Xijiao Dihuang Decoction(犀角地黄汤)and Rehmannia glutinosa Libosch.Protect Mice against Lipopolysaccharide and Tumor Necrosis Factor Alpha-Induced Acute Liver Failure

Objective: To investigate the hepatoprotective effect of Xijiao Dihuang Decoction(犀角地黄汤,XJDHD) on lipopolysaccharide(LPS)-and tumor necrosis factor alpha(TNF-α)-induced acute liver failure(ALF)as well as the underlying mechanism of action, and to clarify the key herbs and components of XJDHD. Methods:LPS/D-galactosamine(D-GalN) or TNF-α/D-GalN were intraperitoneally injected into C57BL/6J mice to induce ALF. Simultaneously, XJDHD or its individual herbs and components were orally administered. Survival rates, transaminase levels in serum, and hepatic histology were examined to evaluate the effects of XJDHD.The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay and real-time polymerase chain reaction were additionally performed to expound the mechanism underlying the anti-apoptotic activity of XJDHD. Results: Oral administration of XJDHD protected mice from lethal liver failure induced by LPS and TNF-α, with notable amelioration of liver injury in histology and a significant decrease in transaminase levels in serum. XJDHD signi?cantly inhibited apoptosis of hepatocytes and enhanced expression of the antiapoptosis genes, c-Flip, Iap1, Gadd45 b and A20(all P<0.05). In addition, Rehmannia glutinosa Libosch. was identi?ed as the key herb of XJDHD and galactose as the effective component of Rehmannia glutinosa Libosch.that protects against ALF. Conclusions: XJDHD inhibits TNF-α-induced apoptosis of hepatocytes by promoting the expression of nuclear factor κB-regulated anti-apoptotic genes. Rehmannia glutinosa Libosch. may be the most effective herb of XJDHD and galactose is an active component in this protection.

Effect of surfactant protein A on lipopolysaccharide-induced tumor necrosis factor-α expression in human proximal tubular epithelial cells

Background Surfactant protein A （SP-A） contributes to the regulation of sepsis-induced acute lung injury.In a previous study,we demonstrated the expression and localization of SP-A in the kidneys.The present study evaluated the effect of SP-A on lipopolysaccharide （LPS）-induced tumor necrosis factor-α （TNF-α） expression and its underlying mechanisms in the human renal tubular epithelial （HK-2） cells.Methods Indirect immunofiuorescence assay was used to detect SP-A distribution and expression in HK-2 cells.HK-2 cells were treated with various concentrations of LPS （0,0.1,1,2,5,and 10 mg/L） for 8 hours and with 5 mg/L LPS for different times （0,2,4,8,16,and 24 hours） to determine the effects of LPS on SP-A and TNF-α expression.Then,HK-2 cells were transfected with SP-A siRNA to analyze nuclear factor κB （NF-κB） P65 and TNF-α expression of HK-2 cells after LPS-treatment.Results Indirect immunofluorescence assay revealed that SP-A is localized to the membrane and cytoplasm of HK-2 cells.Interestingly,SP-A1/SP-A2 and TNF-α expression were found to be significantly increased in HK-2 cells upon LPS treatment.Transfection of LPS-treated HK-2 cells with SP-A siRNA resulted in significant increases in the levels of NF-κB P65 protein and TNF-α mRNA and protein compared to those in non-transfected LPS-treated HK-2 cells.Conclusion SP-A plays an important role in protecting cells against sepsis-induced acute kidney injury by inhibiting NF-κB activity to modulate LPS-induced increase in TNF-α expression.

Activation of p^(38) mitogen activated protein kinase induced by lipopolysaccharide and its role in TNF a gene expression

Objective: To study the molecular mechanisms of TNF--a expression induced by lipopolysaccharide (LPS) for exploring novel methods to prevent or treat clinical patients with endotoxic shock. Methods:Protein kinase assay was used to detect the kinase activity stimulated by LPS; Con focal laser scan technique was used to show the translocation of p38 on the activation; RT PCR and reporter gene system were used to study the molecular mechanism of TNF -a gene transcription. Results: In RAW cells it was found that p38 was activated on the stimulation of LPS. and activated p38 moved into nucleus from cytosol; TNF--a mRNA increased on the stimulation of LPS and the increased promoter transactivity induced by LPS could be inhibited significantly by specific inhibitor for p38. Conclusion: p38 mitogen activated protein kinase (MAPK ) was activated by the stimulation of LPS,which brought about its entry to the nucleus to act on transcription factors to regulate cellular processes. p38 MAPK Is an important regulator of TNF--a gene expression induced by LPS.

Naofen promotes TNF-α-mediated apoptosis of hepatocytes by activating caspase-3 in lipopolysaccharide-treated rats

AIM:To investigate whether naofen is involved in tumor necrosis factor(TNF)-α-mediated apoptosis of hepatocytes induced by lipopolysaccharide(LPS).METHODS:In vivo,rats were treated with LPS or antiTNF-αantibody,whereas in vitro,primary hepatocytes and Kupffer cells(KCs)were separately isolated from rat livers using collagenase perfusion,and primary hepatocytes were cultured in medium containing LPS or TNF-α,or in conditioned medium from LPS-treated KCs(KC-CM)/KC-CM+anti-TNF-αantibody.Naofen and TNF-αmRNA expression was examined by realtime reverse transcription-polymerase chain reaction.Immunoblotting was used to measure protein expression.Hepatocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assay.RESULTS:LPS significantly induced both naofen expression and caspase-3 activity in the rat liver,which coincided with an increase in the number of TUNELpositive hepatocytes.The increase of TNF-αexpression induced by LPS was preceded by increases in naofen and caspase-3 activity.Elevation of naofen expression and caspase-3 activity was abrogated by pretreatment with anti-TNF-αantibody.In KCs,LPS caused an increase in TNF-αthat was almost consistent with that in the liver of LPS-treated rats.In hepatocytes,neither LPS nor TNF-αalone affected either naofen expression or caspase-3 activation.The incubation of hepatocytes with KC-CM significantly enhanced both naofen expression and caspase-3 activity.Moreover,the effects of the KC-CM-induced increase in naofen expression and caspase-3 activity were blocked by anti-TNF-αantibody.CONCLUSION:TNF-αreleased from KCs treated with LPS may induce hepatic naofen expression,which then stimulates hepatocellular apoptosis through activation of caspase-3.

Influence of simvastatin on dopaminergic neurons of lipopolysaccharide—induced rat model of Parkinson's disease

Objective::To investigate the neuroprotective effects of simvastatin on lipopolysaccharide(LPS)-indueed rat model of Parkinson's disease(PD) and the mechanisms involved.Methods:Hemiparkinsonian rat models were induced by stereotaxieal injection of LPS in the right substantia nigra compacts.After 2 weeks of simvastatin treatment,rotational behavior test was performed after the intraperitoneal injection of apomorphine.Expression of tyroxine hydroxylase(TH) and glial fibrillan acidic protein were analyzed through immunohistochemical staining of substantia nigra and striatum,and the level of TNF-α was evaluated using enzyme-linked immunosorbent assay.Results:Comparing with untreated group,behavioral symptoms of the rats were significantly less in the rats that received simvastatin treatment.The TH positive cell count in substantia nigra and striatum were significantly increased(P<0.05) and TNF- α expression was significantly decreased(P<0.05) in simvastatin group compared to untreated group.Conclusions:Simvastatin could effectively inhibit the activation of astrocytes,reduce TNF-α expression,and exert anti-inflammatory effects,and thus protect the dopaminergic neurons in substantia nigra and striatum of the rat model of PD.

Tea polyphenols inhibit expressions of iNOS and TNF-a and prevent lipopolysaccharide-induced liver injury in rats

BACKGROUND: Tea polyphenols have been shown to protect against carbon tetrachloride ( CCl4) -induced liver injury, liver fibrosis, hepatic ischemia-reperfusion injury. In this study, we examined the effect of tea polyphenols on lipopolysaccharide ( LPS ) -induced liver injury, and explored its mechanisms. METHODS: Sprague-Dawley rats received tea polyphenols (100 mg · kg-1·d-1) or vehicle (water) intragastrically by gavage for 14 days, followed by LPS (5 mg/kg) or saline injection intraperitoneally. Liver injury was assessed by biochemical assay and pathological analysis. Serum tumor necrosis factor-α (TNF-α) levels and liver malondialdehyde (MOA) contents were determined. Inducible nitric oxide synthase (iNOS) protein and TNF-α, iNOS and en-dothelial nitric oxide synthase (eNOS) mRNA expressions in the liver were detected by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: Administration of LPS resulted in liver injury in rats, evidenced by elevated activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), hepatocellular necrosis, and neutrophil infiltration in the liver. These responses were associated with increased serum TNF-α levels, induced iNOS protein, expressions of TNF-α, iNOS mRNA in the liver and elevated lipid peroxidation at 90 minutes or 6 hours after LPS injection. Pretreatment with tea polyphenols attenuated LPS-induced liver injury, and blunted the rises of serum TNF-α levels and lipid peroxidation and the induction of expressions of TNF-α, iNOS in the liver. CONCLUSION: Tea polyphenols prevent LPS-induced liver injury, and the mechanisms may involve the reduction of serum TNF-α levels and lipid peroxidation and the suppression of TNF-α, iNOS expressions in the liver.

Temporal dynamic changes of connexin 43 expression in C6 cells following lipopolysaccharide stimulation

Connexin 43, a gap junction protein, is expressed mainly in glia in the central nervous system. Neuroinflammation plays an important role in central nervous system injury. Changes to glial connexin 43 levels and neuroinflammation may trigger brain injury and neurodegenerative diseases To illustrate the relationship between connexin 43 and neuroinflammation, this study investigated how connexin 43 expression levels change in lipopolysaccharide-stimulated rat C6 glioma cells. C6 cells were treated with 0.05, 0.25, 0.5, 1,2.5 and 5 IJg/mL lipopolysaccharide for 24 hours. The nitrite estimation-detected nitric oxide release level was elevated substantially after lipopolysaccharide stimulation. To test the transcriptional level changes of inducible nitric oxide synthase, tumor necrosis factor-a and connexin 43 mRNA, C6 cells were treated with 5 pg/mL lipopolysaccharide for 3 48 hours. Reverse transcription-PCR showed that the expression of inducible nitric oxide synthase and tumor necrosis factor-a mRNA increased over time, but connexin 43 mRNA levels increased in lipopolysaccharide-stimulated C6 cells at 3 and 6 hours, and then decreased from 12 to 48 hours. Connexin 43 protein expression was detected by immunofluorescence staining, and the protein levels matched the mRNA expression levels. These results suggest that connexin 43 expression is biphasic in lipopo^ysacchadde-induced neuroinflammation in C6 cells, which may be correlated with the connexin 43 compensatory mechanism.

Effects of morphine and fentanyl on tumor necrosis factor-α and interleukin-6 concentrations in human whole blood in vitro

Cytokines are essential for hematopoiesis and immune responses, and play a key role in the defense against infections. Lipopolysaccharide ( LPS) is a potent inducer of the agents involved in the pathogenesis of inflammation responses. Tumor necrosis factor-α ( TNF-α ) and interleukin-6 (IL-6) are two important proinflammatory

Characterization of a small molecule inhibitor of tumor necrosis factor-alpha production

Background Numerous studies have shown that reducing the level of tumor necrosis factor-alpha （TNFα） through the use of anti-TNF antibodies or soluble TNF receptor is a safe and efficacious treatment to inflammatory diseases such as rheumatoid arthritis. Therefore, novel approaches to achieve this outcome are desired. The aim of this study was to investigate the characterization of a small molecule inhibitor, Y316, which blocks TNF mRNA upregulation and TNF production by lipopolysaccharides （LPS） stimulated monocytes. Methods Peripheral blood mononuclear cells （PBMC） from healthy volunteers were plated in 24-well plates and stimulated with LPS （1 pg/ml）, phorbol-12-myristate-13-acetate （PMA） （100 ng/ml）, zymosan （10 IJg/ml） and Tsst （100 ng/ml）. Supernatants were collected after 4-hour culture at 37℃, and quantitative determination of TNFα, interleukin-113 （IL-1β）, IL-6, IL-8, IL-10 and IL-2 production in the supernatants was performed by colorimetric enzyme-linked immunosorbent assay （ELISA）. Total RNA of PBMC was isolated and cytokine mRNA quantitation was performed by using a RNA level measuring kit （R ＆ D Systems）. PBMC were pretreated with Y316 （10 μmol/L, 1 μmol/L, 0.1 μmol/L, 0.01 μmol/L and 0.001 μmOl/L） or dimethyl sulfoxide at 37℃ for 10 minutes, and then stimulated with LPS or PMA, protein concentrations of p44.42, IKBa, P38 and Jun NH2-terminat kinase were determined by Western blotting. Cyclic adenosine-3＇,5＇-monophosphate （cAMP） of PBMC was measured by enzyme immunoassay kit （Amersham Pharmacia Biotech）. Results Y316 blocked TNF production and inhibited the upregulation of TNF mRNA levels in response to LPS, and also prevented the production of IL-1 and IL-6. In contrast, Y316 augmented the production of IL-10 in LPS-stimulated monocytes. Y316 failed to prevent the production of IL-2 and TNF in antigen-stimulated T cells, suggesting that its effects may be cell-type specific. Y316 prevented the phosphorylation and activation of the MAPK, ERK, and therefore appeared to mediate its effects on TNF by acting at an early point in the signaling cascade induced in response to LPS. There was no effect of Y316 on cAMP levels either alone or in the presence of LPS. Conclusions Y316 appears to be a small molecule inhibiting TNF production, which may act via a novel mechanism. Identification of the target of Y316 may lead to the development of alternative strategies for achieving selective cytokine inhibition.

三七总皂苷通过p38 MAPK通路抑制内毒素诱导的小胶质细胞活化

目的:探讨三七总皂苷(PNS)通过p38丝裂原活化蛋白激酶(p38 MAPK)通路对脂多糖(LPS)诱导活化的BV2小胶质细胞中肿瘤坏死因子-α(TNF-α)表达的影响。方法:将BV2小胶质细胞分为空白对照组(Control)、LPS激活组和LPS+三七总皂苷干预组(LPS+PNS)。CCK-8法检测BV2小胶质细胞的活力,确定最适合的药物干预浓度。利用Western Blot和免疫荧光检测BV2小胶质细胞中p38 MAPK和TNF-α的表达及p38 MAPK磷酸化水平(p-p38 MAPK)变化。结果:与空白对照组相比,PNS对BV2小胶质细胞的细胞活力无显著差异,最终选定100 mg/L作为药物干预浓度。Western Blot、免疫荧光结果提示,LPS激活后,BV2小胶质细胞中TNF-α的表达显著升高,p38 MAPK磷酸化水平增加(P<0.05);PNS干预后,与LPS激活组相比,TNF-α表达显著下降,p38 MAPK磷酸化水平降低(P<0.05)。使用p38 MAPK通路抑制剂(SB203580)作用后,PNS联合SB203580组(LPS+PNS+SB203580)中,TNF-α表达及p38 MAPK磷酸化水平变化与LPS+PNS组相比无显著差异(P>0.05)。此外,p38 MAPK在各组的变化无显著性差异(P>0.05)。结论:PNS可能通过p38 MAPK通路抑制活化的BV2小胶质细胞分泌的炎性因子TNF-α的表达。

Cytokine and nitric oxide production by rat microglia stimulated with lipopolysaccharides in vitro

目的：研究ＬＰＳ刺激体外培养的新生大鼠小胶质细胞产生ＩＬ１，ＩＬ２，ＴＮＦα和ＮＯ的特征．方法：小胶质细胞与ＬＰＳ（０－１０ｍｇ·Ｌ－１）孵育０－７２ｈ，分别测定细胞外和细胞内的ＩＬ１，ＩＬ２和ＴＮＦα的生物活性和细胞外ＮＯ水平．结果：ＩＬ１，ＴＮＦα和ＮＯ分别在ＬＰＳ刺激后１，４，和８ｈ检测到，并在８，２４和２４ｈ达到峰值．ＬＰＳ１ｍｇ·Ｌ－１刺激细胞外ＩＬ１，ＴＮＦα和ＮＯ的产生最高，但细胞内ＴＮＦα水平极低，ＬＰＳ未能刺激ＩＬ２产生．结论：体外ＬＰＳ刺激大鼠小胶质细胞产生大量炎性细胞肽和ＮＯ．

脂多糖和肿瘤坏死因子α对人牙髓干细胞增殖和成骨分化的影响

目的探讨脂多糖(LPS)和肿瘤坏死因子α(TNF-α)对人牙髓干细胞(hDPSCs)增殖和分化的影响。方法体外分离培养hDPSCs,分别用不同浓度LPS(0,0.1,1,10μg/mL)和TNF-α(0,1,10,100 ng/mL)培养细胞。培养24,48,72 h,应用细胞计数试剂盒-8(CCK-8)检测hDPSCs增殖活性变化;培养7,14,21 d应用茜素红(AR)染色试剂盒和5-溴-4-氯-3-吲哚-磷酸盐(BCIP)/氯化硝基四氮唑蓝(NBT)碱性磷酸酯酶(ALP)显色试剂盒检测hDPSCs肉眼观AR染色变化、钙结节定量、ALP染色和ALP活性等成骨分化指标。结果①CCK-8实验显示1,10,100 ng/mL TNF-α作用于hDPSCs 24,48,72 h后细胞增殖活力降低(P<0.05)。AR成骨诱导染色结果显示培养7,14 d,TNF-α各浓度组肉眼观AR染色无明显差异;培养21 d,10 ng/mL和100 ng/mL TNF-α组矿化程度相比0 ng/mL组低(P<0.05)。ALP染色和ALP活性试剂盒分析显示诱导培养7,14,21 d后,相比0 ng/mL组,1 ng/mL,10 ng/mL和100 ng/mL TNF-α组ALP染色变浅,且ALP活性降低(P<0.05)。②CCK-8实验显示不同浓度LPS作用hDPSCs 24 h和48 h后细胞增殖活性没有明显差异,而1μg/mL和10μg/mL组LPS作用hDPSCs 72 h后OD_(450)值大于0μg/mL组(P<0.05)。ALP成骨诱导染色显示培养7,14,21 d,不同浓度LPS作用后ALP染色和ALP活性变化不明显。AR成骨诱导染色显示培养7 d,LPS各浓度组的矿化程度不明显;10μg/mL LPS培养14,21 d矿化程度较0μg/mL低(P<0.05)。结论LPS对hDPSCs成骨分化的影响取决于LPS浓度和作用时间,高浓度LPS促进hDPSCs增殖。TNF-α抑制hDPSCs的增殖和成骨分化。

鹰嘴豆芽素A对P38αMAPK抑制活性的研究

目的 探讨鹰嘴豆芽素A对靶酶丝裂原激活蛋白激酶P38α亚型(P38αMAPK)的抑制作用。方法 应用软件Autodock Vina将鹰嘴豆芽素A与P38αMAPK晶体三维结构1KV2的活性位点进行分子对接,而后利用脂多糖(LPS)诱导的小鼠RAW 264.7细胞炎症模型验证鹰嘴豆芽素A对P38αMAPK的抑制活性。结果 鹰嘴豆芽素A可结合于靶酶晶体结构1KV2的活性位点以干扰正常底物的进入,从而对P38αMAPK的生物学功能起到抑制作用;细胞炎症模型实验证明,与LPS模型组比较,阳性对照药地塞米松和鹰嘴豆芽素A单体浓度各剂量(25、50、100μmol/L)干预后可明显下调细胞上清液中一氧化氮和肿瘤坏死因子-α水平(P<0.05)。结论 鹰嘴豆芽素A可作用于P38αMAPK活性位点而发挥抗炎的药理活性。

金合欢素对丝裂原蛋白激酶p38α活性抑制作用的研究

目的探讨金合欢素对丝裂原蛋白激酶p38α(p38αMAPK)的抑制作用。方法应用分子对接软件Autodock Vina将金合欢素与靶酶三维晶体结构1KV2的活性位点进行分子对接,而后利用脂多糖诱导的小鼠RAW 264.7细胞炎症模型验证金合欢素对p38αMAPK的抑制作用。结果金合欢素可结合于1KV2的活性位点处而阻碍底物三磷腺苷的进入,对p38αMAPK的生物学功能产生抑制作用。与模型组相比,阳性对照药地塞米松和金合欢素不同浓度干预后可明显下调小鼠RAW 264.7细胞炎症模型细胞上清液中一氧化氮和肿瘤坏死因子-α水平(P<0.05)。结论金合欢素可能是通过作用于p38αMAPK活性位点而发挥抗炎的药理活性。

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.

Lipopolysaccharide “Two-hit” Induced Refractory Hypoxemia Acute Respiratory Distress Model in Rats

To establish a stable and reliable model of refractory hypoxemia acute respiratory distress syndrome （ARDS） and examine its pathological mechanisms, a total of 144 healthy male Wistar rats were randomized into 4 groups： group Ⅰ （saline control group）, group Ⅱ （LPS intravenous ＂single-hit＂ group）, group Ⅲ （LPS intratracheal ＂single-hit＂ group） and Group IV （LPS ＂two-hit＂ group）. Rats were intravenously injected or intratracheally instilled with a large dose of LPS （10 mg/kg in 0.5 mL） to simulate a single attack of ARDS, or intraperitoneally injected with a small dose of LPS （1 mg/kg） followed by tracheal instillation with median dose of LPS （5 mg/kg） to establish a ＂two-hit＂ model. Rats in each group were monitored by arterial blood gas analysis and visual inspection for three consecutive days. Arterial blood gas values, lung wet/dry weight ratio and pathological pulmonary changes were analyzed to determine the effects of each ALI/ARDS model. Concentrations of TNF-α, IL-1 and IL-10 in the bronchoalveolar lavage fluid （BALF） and blood plasma were meastired by using enzyme-linked immunosorbent assays （ELISA）. Our resulsts showed that single LPS-stimulation, whether through intravenous injection or tracheal instillation, could only induce ALl and temporary hypoxemia in rats. A two-hit LPS stimulation induces prolonged hypoxemia and specific pulmonary injury in rats, and is therefore a more ideal approximation of ARDS in the animal model. The pathogenesis of LPS two-hit-induced ARDS is associated with an uncontrolled systemic inflammatory response and inflammatory injury. It is concluded that the rat ARDS model produced by our LPS two-hit method is more stable and reliable than previous models, and closer to the diagnostic criteria of ARDS, and better mimics the pathological process of ARDS.

微小RNA-142-3p靶向肿瘤坏死因子-α对脂多糖诱导的人鼻黏膜上皮细胞炎症反应的影响

目的探讨微小RNA(miR)-142-3p对脂多糖(LPS)诱导的人鼻黏膜上皮细胞(HNEPC)炎症反应的影响及其与TNF-α的靶向关系。方法选取HNEPC为研究对象,使用LPS诱导建立炎症反应模型。将miR-142-3p上调、miR-142-3p下调、空质粒转染至细胞,分为对照组、LPS组、miR-142-3p上调组、miR-142-3p下调组、空质粒组;将TNF-α下调、下调对照质粒分别与miR-142-3p上调质粒共同转染至细胞,记为miR-142-3p上调+TNF-α下调组、空质粒+TNF-α下调组、miR-142-3p上调组和空质粒组。采用四甲基偶氮唑蓝(MTT)检测细胞增殖能力;采用实时荧光定量聚合酶链反应(RT-qPCR)检测miR-142-3p和白细胞介素(IL)-6、IL-10、干扰素γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)mRNA水平;采用酶联免疫吸附试验检测细胞上清液中IL-6、IL-10、IFN-γ、TNF-α含量;通过TargetScan网站、荧光素酶报告实验分析miR-142-3p与TNF-α的靶向关系。结果LPS组细胞增殖能力高于对照组,miR-142-3p上调组细胞增殖能力高于空质粒组,miR-142-3p下调组细胞增殖能力低于空质粒组,差异有统计学意义(P<0.05)。LPS组细胞IL-6、IL-10、IFN-γ及TNF-α蛋白及其mRNA表达水平高于对照组,miR-142-3p上调组细胞相关炎性因子表达水平高于空质粒组,miR-142-3p下调组细胞相关炎性因子表达水平低于空质粒组,差异有统计学意义(P<0.05)。TNF-α下调组细胞IL-6、IL-10、IFN-γ及TNF-αmRNA及细胞上清液中蛋白表达水平低于下调对照组,差异有统计学意义(P<0.05)。miR-142-3p与TNF-α具有良好的靶向关系。结论miR-142-3p在慢性鼻窦炎伴鼻息肉患者鼻黏膜组织中高表达,过表达miR-142-3p可促进LPS诱导HNEPC炎性反应,可能与上调TNF-α基因表达有关。

宫颈炎症状态对HPV感染的影响研究

目的:探讨宫颈炎症状态对人乳头瘤病毒(human papilloma virus,HPV)感染的影响。方法:运用E6-c-Myc-P2A-E7-c-3Flag基因慢病毒分别感染对照组(未处理的细胞)及实验组[经脂多糖(1μg/mL)诱导24 h的细胞],观察宫颈炎症状态对Myc-E6、Flag-E7标签蛋白表达的影响。结果:与对照组比较,实验组中人白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、IL-1β、IL-17A mRNA的表达显著升高,差异有统计学意义(均P<0.05)。E6-c-Myc-P2A-E7-c-3Flag基因慢病毒感染72 h后,实验组和对照组转染效率分别是(89.05±0.16)%和(79.30±1.97)%,差异有统计学意义(P<0.05);实验组中Myc-E6和Flag-E7标签蛋白表达水平比对照组显著增加,差异有统计学意义(均P<0.05)。结论:宫颈上皮细胞处于炎症状态下更易感染HPV。

过表达AT2R对LPS诱导的AML12细胞炎症反应的影响

目的探究过表达血管紧张素Ⅱ2型受体(AT2R)对脂多糖(LPS)诱导的小鼠肝细胞(AML12细胞)炎症反应的影响。方法以乳鼠组织器官为样本,通过PCR扩增出含有EcoRⅠ和HindⅢ双酶切位点的AT2R目的基因片段,通过酶切、连接得到最终产物,挑选出的阳性克隆经测序鉴定;将pCMV-Flag-N-AT2R质粒转染至HEK 293T细胞,24 h后采用Western blot法检测Flag蛋白的表达;通过Western blot和激光共聚焦显微镜观察AML12细胞上的AT2R表达;对AML12细胞进行不同的处理,分为对照组、LPS处理组、pCMV-Flag-N-AT2R组和pCMV-Flag-N-AT2R+LPS组,通过CCK-8检测细胞活力;Western blot检测PCNA蛋白,观察细胞增殖;qPCR检测细胞炎性因子水平;Western blot检测细胞核中转录因子NF-кB(p65)表达水平。结果EcoRⅠ和HindⅢ双酶切鉴定以及测序结果表明pCMV-Flag-N-AT2R质粒构建成功,Western blot法的检测结果表明AT2R蛋白成功表达;激光共聚焦观察到AML12细胞上有AT2R受体,AT2R重组质粒可以在AML12上表达;与对照组比较,LPS处理的AML12细胞中细胞的活力和增殖能力减弱,而白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平升高,核转录因子NF-кB(p65)表达水平升高;与LPS组比较,pCMV-Flag-N-AT2R和LPS联合处理的AML12细胞中细胞的活力和增殖能力增强,而IL-6和TNF-α水平降低,核转录因子NF-кB(p65)表达水平降低。结论成功构建了AT2R过表达质粒,并在AML12细胞上成功表达,且AT2R可以抑制LPS诱导的AML12细胞的炎症反应。