作用及机制探讨

目的观察梗死后高迁移率族蛋白B1(HMGB1);晚期糖基化终产物(RAGE);Toll样受体4(TLR4);核因子-κB(NF-κB);紧密连接蛋白5(claudin-5)的表达变化,并观察行为学评分、脑水肿及梗死体积,探讨甘草甜素(Gly)对脑缺血的保护机制。方法线栓法制备大鼠大脑中动脉永久性闭塞(pMCAO)模型。Gly进行干预,比较各组之间神经功能缺损评分、患侧脑水肿和脑梗死体积的变化。同时用免疫组化、实时定量PCR和Western blot方法,分别在梗死后24 h观察HMGB1、RAGE、TLR4、NF-κB和claudin-5蛋白表达变化,以及TLR4、NF-κB和claudin-5的基因表达变化。结果 Gly显著改善神经功能缺失,减轻梗死后患侧脑水肿,减小脑梗死体积(P<0.05);Gly能明显降低HMGB1、RAGE、TLR4和NF-κB的表达(P<0.05),升高claudin-5的表达(P<0.05)。结论 Gly对缺血性脑组织发挥保护作用可能是通过下调HMGB1、RAGE、TLR4、NF-κB的表达,升高claudin-5的表达实现的。

Ethyl pyruvate protects against experimental acute-on-chronic liver failure in rats

AIM： To investigate the protective effects of ethyl py- ruvate （EP） on acute-on-chronic liver failure （ACLF） in rats. METHODS： An ACLF model was established in rats, and animals were randomly divided into normal, mod- el and EP treatment groups. The rats in EP treatment group received EP （40 mg/kg） at 3 h, 6 h, 12 h and 24 h after induction of ACLF. Serum endotoxin, high mobility group box-1 （HMGB1）, alanine transaminase （ALT）, tumor necrosis factor-α （TNF-α）, interferon-α （IFN-γ）, interleukin （IL）-10 and IL-18 levels, changes of liver histology and HMGB1 expressions in liver tis- sues were detected at 48 h after induction of ACLF. The effects of EP on the survival of ACLF rats were also observed.RESULTS： Serum levels of endotoxin （0.394 ± 0.066 EU/mL vs 0.086±0.017 EU/mL, P 〈 0.001）, HMGB1 （35.42±10,86 μg/L vs 2.14 ± 0.27 μg/L, P 〈 0.001）, ALT （8415.87 ± 3567.54 IU/L vs 38.64 ± 8.82 IU/L, P 〈 0.001）, TNF-α （190.77 ± 12.34 ng/L vs 124.40 ± 4.12 ng/L, P 〈 0.001）, IFN-γ （715.38 ± 86.03 ng/L vs 398.66 ± 32.91 ng/L, P 〈 0.001）, IL-10 （6.85 ± 0.64 ng/L vs 3.49 ± 0.24 ng/L, P 〈 0.001） and IL-18 （85.19 ±3.49 ng/L vs 55.38 ±1.25 ng/L, P 〈 0.001） were significantly increased, and liver tissues presented se- vere pathological injury in the model group compared with the normal group, Howeverr EP administration significantly improved hepatic histopathology and re- duced the serum levels of endotoxin （0.155±0.045 EU/mL vs 0.394 ± 0.066 EU/mL vs P 〈 0.001） and in- flammatory cytokines （11.13 ± 2.58 μg/L vs 35.42 ± 10.86 μg/L for HMGB1, 3512.86 ± 972.67 IU/L vs 8415.87 ± 3567.54 IU/L for ALT, 128.55 ± 5.76 ng/L vs 190.77 ± 12.34 ng/L for TNF-α 438.16 ± 38.10 ng/L vs 715.38 ± 86.03 ng/L for IFN-γ 3.55 ± 0.36 ng/L vs 6.85 ± 0.64 ng/L for IL-10, and 60.35 ± 1.63 ng/L vs 85.19 ± 3.49 ng/L for IL-18, respectively, P 〈 0.001）, and the levels of HMGB1 in liver tissues re- gardless of treatment time after induction of ACLF. EP treatment at the four time points prolonged the me- dian survival time of ACLF rats （60 h） to 162 h, 120 h, 102 h and 78 h, respectively （χ2 = 41.17, P 〈 0.0001）. CONCLUSION： EP administration can protect against ACLF in rats, and is a potential and novel therapeutic agent for severe liver injury.

Sodium butyrate protects against toxin-induced acute liver failure in rats

BACKGROUND: Acute liver failure(ALF) is a serious clinical syndrome with high mortality. Sodium butyrate has been shown to alleviate organ injury in a wide variety of preclinical models of critical diseases. The aim of this study was to investigate the protective effect of sodium butyrate on ALF in rats.METHODS: All rats were randomly divided into control,model and sodium butyrate treatment groups. Except the control group, the rats were induced ALF animal model by subcutaneous injection of human serum albumin+D- galactosamine+lipopolysaccharide. After induction of ALF,the rats in the treatment group received sodium butyrate(500mg/kg) at 12-hour or 24-hour time point. Fourty-eight hours after ALF induction, the animals were sacrificed and samples were harvested. Serum endotoxin, high mobility group box-1(HMGB1), liver function parameters, tumor necrosis factoralpha(TNF-α) and interferon-gamma(IFN-γ) were measured.The expression of HMGB1 and nuclear factor-kappa B(NF-κB)p65 protein in liver tissue was detected by Western blotting. The histological changes of liver and intestine were examined. The survival duration was also observed.RESULTS: Serum endotoxin, alanine aminotransferase, HMGB1,TNF-α and IFN-γ were significantly increased and the liver histology showed more severe histopathological injury in the model group compared with the control group(P<0.05).Compared to the model group, sodium butyrate treatment significantly improved the histopathological changes in the liver and intestine, reduced serum endotoxin and inflammatory cytokines, suppressed HMGB1 and NF-кB p65 proteins in liver tissue, and prolonged the survival duration regardless of treatment at 12 hours or 24 hours after induction of ALF(P<0.05).CONCLUSIONS: Sodium butyrate protected the liver from toxin-induced ALF in rats. The mechanisms may be due to direct hepatoprotection and decreased intestinal permeability.

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.