Effect of Jianpi Huoxue Decoction(健脾活血方)-containing Serum on Tumor Necrosis Factor-α Secretion and Gene Expression of Endotoxin Receptors in RAW264.7 Cells Induced by Lipopolysaccharide

Objective:To evaluate the effect of Jianpi Huoxue decoction(健脾活血方,JHD)-containing serum on tumor necrosis factor-α(TNF-α) secretion and endotoxin receptor gene expression in RAW264.7 cells induced by lipopolysaccharide(LPS).Methods:The cytotoxicity of blank-control serum and JHD-containing serum at different concentrations were evaluated through the lactate dehydrogenase(LDH) assay in RAW264.7 cells.RAW264.7 cells were divided into six groups:5%blank-control serum group(C1,n=3),5%blank-control serum plus LP...

Effects of endotoxin on endothelin receptor in hepatic and intestinal tissues after endotoxemia in rats

INTRODUCTIONEndothelins(ETs) has a potent and sustainedvasoconstrictive effect on a variety of blood vessels.The vascular smooth muscle cell (VSMC)is thetarget for ETs.VSMC of the whole body containsendothelin receptor (ETR).A great number ofexperiments have shown that three distinctcomplementary DNAs of ETR have been identifiedi.e.,endothelin A receptor(ET_A receptor),endothelin B receptor(ET_B receptor)

Changes of pulmonary α_1-and β-adrenergic receptors after endotoxininduced acute lung injury in rats

Changes of pulmonary α1-and βadrenergic receptors (α1- ARs and β-ARs) after endotoxin-induced lung injury were dynamically observed with radioligand binding assay and the pulmonary rnicrovascular permeability and histopathology were also studied in rats to investigate the relationship between changes of pu1monary ARs and acute lung injury. It was found that the contents of both α1-ARs and β-ARs were significantly decreased after endotoxin-induced acute lung injury. On the basis of these findings, the authors inferred that the down regulation of β-ARs might be one of the factors to increase pulmonary microvascular permeability and that of α1-ARs seems to be a protective reaction. This inference might serve as the theoretical basis to treat pulmonary edema with α1-antagonists and β-agonists clinically.

Changes of pulmonary beta-adrenergic receptors and their relationship with membranous phospholipid metabolism in endotoxin-induced lung injury in rats

The changes of beta-adrenergic receptors (AARs) in lung tissue in endotoxin-induced acute lung injury was investigated with radioligand bindig assay in rats. The lipid fluidity and phospholipid content of the cellular membrane of lung tissue were measured with fluorescent polarization and high performance liquid chromatography respectively. The findings were as follows:1- Four hours after endotoxin injection, there was a 47% decrease of the maximal binding capacity of fyARsas compared with the control.2. Endotoxin was able to decrease the lipid fluidity and phospholipid content of the pulmonary cellular membrane markedly and at the same time. There was an elevated activity of phospholipase A2 in the pulmonary tissueThese findings suggest that the decrease of the binding capacity of &ARs results in a decrease of the PAR mediated functions, which plays a ro1e in the pathogensis of endotoxin-induced acute lung injury and the activation of phospholipase A2 which is an important factor to reduce the phospholipid content of cell membrane and subsequently to decrease its lipid fluidity, can result in a reduction of the lateral diffusion and rotatory movement of β-ARs and to decrease the chances of β-ARs to bind with the ligands.

Correlation between Endotoxin Tolerance in Human Monocyte Leukemia Cell Line THP-1 with Glucocorticoid Receptor-α

Human monoeyte leukemia cell line THP-1 was stimulated with lipopolysaccharide （LPS） to simulate the sepsis model and the expression of human glucocorticoid receptor-α （GR-α） mRNA in montocytes with endotoxin tolerance was investigated. THP-1 cells were cultured in serum-free medium, randomly divided into groups A, B, C, D and E, and stimulated with 0, 10, 10, 100, 0 ng/mL LPS for 24 h followed with 100, 100, 10, 100, 0 ng/mL LPS for another 24 h respectively. The expression of GR-α mRNA was detected by semi-quantitative reverse transcriptional polymerase chain reaction. Tumor necrosis factor-α （TNF-α） was determined by enzyme linked immunosorbent assay （ELISA）. The results showed that the A values of GR-α/β-actin in groups A, B, C, D and E was 0. 607±0. 006, 0. 368±0. 005, 0. 484±0. 008, 0. 509±0. 004 and 0. 564±0. 014 respectively with the difference being significant among the groups （P〈0. 05）. The GR-α mRNA expression was negatively correlated with the TNF-α expression （P〈0. 01）. It was concluded that the down-regulation of the expression of GR-α mRNA in endotoxin tolerance THP-1 cells might play an important role in the development of endotoxin tolerance in THP-1 cells.

多囊卵巢综合征患者血清内毒素、TLR4表达水平及临床意义

目的探究多囊卵巢综合征(PCOS)患者血清内毒素、Toll样受体4(TLR4)的表达水平和临床意义。方法选取2021年3月至12月唐山市妇幼保健院收治的50例PCOS伴有月经紊乱或既往检测排卵提示稀发排卵甚至排卵障碍的患者作为研究组,选取同期同一医院体检的50例非PCOS女性作为对照组。采用酶联免疫吸附试验(ELISA)法检测两组血清内毒素、TLR4水平;绘制受试者工作特征(ROC)曲线分析血清内毒素、TLR4水平对PCOS的诊断价值。结果与对照组相比,研究组PCOS患者体重指数(BMI)、血清内毒素、TLR4、睾酮(T)、黄体生成素(LH)、胰岛素抵抗指数(HOMA-IR)水平显著升高(P<0.05)。血清内毒素、TLR4诊断PCOS的曲线下面积(AUC)优于二者单独诊断(Z_(二者联合-内毒素)=1.992、Z_(二者联合-TLR4)=2.814,P<0.05)。结论PCOS患者血清中内毒素、TLR4水平显著升高,二者联合检测对PCOS有较高的诊断价值。

Toll-like receptor 4 involvement in hepatic ischemia/reperfusion injury in mice

BACKGROUND: Toll-like receptor 4 (TLR4) is involved in innate immunity by recognizing endotoxin resulting in a burst of inflammatory cascade. We investigated the relation between activation of TLR4 and liver injury in partial hepa- tic ischemia/reperfusion (I/R) injury in mice. METHODS: TLR4-deficient mice ( C3H/Hej) and wild type mice (WT, C3H/Heouj) were used in the model of I/R injury. Partial hepatic ischemia was produced by oc- clusion of inflow to the median and left lobes for 45 mi- nutes. Blood was drawn at 1 and 3 hours after reperfusion. The blood was analyzed for aspartate aminotransferase (AST) and tumor necrosis factor alpha (TNF-α). TNF-α mRNA expression and myeloperoxidase (MPO) level in the ischemic lobes were examined by northern blot and myeloperoxidase assay respectively. RESULTS: AST levels were significantly decreased in TLR4- deficient mice compared with WT mice at both time points (WT: 1215.5 ±174. 03, 2958. 17 ± 186. 81 IU/L at 1 and 3 hours respectively vs TLR4def: 661.83±106.09, 1145.17± 132.43 IU/L at 1 and 3 hours, mean ± SD, 6 mice/group, (=-6.65 and -5.57, P <0.001). Consistent with the role of TNF-α in hepatic I/R, serum TNF-α was decreased in TLR4 deficient mice at 3 hours after reperfusion compared with WT (152.39±43.3 vs 249.12 ± 51.89, n=6, t=-3.13, P<0.05). MPO level in the ischemic lobes in TLR4 defi- cient mice at 3 hours after reperfusion was significantly low- er than that in WT mice (0.059±0.004 vs 0.173±0.025, n=6, F=33.49, P<0.001). This difference appears to be mediated at the gene level since TLR4 deficient mice had decreased TNF-α mRNA expression at 1 hour after reperfu- sion compared with WT mice (80.3±28.8 vs 189.4±24.6, t=-3.25, P<0.05). CONCLUSIONS: Compared with WT mice, TLR4-defi- cient mice appear to have a mild I/R injury. Regulation of TNF-a at mRNA level seems to have a critical effect. These suggest TLR4 be involved in the mechanism of he-patic I/R injury in mice.

Effects of anisodamine on pulmonary α_1-adrenergic receptor and phospholipase A_2 in acute lung injury

Objective: To imastigate the effects of anisodamine on pulmonary α1- adrenergic receptor andphospholipase A, in acute lung injurg. Methods: Change of α1--adrenergic receptor (al AR ) in lung tissllesduring endotoxin--induced rat acute lung injury was measured with radioligand biding assay. The effects ofanisodamine on pulmonary α1--AR and phospholipase A2 (PLA2 ) were observed. Results: 1. 4 h after theendotoxin injection, there was a significant decrease in the maximal binding capacity of α1--AR by 34% ascompared with the control group. meanwhile elevated activity of PLA2 in rat lung and reduction of thephospholipids content of cell membrane was found. 2. Anisodamine could attenuate endotoxin--induced acutelung injury in rats. Conclusion: This effect might be related to anisodamine’s blockage of α1--AR andsuppression of PLA2, prevention of membranous phospholipids from degradation. and the reduction ofarachidonic acid release.

sTREM-1、HMGB1和内毒素对胸腔镜肺癌根治术后肺部感染的预测价值

目的研究血清高迁移率族蛋白B1(HMGB1)、可溶性髓系细胞触发受体-1(sTREM-1)、内毒素对胸腔镜肺癌根治术后肺部感染的预测价值。方法采用回顾性分析,观察对象为2020年1月至2022年1月入海南医学院第二附属医院的400例非小细胞肺癌(NSCLC)患者,均接受胸腔镜肺癌根治术治疗,参考术后肺部感染与否分为研究组(肺部感染,36例)与对照组(肺部未感染,364例)。记录肺部感染病原菌分布状况,比较两组患者手术前后HMGB1、sTREM-1、内毒素水平,采用受试者工作特征(ROC)曲线分析术后1 d血清HMGB1、sTREM-1、内毒素对术后肺部感染的预测价值。结果研究组共检出56株病原菌,且革兰阴性菌(76.79%)占比最高。研究组患者手术后1 d的HMGB1、sTREM-1、内毒素水平为(56.08±14.69)μg/L、(20.73±6.54)pg/mL、(0.32±0.18)EU/mL,手术后3 d的HMGB1、sTREM-1、内毒素水平为(76.76±16.57)μg/L、(31.42±6.56)pg/mL、(0.42±0.17)EU/mL,均明显高于对照组[手术后1 d:(56.08±14.69)μg/L、(20.73±6.54)pg/mL、(0.32±0.18)EU/mL;手术后3 d:(35.93±6.51)μg/L、(13.69±2.63)pg/mL、(0.11±0.02)EU/mL],差异均有统计学意义(P<0.05)。ROC曲线显示,以46.88μg/L为截断值,术后1 d HMGB1诊断胸腔镜肺癌根治术后肺部感染的曲线下面积(AUC)为0.875(95%CI:0.773~0.977),敏感度为72.23%,特异度为94.60%;以18.10 pg/mL为截断值,术后1 d sTREM-1诊断胸腔镜肺癌根治术后肺部感染的AUC为0.812(95%CI:0.658~0.966),敏感度为77.79%,特异度为90.55%;以0.23 EU/mL为截断值,术后1 d内毒素诊断胸腔镜肺癌根治术后肺部感染的AUC为0.728(95%CI:0.559~0.896),敏感度为61.00%,特异度为100.00%。结论HMGB1、sTREM-1和内毒素检测在胸腔镜肺癌根治术后肺部感染诊断中有较高应用价值,均可作为预测早期术后肺部感染的诊断指标。

内毒素受体TLR4基因多态性“Asp299Gly”的分型检测

目的 :建立一种用于内毒素受体 TL R4基因多态性“Asp2 99Gly”检测的方法 ,并在中国汉族人中进行该位点的检测。 方法 :以等位基因特异性 PCR原理为基础 ,在两个等位基因特异性引物的 3′端第 3位引入不配对碱基以增加特异性 ,与共同的下游引物分别构成两个 PCR反应体系。采用 PCR定点突变方法得到含和不含“Asp2 99Gly”突变位点的 DNA片段插入质粒作为标准模板 ,用以验证这一方法的鉴别效果 ,并采用 PCR- RFL P分析和测序加以验证。 结果 :该方法用于“Asp2 99Gly”检测的结果 ,与 PCR- RFL P分析的结果及测序的结果完全相符 ,采用我们建立的已知基因型的突变型和野生型重组质粒作模板验证 ,结果也完全相符。采用该方法共检测 92份汉族人群 DNA样本 ,未发现“Asp2 99Gly”的携带者。结论 :以等位基因特异性 PCR原理为基础的检测方法 ,是一种可用于 TL R4基因多态性“Asp2 99Gly”检测的简便、快速、准确、适合于一般实验室的好方法。中国汉族人群携带这一突变的基因频率可能很低。

急性前脑缺血致多器官功能障碍综合征动物模型的建立及内毒素受体CD14的表达

目的 探讨建立大鼠急性前脑缺血致多器官功能障碍综合征模型的可能性,研究脑缺血模型内毒素受体CD14 mRNA在肺、肝、肠和肾组织的表达变化规律与导致多器官功能障碍综合征(MODS)的发生机制。 方法 通过阻断双侧颈总动脉30 min建立急性前脑缺血模型,随机将54只大鼠分为正常对照组(6只)、假手术组(8只大鼠)及缺血后5个亚组(12、24、36、48及72 h组,每组8只大鼠),依据全身炎症反应综合征(SIRS)、MODS的诊断标准判断SIRS、MODS的发生率,采用原位杂交技术检测缺血后肺、肝、肠和肾脏器CD14的基因表达。 结果 (1)缺血后12 h肺、肝、肠和肾组织CD14 mRNA表达升高,24-36 h达高峰,48 h后下降,以肺脏变化最显著(P<0.001);(2)缺血后12、24、36、48及72 h时相点动物肺、肝、肠和肾组织均有不同程度的损害,但以24-48 h时脏器病理变化较显著,其中以肺脏改变为著,与CD14在各器官的基因表达峰值一致;(3)大鼠急性前脑缺血后SIRS发生率为100%,MODS发生率为53.1％;(4)正常对照组和假手术组中肺、肝、肠和肾组织CD14 mRNA均有不同程度的表达,其中两组肺脏CD14 mRNA的表达差异有显著意义(P<0.01)。结论 (1)大鼠急性前脑缺血可成功建立脑缺血致MODS的动物模型;(2)脑缺血致MODS动物模型的肺、肝、肠和肾各脏器CD14

大鼠内毒素性肺损伤中肺α_1和β受体的变化

本研究动态观察大鼠内毒素性肺损伤中肺α１－和β－ＡＲ的变化，以探讨肺ＡＲ变化与急性肺损伤的关系及其机制。结果表明，内毒素诱导大鼠急性肺损伤过程中，肺α１－ＡＲ和β－ＡＲ的Ｂｍａｘ值均明显降低（下调），分别较对照组下降３５和４３％。β－ＡＲ下调可能是导致急性肺损伤的原因之一；α－ＡＲ下调则是一种保护性反应。活性氧在肺ＡＲ下调中起重要作用，而循环血去甲肾上腺素和肾上腺素水平的升高不是其主要因素。

TLR4在创伤后人外周血单核细胞内的表达变化

目的 :研究人体在创伤条件下 ,外周血单核细胞内内毒素复合受体TLR4mRNA的表达变化 ,观察它们与临床预后的关系。方法 :35例创伤患者 (ISS≥ 9分 )和 14例正常对照 ,采用RT -PCR法扩增TLR4 ,以 β肌动蛋白 (β-actin)的表达作为内参照 ,比较TLR4的表达水平 ;采用鲎试剂法测血浆LPS浓度 ;ELISA法检测血浆TNF -α、IL - 10 ,同时观察患者的预后情况。结果 :所观察的创伤患者中 ,血浆LPS浓度均发生不同程度的升高 ;血浆TNF -α、IL - 10升高 ,分别于创伤后第 3天、第 5天达到峰值 ;TLR4mRNA的表达均降低 ,但预后良好的可在伤后第 5天恢复正常水平 ,预后差的在伤后第 5天仍处于低水平。结论 :创伤可引起内毒素血症 ;创伤后人外周血单核细胞内TLR4mRNA的表达与患者的免疫机能、预后情况密切相关。

肝纤维化过程中Toll样受体4在肝脏的表达分布及意义

目的动态观察大鼠肝纤维化过程中Toll样受体4(Toll-like receptor4,TLR4)蛋白在肝脏的表达,探讨TLR4与肝纤维化发生发展的关系。方法以四氯化碳皮下注射复制大鼠肝纤维化模型,设立正常对照组和模型1周组、2周组、4周组、6周组。常规HE染色和天狼猩红胶原染色观察肝脏病变;检测肝组织羟脯氨酸和血浆内毒素含量;免疫组化和Western blot检测TLR4在肝组织中的表达,检测α-SMA观察活化的肝星状细胞(HSCs)。结果与正常对照组比较,CCl4作用2周时,肝组织羟脯氨酸含量开始明显增多(P<0.01);模型组各组血浆内毒素含量呈梯度上升(P<0.01),且与肝组织羟脯氨酸含量呈显著正相关关系(P<0.01);CCl4作用1周后肝组织TLR4的表达即明显增强(P<0.01),4周时和6周时有所下降(与2周组相比,P<0.05),但仍高于正常对照组(P<0.01)。TLR4阳性细胞包括枯否细胞、活化的HSCs及少量的肝细胞和内皮细胞。结论内毒素及其受体TLR4的改变可能在肝纤维化中起重要作用。

严重腹腔感染大鼠组织Toll样受体2/4基因表达及其调节机制

目的 :研究腹腔感染后主要脏器 Toll样受体 (TL R) 2 / 4 m RNA表达的变化规律及组织分布特点 ,并对其诱生机制进行初步探讨。方法 :采用大鼠盲肠结扎穿孔 (CL P)造成严重脓毒症模型。动物分为正常对照组(10只 )、假手术组 (10只 )、CL P组 (6 0只 )及杀菌 /通透性增加蛋白 (BPI)治疗组 (2 0只 )。分别检测肝、肺、肾、小肠组织 TL R2 / 4 m RNA表达及血浆肿瘤坏死因子α(TNFα)和白介素 10 (IL 10 )水平。结果 :CL P后 2 h肝、肺、肾及小肠组织中 TL R2 / 4 m RNA表达开始增高 ,伤后 6～ 12 h各组织 TL R2 / 4 m RNA表达迅速达峰值。 TL R4 m RNA表达于伤后 2 4～ 4 8h开始下降 ,CL P后 72 h趋于伤前范围甚至阴性 ,而 TL R2 m RNA表达则持续增高至 72 h。早期给予 BPI治疗后 ,动物 12～ 2 4 h肝、肺、肾及小肠组织 TL R2 m RNA水平均显著降低(P<0 .0 5或 P<0 .0 1) ,同时 CL P后 12 h各组织 TL R4 m RNA表达亦不同程度下调 (P<0 .0 5或 P<0 .0 1)。BPI治疗组伤后 12 h血浆 TNFα水平显著降低 (P<0 .0 5 ) ,恢复至伤前正常范围 ,但伤后 2 4 h血浆 IL 10水平显著升高 (P<0 .0 1)。结论 :严重腹腔感染可迅速上调体内多器官 TL R2 / 4的基因表达 ,诱导促炎细胞因子产生 ,TL

内毒素对妊娠期糖尿病患者外周血单核细胞TLR4 mRNA及NFκ-B mRNA表达的影响

目的研究妊娠期糖尿病(GDM)患者外周血单核细胞在内毒素(LPS)作用下Toll样受体4(TLR4)mRNA及核因子-κB(NFκ-B)mRNA的表达,探讨GDM的发病机制。方法正常妊娠孕妇20例为对照组,GDM患者20例为GDM组,受试者静脉血分离出单核细胞,用LPS刺激并培养,RT-PCR法检测单核细胞TLR4 mRNA及NFκ-B mRNA的表达,ELISA法检测培养液中白介素-1(IL-1)、IL-10、肿瘤坏死因子-α(TNFα-)水平。结果与对照组相比,GDM组TLR4 mRNA及NFκ-B mRNA表达增加,差异具有统计学意义(P<0.01)。两组TLR4 mRNA与NFκ-B mRNA表达水平存在相关性(rsGDM=0.768,rs对照=0.833,P<0.05)。与对照组比较,GDM组IL-1、IL-10及TNFα-水平升高,差异具有统计学意义(P<0.05)。结论 GDM组孕妇体内TLR4 mRNA、NF-κB mRNA呈高表达,炎症因子IL-1、IL-10、TNFα-升高,提示存在免疫炎症,LPS-TLR4-NFκ-B通路介导了大量炎症因子释放,参与了GDM的发病。

山莨菪碱对肺损伤肺α_1受体及磷脂酶A_2的影响

用放射性配基结合分析法，检测大鼠内毒素性肺损伤中肺α１肾上腺素受体（α１受体）的变化，并观察山莨菪碱对肺α１受体和磷脂酶Ａ２（ＰＬＡ２）的影响。结果表明，（１）ｉｖ内毒素（ＥＴ）后４ｈ，大鼠肺α１受体的最大结合容量明显降低，较对照组减少３４％，同时肺组织ＰＬＡ２激活及膜磷脂含量减少。（２）山莨菪碱能减轻ＥＴ诱导的大鼠急性肺损伤，其作用机制与阻滞α１受体，抑制ＰＬＡ２、防止膜磷脂降解、减少花生四烯酸的释放等有关。

内毒素血症时小鼠肺泡巨噬细胞CD14和清道夫受体的表达

目的 探讨内毒素肺损伤时 ,肺泡巨噬细胞逐步由免疫防御型转变为效应型的分子机制。方法 建立内毒素肺损伤动物模型 ,免疫组化法观察肺泡巨噬细胞CD14及清道夫受体 (SR)表达的变化 ,并辅以计算机图像分析 ;同时检测肺体指数 ,肺组织的病理变化及动物存活率。结果 内毒素对CD14的表达呈时间依赖性升高 ,但加大剂量并未使其进一步增加 ;SR呈时间及剂量依赖性降低。肺泡巨噬细胞SR、CD14的表达变化与小鼠肺损伤程度及存活率呈平行关系。结论 内毒素致小鼠肺损伤过程中 ,肺泡巨噬细胞表面SR表达下调以及CD14表达上调可能是肺泡巨噬细胞由免疫防御细胞转化为致炎效应细胞的机制之一。

大黄对烫伤大鼠肠粘膜上皮细胞肿瘤坏死因子受体基因表达的影响

目的 观察烫伤和内毒素二次打击后肠粘膜上皮细胞表达肿瘤坏死因子受体TNFR1和TNFR2 的情况 ;研究大黄对肠粘膜上皮细胞内TNFR1、TNFR2 基因表达的影响。方法 应用随机、前瞻性研究方法。采用大鼠烫伤和内毒素二次打击模型。应用原位杂交法检测肠粘膜上皮细胞TNFR1和TNFR2 的基因表达情况 ,以及血浆内TNF -α浓度。结果 原位杂交显示烫伤和内毒素二次打击后肠粘膜上皮细胞内TNFR大量表达 ,以TNFR1为主 ,TNFR2 仅小量表达 ,大黄 ( 50mg/kg)治疗后两种受体表达均明显减少。烫伤和内毒素二次打击后血浆内TNF -α浓度明显增高。大黄治疗后TNF -α浓度明显降低 (P<0 0 1)。结论 大黄可抑制病理情况下TNFR1、TNFR2 表达 ,降低血浆内TNF -α水平 ,抑制TNF

急性肺损伤大鼠肺组织β和β_2受体及β_2受体mRNA表达的变化

目的：观察急性肺损伤（ＡＬＩ）大鼠肺组织β、β２受体和β２受体ｍＲＮＡ含量的变化，探讨上述变化与ＡＬＩ的关系及β２受体变化可能的分子机制。方法：静注内毒素复制大鼠ＡＬＩ模型，肺组织β、β２受体及β２受体ｍＲＮＡ含量，分别用放射性配基结合分析法和斑点杂交技术测定。结果：静注内毒素后１、４、６ｈ，大鼠肺组织β和β２受体的最大结合容量（Ｂｍａｘ）均明显低于正常对照组（Ｐ＜０．０１），尤以β２受体为甚；静注内毒素后４及６小时，大鼠肺组织β２受体ｍＲＮＡ含量分别下降至正常对照组的７６４％±１９６％（Ｐ＜０．０１）和５２８％±２０４％（Ｐ＜０．０１）。结论：肺组织β受体数目的减少在ＡＬＩ发生发展中可能起一定的作用，β２受体数目的减少可能与ＡＬＩ关系更密切；β２受体ｍＲＮＡ含量的减少可能是ＡＬＩ后一定阶段肺组织β２受体数目减少的原因之一。