雷帕霉素抑制LPS诱导的RAW264.7细胞表达和释放HMGB1的机制

目的：探讨雷帕霉素抑制LPS诱导小鼠RAW264.7细胞表达和释放HMGB1的作用机制。方法:传代培养的小鼠RAW264.7细胞分5组接种于6孔板，即仅加培养液的对照组；加250 μg/L LPS的诱导组； 诱导组基础上加100 μg/L雷帕霉素的干预组；干预组基础上加rTNF-α 50 μg/L的抗干预组；及抗干预组基础上加抗鼠TNF-α 100 μg/L的抗体中和组。培养4 h后，ELISA法检测对照组、诱导组和干预组上清液中TNF-α水平；培养24 h后，RT-PCR和Western blotting法分别检测各组细胞内HMGB1 mRNA表达水平和上清液中HMGB1含量。结果:培养4 h，干预组上清液中TNF-α水平与对照组比无显著差异（P>0.05），但明显低于诱导组（P<0.05）；培养24 h, 与对照组比，诱导组细胞内HMGB1 mRNA表达明显增强（P<0.05），上清液中HMGB1含量也明显增多（P<0.05）；干预组明显减少了HMGB1 mRNA表达及上清液中HMGB1含量（P<0.05）； 与干预组比，抗干预组细胞HMGB1 mRNA表达及上清液中HMGB1含量明显增加（P<0.05）；抗体中和组细胞HMGB1 mRNA表达水平及上清液中HMGB1含量和干预组无显著差异（P>0.05）。 结论:雷帕霉素抑制LPS诱导RAW264.7细胞表达和释放HMGB1，可能部分地与其抑制TNF-α的表达有关。     

Effect of Honghua （Flos Carthami） on nitric oxide production in RAW 264.7 cells and a.glucosidase activity

OBJECTIVE： To study the effects of extracts from Honghua （Flos Carthaml~ on lipopolysaccharide in- duced nitric oxide （NO） production in RAW 264.7 cells and the influence of the extracts on yeast a-glucosidase activity. The total flavonoid content of the extracts was also determined. METHODS： Cytotoxicity of the extracts to RAW 264.7 cells was evaluated by the ATPliteTM method. Inhibitory effects of the extracts on NO production were evaluated by Griess assay. Curcumin was used as a positive control. Screening of extracts for po- tential a-glucosidase inhibitors was done by a fiuo- rometric assay. The assay was based on the hydroly- sis of 4-methylumbelliferyl-a-D-glucopyranoside toform the fluorescent product, 4-methylumbellifer- one. Acarbose was used as a positive control. The total t3avonoid content was tested using kaempfer- ol as the standard. RESULTS： There were significant inhibitory effects on NO production when the extracts were 25-100 μg/ mL （P〈0.05） and curcumin was 2-4 μg/mL （P〈 0.001）. The extracts showed an inhibitory effect on a-glucosidase activity at the concentrations of 15.6-125 μg/mL with a half maximal （50%）inhibito- ry concentration （ICs0） of （32.8± 5.7） μg/mL, com- pared with the ICs0 of acarbose at （1.8±0.4） μg/mL. There was a significant difference between the two IC50 values （P〈0.001）. The total content of flavo- noids per gram of dried herb was 1.14 mg. CONCLUSION： Honghua （Flos Carthami） showed in- hibitory effects on NO production in activated RAW 264.7 macrophage cells and an inhibitory effect on yeast a-glucosidase. There might be a relationship between these pharmacological effects and its fla- vonoid content.     

X射线照射对RAW264·7细胞TNF-α和NF-κB表达的影响

目的探讨X射线照射诱发小鼠巨噬细胞分泌TNF-α和NF-κB的规律。方法以8Gy X射线单次照射小鼠巨噬细胞系RAW264.7细胞,并于照后不同时间收集细胞培养上清液,采用ELISA法检测RAW264.7细胞TNF-α分泌水平;以8Gy X射线单次照射小鼠巨噬细胞系RAW264.7细胞,并于照后不同时间收集细胞并进行裂解,采用Western blot实验检测NF-кB（p65）的表达。结果 X射线照射能使小鼠巨噬细胞分泌TNF-α增加,在照射后34h达到一次分泌峰值;细胞核中NF-κB（p65）分布增多,并且随着时间的延长其核移位明显增多,照射后4h达到一次最高值。结论 X射线照射能诱发小鼠巨噬细胞TNF-α和NF-κB表达增强。     

复方甘草酸苷制剂对脂多糖诱导小鼠RAW264.7细胞分泌炎症因子的调节作用

目的:研究复方甘草酸苷片剂和注射剂对脂多糖诱导小鼠巨噬细胞RAW 264.7分泌炎症因子的调节作用。方法:采用脂多糖诱导的小鼠巨噬细胞RWA264.7,建立体外炎症模型。取复方甘草酸苷片剂和注射剂,制备供试药液。分别通过,MTT、Griess和双抗体夹心ABC-ELISA等实验,测定在不同浓度的供试药液作用下RAW264.7细胞活力以及经脂多糖诱导后的RAW264.7细胞的一氧化氮、肿瘤坏死因子TNF-α及白介素IL-1β、-6和-10等炎症因子分泌量的变化。结果:复方甘草酸苷的2种制剂药液在0～300μmol.L-1浓度范围内对RAW264.7细胞活力无影响。复方甘草酸苷注射剂药液在75、150和300μmol.L-1浓度下对经脂多糖处理的RAW264.7细胞的一氧化氮分泌抑制率分别为13.8%、40.4%和53.8%,并致细胞的TNF-α分泌量降低29.8%、41.5%和52.1%,IL-1β分泌量降低39.5%、55.6%和69.6%,IL-6分泌量降低18.3%、29.5%和39.1%,但IL-10分泌量却提高了8.2%、23.8%和30.8%;而复方甘草酸苷片药液在相同浓度下对同样细胞的一氧化氮分泌抑制率分别为9.8%、27.1%和37.8%,致细胞的TNF-α分泌量降低16.6%、37.0%和48.4%,IL-1β分泌量降低28.1%、47.9%和57.9%,IL-6分泌量降低13.5%、19.9%和28.2%,IL-10分泌量提高了3.9%、14.8%和23.4%。复方甘草酸苷的2种制剂对细胞分泌炎症因子的调节作用与阳性对照药地塞米松相似,其中注射剂的作用强于片剂。结论:复方甘草酸苷可剂量依赖性地显著抑制脂多糖诱导小鼠RAW 264.7细胞产生促炎因子一氧化氮、TNF-α及IL-1β和-6并促进抗炎因子IL-10的表达,这可能为其抗炎作用机制。     

The Effect of l‐Arginine on Porphyromonas gingivalis‐Induced Phagocytosis of a Murine Macrophage‐like Raw 264.7 Cell Line

The aim of this study was to determine the effect of l‐arginine on Porphyromonas gingivalis‐induced phagocytosis by RAW 264.7 cells. The cells were pretreated with l‐arginine or d‐arginine prior to incubation with either unopsonized or opsonized P. gingivalis. In other experiments, the cells were pretreated with l‐arginine and various concentrations of NMLA (N^G‐monomethyl‐l‐arginine) prior to incubation with the bacteria. The phagocytosis was microscopically assessed and determined by the phagocytic index. The results showed that l‐arginine, but not d‐arginine enhances the ability of RAW264.7 cells to engulf the bacteria. The upregulatory effect of l‐arginine on P. gingivalis‐induced phagocytosis was abolished by NMLA. The results of the present study suggest that l‐arginine may upregulate the P. gingivalis‐induced phagocytic activity of RAW264.7 cells, perhaps, via modulation of nitric oxide synthase.     

染料木素通过TIPE 2负性调控Akt活性促进脂多糖活化的巨噬细胞凋亡

目的探究染料木素(genistein,GEN)对脂多糖(lipopolysaccharide,LPS)活化的RAW264.7细胞凋亡的影响及其可能的药理学作用机制。方法GEN预孵育RAW264.7细胞或慢病毒介导的肿瘤坏死因子α诱导蛋白8样分子2(tumor necrosis factor-α-induced protein 8-like 2,TIPE 2)过表达细胞2 h,再与LPS共孵育24 h,采用CCK 8试剂盒检测细胞活力,Annexin V-FITC/PI试剂盒检测细胞凋亡水平,qRT-PCR检测TNF-α、IL-6、caspase-8、caspase-3和TIPE 2 mRNA,Western blot检测iNOS、COX-2、caspase-8、caspase-3、TIPE 2、Akt和p-Akt蛋白表达。结果LPS促进RAW264.7细胞TNF-α、IL-6、iNOS、COX-2合成;GEN抑制LPS活化的RAW264.7细胞活力,凋亡细胞增多,并上调caspase-8、caspase-3、TIPE 2 mRNA及蛋白表达;TIPE 2过表达上调活化RAW264.7细胞caspase-8、caspase-3 mRNA及蛋白表达,减少Akt磷酸化,且与GEN具有协同作用。结论GEN可能通过上调TIPE 2抑制Akt活性,激活外源性凋亡途径,促进LPS活化的RAW264.7细胞凋亡。     

Inhibitors of TLR-4, NF-κB,and SAPK/JNK signaling reduce the toxic effect of lipopolysaccharide on RAW 264.7 cells

The present study was designed to examine and compare the effects of three suppressors on the cytokine response in tandem with examining: the synthesis of inducible forms of heat shock proteins; HSP72 and HSP90α; activities of NF-κB and SAPK/JNK signaling pathways; and TLR4 expression. Pre-treatment with inhibitors offers promise as protective means to lower the activity of these cascades, thereby circumventing the formation of excessive amounts of pro-inflammatory molecules. Three inhibitors of TLR4, SAPK/JNK, and NF-κB signaling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before the Escherichia coli lipopolysaccharide (LPS) application. Treatments of RAW 264.7 cells with each of the inhibitors resulted in a reduced response to LPS as was visualized by a decrease of TNF-α, IL-1, and IFN-γ production. In addition, inhibitors of the NF-κB and SAPK/JNK signaling reduced IL-6 production in LPS-treated cells, whereas the IKK inhibitor XII also decreased IL-10 production. Further, the NO production in LPS-stimulated macrophages was significantly reduced following application of CLI-095 or IKK inhibitor XII. The results also showed that the inhibitors suppressed TLR4 production and decreased phosphorylation of NF-κB and SAPK/JNK proteins, thereby preventing the activation NF-κB and SAPK/JNK signaling pathways in LPS-activated cells. In addition, the production of inducible heat shock proteins, HSP72 and HSP90-α, was reduced in LPS-stimulated RAW 264.7 cells pre-treated with inhibitors. These results suggest that inhibitors CLI-095, SP600125, and IKK inhibitor XII demonstrate potential effectiveness in the reduction of the inflammatory response by mechanisms involving both the cellular defense system and cellular signaling. In conclusion, suppressor of NF-κB cascade, IKK inhibitor XII, seems to be the most effective anti-toxic agent among studied inhibitors.     

TG2和Mertk基因共沉默腺病毒干扰载体的构建及其基因沉默作用

目的构建转谷氨酰胺酶2（TG2）和Mer受体酪氨酸激酶（Mertk）基因共沉默腺病毒干扰载体,并检测其基因沉默作用。方法首先构建能干扰TG2和Mertk蛋白表达的质粒载体pSUPER/TG2及pSUPER/Mertk,再将其干扰序列和H1启动子序列切下并连接到pAdTrack上,构建成pAdTrack/TG2/Mertk载体。将其转入含有pAdEasy-1的BJ5183感受态细菌中,回收并酶切鉴定重组腺病毒载体。将阳性腺病毒载体感染HEK293细胞,收集病毒,反复扩增后测定病毒滴度。分别以pAdTrack/TG2/Mertk、pAdTrack/TG2、pAdTrack/Mertk及pAdTrack/绿色荧光蛋白（GFP）感染RAW264.7细胞,采用免疫印迹法检测其TG2蛋白和Mertk蛋白的表达水平。结果pAdTrack/TG2/Mertk载体的病毒滴度为6.13×1010GFU/mL。经酶切鉴定,pAdTrack/TG2/Mertk含有插入的2个启动子和2个干扰序列,载体构建成功。pAdTrack/TG2和pAdTrack/TG2/Mertk组TG2蛋白的表达水平比较差异无统计学意义（P〉0.05）,但均低于pAdTrack/GFP和pAdTrack/Mertk组（P〈0.01）,后两者比较差异无统计学意义（P〉0.05）。pAdTrack/Mertk和pAdTrack/TG2/Mertk组Mertk蛋白的表达水平比较差异无统计学意义（P〉0.05）,但均低于pAdTrack/GFP和pAdTrack/TG2组（P〈0.01）,后两者比较差异无统计学意义（P〉0.05）。结论 TG2和Mertk基因共沉默腺病毒干扰载体pAdTrack/TG2/Mertk构建成功,其能显著降低小鼠巨噬细胞样RAW246.7细胞中TG2蛋白和Mertk蛋白的表达水平。     

Toll-like receptor interactions: tolerance of MyD88-dependent cytokines but enhancement of MyD88-independent interferon-beta production

Toll-like receptors (TLRs) signal through two main pathways: a myeloid differentiation factor (MyD)88-dependent pathway that acts via nuclear factor kappaB (NF-kappaB) to induce proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and a MyD88-independent pathway that acts via type I interferons to increase the expression of interferon-inducible genes. Repeated signalling through TLR4 and a number of other TLRs has been reported to result in a reduction in the subsequent proinflammatory cytokine response, a phenomenon known as TLR tolerance. In this study we have shown that, whilst NF-kappaB activation and production of TNF-alpha and interleukin-12 by murine RAW264.7 and J774.2 cells in response to stimulation by TLR4, -5, -7 or -9, was reduced by prior stimulation with TLR4, -5, -7 or -9 ligands, the primary stimulation of TLR3, which does not use the MyD88 pathway, did not reduce the TNF-alpha or interleukin-12 responses to subsequent TLR stimulation. The response to TLR3 stimulation was not diminished by prior TLR ligand exposure. Furthermore, the production of interferon-beta (IFN-beta) following stimulation of TLR3 or -4, which is MyD88-independent, was increased by prior activation of TLR4, -5, -7 or -9. In contrast, TLR9 ligand-induced IFN-beta production, which is MyD88-dependent, was tolerized by prior TLR stimulation. These results are consistent with differential regulation of MyD88-dependent and MyD88-independent cytokine production following serial activation of TLRs.