以细胞因子为研究指标的光致敏体外评价方法的建立

目的 研究和建立以细胞因子水平变化作为评价指标的光致敏体外评价方法.方法 THP-1细胞分别与光致敏剂6-甲基香豆素(6-MC)和对氨基苯甲酸(PABA)、光致敏剂兼光刺激剂硫氯酚(bithionol)、光致敏兼皮肤致敏剂对苯二胺(PPD)和二苯甲酮(BP)、单纯皮肤致敏剂盐酸双氯苯双胍己烷(CHD)和二硝基氯苯(DN...     

以CD54为评价指标的THP-1细胞光致敏体外评价方法的确定和验证

目的 确定并验证以细胞表面标志物CD54作为评价指标的光致敏体外评价方法。方法 将THP-1细胞与多种光致敏剂、光刺激剂、皮肤致敏剂、皮肤刺激剂和阴性受试物分别孵育,在光照射或避光处理后,用流式细胞仪测定细胞表面标志物CD54和CD86的表达水平并统计分析,进一步确定具体的评价指标,并对该方法的准确性、特异性、灵敏度、重复性进行验证。结果 19种光致敏剂中有15种引起照射组THP-1细胞表达CD54的平均荧光强度（MFI）较非照射组显著增加（P<0.05、0.01） ,且照射组细胞表达CD54的相对荧光强度（RFI）值均在1.5以上。光刺激剂经光照射后也可引起细胞表达CD54的MFI显著增加（P<0.01） ,但经过预照射处理后,CD54的表达水平较直接照射组显著下降（P<0.01）。未经光照射条件下,皮肤致敏剂即可引起THP-1细胞表达CD54和CD86的MFI比对照组显著增加（P<0.01） ,光照后CD54的表达反而略有下降。在光照和避光条件下,皮肤刺激剂、阴性受试物（乳酸）均未引起细胞表达CD54或CD86的显著性变化。以CD54为评价指标的THP-1细胞光致敏评价方法检测光致敏剂的准确性、特异性和灵敏度分别是85.2%、100%和78.9%,具有良好的重复性。结论 确定了THP-1细胞光致敏评价方法的细胞表面标志物评价指标为CD54,判定标准为：（1）光照后THP-1细胞表达CD54的MFI较照射前显著性增加;（2）光照后THP-1细胞表达CD54的RFI≥1.5;（3）当上述条件均满足时,对受试物进行预照射处理,结果仍然满足前2条标准。     

支气管哮喘患者血清及痰中IL-8、TNF-α和IL-1-β水平变化的探讨

目的探讨和比较IL-8、TNF-α、IL-1-β等细胞因子在支气管哮喘发病机制中的作用。方法哮喘急性发作期患者31例、正常对照组30例的血清和诱导痰标本,于病情缓解后作自身对照。用放免法分别测定血清和痰液中IL-8、TNF-α、IL-1-β水平。结果急性发作期血清及痰液中IL-8水平显著高于稳定期和对照组(P均<0.01);急性发作期TNF-α与缓解期比较无明显变化(P>0.05),但仍高于对照组;缓解期IL-8、TNF-α显著高于对照组(P均<0.05)。血清及痰液中IL-1-β差异无显著性(P均>0.05)。急性发作期血清中IL-8与TNF-α呈显著正相关(r=0.461,P<0.01),痰液中IL-8与TNF-α也呈显著正相关(r=0.572,P<0.01)。血清和痰液中IL-8呈显著正相关(r=0.553,P<0.01)。结论IL-8、TNF-α参与了气道炎症的形成,在哮喘的发病机制中起重要作用。     

夏枯草对痤疮患者唾液中IL-1α、IL-4、IL-8和TNF-α表达的影响

目的检测夏枯草对中、重度痤疮患者唾液中白细胞介素1α(interleukin-1α,IL-1α)、白细胞介素4(interleukin-4,IL-4)、白细胞介素8(interleukin-8,IL-8)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达的影响。方法对100例中、重度痤疮患者口服夏枯草水煎液治疗,其中Ⅱ级38例,Ⅲ级27例,Ⅳ级35例,每人每日15 g,连续1周。采集非刺激性全唾液,运用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)分别检测患者治疗前后及40名正常对照者唾液中IL-1α、IL-4、IL-8和TNF-α的表达水平。结果痤疮患者唾液中IL-1α、IL-4、IL-8和TNF-α水平较对照组有不同程度的升高,其变化程度均为Ⅳ级>Ⅲ级>Ⅱ级,治疗后IL-1α、IL-4、IL-8和TNF-α水平较治疗前明显降低。结论痤疮的发病与唾液中IL-1α、IL-4、IL-8和TNF-α水平的表达密切相关,说明免疫因素是导致痤疮发生发展的重要环节,夏枯草可能通过调节痤疮患者外周免疫因素起到治疗痤疮的作用。     

脑梗死患者血清TNF-α、IL-6和IL-8水平变化的临床意义分析

目的 分析脑梗死患者急性期与恢复期血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-8(IL-8)水平变化的临床意义。方法 65例脑梗死患者和同期30例健康体检者(对照组),采用双抗体夹心酶联免疫吸附法(ELISA)检测脑梗死患者急性期、恢复期和对照组的血清TNF-α、IL-6和IL-8水平,比较三组间差异。结果 脑梗死急性期患者血清TNF-α、IL-6和IL-8水平均明显高于恢复期和对照组,三组间差异有统计学意义(F值分别为4.654、5.382和3.386,均P<0.05)。结论 脑梗死患者急性期血清TNF-α、IL-6和IL-8水平明显升高,随着病情的恢复呈逐渐下降趋势,有助于判断病情的预后。     

脑梗死患者血清TNF-α、IL-6和IL-8水平变化的临床意义分析

目的 分析脑梗死患者急性期与恢复期血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-8(IL-8)水平变化的临床意义。方法 65例脑梗死患者和同期30例健康体检者(对照组),采用双抗体夹心酶联免疫吸附法(ELISA)检测脑梗死患者急性期、恢复期和对照组的血清TNF-α、IL-6和IL-8水平,比较三组间差异。结果 脑梗死急性期患者血清TNF-α、IL-6和IL-8水平均明显高于恢复期和对照组,三组间差异有统计学意义(F值分别为4.654、5.382和3.386,均P<0.05)。结论 脑梗死患者急性期血清TNF-α、IL-6和IL-8水平明显升高,随着病情的恢复呈逐渐下降趋势,有助于判断病情的预后。     

脑梗死患者血清TNF-α、IL-6和IL-8水平变化的临床意义分析

目的 分析脑梗死患者急性期与恢复期血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-8(IL-8)水平变化的临床意义。方法 65例脑梗死患者和同期30例健康体检者(对照组),采用双抗体夹心酶联免疫吸附法(ELISA)检测脑梗死患者急性期、恢复期和对照组的血清TNF-α、IL-6和IL-8水平,比较三组间差异。结果 脑梗死急性期患者血清TNF-α、IL-6和IL-8水平均明显高于恢复期和对照组,三组间差异有统计学意义(F值分别为4.654、5.382和3.386,均P<0.05)。结论 脑梗死患者急性期血清TNF-α、IL-6和IL-8水平明显升高,随着病情的恢复呈逐渐下降趋势,有助于判断病情的预后。     

乙型肝炎患者血清TNFα、IL-6和IL-8检测及临床意义

目的：研究血清肿瘤坏死因子α（TNFα）白细胞介素-6（IL-6）和白细胞介素-8（IL-8）水平与慢性乙型肝炎（CHB）关系。方法：采用放射免疫分析法（RIA）检测了65例CHB患者及30例健康人血清IL-1β、IL-6及IL-8的浓度。结果：CHB患者血清TNFα、IL-6和IL-8水平均显著高于正常对照组（P〈0．01），CHB患者TNFα、IL-6和IL-8水平增高与病情严重程度呈正相关（P〈0．01）。结论：TNFα、IL-6和IL-8参与CHB免疫病理损伤过程，检测血清TNFα、IL-6和IL-8在判断CHB患者肝损伤程度及预后有临床实用价值。     

急性胰腺炎患者血清IL-1β和TNF-α的水平变化的研究

目的:通过检测急性胰腺炎(acute pancreatitis,AP)患者血清IL-1β和TNF-α的含量的变化,探讨二者与AP的关系。方法:选择AP患者20例,另选10例健康体检者做对照。均于入院后第1天及第7天抽取外周静脉血2mL,采用双抗体夹心ELISA法检测血清IL-1β和TNF-α的含量。结果:入院第1天AP组IL-1β和TNF-α的含量明显增高,与正常对照组比较有显著性差异(P<0.01);治疗7d后,IL-1β和TNF-α含量均下降。结论:IL-1β和TNF-α参与了急性胰腺炎的发生发展过程;急性胰腺炎患者血清中IL-1β和TNF-α的含量均增高。     

慢性支气管炎患者血清IL-6、IL-8和TNF-α水平动态变化研究

目的:研究慢性支气管炎患者血清白细胞介素-6(IL-6)、白细胞介素-8(IL-8)和肿瘤坏死因子-α(INF-α)水平的动态变化及其在慢性支气管炎发病中的作用。方法:检测住院慢性支气管炎患者52例(治疗组)急性期与恢复期血清的IL-6、IL-8和TNF-α水平,并与正常对照组进行比较。结果:治疗组急性期血清IL-6、IL-8和TNF-α水平显著高于恢复期和对照组(P<0.01),恢复期与健康对照组比较差异无显著性(P>0.05);慢性支气管炎并发呼吸衰竭和慢性支气管炎急性发作期患者血清IL-6、IL-8和INF-α的含量明显高于无呼吸衰竭的急性发作期患者(P<0.01),临床症状缓解患者的血清IL-6、IL-8和TNF-α含量仍明显高于对照组(P<0.01),低于急性发作期患者(P<0.01)。结论:血清IL-6与IL-8和TNF-α含量测定可作为评估慢性支气管炎患者病情轻重程度及判断预后的参考指标。     

An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.     

Establishment of an in vitro photoassay using THP-1 cells and IL-8 to discriminate photoirritants from photoallergens

At present, there are no in vivo or in vitro methods developed which has been adopted by regulatory authorities to assess photosensitization induced by chemicals. Recently, we have proposed the use of THP-1 cells and IL-8 release to identify the potential of chemicals to induce skin sensitization. Based on the assumption that sensitization and photosensitization share common mechanisms, the aim of this work was to explore the THP-1 model as an in vitro model to identify photoallergenic chemicals.THP-1 cells were exposed to 7 photoallergens and 3 photoirritants and irradiated with UVA light or kept in dark. Non phototoxic allergens or irritants were also included as negative compounds. Following 24 h of incubation, cytotoxicity and IL-8 release were measured. At subtoxic concentrations, photoallergens produced a dose-related increase in IL-8 release after irradiation. Some photoirritants also produced a slight increase in IL-8 release. However, when the overall stimulation indexes of IL-8 were calculated for each chemical, 6 out of 7 photoallergens tested reached a stimulation index above 2, while the entire set of negative compounds had stimulation indexes below 2. Our data suggest that this assay may become a useful cell-based in vitro test for evaluating the photosensitizing potential of chemicals.     

Synergistic effect of PGD2 via prostanoid DP receptor on TNF-alpha-induced production of MCP-1 and IL-8 in human monocytic THP-1 cells

Prostaglandin (PG) D₂, a major cyclooxygenase metabolite generated predominantly from immunologically stimulated mast cells, is thought to contribute to the pathogenesis of allergic diseases via the two PGD₂ receptors, prostanoid DP receptor and chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2). Monocytes are known to express the prostanoid DP receptor, however, the role of it in inflammatory responses is still unclear. In the present study, to clarify the functional roles of prostanoid DP receptor on monocytes, we examined the effect of PGD₂ on the production of monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 from a human monocytic cell line, THP-1. Single activation of prostanoid DP receptor hardly produced any cytokines or chemokines. However, activation with PGD₂ in the presence of tumor necrosis factor (TNF)-α mediated significant production of MCP-1 and IL-8, but not the other cytokines and chemokines, in comparison to single stimulation with TNF-α. In addition, the selective prostanoid DP receptor antagonist, pinagladin ((Z)-7-[(1R,2R,3S,5S)-2-(benzothiophen-3-ylcarbonylamide)-10-norpinan-3-yl]hept-5-enoic acid) inhibited the production of MCP-1 and IL-8 upon combined stimulation with PGD₂ and TNF-α. The synergistic production of MCP-1 and IL-8 by PGD₂ was mimicked by dibutyryl cAMP (db-cAMP) and was inhibited by a protein kinase A (PKA) inhibitor. Our findings suggest that activation of the prostanoid DP receptor on THP-1 cells enhances TNF-α-induced MCP-1 and IL-8 production via the cAMP/PKA signaling pathway.     

Isoeugenol destabilizes IL-8 mRNA expression in THP-1 cells through induction of the negative regulator of mRNA stability tristetraprolin

We previously demonstrated in the human promyelocytic cell line THP-1 that all allergens tested, with the exception of the prohapten isoeugenol, induced a dose-related release of interleukin-8 (IL-8). In the present study, we investigated whether this abnormal behavior was regulated by the AU-rich element–binding proteins HuR and tristetraprolin (TTP) or by the downstream molecule suppressor of cytokine signaling (SOCS)-3. The contact allergens isoeugenol, diethylmaleate (DEM), and 2,4-dinitrochlorobenzene (DNCB), and the irritant salicylic acid were used as reference compounds. Chemicals were used at concentrations that induced a 20% decrease in cell viability as assessed by propidium iodide staining, namely 100 μg/ml (0.61 mM) for isoeugenol, 100 μg/ml (0.58 mM) for DEM, 3 μg/ml (14.8 μM) for DNCB, and 250 μg/ml (1.81 mM) for salicylic acid. Time course experiments of IL-8 mRNA expression and assessment of IL-8 mRNA half-life, indicated a decreased IL-8 mRNA stability in isoeugenol-treated cells. We could demonstrate that a combination and regulation of HuR and TTP following exposure to contact allergens resulted in a different modulation of IL-8 mRNA half-life and release. The increased expression of TTP in THP-1 cells treated with isoeugenol results in destabilization of the IL-8 mRNA, which can account for the lack of IL-8 release. In contrast, the strong allergen DNCB failing to up-regulate TTP, while inducing HuR, resulted in longer IL-8 mRNA half-life and protein release. SOCS-3 was induced only in isoeugenol-treated cells; however, its modulation did not rescue the lack of IL-8 release, indicating that it is unlikely to be involved in the lack of IL-8 production. Finally, the destabilization effect of isoeugenol on IL-8 mRNA expression together with SOCS-3 expression resulted in an anti-inflammatory effect, as demonstrated by the ability of isoeugenol to modulate LPS or ionomycin-induced cytokine release.     

Identification of herbal components as TRPA1 agonists and TRPM8 antagonists

Transient receptor potential (TRP) channels are non-selective cation channels that are implicated in analgesia, bowel motility, wound healing, thermoregulation, vasodilation and voiding dysfunction. Many natural products have been reported to affect the activity of TRP channels. We hypothesize that numerous traditional herbal medicines (THMs) might exert their pharmacological activity through modulating the activity of TRP channels. The present study aimed to evaluate the effects of flavonoid aglycones and their glycosides, which are the main components of many THMs, on the TRP channel subtypes. A Ca²⁺ influx assay was performed using recombinant human TRPA1, TRPV1, TRPV4 and TRPM8 cell lines. Our findings showed that flavonoid aglycones and glycycoumarin activated TRPA1. In particular, isoflavone and chalcone compounds displayed potent TRPA1 agonistic activity. Furthermore, flavone aglycones showed concomitant potent TRPM8 inhibiting activity. Indeed, flavone, isoflavone aglycones, non-prenylated chalcones and glycycoumarin were found to be TRPM8 inhibitors. Hence, flavonoid aglycones metabolized by lactase-phlorizin hydrolase and β-glucosidase in the small intestine or gut microbiota of the large intestine could generate TRPA1 agonists and TRPM8 antagonists.

     

Effect of Mahonia aquifolium active compounds on interleukin-8 production in the human monocytic cell line THP-1

The effect of the crude extract and of two alkaloid fractions prepared from Mahonia aquifolium on interleukin-8 (IL-8) production in the lipopolysaccharide (LPS)-stimulated human monocytic cell line THP-1 was studied. The production of IL-8 by cells stimulated with 20 ng/ml LPS after 48 h treatment with 20 microg/ml crude extract was inhibited by about 30 %. LPS-stimulated cells treated with 0.1 microg/ml bisbenzylisoquinoline alkaloid fraction exhibited a 40% inhibition of IL-8 production in comparison with control cells. The fraction of protoberberine alkaloids had no significant inhibitory activity. Weak or no inhibition of IL-8 production was found after treatment with 0.5 microg/ml of protoberberine alkaloids berberine and jatrorrhizine and with berbamine from the group of BBI alkaloids. In contrast, the production was inhibited after treatment with BBI alkaloids baluchistine (about 20%) and aromoline (up to 30%).     

Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro

The local lymph node assay (LLNA) has been developed to assess skin sensitization, and based on the EC3 value, it can also be used to evaluate allergen potency. Therefore, in the development of in vitro alternatives to the LLNA assay, one should not only consider the hazard identification but also the possibility to classify allergens relatively to their potency.We have recently described a selective release of interleukin-8 (IL-8) by chemical allergens in THP-1 cell line, and identified the activation of p38 mitogen-activated protein kinase (p38 MAPK) as a common pathway. Therefore, the purpose of this study was to expand the number of chemicals tested and to investigate whether IL-8 production and p38 MAPK activation can be used to classify allergens according to their potency.THP-1 cells were exposed to the contact allergens (p-benzoquinone, 2-aminophenol, isoeugenol, diethyl maleate, citral and imidazolidinyl urea), selected according to their potency in the LLNA, and to lactic acid and propylene glycol as non-sensitizers. p38 MAPK activation was evaluated 5–15 min after treatment by FACS analysis, while IL-8 release was assed by ELISA following 24 h of incubation. p38 MAPK was activated by all contact allergens, including the pro-apten isoeugenol, whereas IL-8 release was significantly increased after stimulation with all allergens tested, except for isoeugenol. The failure of isoeugenol may be due to decrease in the stability of IL-8 mRNA. Irritants exposure, as expected, failed to induce both p38 MAPK activation and IL-8 release.A significant correlation between IL-8 release and the LLNA EC₃ was found (Pearson correlation r = 0.743, p = 0.0036, n = 12). On the contrary, the activation of p38 MAPK showed no significant correlation between LLNA data and vigor of p38 MAPK activation.Overall, data presented confirm our previous observations and reveal IL-8 as potential tool not only to identify sensitizers, with the exception of pro-haptens, but also to classify them according to their potency, while p38 MAPK activation allows the identification of all sensitizers, including pro-haptens, but was not useful for potency classification.     

黄芩苷对单核THP-1细胞趋化功能的影响

目的:观察黄芩苷对单核细胞THP-1趋化功能的影响。方法:采用流式细胞仪分析单核THP-1细胞上趋化因子CXCR4,CCR1受体的表达,用细胞趋化实验观察黄芩苷对单核细胞THP-1趋化功能的影响。结果:单核THP-1细胞表面表达趋化因子CXCR4,CCR1受体,30μmol·L~(-1)和100μmol·L~(-1)黄芩苷明显抑制趋化因子SDF-1α引起的细胞趋化,而对趋化因子RANTES诱导的细胞趋化无影响,对血清诱导的细胞趋化也没有明显的影响,黄芩苷本身对THP-1细胞没有明显的趋化作用。结论:黄芩苷对趋化因子SDF-1α/CXCR4诱导的单核细胞趋化有明显的抑制作用。