铁皮石斛多糖对RAW264.7细胞分泌TNF-α的影响

目的研究铁皮石斛多糖(polysaccharides from Den-drobium officinale,DOP)对小鼠巨噬细胞系RAW264.7细胞分泌TNF-α的影响并探讨其作用机制。方法 MTT法检测细胞活性;ELISA法检测TNF-α的分泌水平;反转录聚合酶链反应(RT-PCR)检测TNF-αmRNA表达量;Western blot技术检测胞质I-κBα蛋白的表达量。结果 DOP在20～160mg·L-1范围内明显促进RAW264.7细胞增殖,无细胞毒性;与空白对照组比较,DOP剂量依赖性地促进TNF-α的分泌;上调TNF-αmRNA表达;Western blot结果显示DOP能明显降低胞质内I-κBα蛋白水平。结论 DOP能增强RAW264.7细胞分泌TNF-α,其作用机制可能与降低胞质内I-κBα蛋白水平,活化核转录因子NF-κB,诱导TNF-αmRNA的表达有关。     

Biocompatible hydrogel based on aldehyde-functionalized cellulose and chitosan for potential control drug release

The present study aimed to prepare cellulose-based biodegradable hydrogel that revealing a potential control of drug release. To achieve this purpose, activation of cellulose was achieved firstly by pretreatment stage using ZnCl₂. Then, selective oxidation of activated cellulose was performed by using sodium periodate to achieve cleavage of the bond between C2 – C3 in the glucose unit generating two active aldehyde groups. The modified cellulose (Dialdehyde cellulose, DAC) was reacted with chitosan, in different molar ratios, via condensation between aldehyde and primary amine groups situated onto cellulose and chitosan, respectively. The activated cellulose, DAC, and produced hydrogels were characterized via FTIR, XRD, and SEM to detect the emerging functional groups as well as the variation of crystallinity and surface morphology features of activated cellulose, DAC, and hydrogels. The swelling capacities of hydrogels showed the optimum value at pH = 7.0. The prepared hydrogels cytotoxicity was investigated toward normal skin fibroblast by MTT assay. It was found that the cells treated with chitosan/DAC hydrogels with ratio 1:1, 1:2, and 1:3 affected by 1.3, 1.8, and 0%, respectively. Streptomycin was incorporated in the prepared hydrogels and its release was evaluated. The DAC ratio was played a key role in controlling the release process. Moreover, further studies were carried out on chitosan (PDB: 2RVA) to evaluate its potential interaction with DAC and streptomycin with binding energies ?4.4 and - 4.3 kcal/mol with short bond lengths 1.3 and 1.944 Å, respectively.     

Psidium guajava extract inhibits thymus and activation-regulated chemokine (TARC/CCL17) production in human keratinocytes by inducing heme oxygenase-1 and blocking NF-κB and STAT1 activation

Psidium guajava (P. guajava) is a food and medicinal plant with antioxidant, anti-inflammatory, and anti-allergic activities that support its traditional uses. The aim of this study was to determine the effects of P. guajava ethyl acetate extract (PGEA) on atopic dermatitis and to investigate the possible mechanisms by which PGEA inhibits cytokine-induced Th2 chemokine expression in HaCaT human keratinocyte cells. We found that PGEA suppressed the IFN-γ/TNF-α-co-induced production of thymus and activation-regulated chemokine (TARC) protein and mRNA in HaCaT cells. Additionally, PGEA inhibited the TNF-α/IFN-γ-co-induced activation of NF-κB and STAT1 and increased the expression of heme oxygenase-1 (HO-1) protein and mRNA. HO-1 inhibitor enhanced the suppressive effects of PGEA on TNF-α/IFN-γ-co-induced TARC production and gene expression. Collectively, these data demonstrate that PGEA inhibits chemokine expression in keratinocytes by inducing HO-1 expression and it suggests a possible therapeutic application in atopic dermatitis and other inflammatory skin diseases.     

Protective effect of diosmin on LPS-induced apoptosis in PC12 cells and inhibition of TNF-α expression

Several studies have demonstrated a link between increased pro-inflammatory mediators and apoptosis in neurodegenerative diseases. It has been reported that lipopolysaccharide (LPS) induces apoptosis mostly through the production of TNF-α. In this study, we investigated the possible protective and anti-inflammatory mechanisms of diosmin, a natural flavone glycoside, on LPS-induced PC12 cells death through inhibition of TNF-α production. PC12 Cells were pretreated with diosmin for 2 h prior to LPS treatment for 48 h to assess PC12 cells viability, TNF-α expression, and cell death mechanisms. Diosmin significantly increased cells survival and suppressed LPS-induced TNF-α in a concentration-dependent manner. Diosmin also significantly reduced the DNA fragmentation of LPS-induced cells, and its anti-apoptotic effect was confirmed by the decrease in the expression of pro-apoptotic protein Bad and the increase in the expression of anti-apoptotic protein Bcl-2 on Western blot analysis. Furthermore, diosmin inhibited LPS-induced caspase-3 activation further confirming its anti-apoptotic effects. This is the first study to report the anti-inflammatory and anti-apoptotic effects of diosmin via inhibition of TNF-α and a caspase-dependent pathway in neuronal PC12 cells. These results support the potential for diosmin to be investigated as a potential agent for the treatment of neurodegenerative diseases.     

Different involvement of DNA methylation and histone deacetylation in the expression of solute-carrier transporters in 4 colon cancer cell lines

The purpose of this study on the involvement of epigenetic control of the expression of solute carrier (SLC) transporters by DNA methylation and histone deacetylation in 4 colon cancer cells is to find the epigenetic control mechanisms of drug transporters in colon cancers. Human colon cancer cell lines (HCT116, HT29, SW48, SW480) were treated with 5-aza-2'-deoxycytidine (DAC), as a DNA methyltransferase inhibitor, followed by trichostatin A (TSA), as a histone deacetylase inhibitor. The mRNA expression and DNA methylation of several SLC transporters were analyzed by real-time polymerase chain reaction (PCR) and methylation-specific PCR, respectively. Among 12 SLC transporters possessing cytosine-phosphate-guanine (CpG) islands, thiamine transporter 2 (THTR2) (SLC19A3) gene showed a correlation between its mRNA expression level and DNA methylation status. TSA treatment increased histone H3 acetylation of THTR2 promoter region in all 4 colon cancer cell lines examined. HCT116 and SW48 cells showed a lack of THTR2 mRNA expression and methylation of its promoter, and DAC treatment induced its re-expression. In addition, the co-treatment with DAC and TSA increased THTR2 mRNA expression more markedly than DAC treatment in HCT116 and SW48 cells. In HT29 and SW480 cells that showed little methylation of THTR2 promoter, TSA treatment induced THTR2 mRNA expression markedly, but DAC treatment did not. In the 4 colon cancer cells examined, THTR2 mRNA expression is down-regulated by DNA methylation and/or histone deacetylation.     

丹皮酚对TNF-α诱导真皮成纤维细胞MMP-9 mRNA及细胞因子表达的影响

目的通过研究丹皮酚对TNF-α诱导的真皮成纤维细胞MMP-9 mRNA及细胞因子表达的影响,探讨丹皮酚抗皮肤炎症的可能机制。方法采用RT-PCR法考察正常人真皮成纤维细胞中MMP-9 mRNA表达情况以及丹皮酚对TNF-α诱导的真皮成纤维细胞MMP-9 mRNA表达的影响。采用ELISA法测定丹皮酚对TNF-α诱导的真皮成纤维细胞表达IL-1β、IL-6及IL-8的影响。结果MMP-9在正常真皮成纤维细胞上表达极弱,TNF-α可以诱导MMP-9 mRNA的表达,丹皮酚可以抑制TNF-α诱导引起的MMP-9 mRNA表达的上调。成纤维细胞可分泌少量的IL-1β、IL-6及IL-8,TNF-α可以明显增加成纤维细胞IL-1β和IL-8的分泌量,丹皮酚可以抑制TNF-α诱导的IL-1β和IL-8的产生,但对IL-6的分泌无影响。结论丹皮酚可以抑制TNF-α诱导真皮成纤维细胞引起的MMP-9 mRNA及炎症因子IL-1β和IL-8表达水平的上调。     

Approach to new therapeutics: investigation by the use of MDS-derived cell lines

Myelodysplastic syndromes (MDS) are a group of aquired hematopoietic disorders characterized by ineffective hematopoiesis, and increased risk of progression of acute myeloid leukemia. For a long period of time, the standard therapy for MDS was hematopoietic stem cell transplantation, however DNA methyltransferase inhibitors (DNMT inhibitors) including decitabine (DAC) and azacitidine (AZA), and lenalidomide, a derivative of thalidomide have been highlighted as new chemotherapeutic agents for MDS. However, the underlying mechanisms of action of these drugs have not been fully defined yet. Therefore, we investigated the in vitro effects of DNMT inhibitors and lenalidomide on an MDS-derived cell line, MDS92 and its blastic subline MDS-L, both of which carry del(5q). MDS-L cells were found to be quite sensitive to DAC, which induced to cell death through DNA damage-mediated G2 arrest via p53- independent pathways. Gene expression profiling suggested that DAC affects biogroups representing hematological systems, cellular development, cell death and apoptosis. Next, we examined the effects of lenalidomide on MDS-L. Cell growth was inhibited and multinucleated cells were frequently formed prior to cell death by lenalidomide treatment. Time-lapse microscopic observation and DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis itself but inhibits cytokinesis of MDS-L cells. The gene expression profiling showed decreased expression of M phase-related genes. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q).     

Tumor necrosis factor alpha increases P-glycoprotein expression in a BME-UV in vitro model of mammary epithelial cells

P-glycoprotein is an efflux pump belonging to the ATP-binding cassette super-family that influences the bioavailability and disposition of many drugs. Mammary epithelial cells express various drug transporters including P-glycoprotein, albeit at low level during lactation. During inflammatory reactions, which can be associated with changes in epithelial barrier functions, pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) are elevated in milk and serum. In this study, the role of TNF-α in the regulation of P-glycoprotein was determined in cultured BME-UV cells, an immortalized bovine mammary epithelial cell line. The protein production of P-glycoprotein and mRNA expression of bABCB1, the gene encoding P-glycoprotein, were increased after 24?h of TNF-α exposure. The highest observed effects for TNF-α on the regulation of P-glycoprotein was after 72?h of exposure. Protein and mRNA expression also increased significantly after 120?h of TNF-α exposure, but was lower than the level observed in the cells exposed to TNF-α for 72?h. The apical to basolateral flux of digoxin, a P-glycoprotein substrate, was decreased in the TNF-α-exposed epithelium. This effect was reversed when verapamil or ketoconazole, compounds known to interact with P-glycoprotein, were added together with digoxin into the donor compartment. Probenecid, a compound known to interact with organic anion transporters, but not P-glycoprotein, did not increase the flux of digoxin. This model has important implications for understanding the barrier function of the mammary epithelium and provides insight into the role of P-glycoprotein in the accumulation and/or removal of xenobiotics from milk and/or plasma.     

小檗碱对高果糖饲养诱导大鼠胰岛素抵抗和肝脏TNF-α表达的影响

目的研究小檗碱对高果糖饲养诱导胰岛素抵抗大鼠的作用。方法高果糖饲料喂养SD大鼠6wk后,分为3组,模型组,小檗碱组(187.5mg.kg-1.d-1灌服)、二甲双胍组(184mg.kg-1.d-1灌服)作为对照,继续高果糖饮食。实验同时设立正常对照组,普通饮食喂养。4wk后处死大鼠;测定血糖、血清胰岛素、胰岛素抵抗指数及血脂的变化,用RT-PCR方法观察肝脏TNF-αmRNA的表达。结果模型组胰岛素、胰岛素抵抗指数、游离脂肪酸(free fatty acids,FFA)及甘油三酯(triglycerides,TG)水平均明显升高;肝脏TNF-αmRNA的表达与正常对照组比较明显升高。小檗碱降低了胰岛素抵抗大鼠的血糖、血清胰岛素、胰岛素抵抗指数、TG、FFA及TNF-αmRNA的表达。二甲双胍降低了大鼠的血糖、血清胰岛素、胰岛素抵抗指数及TG。结论小檗碱可以改善胰岛素抵抗,其机制可能包括抑制肝脏TNF-αmR-NA的表达与改善脂代谢紊乱。     

联合使用脱甲基化药物和紫杉醇对肾癌细胞的侵袭能力影响机制研究

目的：观察联合使用脱氧杂胞苷（ DAC）与紫杉醇（ PTX）对肾患细胞是否存在协同作用,并探究其机制。方法离体培养肾透明细胞癌ACHN细胞株,将120培养皿随机分为空白对照组、DAC组、PTX组、联合组,每组30个。 DAC组：用DAC：0．5、1、2、4、8μmol／L处理3 d;PTX组：用PTX：1、2、4、8、16 nmol／L处理3 d;联合组：用DAC＋PTX处理3 d。使用高速分选流式细胞仪观察T肽协同树突状细胞对肾癌细胞的凋亡率;通过ELISA检测淋巴细胞增强因子（LEF1）和E－cadherin在肾癌细胞中的表达;通过ELISA技术检测凋亡蛋白caspass－3在肾癌细胞中的表达。结果 DAC组、PTX组以及联合组对肿瘤细胞的抑制作用明显,其中联合组对肿瘤细胞的抑制作用最强（ P＜0．05）,而且DAC和PTX对肿瘤细胞的抑制作用与药物浓度成正比（P＜0．05）。与DAC组、PTX组比较,联合组中LEF1的表达水平最低（P＜0．05）,E－cadherin、caspass－3的表达水平最高（P＜0．05）,而且DAC和PTX对LEF1表达的抑制作用、对E－cadherin、caspass－3的表达的促进作用与药物浓度成正比（P＜0．05）。结论 DAC与 PTX联合干预肾癌细胞,具有协同作用,而且LEF1是新的肾癌治疗靶点,DAC与 PTX合用可以提高肾癌细胞的凋亡率,并且降低肾癌细胞的侵袭转移能力。     

塞隆骨提取物对牛Ⅱ型胶原诱导的小鼠关节炎的治疗作用及机制研究

目的了解塞隆骨水提物(SLG-B)对胶原诱导的小鼠关节炎的治疗作用及治疗机制。方法利用牛Ⅱ型胶原诱导DBA/1小鼠类风湿性关节炎模型即CIA。SLG-B口服给药观察小鼠关节炎指数的变化情况,体外培养关节炎小鼠脾细胞及巨噬细胞,ELISA法测定IL-12、IL-2、IFN-γ、TNF-α几种细胞因子。RT-PCR测定SLG-B对关节炎小鼠巨噬细胞IL-1β、IL-6、iNOS mRNA表达的影响。结果SLG-B能明显的抑制牛Ⅱ型胶原诱导关节炎的发生,能够减轻关节炎的各种症状。SLG-B在加抗原和不加抗原的情况下都能够明显的抑制关节炎小鼠脾细胞的增殖同时发现SLG-B能够抑制IL-2、IFN-γ、L-12p40、TNF-α细胞因子的产生还能够抑制小鼠腹腔巨噬细胞IL-1、IL-6及iNOS mRNA的表达。这可能是SLG-B在小鼠关节炎中表现治疗效果的分子基础。结论SLG-B对小鼠关节炎有很好的治疗效果,其作用机制主要是通过抑制巨噬细胞及脾细胞产生炎性细胞因子和抑制炎性细胞因子mRNA的表达来达到治疗类风湿性关节炎的作用。     

Camptothecin fails to induce apoptosis in tumor necrosis factor-alpha-treated HaCaT cells

Camptothecin (CPT), a DNA topoisomerase I inhibitor, was originally isolated from the fruits of the Chinese Camptotheca acuminata tree. CPT and its derivatives have been used in the treatment of psoriasis and cancer in China for decades. It is well known that tumor necrosis factor-α (TNF-α) is a key proinflammatory cytokine in the pathogenesis of psoriasis. In this study, we investigated the effect of CPT on TNF-α-treated HaCaT cells. The results indicated that CPT in the concentration range of 0.5-2.0 μg·ml(-1) failed to show any proapoptotic effect in HaCaT cells. It was found that both CPT and TNF-α up-regulated the expression of TRAIL receptor 1/2 but not TRAIL in HaCaT cells. Furthermore, the expression of antiapoptotic proteins (IAP1, IAP2, and Bcl-X(L)) was up-regulated by TNF-α and suppressed by CPT in HaCaT cells. Because these gene products are known to be regulated by nuclear factor-kappa B (NF-κB), we examined the role of CPT on NF-κB activation. It was found that CPT not only failed to inhibit TNF-α-induced NF-κB activation but also contributed to NF-κB activation. In addition to these effects, CPT also promoted the production of interleukin-6, similar to TNF-α, in HaCaT cells. In conclusion, despite ample evidence supporting CPT-induced carcinoma cell apoptosis, our study clearly shows that CPT fails to show any proapoptotic effects in HaCaT cells, even though it enhanced TRAIL receptor 1/2 expression and inhibited the expression of TNF-α-induced antiapoptotic proteins. Taken together, this study demonstrates that CPT fails to block the activity of TNF-α. With respect to the NF-κB-activating role of CPT, we suggest that the benefit of CPT in the treatment of psoriasis should be reevaluated.     

三甲氧基二苯乙烯对小鼠巨噬细胞产肿瘤坏死因子-α及细胞核因子-κB活性的作用研究

目的研究三甲氧基二苯乙烯（BTM）对小鼠巨噬细胞经脂多糖（LPS）诱导后产生肿瘤坏死因子-α（TNF-α）及细胞核因子-κB（NF-κB）活化的调控作用。方法培养RAW246．7小鼠巨噬细胞,加入不同浓度的BTM或白藜芦醇,再加入LPS活化细胞,收集细胞上清液用L929细胞结晶紫染色法检测TNF-α的活性,制作细胞爬片用免疫细胞化学法检测巨噬细胞中NF-κB的表达。结果BTM和白藜芦醇本身对L929细胞无细胞毒性;经不同浓度BTM和白藜芦醇处理后巨噬细胞产生TNF-α的水平及表达NF-κB的阳性率呈剂量依赖性减少,BTM抑制巨噬细胞产生TNF-α及表达NF-κB的能力大于白藜芦醇,巨噬细胞产生TNF-α的活性及NF-κB的阳性细胞率均与药物浓度呈负相关,TNF-α的活性和NF-κB的阳性细胞率呈正相关。结论BTM和白藜芦醇在浓度为5～80μmol／L范围内,呈浓度依赖性地通过抑制NF-κB的活化来抑制TNF-α的产生,且BTM抑制巨噬细胞产生TNF-α和NF-κB活化的能力犬于白藜芦醇。     

复方丹参方对TNF-α刺激血管平滑肌细胞影响的蛋白质组学研究

目的观察复方丹参方对TNF-α刺激血管平滑肌细胞的影响,在分子水平上探讨复方丹参方治疗动脉粥样硬化的作用机制。方法用TNF-α刺激的血管平滑肌细胞来建立体外平滑肌细胞功能失常的细胞模型。用MTT和流式细胞术测定血管平滑肌细胞的增殖活力,用双向电泳、图像分析、质谱鉴定等蛋白质组学技术测定复方丹参方对血管平滑肌细胞中有影响的蛋白质。结果复方丹参方抑制了TNF-α引起的血管平滑肌细胞的增殖,TNF-α作用血管平滑肌细胞后有16个蛋白质下调和24个蛋白质上调,复方丹参方作用血管平滑肌细胞后可使这些改变的蛋白质水平有所恢复。复方丹参方可以下调钙调蛋白依赖蛋白激酶、N-ras、基质金属蛋白酶9的水平,上调p53肿瘤抑制因子、周期素依赖激酶抑制因子p21的水平。结论复方丹参方通过抑制基质金属蛋白酶9的分泌和升高周期素依赖激酶抑制因子的水平进而抑制血管平滑肌细胞的增殖与迁移是其治疗动脉粥样硬化的可能机制。     

蟛蜞菊内酯对脂多糖诱导RAW264.7细胞环氧化酶2、NO及TNF-α的影响

目的:研究蟛蜞菊内酯对脂多糖(lipopo-lysaccharide,LPS)诱导RAW264.7巨噬细胞环氧化酶2(COX-2)、NO及TNF-α的作用。方法:ELISA方法检测0.2、2、20μmol/L不同浓度蟛蜞菊内酯对终浓度为10μg/mL LPS诱导RAW264.7细胞产生TNF-α、NO及前列腺素E2(PGE2)的影响,Western blot方法检测蟛蜞菊内酯对LPS诱导COX-2酶蛋白表达的影响。结果:LPS能够明显诱导小鼠RAW264.7细胞产生的COX-2酶蛋白,蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的COX-2酶蛋白表达。PGE2可以被LPS诱导增加,与空白组比有显著差异。蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的PGE2、NO和TNF-α,呈现剂量依赖性。结论:蟛蜞菊内酯抗炎的作用机制可能为抑制COX-2的蛋白表达,进而抑制PGE2的生成,也可能与抑制NO和TNF-α生成有关。     

灵芝多糖对小鼠巨噬细胞白介素1α和肿瘤坏死因子αmRNA表达的影响

采用 RT- PCR和凝胶图像吸光度分析系统检测灵芝多糖 (GLB7)对小鼠腹腔巨噬细胞白介素 1α(IL- 1α) ,肿瘤坏死因子α(TNF-α) m RNA表达的影响 .加入不同浓度的 GLB7(5,1 0 ,2 0 ,40 mg·L-1) ,培养 3及 3.5h后观察 GLB7浓度与 IL- 1 α和 TNF- α m RNA表达之间的量效关系 .发现GLB7能浓度依赖性诱发 IL- 1 α和 TNF- α m RNA的表达 ,而对照组未见有 IL- 1 α和 TNF- α m RNA的表达 ;3及 3.5h培养上清中 IL- 1 α和 TNF- α活性与对照组比较亦有明显升高 ,并具有一定的浓度依赖关系 .说明 GLB7免疫增强和抗肿瘤作用的基础与其在转录水平诱发 IL- 1 α和 TNF- α m RNA的表达有关 .     

OHM3597: A novel fentanyl derivative with morphine-like behavioral effects in rhesus monkeys

OHM3597, a fentanyl-like piperidine with a thalidomide-like moiety, was studied in rhesus monkeys for its behavioral effects and for its effects on lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-α, OHM3597 had morphine-like discriminative stimulus effects that were antagonized by naltrexone in a manner that was consistent with μ receptor mediation. OHM3597 also had antinociceptive effects, producing a maximal (20 sec) antinociceptive effect in a tail withdrawal procedure with a 50°C stimulus. This effect of OHM3597 also was antagonized by naltrexone in a dose-related manner. Behavioral effects of this fentanyl derivative had a rapid onset and a relatively short duration of action; discriminative stimulus effects were evident 3 min after subcutaneous (sc) administration of 0.32 mg (0.58 μM)/kg of OHM3597 and the duration of antinociceptive effects produced by 1.0 mg (1.6 μM)/kg (sc) was less than 90 min. OHM3597 also was compared to thalidomide for its effects on LPS-induced TNF-α production in peripheral blood mononuclear cells that were obtained from drug-naive rhesus monkeys. Thalidomide suppressed the production of TNF-α in a concentration-dependent manner with concentrations of 10 nM and 1 μM of thalidomide decreasing TNF-α levels to 81 and 65%, respectively, of control (saline) values. In contrast, up to a concentration of 1 μM, OHM3597 failed to suppress LPS-induced TNF-α production. These results demonstrate OHM3597 to be a potent, morphine-like opioid with a relatively short duration of action. Although OHM3597 did not alter TNF-α production, a compound with both antinociceptive and immunomodulatory effects might be available within this chemical series and could provide a unique approach to the concurrent treatment of pain and infectious disease. © 1995 Wiley-Liss, Inc.     

枸杞多糖对小鼠海马神经元细胞系缺糖缺氧损伤保护作用及机制研究

目的探讨枸杞多糖(LBP)对小鼠海马神经元细胞系HT22细胞缺糖缺氧损伤保护作用的机制。方法采用含1 mmol/L连二亚硫酸钠的无糖DMEM培养基建立HT22细胞缺糖缺氧损伤模型,设对照组、模型组和LBP高、中、低剂量(100、50、25 mg/L)组,以CCK8法检测细胞活性,流式细胞术检测检测细胞凋亡情况;Western blotting检测Bcl-2、Bax蛋白表达情况;利用实时荧光定量PCR(RT-qPCR)检测TNF-α、NF-κB、I-κBα、Caspase-9基因转录活性。结果与对照组比较,模型组HT22细胞存活率显著降低、凋亡率显著增加(P<0.05);TNF-α、NF-κB、Caspase-9基因转录活性显著上调,I-κBα基因转录活性显著下调(P<0.05);Bax蛋白表达水平显著升高,Bcl-2的蛋白表达水平显著降低(P<0.05)。与模型组比较,LBP各剂量组细胞存活率显著上升、凋亡率显著下降(P<0.05);TNF-α、NF-κB、Caspase-9基因转录活性显著下调,I-κBα基因转录活性显著上调(P<0.05);Bax蛋白表达水平显著降低,Bcl-2的蛋白表达水平显著增高(P<0.05)。结论 LBP可能是通过下调TNF-α、NF-κB、Caspase-9基因转录活性以及Bax蛋白表达,增强I-κBα基因转录活性及Bcl-2的蛋白表达,抑制缺糖缺氧损伤诱导神经细胞凋亡的发生。     

De-risking bio-therapeutics for possible drug interactions using cryopreserved human hepatocytes

Inflammatory diseases such as rheumatoid arthritis and psoriasis are characterized by increases in circulating cytokines, which play an important role in modulation of the disease state. Several marketed bio-therapeutics target cytokines and act as effective treatment strategies. Previous in-vitro and in-vivo studies have suggested that cytokines may have both direct and indirect effects on drug metabolizing enzyme levels in the liver. Few studies have characterized models to evaluate the risk of potential drug interactions that might be mediated by changes in cytokine levels. In the present studies the potential of three cytokines (IL-2, IL-6 and TNF-α) to modulate gene expression and activity of the major human cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4) in cryopreserved human hepatocytes (CHH) was investigated. Significant decreases in the activity of all 6 CYP isoforms occurred in hepatocytes incubated with TNF-α or IL-6 (17-85%; and 22-76% of untreated control values, respectively). TNF-α down-regulated the gene expression of CYP1A2, 2D6 and 3A4 only, whereas IL-6 down-regulated gene expression of all of the tested CYP isoforms except 2D6. IL-2 had only mild effects on CYP activity and mRNA levels of examined isoforms. In CHH exposed to TNF-α, changes in CYP activity were not always paralleled by gene expression alterations for three of the examined CYP isoforms. These studies highlight several potential pitfalls in using isolated human hepatocytes for determination of drug interactions by bio-therapeutics including lack of correlation of mRNA and activity measurements for some CYP isoforms when using single time point determinations, and appropriateness of the model for indirect acting cytokine and cytokine modulators.