Crotalaria ferruginea extract attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting MAPK/NF-κB signaling pathways

Objective:To evaluate the anti-inflammatory activity of Crotalaria ferruginea extract(CFE)and its mechanism.Methods:An intratracheal lipopolysaccharide(LPS)instillationinduced acute lung injury(ALI)model was used to study the antiinflammatory activity of CFE in vivo.The LPS-induced shock model was used to analyze the effect of CFE on survival.LPS-stimulated RAW264.7 cell model was used to investigate the anti-inflammatory activity of CFE in vitro and the effects on mitogen-activated protein kinase(MAPK)or nuclear factor-κB(NF-κB)signaling pathways.Results:CFE administration decreased the number of inflammatory cells,reduced the levels of tumor necrosis factor-α(TNF-α),monocyte chemotactic protein-1(MCP-1),interleukin-6(IL-6),and interferon-γ,and diminished protein content in the bronchoalveolar lavage fluid of mice.CFE also reduced lung wet-to-dry weight ratio,myeloperoxidase,and lung tissue pathological injury.CFE preadministration improved the survival rate of mice challenged with a lethal dose of LPS.CFE reduced LPS-activated RAW264.7 cells to produce nitric oxide,TNF-α,MCP-1,and IL-6.Furthermore,CFE inhibited nuclear translocation and phosphorylation of NF-κB P65,extracellular signal-regulated kinase,c-Jun N-terminal kinases,and P38 MAPKs.Conclusions:CFE exhibits potent anti-inflammatory activity in LPS-induced ALI mice,LPS-shock mice,and RAW264.7 cells,and its mechanism may be associated with the inhibition of NF-κB and MAPK signaling pathways.Crotalaria ferruginea may be a useful therapeutic drug for the treatment of ALI and other respiratory inflammations.

Ethanol extracts of Scutellaria baicalensis protect against lipopolysaccharide-induced acute liver injury in mice

Objective: To investigated the protective potential of ethanol extracts of Scutellaria baicalensis(S. baicalensis) against lipopolysaccharide(LPS)-induced liver injury. Methods: Dried roots of S. baicalensis were extracted with ethanol and concentrated to yield a dry residue. Mice were administered 200 mg/kg of the ethanol extracts orally once daily for one week. Animals were subsequently administered a single dose of LPS(5 mg/kg of body weight, intraperitoneal injection). Both protein and m RNA levels of cytokines, such as tumor necrosis factor alpha, interleukin-1β, and interleukin-6 in liver tissues were evaluated by ELISA assay and quantitative PCR. C yclooxygenase-2, inducible nitric oxide synthase, and nuclear factor-κB protein levels in liver tissues were analyzed by western blotting. Results: Liver injury induced by LPS signifi cantly increased necrosis factor alpha, interleukin-1β, interleukin-6, cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor-κB in liver tissues. Treatment with ethanol extracts of S. baicalensis prevented all of these observed changes associated with LPS-induced injury in liver mice.Conclusions: Our study showed that S. baicalensis is potentially protective against LPS-induced liver injury in mice.

Role of Nuclear Transcription Factor-кB in Endotoxin induced Shock in Rats

Summary: To investigate the role of NF-κB in endotoxic shock in rats, the model of endotoxin-shock rats was induced by intravenous infusion of lipopolysaccharide (LPS). 1 h, 2 h, 4 h and 6 h after LPS injection, the activation of NF-κB in blood mononuclear cells and the content of TNF-α and IL-6 in plasma was detected by enzyme-linked immunoadsordent assay (ELISA). The level of mean arterial pressure (MAP) and the histopathological changes of lung and liver were also observed. The activation of NF-κB in mononuclear cells increased 1 h after LPS injection and reached its peak 2 h after the injection, and its level was higher than that of normal group. The level of TNF-α was increased 1 h after the infusion and peaked 2 h after the injection, and its level was higher than that of normal group after LPS infusion. The content of IL-6 increased gradually with time, the IL-6 level was higher than that of normal group after LPS injection. MAP was decreased gradually with time and its level was lower than that of normal group after LPS injection. Pathological examination showed that endotoxic shock could cause pulmonary alveolar hemorrhage, edema and infiltration of inflammatory cell in lung tissue and congestion, edema, capillary dilation and inflammatory cell infiltration in liver tissue. It is concluded that NF-κB can up-regulate the expression of TNF-α and IL-6 in plasma and play an important role in endotoxin-induced shock in rats.

Protective Effect of Emodin against Lipopolysaccharides-induced Corneal Injury in Rats

Objective To investigate the effect of emodin on lipopolysaccharides （LPS）-induced corneal injury in rats. Methods Three parallel incisions on the central surface of corneal epithelium were made and LPS was applied on them to induce corneal injury in Wistar rats. All rats were randomly divided into emodin group （n=40） and keratitis group （n=40）. Rats in the emodin group received subconjunctival injection of emodin and rats in the keratitis group received its vehicle 30 minutes before LPS exposure. At different time points-1 3, 6, 12, and 24 hours after LPS exposure, the symptoms of all rats were observed and the severity of their ocular inflammation was examined with a slit lamp microscope, then 8 rats in each group were killed through cervical dislocation and their eyes were enucleated and prepared to observe pathological changes of corneal tissue under a light microscope. The activation of nuclear factor-loB （NF-κB） under different condi- tions was determined by Western blot. Immunocytochemistry staining with an antibody against intercellular adhesion molecule-1 （ICAM-1） was performed to identify positive cells in corneal tissues. Results The model of acute keratitis was successfully established in Wistar rats. LPS could induce a typical corneal inflammatory response, such as hyperemia, corneal edema and opacity, which were observed in model rats. Compared with keratitis group, both ocular behaviors and damages of the corneal structure were improved in emodin group. Furthermore, the activation of NF-κB and the expression of ICAM-1 induced by LPS were markedly inhibited in emodin group. Conclusion Emodin can inhibit the activation of NF-κB and the expression of ICAM-I induced by LPS in corneas, protect against acute corneal injury, and improve symptoms in rats.

Dexamethasone impairs the differentiation and maturation of murine dendritic cells by Toll-like receptor 4-nuclear factor-κB pathway

Background Recent studies have demonstrated that dexamethasone （DEX） interferes with immune responses by targeting key functions of dendritic cells （DCs） at the earliest stage. However, the cellular and molecular mechanisms are still incompletely understood. This study aimed to explore the possible mechanisms by investigating the roles of DEX on differentiation, maturation ＆ function of murine DCs and the effects of DEX on DCs via Toll-like receptor 4 （TLR4）-nuclear factor （NF）-KB mediated signal pathway. Methods Immature DCs （imDCs） were cultured from murine bone marrow （BM） cells. We added DEX into culture medium at different time. The expression of CD11c, CD86 and I-Ab （mouse MHC class II molecule） was determined by flow cytometry. We determined the expression of NF-κB and its inhibitory protein I-κBα by electrophoretic mobility shift assay （EMSA） and Western blotting, respectively. The productions of interleukin （IL）-12p70 and IL-10 in cell culture supernatants were determined by enzyme-linked immunosorbent assay （ELISA）. Results DEX impaired differentiation of DCs from murine bone marrow progenitors, and inhibited lipopolysaccharide （LPS） induced maturation of DCs. DEX significantly inhibited NF-κB expression of normal DCs, the higher the DEX concentration or the longer the DEX treatment time, the more obvious the effect. However, DEX had little effect on LPS-induced NF-KB activation, and partially impaired LPS-induced I-κBα degradation. DEX significantly decreased LPS induced IL-12p70 production by DCs. Interestingly, our results showed a synergistic effect between DEX and LPS on the production of IL-10 by DCs. Conclusions DEX inhibits the differentiation and maturation of murine DCs involved in TLR4-I-κB-NF-κB pathway, and also indirectly impairs Thl development and interferes with the Thl-Th2 balance through IL-12 and/or IL-10 secretion by DCs.

Lipopolysaccharide-enhanced early proliferation of insulin secreting NIT-1 cell is associated with nuclear factor-kappaB- mediated inhibition of caspase 3 cleavage

Background Increased levels of plasma lipopolysaccharide （LPS） have been found in obesity and diabetes patients. This study was to investigate the effect of LPS on pancreatic beta-cell viability and the involvement of caspase 3 in NIT-1 cell line. Methods Mouse insulinoma NIT-1 cells were treated with LPS for the indicated time and dose. Cell viability was measured by cell counting kit-8 reagent. Toll-like receptor 4 （TLR4）, caspase 3 and cleaved caspase 3 were detected by Western blotting. Insulin was determined by radioimmunoassay （RIA）. Results LPS promoted NIT-1 cell proliferation at 1 μg/ml, peaked at 72 hours of incubation. A reduction in cleavage of caspase 3 was observed upon LPS treatment. Bay11-7082, a specific inhibitor of nuclear factor （NF）-κB, blunted LPS-induced inhibition of caspase 3 cleavage. Reduction in chronic insulin secretion was observed after treatment with LPS at 1 μg/ml for 48 and 72 hours, not for 24 hours. TLR4 protein was upregulated when NIT-1 cells were treated with LPS at 1 μg/ml for 24 hours. Conclusions LPS promotes early NIT-1 cell proliferation in association with NF-KB-mediated inhibition of caspase 3 cleavage. LPS exerts a time-dependent inhibitory effect on chronic insulin secretion from NIT-1 cells.

阻断 NF -κB 信号途径防治急性肺损伤的研究进展

目的：探讨以核因子κB（NF-κB）为靶点防治急性肺损伤（ALI）的分子生物学机制。方法应用CNKI、Medline、ScienceDirect 等数据库，查阅近年来相关文献，总结NF-κB在ALI中的作用机制与干预手段。结果 NF-κB在ALI中的信号转导途径及作用机制逐渐被揭示，大量研究证实，通过干预NF-κB上游信号通路、NF-κB抑制蛋白（ IκB）、IκB激酶（ IKK）等途径，能在一定限度内阻断细胞因子和炎症介质的释放，缓解ALI的炎症反应。结论阻断靶细胞中NF-κB通路、特异性抑制目的蛋白和基因表达，或许能成为未来治疗ALI的研究方向；不恰当的药物干预会破坏机体的免疫平衡状态，虽然NF-κB阻断剂已进入临床试验阶段，但多数仅局限于动物实验和细胞水平，实现临床防治尚需进行大量实验研究。

甘草酸二铵与地塞米松联用对内毒素诱导ALI大鼠肺组织糖皮质激素受体表达的影响

目的探讨联合应用甘草酸二铵（DG）和地塞米松（DXM）对内毒素（LPS）所致急性肺损伤（Au）大鼠肺组织糖皮质激素受体（GR）及核因子-κB（NF-κB）表达的影响。方法将40只雄性SD大鼠随机分为生理盐水组、ALI组、DG干预组、DXM干预组和联合用药组（DG＋DXM）。除正常对照组外，其余各组均给予LPS5mg／kg尾静脉注射；LPS注射前1h，DG干预组给予DG20mg／kg静脉注射，DXM干预组给予DXM2mg／kg静脉注射，联合用药组同时给予DG20mg／kg和DXM2mg／kg静脉注射。采用逆转录-PCR反应检测肺组织GRmRNA及NF-KBmRNA的表达；ELISA法测定血清TNF-01、IL-10浓度，同时对大鼠肺组织病理、肺湿干比（W／D）及PaO：等指标进行比较分析。结果①Au组GRmRNA表达明显低于生理盐水组（P〈0．01），而DG干预组及联合用药组GRmRNA表达则明显高于Au组（分别P〈0．05，P〈0．01）；DG干预组、DXM干预组及联合用药组NF-κBmRNA表达均明显低于Au组（均P〈0．01）。②ALI组TNF-α明显高于生理盐水组、DG干预组、DXM干预组和联合用药组（均P〈0．01）。ALI组IL-10明显高于生理盐水组（P〈0．01），但低于DG干预组和联合用药组（均P〈0．01）。③Au组大鼠肺组织水肿明显、中性粒细胞部分浸润，而DG干预组、DXM干预组和联合用药组肺部炎症较Au组减轻。结论甘草酸二铵和地塞米松联用可上调大鼠GR及IL-10的表达，抑制NF-κB及TNF-α的过度表达，从而起到减轻ALI的作用。

Influence of Silencing TRAF6 with shRNA on LPS/TLR4 Signaling in vitro

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide(LPS)/toll-like receptor(TLR)-4 signaling pathway in vitro.Four plasmids(pGCsi-TRAF6-shRNA1,2,3,4) containing different shRNA sequences were designed and synthesized.The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay.Inflammatory cellular models were established by LPS stimulation.Levels of TNF-α,IL-1β and TGF-β1 in the supernatants,mRNA expressions of TRAF6,IL-6 and COX-2,protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA,real-time quantitative PCR and Western blotting,respectively.The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h.The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1,2 groups than in the TRAF6-shRNA3,4 groups.Therefore,pGCsi-TRAF6-shRNA1,2 were selected for the subsequent experiments.Our results still showed that pGCsi-TRAF6-shRNA1,2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α,IL-1β,IL-6 and COX-2,and inhibit NF-κB nuclear translocation.Moreover,pGCsi-TRAF6-shRNA1,2 could suppress the release of TGF-β1 at the protein level.It was concluded that the recombinant plasmid pTRAF6-shRNA can,to some extent,inhibit inflammatory response stimulated by LPS at the initial phase.TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

MyD88/PI3-K/NF-κB pathway mediates the antagonism of lipoxin A_4 on LPS-induced synthesis of interleukins in endothelial cells

In order to investigate whether lipoxin A4 （LXA4） has an antagonistic effect on lipopolysaccharide （LPS）-induced synthesis of interleukin （IL）-β3, IL-6 and IL-8 in rat pulmonary microvascular endothelial cells （PMVEC）, and to explore the molecular mechanisms of signal pathway in LXA4 actions, cultured PMVEC were treated with LPS, with or without preincubation with LXA4. Proteins of IL-β3, IL-6 and IL-8 in supernatant were analyzed by enzyme-linked immunosorbent assay （ELISA）. Expressions of mRNA of IL-β3, IL-6 and IL-8 were determined by RT-PCR. Expressions of phosphorylation of phosphoinositide 3-kinase （PI3-K） and myeloid differentiation factor 88 （MyD88） were analyzed by Western blot. Activities of DNA-binding of nuclear factor-kappa B （NF-κB） and activator protein-1 （AP-1） were measured by electrophoretic mobility shift assay （EMSA）. The results showed that LPS induced production of IL-β3, IL-6 and IL-8 in rat PMVEC via MyD88/PI3-K/NF-κB and AP-1 pathway-dependent signal transduction. LPS-stimulated expression of PI3-K, activities of NF-κB and AP-1, secretion of protein and expression of mRNA of IL-β3, IL-6 and IL-8 but not MyD88 expression in PMVEC were inhibited by LXA4 in a dose-dependent manner. In conclusion, LXA4 inhibits synthesis of IL-β3, IL-6 and IL-8 by down-regulation of PI3-K/NF-κB and AP-1 signal pathway in PMVEC.

Perfluorocarbon attenuates lipopolysaccharide-mediated inflammatory responses of alveolar epithelial cells in vitro

Background Toll-like receptor-4 （TLR-4） is integrally involved in lipopolysaccharide （LPS） signaling and has a requisite role in the activation of nuclear factor-κB （NF-κB）. The exact mechanisms that lend perfluorocarbon （PFC） liquids a cytoprotective effect have yet to be elucidated. Therefore we examined in an in vitro model the cytoprotective effect of PFC on LPS-stimulated alveolar epithelial cellls （AECs）. Methods AECs （A549 cells, human lung adenocarcinoma cell line） were divided into four groups： control, PFC, LPS and LPS ＋ PFC （coculture group） groups. Intercellular adhesion molecule-1 （ICAM-1） was detected by ELISA, tumor necrosis factor-g （TNF-a） and interleukin-8 （IL-8） were detected by radioimmunological methods. The expression of TLR-4 mRNA and protein was detected by real time PCR and Western blotting, respectively. The activation of NF-κB was detected by Western blotting （proteins of I-κBa and NF-κB p65）. Results ICAM-1, TNF-a and IL-8 were significantly increased in LPS-stimulated AECs groups. The expression of TLR-4 mRNA and protein in LPS-stimulated groups was markedly increased. Meanwhile, NF-κB was activated as indicated by the significant degradation of IKB-a and the significant release of NF-κB P65 and its subsequent translocation into the nucleus. There were no significant effects of PFC alone on any of the factors studied while the coculture group showed significant downregulation of the secretion of ICAM-1, TNF-a and IL-8, the expression of TLR-4 rn^NA and the activity of NF-κB. Conclusions Taken together, our results demonstrate that LPS can induce AEC-related inflammatory injury via the activation of TLR-4 and subsequent activation of NF-κB. PFC is able to protect AECs from LPS-induced inflammatory injury by blocking the initiation of the LPS signaling pathway, which is indicated by the significant decrease of TLR-4 expression and NF-κB activation.

Effect of Palrnatine on lipopolysaccharide-induced acute lung injury by inhibiting activation of the Akt/NF-κB pathway

Inflammation plays an important role in the development of acute lung injury(ALI).Severe pulmonary inflammation can cause acute respiratory distress syndrome(ARDS)or even death.Expression of proinflammatory interleukin-1β(IL-1β)and inducible nitric oxide synthase(iNOS)in the process of pulmonary inflammation will further exacerbate the severity of ALI.The purpose of this study was to explore the effect of Palrnatine(Pa)on lipopolysaccharide(LPS)-induced mouse ALI and its underlying mechanism.Pa,a natural product,has a wide range of pharmacological activities with the potential to protect against lung injury.Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)assays were performed to detect the expression and translation of inflammatory genes and proteins in vitro and in vivo.Immunoprecipitation was used to detect the degree of P65 translocation into the nucleus.We also used molecular modeling to further clarify the mechanism of action.The results showed that Pa pretreatment could significantly inhibit the expression and secretion of the inflammatory cytokine IL-1β,and significantly reduce the protein level of the proinflammatory protease iNOS,in both in vivo and in vitro models induced by LPS.Further mechanism studies showed that Pa could significantly inhibit the activation of the protein kinase B(Akt)/nuclear factor-κB(NF-κB)signaling pathway in the LPS-induced ALI mode and in LPS-induced RAW264.7 cells.Through molecular dynamics simulation,we observed that Pa was bound to the catalytic pocket of Akt and effectively inhibited the biological activity of Akt.These results indicated that Pa significantly relieves LPS-induced ALI by activating the Akt/NF-κB signaling pathway.

地塞米松对脓毒症大鼠急性肺损伤影响的实验研究

目的 观察急性肺损伤(ALI)肺组织核因子(NF)-κB的活性变化及地塞米松(Dex)的干预作用。方法 采用LPS诱导的急性肺损伤动物模型,将30只SD大鼠随机分为实验组(ALI+Dex,10只)、模型组(ALI+NS,10只)和对照组(NS,10只)三组,实验组在建模成功后腹腔注射地塞米松,模型组在建模成功后腹腔注射相同剂量的生理盐水,对照组仅腹腔注射相同剂量的生理盐水。注药6小时后留取肺组织,用免疫组织化学法结合图像分析仪检测其NF-κBP65、IκB-α蛋白的相对含量,并进行病理学光镜检查。结果 ALI大鼠肺组织可见大量出血和炎性细胞浸润,NF-κBP65的蛋白表达明显升高,IκB-α表达显著降低。Dex能明显下调NF-κBP65的蛋白表达,上调IκB-α表达,并能减轻肺组织的损伤程度。结论 NF-κBP65活化在ALI的发生、发展过程中起着重要作用。Dex具有明显的抗炎作用,减少了肺组织损害。

Anti-inflammatory effects of Eucommia ulmoides Oliv. male flower extract on lipopolysaccharide-induced inflammation

Background:Eucommia ulmoides Oliv. is a medicinal plant native to China, with its bark (Eucommiae Cortex) traditionally being used for medicinal purposes. Previous research has shown that Eucommia male flowers can exert anti-inflammatory, analgesic, antibacterial, and other pharmacological effects, including immune regulation. This study explored the anti-inflammatory effects of the 70% ethanol extract of male flowers (EF) of E. ulmoides in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and LPS-administered mice.Methods:Cytotoxicity of EF for RAW 264.7 cells was investigated using Cell Counting Kit-8. The production of proinflammatory mediators, nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 was determined using enzyme-linked immunosorbent assays. IL-17, IL-23, and IL-10 mRNA levels were determined using quantitative real-time polymerase chain reaction. Activation of the nuclear factor (NF)-κB pathway in RAW 264.7 cells was investigated via Western blotting. In vivo antiinflammatory effects of EF were studied in an LPS-induced acute inflammation mouse model by analyzing lung tissue histopathology, serum TNF-α and IL-6 levels, and myeloperoxidase (MPO) activity in lung tissue.Results:EF showed no significant cytotoxicity at concentrations from 10 to 60 μg/mL (cell viability > 80%) in the CCK-8 cell viability assay. EF inhibited the RAW 264.7 cell proliferation (EF 60 μg/mL, 120 μg/mL, and 250 μg/mL vs. negative control: 87.31±2.39% vs. 100.00±2.50%, P=0.001;79.01±2.56 vs. 100.00±2.50%, P<0.001;and 64.83±2.50 vs. 100.00±2.50%, P<0.001), suppressed NO (EF 20 μg/mL and 30 μg/mL vs. LPS only, 288.81±38.01 vs. 447.68±19.07 μmol/L, P=0.004;and 158.80±45.14 vs. 447.68±19.07 μmol/L, P<0.001), TNF-α (LPS+EF vs. LPS only, 210.20±13.85 vs. 577.70±5.35 pg/mL, P<0.001), IL-1β (LPS+EF vs. LPS only, 193.30±10.80 vs. 411.03±42.28 pg/mL, P<0.001), and IL-6 (LPS+EF vs. LPS only, 149.67±11.60 vs. 524.80±6.24 pg/mL, P<0.001) secretion, and downregulated the mRNA expression of IL-17 (LPS+EF vs. LPS only, 0.23±0.02 vs. 0.43±0.12, P<0.001), IL-23 (LPS+EF vs. LPS only, 0.29±0.01 vs. 0.42±0.06, P=0.002), and IL-10 (LPS+EF vs. LPS only, 0.30±0.01 vs. 0.47±0.01, P=0.008) in LPS-stimulated RAW 264.7 cells. EF inhibited the LPS-induced NF-κB p65 (LPS+EF 20 μg/mL and 30 μg/mL vs. LPS only: 0.78±0.06 vs. 1.17±0.08, P<0.001;and 0.90±0.06 vs. 1.17±0.08, P=0.002) and inhibitor of kappa B (IκBα) phosphorylation (LPS+EF 20 μg/mL and 30 μg/mL vs. LPS only: 0.25±0.01 vs. 0.63±0.03, P<0.001;and 0.31±0.01 vs. 0.63±0.03, P<0.001), LPS+EF 30 μg/mL inhibited IκB kinase (IKKα/β) phosphorylation (LPS+EF 30 μg/mL vs. LPS only, 1.12±0.14 vs. 1.71±0.25, P=0.002) in RAW 264.7 cells. Furthermore, EF 10 mg/kg and EF 20 mg/kg inhibited lung tissue inflammation in vivo and suppressed the serum TNF-α (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 199.99±186.49 vs. 527.90±263.93 pg/mL, P=0.001;and 260.56±175.83 vs. 527.90±263.93 pg/mL, P=0.005), and IL-6 (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 41.26±30.42 vs. 79.45±14.16 pg/ml, P=0.011;and 42.01±26.26 vs. 79.45±14.16 pg/mL, P=0.012) levels and MPO (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 3.19±1.78 vs. 5.39±1.51 U/g, P=0.004;and 3.32±1.57 vs. 5.39±1.51 U/g, P=0.006) activity in lung tissue.Conclusions:EF could effectively inhibit the expression of inflammatory factors and overactivation of neutrophils. Further investigation is needed to evaluate its potential for anti-inflammation therapy.

Hwangryunhaedoktang exerts anti-inflammation on LPS-induced NO production by suppressing MAPK and NF-κB activation in RAW264.7 macrophages

OBJECTIVE： This study aimed to evaluate whether Hwangryunhaedoktang （HHT）, a herbal compound, has an inhibitory effect on lipopolysaccharide （LPS）-induced inflammation in RAW264.7 macrophages. METHODS： The effects of HHT were evaluated by confirming nitric oxide （NO） production and expression of inducible NO synthase （iNOS） and mitogen-activated protein kinases （MAPKs） in LPS-stimulated RAW264.7 macrophages via the Griess assay, Western blotting, and real-time reverse transcription quantitative polymerase chain reaction. Western blot analyses and luciferase assays were used to evaluate whether HHT has an effect on the phosphorylation and translocation of nuclear factor-κB （NF-κB）. The secretion and expression of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were determined via enzyme-linked immunosorbent assay and Western blot analyses. RESULTS： HHT suppressed LPS-induced NO production and expression of iNOS in a dose-dependent manner. Additionally, MAPKs activation was also attenuated via inhibition of phosphorylation of extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinase and p38 which were related to inflammatory pathway. Furthermore, HHT also effectively attenuated NF-κB activation and its translocation to the nucleus, a process that is closely linked to inflammation. LPS normally induced the expression of inflammatory cytokines such as TNF-α and IL-6, but the secretion and expression of TNF-α and IL-6 were significantly attenuated by pretreating the cells with HHT. CONCLUSION： HHT suppressed LPS-induced NO production by blocking the activation of NF-κB and MAPK signaling pathways in RAW264.7 macrophages. Furthermore, HHT may have an anti-inflammatory effect by suppressing the LPS-induced secretion of TNF-α and IL-6. Therefore, the traditional herbal formula HHT might be a useful potential therapeutic agent for inflammation.

Role of Shenfu Injection(参附注射液) in Rats with Systemic Inflammatory Response Syndrome

Objective： To investigate the role of Shenfu Injection （参附注射液, SFI） in rats with systemic inflammatory response syndrome （SIRS）. Methods： The SIRS rat model was induced by the intravenous injection of lipopolysaccharide （LPS）. Forty-five male Wistar rats were randomly divided into 3 groups, the sham operative control group （control group, n=5）, the SIRS model group （model group, n=20） and the SFI treatment group （SFI group, n=20）. LPS was injected through the external jugular vein （12 mg/kg, 6 mg/mL） to all rats except for those in the control group, and SFI （10 mL/kg） was given to those in the SF group only once through intraperitoneal injection, while the normal saline （10 mL/kg） was given to those in the model group. For those in the control group, normal saline was given through the external jugular vein （2 mL/kg） and intraperitoneal injection （10 mL/kg）. Then, rats in the model group and SFI group were divided into 4 subgroups according to the time points, i.e., 1 h, 2 h, 4 h and 6 h subgroups, 5 rats in each group. The activity of nuclear factor of κB （NF-κB） of in blood mononuclear cells and the plasma levels of tumor necrosis factor- α （TNF- α ） and interleukin 6-（IL-6） were determined using enzyme-linked immunoabsordent assay （ELISA） at 1 h, 2 h, 4 h and 6 h after modeling. Histopathologic changes of the lung and liver were observed under a light microscope. Results： Compared with the control group, the activity of NF-κB in mononuclear cells and the plasma level of TNF-α were obviously increased at each time points （all P〈0.01）, reaching the peaks at 2 h after modeling. The plasma level of IL-6 increased gradually as time went by in the model group （P〈0.01）. Pathological examination showed pulmonary alveoli hemorrhage, edema and inflammatory cell infiltration in the lung tissue, and angiotelectasis, congestion, and local necrosis in the liver tissue in the model group. Compared with the model group, the activity of NF- κB and the levels of TNF-α and IL-6 in plasma decreased significantly in the SFI group （P〈0.01）, and the pathological injury in the lungs and liver was significantly alleviated. Conclusion： SFI plays a protective role by inhibiting the activity of NF-κB, and reducing the expressions of TNF-α and IL-6 in SIRS rats.