Acupuncture stimulation of ST-36 (Zusanli) significantly mitigates acute lung injury in lipopolysaccharide-stimulated rats

BACKGROUND: We sought to investigate the potential therapeutic effects of acupuncture stimulation of ST-36 (Zusanli) on endotoxemia-induced acute lung injury in lipopolysaccharide (LPS)-stimulated rats. METHODS: Sixty rats were randomized into six groups (n = 10): (i) lipopolysaccharide (LPS) control group, (ii) normal saline (N/S) control group, (iii) LPS plus ST-36 group, (iv) N/S plus ST-36 group, (v) LPS plus sham point (Sham) group, and (vi) N/S plus Sham group. Manual acupuncture stimulation of ST-36 (designated as 'ST-36') or a 'non-acupoint' (designated as 'Sham') was performed in lightly immobilized rats for 30 min. Then, LPS injection was employed to induce sepsis. Rats were killed at 6 h after LPS injection and lung injury, nitric oxide (NO) biosynthesis and inducible NO synthase (iNOS) expression were assayed. RESULTS: Significant lung injury, pulmonary iNOS expression and systemic and pulmonary NO biosynthesis were noted in the LPS groups. Rats in the LPS plus Sham group had lung injury, pulmonary iNOS expression, systemic and pulmonary NO biosynthesis similar to those observed in the LPS group. However, the degree of lung injury, pulmonary iNOS expression and pulmonary NO biosynthesis, but not systemic NO biosynthesis, were significantly attenuated in the LPS plus ST-36 group as compared with those in both the LPS group and the LPS plus Sham group. CONCLUSION: Acupuncture stimulation of ST-36 may be effective as a prophylaxis measure against sepsis. However, results from this study do not support the use of acupuncture for the treatment of sepsis.     

杀菌性/通透性增加蛋白模拟肽对内毒素/脂多糖致小鼠急性肺损伤的保护作用

目的　观察杀菌性/通透性增加蛋白模拟肽(BNEP)对内毒素/脂多糖(LPS)致小鼠急性肺损伤(ALI)的作用。　方法　将BALB/c小鼠随机分为对照组、LPS组、BNEP组,每组20只。LPS组和BNEP组分别经鼻滴注等渗盐水(NS) LPS和NS LPS 尾静脉注射BNEP复制小鼠ALI模型。对照组处理方式类似,但仅滴注NS.检测各组小鼠肺脏湿干重比、肺血管通透性、肺组织病理学变化,应用免疫组织化学法检测肺组织中Toll样受体(TLR) 2、4表达水平的变化。　结果　BNEP组与LPS组比较,肺湿干重比减小,肺血管通透性降低,肺内以中性粒细胞为主的炎性细胞浸润减轻, TLR2、4在肺组织中的表达减弱(两组TLR2分别为128±10、214±12,P<0. 01).　结论　BNEP对由LPS引起的小鼠ALI有较好的保护作用。     

NF-kappaB involvement in the induction of high affinity CAT-2 in lipopolysaccharide-stimulated rat lungs

BACKGROUND: Endotoxemia stimulates nitric oxide (NO) biosynthesis through induction of inducible NO synthase (iNOS). Cellular uptake of L-arginine, the sole substrate for iNOS, is an important mechanism regulating NO biosynthesis by iNOS. The isozymes of type-2 cationic amino acid transporters, including CAT-2, CAT-2A, and CAT-2B, constitute the most important pathways responsible for trans-membrane L-arginine transportation. Therefore, regulation of CAT-2 isozymes expression may constitute one of the downstream regulatory pathways that control iNOS activity. We investigated the time course of enzyme induction and the role of nuclear factor-kappaB (NF-kappaB) in CAT-2 isozymes expression in lipopolysaccharide-(LPS) treated rat lungs. METHODS: Adult male Sprague-Dawley rats were randomly given intravenous injections of normal saline (N/S), LPS, LPS plus NF-kappaB inhibitor pre-treatment (PDTC, dexamethasone, or salicylate), or an NF-kappaB inhibitor alone. The rats were sacrificed at different times after injection and enzyme expression and lung injury were examined. Pulmonary and systemic NO production were also measured. RESULTS: LPS co-induced iNOS, CAT-2, and CAT-2B but not CAT-2A expression in the lungs. Furthermore, NF-kappaB actively participated in LPS-induction of iNOS, CAT-2, and CAT-2B. LPS induced pulmonary and systemic NO overproduction and resulted in lung injuries. Attenuation of LPS-induced iNOS, CAT-2, and CAT-2B induction significantly inhibited NO biosynthesis and lessened lung injury. CONCLUSION: NF-kappaB actively participates in the induction of CAT-2 and CAT-2B in intact animals. Our data further support the idea that CAT-2 and CAT-2B are crucial in regulating iNOS activity.     

Effects of human cathelicidin antimicrobial peptide LL-37 on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro

BACKGROUND: Lipopolysaccharides (LPS), released by Gram-negative bacteria, cause vascular expression of inducible nitric oxide synthase (iNOS) leading to nitric oxide (NO) production and septic shock. Human cathelicidin antimicrobial peptide (LL-37) can bind and neutralize LPS. We wanted to study whether LL-37 affects LPS or interleukin-1beta (IL-1beta)-induced production, release and function of NO in intact rat aorta rings and cultured rat aorta smooth muscle cells. METHODS: Isolated segments of thoracic aorta and cultured cells were incubated in the presence of LPS, LL-37, LPS + IL-37, IL-1beta, IL-1beta + IL-37 or in medium alone. Smooth muscle contraction in response to phenylephrine and accumulation of the sdegradation products of NO, nitrate and nitrite, were measured on aorta segments. Levels of iNOS were assessed by Western blot and cytotoxic effects were detected by measurement of DNA fragmentation in cultured cells. Number of viable cells were determined after Trypan blue treatment. RESULTS: Both LPS and IL-1beta reduced contractility in response to phenylephrine and increased NO production as well as iNOS expression. LL-37 inhibited the LPS depression of vascular contractility induced only by LPS. LL-37 reduced both the LPS- and IL-1beta-induced NO production and iNOS expression. LL-37 at high concentrations induced DNA fragmentation and decreased the number of living cells. CONCLUSION: IL-37 reduces NO production induced by LPS and IL-1beta. The reduction does not seem to result only from neutralization of LPS but also from a cytotoxic effect, possibly via induction of apoptosis.     

Enhancement of acute lung injury related to bacterial endotoxin by components of diesel exhaust particles

BACKGROUND: Diesel exhaust particles (DEP) synergistically aggravate acute lung injury related to lipopolysaccharide (LPS) in mice, but the components in DEP responsible for this have not been identified. A study was undertaken to examine the effects of the organic chemicals (DEP-OC) and residual carbonaceous nuclei (washed DEP) derived from DEP on LPS related lung injury. METHODS: ICR mice were divided into experimental groups and vehicle, LPS, washed DEP, DEP-OC, washed DEP+LPS, and DEP-OC+LPS were administered intratracheally. The cellular profile of the bronchoalveolar lavage (BAL) fluid, pulmonary oedema, lung histology, and expression of proinflammatory molecules and Toll-like receptors in the lung were evaluated. RESULTS: Both DEP-OC and washed DEP enhanced the infiltration of neutrophils into BAL fluid in the presence of LPS. Washed DEP combined with LPS synergistically exacerbated pulmonary oedema and induced alveolar haemorrhage, which was concomitant with the enhanced lung expression of interleukin-1beta, macrophage inflammatory protein-1alpha, macrophage chemoattractant protein-1, and keratinocyte chemoattractant, whereas DEP-OC combined with LPS did not. Gene expression of Toll-like receptors 2 and 4 was increased by combined treatment with washed DEP and LPS. The enhancement effects of washed DEP on LPS related changes were comparable to those of whole DEP. CONCLUSIONS: These results suggest that the residual carbonaceous nuclei of DEP rather than the extracted organic chemicals predominantly contribute to the aggravation of LPS related lung injury. This may be mediated through the expression of proinflammatory cytokines, chemokines, and Toll-like receptors.     

Human renal epithelial cells express iNOS in response to cytokines but not bacteria

BACKGROUND: Epithelial cells form the mucosal barriers that prevent the entry of mucosal pathogens, and respond to bacterial infections by producing various host defense molecules. In this study, we examined the inducible nitric oxide synthase (iNOS) response of primary human renal tubular epithelial cells (HRTEC) following infection with uropathogenic Escherichia coli Hu734, or stimulation with lipopolysaccharide (LPS) or cytokines. METHODS: Induction of iNOS was examined by RT-PCR, Western blot, immunohistochemistry and nitrite measurements. The effects of endogenously produced nitric oxide (NO), and exogenously applied DETA/NO, SIN-1 and H2O2 on cell viability were analyzed using a respiration assay. RESULTS: HRTEC did not produce NO following infection with E. coli Hu734, LPS alone, or in combination with interferon-gamma (IFN-gamma), even though these agents caused a marked increase in iNOS expression by RAW 264.7, a macrophage cell line. In contrast, iNOS protein and mRNA expression by HRTEC increased after exposure to a cytokine mixture consisting of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. This was due to the combination of IL-1beta and IFN-gamma, but the individual cytokines had no effect. Inducible NOS-expressing cell cultures showed reduced viability, and this effect was inhibited with the NOS inhibitor L-NMMA in RAW 264.7 cells, but not in HRTEC. HRTEC were more sensitive to oxidative stress induced by H2O2 than to nitrogen stress induced by DETA/NO. CONCLUSIONS: We conclude that uropathogenic E. coli that attach to HRTEC fail to directly activate iNOS expression, and that iNOS expression during bacterial infection is more likely to result from stimulation by local cytokines such as IL-1beta and IFN-gamma.     

Differential expression of inducible nitric oxide synthase in septic shock

During cardiopulmonary bypass (CPB), the septic patient has markedly decreased peripheral vascular resistance as a consequence of endotoxin release from microorganisms. This decrease in peripheral vascular resistance is the result of endotoxin-induced nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). iNOS and eNOS are responsible for the synthesis of NO because of various stimuli, including the bacterial endotoxin, lipopolysaccharide (LPS). We tested the hypothesis that a differential expression of iNOS among human endothelial cells and murine macrophage is dependent upon exposure to endotoxin and various pro-inflammatory cytokines. Using a human endothelial cell line, ECV-304 and murine macrophage cell line, RAW 264.7, we quantified the expression of iNOS with specific FITC-conjugated antibodies using fluorescence activated cell sorter (FACS) and NO production with a Bioxytech nitric oxide spectrophotometric assay. This in vitro septic model utilized LPS supported with species-specific interferon-gamma, interleukin-1 beta, and tumor necrosis factor-alpha. The cell type were stimulated for 8 hours with combinations of the cytokines mentioned. The FACS data demonstrated a significant stimulus-dependent increase in iNOS expression among the macrophage groups; however, the stimulated endothelial cells showed no significant change in iNOS expression. The nitric oxide production data demonstrated significant increases in NO production among macrophage stimulated groups; whereas, endothelial stimulated groups exhibit no significant change. We conclude that NO secreted during septic shock is the result of activated macrophage, not the endothelium. The clinical relevance is that the more severe the infectious process, the lower the PVR may be during CPB because of increased NO production from activated macrophage.     

Propofol has anti‐inflammatory effects on alveolar type II epithelial cells

Background: We investigated whether lipopolysaccharide (LPS) induced inflammation in alveolar epithelial type II (ATII) cells is through cluster of differentiation 14 (CD14) and Toll‐like receptor 4 (TLR4) and the effect of different dosages of propofol on the inflammation in primary cultured rat ATII cells. Methods: Cultured ATII cells were randomly assigned to one of the following five groups: Group C: untreated group (control) cultured in the absence of propofol and LPS; Group LPS: treated with 1 μg/ml LPS; Group P1: treated with 1 μg/ml LPS and 25 μM propofol; Group P2: treated with 1 μg/ml LPS and 50 μM propofol; Group P3: treated with 1 μg/ml LPS and 100 μM propofol. ATII cells in all groups were cultured at 37 °C for 3 h. CD14 and TLR4 mRNA was detected using real‐time polymerase chain reaction. Western blot was used to detect CD14 and TLR4 protein expression. CD14 and TLR4 expression on the ATII cells was imaged using immunofluorescence. Tumor necrosis factor‐α (TNF‐α) production was determined using an ELISA kit. Results: LPS stimulation resulted in an increased CD14 and TLR4 expression and increased TNF‐α production in ATII cells. Propofol, at concentrations ≥50 μM, significantly (P<0.05) and dose‐dependently decreased CD14 and TLR4 mRNA expression and protein expression in ATII cells. This was accompanied by a decrease in TNF‐α production (P<0.05). Conclusion: These results suggest that propofol, at clinically relevant concentrations, can reduce inflammatory responses in LPS‐induced ATII cells injury through downregulation of CD14 and TLR4 expression.     

Kupffer细胞CD14及Toll样受体4介导大鼠肝移植缺血再灌注损伤的机制

目的研究大鼠肝移植缺血再灌注后Kupffer细胞CD14和Toll样受体4(TLR4)的表达及其参与缺血再灌注损伤的机制。方法建立肝移植缺血再灌注模型,并分为正常对照组、缺血再灌注组、抗CD14抗体组,每组均为10只大鼠。分离培养大鼠肝移植缺血再灌注后的Kupffer细胞。检测Kupffer细胞CD14及TLR4的mRNA、蛋白表达、核转录因子κB(NFκB)活性以及培养上清TNFα的分泌量。结果再灌注后Kupffer细胞CD14及TLR4的mRNA和蛋白表达明显高于正常对照组(P<001),再灌注后核转录因子κB活性、培养上清TNFα表达量明显高于对照组(P<001)。用抗CD14抗体后NFκB活性,TNFα表达量明显下降(与再灌注组相比,P<005),但仍然高于对照组(P<001)。结论缺血再灌注后肠道内毒素(脂多糖)能够上调Kupffer细胞CD14及TLR4的表达,激活NFκB,启动细胞因子的转录和分泌,但除CD14和TLR4以外的其他信号途径参与了缺血再灌注损伤。     

Protective role of metallothionein in acute lung injury induced by bacterial endotoxin

BACKGROUND: Metallothionein (MT) is a protein that can be induced by inflammatory mediators and participate in cytoprotection. However, its role in inflammation remains to be established. A study was undertaken to determine whether intrinsic MT protects against acute inflammatory lung injury induced by bacterial endotoxin in MT-I/II knock out (-/-) and wild type (WT) mice. METHODS: MT (-/-) and WT mice were given vehicle or lipopolysaccharide (LPS, 125 microg/kg) intratracheally and the cellular profile of the bronchoalveolar lavage (BAL) fluid, pulmonary oedema, lung histology, expression of proinflammatory molecules, and nuclear localisation of nuclear factor-kappaB (NF-kappaB) in the lung were evaluated. RESULTS: MT (-/-) mice were more susceptible than WT mice to lung inflammation, especially to lung oedema induced by intratracheal challenge with LPS. After LPS challenge, MT deficiency enhanced vacuolar degeneration of pulmonary endothelial cells and type I alveolar epithelial cells and caused focal loss of the basement membrane. LPS treatment caused no significant differences in the enhanced expression of proinflammatory cytokines and chemokines nor in the activation of the NF-kappaB pathway in the lung between the two genotypes. Lipid peroxide levels in the lungs were significantly higher in LPS treated MT (-/-) mice than in LPS treated WT mice. CONCLUSIONS: Endogenous MT protects against acute lung injury related to LPS. The effects are possibly mediated by the enhancement of pulmonary endothelial and epithelial integrity, not by the inhibition of the NF-kappaB pathway.     

氯化镧对内毒素/脂多糖诱导巨噬细胞株一氧化氮合酶表达的抑制作用

目的了解氯化镧（LaCl3）对内毒素／脂多糖（LPS）刺激的巨噬细胞诱导型一氧化氮合酶（iNOS）表达的影响，并探讨其机制。方法将小鼠巨噬细胞株RAW264．7分为空白对照组、LaCl、组、LPS组和LaCl3＋LPS组。前3组细胞分别用常规培养液、含2．50μmol／L LaCl3的培养液、含1mg／L LPS的培养液培养24h，LaCl3＋LPS组用含2．5μmol／LLaCl，的培养液培养24h后，换为含1mg／L LPS的培养液培养24h。采用免疫细胞化学染色法检测iNOS在各组细胞中的表达强度；蛋白质印迹法检测iNOS的蛋白表达水平；反转录一PCR测定iNOS的mRNA表达水平；硝酸还原酶法测定各组细胞培养上清液中一氧化氮（NO）含量。结果免疫细胞化学染色结果显示，iNOS主要分布于各组细胞的胞质中，空白对照组和LaCl3组荧光强度极弱；LPS组荧光强度最强，阳性细胞百分率为44．4％，明显高于LaCl3＋LPS组（11．8％，P〈0．05）。LPS组iNOS蛋白及其mRNA表达量和细胞培养上清液中NO含量均高于其余各组（P〈0．05）。结论LaCl3可在mRNA水平和蛋白水平抑制LPS诱导的iNOS过度表达，减少NO生成，提示LaCl3能拮抗LPS诱导的iNOS过度活化。     

促红细胞生成素预先给药对大鼠内毒素性急性肺损伤的影响

目的 探讨促红细胞生成素(EPO)预先给药对大鼠内毒素性急性肺损伤的影响.方法 成年雄性SD大鼠32只,体重180～220 g,随机分为4组(n=8),C组腹腔注射生理盐水4 ml/kg(EPO溶剂对照),30 min后静脉注射生理盐水2 ml/kg[脂多糖(LP3)溶剂对照];EPO组腹腔注射EPO3 000 U/kg,30 min后静脉注射生理盐水2 ml/kg;LPS组腹腔注射生理盐水4 ml/kg,30 min后静脉注射LPS 6 mg/kg;EPO+LPS组腹腔注射EPO 3 000 U/kg,30 min后静脉注射LPS 6 mg/kg.于静脉注射LPS后4 h时处死大鼠,观察肺组织病理学结果 ,计算肺组织湿/干重(W/D)比;测定肺组织髓过氧化物酶(MPO)活性和丙二醛(MDA)、一氧化氮(NO)含量;采用Western blot法测定肺组织诱导型一氧化氮合酶(iNOS)和硝基酪氨酸(NT)的表达.结果 与C组相比,LPS组和EPO+LPs组肺组织W/D比、MPO活性、MDA和NO含量升高,iNOS和NT表达上调(P<0.01);与LPS组相比,EPO+LPS组肺组织W/D比、MPO活性、MDA和NO含量降低,iNOS和NT表达下调(P<0.01).结论 EPO预先给药可减轻大鼠内毒素性急性肺损伤,与其下调iNOS表达,减少NO生成有关.     

CD14的表达及其在库普弗细胞激活中的意义

目的　探讨LPS对库普弗细胞 (KC)CD14表达的影响及CD14在LPS激活KC中的意义。　方法　在分离培养大鼠KC的基础上 ,应用免疫组织化学染色、RT PCR等方法分别测定CD14表达的变化、培养KC中上清中TNFα、IL 6和NO浓度。　结果　 (1)不同浓度的LPS使KC中CD14mRNA的表达及其蛋白合成明显增加 ,其表达量与LPS浓度呈剂量依赖性相关 ;(2 )同一浓度的LPS可使KC中CD14mRNA的表达及其蛋白合成明显增加 ,且在 3～ 6h左右达到高峰 ;(3)LPS刺激KC后产生的活性介质能明显上调新培养KC中CD14mRNA的表达及其蛋白合成 ;(4)在血清存在的情况下加入抗CD14单抗或在无血清的情况下单独加入LPS ,可明显降低KCTNFα、IL 6和NO的释放。而后者如果同时加入LBP ,则可明显上调培养KC中的TNFα、IL 6和NO浓度。　结论　 (1)LPS及其刺激KC后产生的活性介质与CD14mRNA的表达及其蛋白合成密切相关 ,并推测在实验 1～ 3h的CD14表达的增强可能主要由LPS引起 ,而此后CD14表达的进一步增强可能与KC释放的细胞因子密切相关 ;(2 )低浓度LPS对KC的激活是CD14依赖的。     

Nitric oxide induces heme oxygenase-1 gene expression in mesangial cells

BACKGROUND: Nitric oxide (NO) release as a result of activation of inducible NO synthase (iNOS) can be sustained and reach cytotoxic concentrations. It is unknown whether cells possess intrinsic systems to attenuate NO-mediated cytotoxicity. One potential system is the heme oxygenase-1 (HO-1) enzyme because it catabolizes heme and therefore may limit synthesis or availability of iNOS. These studies were undertaken to explore whether NO derived from NO donors or from activation of iNOS induces HO-1 in mesangial cells. METHODS: The expression of the HO-1 gene was evaluated at the mRNA (Northern blot analysis) and protein (Western blot analysis) levels in mesangial cells treated with two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine (SNAP), or was stimulated by the combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) to induce iNOS in the presence and absence of NOS inhibitor NG-Monomethyl-L-arginine (L-NMMA). RESULTS: HO-1 was constitutively expressed in mesangial cells. Both SNP and SNAP induced HO-1 mRNA in a dose-dependent manner. The increase in mRNA was associated with an increase in HO-1 protein in SNP-treated cells. The combination of the LPS/IFN-gamma mixture induced iNOS expression and NO production in murine mesangial cells, as assessed by Western blot analysis and measurement of nitrite levels. HO-1 expression was also increased in response to LPS/IFN-gamma. L-NMMA dose dependently attenuated HO-1 mRNA and protein levels. In contrast, iNOS expression was dose dependently enhanced. CONCLUSIONS: Our studies demonstrate that both exogenously or iNOS-derived NO enhance HO-1 expression in mesangial cells and point to regulatory interactions between the iNOS and HO pathways. HO-1 activation may defend against NO-mediated toxicity.