Effects of Phorbol-12,13-dibuterate on Sodium Currents and Potassium Currents in Rat Trigeminal Ganglion Neurons

The effects of phorbol-12,13-dibuterate （PDBu） on total sodium current （INa-total）, tetrodotoxin-resistant sodium current （INa-TFXr）, 4-AP-sensitive potassium current （IA） and TEA-sensitive potassium current （IK） in trigeminal ganglion （TG） neurons were investigated. Whole-cell patch clamp techniques were used to record ion currents in cultured TG neurons of rats. Results revealed that 0.5μmol/L PDBu reduced the amplitude of INa-total by （38.3±4.5）% （n=6, P〈0.05）, but neither the G-V curve （control： V0.5 =-17.1±4.3 mV, k=7.4±1.3; PDBu： V0.5=-15.9±5.9 mV, k=5.9±1.4; n=6, P〉0.05） nor the inactivation rate constant （control： 3.6±0.9 ms; PDBu： 3.6±0.8 ms; n=6, P〉0.05） was altered. 0.5 μmol/L PDBu could significantly increase the amplitude of INa-TFXr by （37.2± 3.2）% （n=9, P〈0.05） without affecting the G-V curve （control： V0.5=-14.7±6.0 mV, k=6.9± 1.4; PDBu： V0.5=- 11.1±5.3 mV, k=8.1± 1.5; n=5, P〉0.05 ） or the inactivation rate constant （control： 4.6±0.6 ms; PDBu： 4.2±0.5 ms; n=5, P〉0.05）. 0.5 μmol/L PDBu inhibited IK by （15.6±5.0） % （n=16, P〈0.05）, and V0.5 was significantly altered from - 4.7±1.4 mV to -7.9 ±1.8 mV （n=16, P〈0.05）. IA was not significantly affected by PDBu, 0.5μmol/L PDBu decreased IA by only （0.3±3.2）% （n=5, P〉0.05）. It was concluded that PDBu inhibited INa-total ：.but enhanced INa-TFXr, and inhibited IK without affecting IA. These data suggested that the activation of PKC pathway could exert the actions.

Comparison of Detoxification Methods on Phorbol Esters in Deoiled Jatropha curcas Meal for Animal Feeds

The deoiled Jatropha curcas meal by hexane extraction was detoxified phorbol esters by two different methods. These two methods were alkali in methanol and only ethanol washing. After both treatments, the PEs （phorbol esters） was decreased by 100%. The crude protein in detoxified meal of alkali in methanol washing was less amount than only ethanol washing. The result showed that treatment by only ethanol washing was a promising way to detoxify deoiled Jatropha curcas meal for animal feeds in industrial scale.

Two potentially specific but relevant patterns of proteomic change Response of SH-SY5Y cells to differentiation with retinoic acid followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, and susceptibility of differentiated cells to dopamine

Dopamine （DA） exposure at a dose of 100 pmol/L for 24 hours causes oxidative stress in SH-SY5Y cells with induction of neuronal differentiation by retinoic acid （RA,10 pmol/L,72 hours） followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate （TPA,80 nmol/L,72 hours）. However,it remains unclear whether the alteration of phenotype observed in response to oxidative stress is associated with protein regulation in this cellular model for Parkinson＇s disease. The present study detected protein regulation affected by oxidative stress at a proteomic level：selection of differentially altered proteins using two dimensional difference in-gel electrophoresis and identification of these proteins using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated significant alterations in expression of six proteins in SH-SY5Y cells following the differentiation and fourteen proteins in the differentiated cells following the exposure,exemplified by an increase of tubulin alpha1 in the former but a decrease of tubulin alpha-ubiquitous chain in the latter. These results suggest that two potentially specific but relevant patterns of proteomic change may be produced in SH-SY5Y cells with the induction of differentiation by RA followed by TPA,and in the differentiated cells after DA exposure.

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

Objective:To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate(PMA)/ionomycine for 72 h.MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern.Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%.Results:The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and78.8 k Da.These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 k Da band was appeared in unstimulated and 72 h PMA stimulated cells.Moreover,we found another seven small size(10–19.5 k Da) proteins in supernatants of48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts.Most of these proteins expression were down regulated following fibroblast activation.This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death.Conclusions:Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation.Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.

The Role of Oxygen Radicals in Rat Acute Lung Injury Induced by Phorbol Myristate Acetate

We tried to clarify the role of oxygen radicals released from granulocytes stimulated byphorbol myristate acetate(PMA) in rat acute lung injury. It was found that DNA strand-breakdamage(DSBD) in peripheral white blood cells (WBC) was significantly increased 40 min after injec-tion of PMA. DSBD in lung tissue of rats treated with PMA was also markedly increased comparedwith the controls. The PMA-treated rats showed significantly higher lipid-peroxide (LPO) level inplasma and lung tissue hemogenate than the controls did. These results suggest that determination ofDSBD, a simple and sensitive indicator for oxygen radical damaging, might be useful in thediagnosis of adult respiratory distress syndrome (ARDS), when it is used together with themeasurement of plasma LPO.

The Influence of Phorbol Ester on the Effect of Tamoxifen in Breast Cancer Cells

To investigate the correlation between the activity of kinases in the growth factor signal transduction pathway and the development of resistance of breast cancer to tamoxifen, reporter gene regulated by the regulating fragment of CCD1 was transfected into the MCF 7 cells, and the influence of tamoxifen on the reporter gene expression was examined under different conditions of TPA treatment. Our results showed that the reporter gene expression was inhibited by tamoxifen and promoted by TPA. Furthermore, tamoxifen exerts an agonist effect on the reporter gene expression when the cells was treated by TPA previously for 12 h. It is concluded that TPA could induce estrogen like effect of tamoxifen on estrogen receptor positive breast cancer cells and it may be one of the mechanisms responsible for the development of tamoxifen resistance.

Relaxin Inhibit Cardiac Fibrosis Induced by Phorbol 12-myristate 13-acetate

Relaxin is known to inhibit cardiac fibrosis. However, it is unclear whether relaxin could regulate the effects of Phorbol 12-myristate 13-acetate （PMA, PKC activator） on cardiac fibrosis. So the influence of relaxin on the cell proliferation and collagen expression induced by PMA in cultured cardiac fibroblasts was studied. It showed that PMA significantly increased cardiac fibroblasts proliferation, Type I pro-collagen protein expression, Type I pro-collagen mRNA expression, and rhRLX absolutely significantly decreased PMA induced effects on cardiac fibroblasts proliferation and Type I pro-collagen expressions, indicating that relaxin could inhibit cardiac fibrosis induced by PMA.

EFFECT OF PHORBOL ESTER ON cAMP－DEPENDENT PROTEIN KINASE ACTIVITY IN CARDIOMYOCYTES

Cardiomyocytes isolated from neonatal rats were treated with phorboll 2-myristatel 3-acetate (PMA ) ranging from 10(-11)to 10-7 mol/L for 20 min, causing cytosol protein kinase A (PKA) activity to decrease while particulate PKA activity increase in a concentration-dependent manner. The change of PKA activity induced by PMA was abolished completely by pretreatment of polymyxin B or depletion of protein kinase C (PKC). Type II PKA activity in particulate fraction was enhanced remarkably, while that of type I PKA was not altered when the cells were treated with 100 nmol/L PMA. The results suggested that subcellular distribution and activity of PKA in cardiomyocytes may be regulated by PKC.

Experimental study on the inhibitory effect of arsenic trioxide combined with phorbol ester on the proliferation of acute promyelocytic leukemia cell line Kasumi-1

Objective:To study the effect of arsenic trioxide (As2O3) combined with phorbol ester (PMA) on the proliferation of acute promyelocytic leukemia cell line Kasumi-1.Methods:Acute promyelocytic leukemia cell lines Kasumi-1 were cultured and randomly divided into control group (treated with the RPMI1640 medium without drugs or serum), As2O3 group (treated with serum-free RPMI1640 medium containing 20 μmol/L As2O3), PMA group (treated with serum-free RPMI1640 medium containing 160 nmol/L PMA) and As2O3+ PMA group (treated with serum-free RPMI1640 medium containing 20 μmol/L As2O3 and 160 nmol/L PMA). After treatment, the cell proliferation activity, cell cycle ratio and the protein expression of related genes were measured.Results: 12 h, 24 h and 48 h after treatment, the cell proliferation activity of As2O3 group, PMA group and As2O3+PMA group were significantly lower than that of control group, and the cell proliferation activity of As2O3+PMA group was significantly lower than that of As2O3 group and PMA group;48 h after treatment, the G1 phase and S phase ratio as well as CDK1 and CyclinB1 expression of As2O3 group, PMA group and As2O3+PMA group were significantly lower than those of control group while the G2 phase ratio as well as Bax, Caspase-3, Caspase-9, p-Chk1 and p-Cdc25C9 expression were significantly higher than those of control group;the G1 phase and S phase ratio as well as CDK1 and CyclinB1 expression of As2O3+PMA group were significantly lower than those of As2O3 group and PMA group while the G2 phase ratio as well as Bax, Caspase-3, Caspase-9, p-Chk1 and p-Cdc25C9 expression was significantly higher than those of As2O3 group and PMA group.Conclusion:As2O3 combined with PMA can inhibit the proliferation of acute promyelocytic leukemia cell line Kasumi-1 by inducing apoptosis and blocking cell cycle.

Synthesis and anti-HIV activities of phorbol derivatives

In this study,37 derivatives of phorbol esters were synthesized and their anti-HIV-1 activities evaluated,building upon our previous synthesis of 51 phorbol derivatives.12-Para-electron-acceptor-trans-cinnamoyl-13-decanoyl phorbol derivatives stood out,demonstrating remarkable anti-HIV-1 activities and inhibitory effects on syncytia formation.These derivatives exhibited a higher safety index compared with the positive control drug.Among them,12-(trans-4-fluorocinnamoyl)-13-decanoyl phorbol,designated as compound 3c,exhibited the most potent anti-HIV-1 activity(EC_(50)2.9 nmol·L^(−1),CC50/EC_(50)11117.24)and significantly inhibited the formation of syncytium(EC_(50)7.0 nmol·L^(−1),CC50/EC_(50)4891.43).Moreover,compound 3c is hypothesized to act both as an HIV-1 entry inhibitor and as an HIV-1 reverse transcriptase inhibitor.Isothermal titration calorimetry and molecular docking studies indicated that compound 3c may also function as a natural activator of protein kinase C(PKC).Therefore,compound 3c emerges as a potential candidate for developing new anti-HIV drugs.

Seco-cyclic phorbol derivatives and their anti-HIV-1 activities

Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C(PKC).The efficacy of phorbol esters in binding with PKC is attributed to the presence of oxygen groups at positions C20,C3/C4,and C9 of phorbol.Concurrently,the lipids located at positions C12/C13 are essential for both the anti-HIV-1 activity and the formation of the PKC-ligand complex.The influence of the cyclopropane ring at positions C13 and C14 in phorbol derivatives on their anti-HIV-1 activity requires further exploration.This research entailed the hydrolysis of phorbol,producing seco-cyclic phorbol derivatives.The anti-HIV-1 efficacy of these derivatives was assessed,and the affinity constant(Kd)for PKC-δprotein of selected seco-cyclic phorbol derivatives was determined through isothermal titration calorimetry.The findings suggest that the chemical modification of cyclopropanols could affect both the anti-HIV-1 activity and the PKC binding affinity.Remarkably,compound S11,with an EC_(50) of 0.27μmol·L^(−1) and a CC_(50) of 153.92μmol·L^(−1),demonstrated a potent inhibitory effect on the intermediate products of HIV-1 reverse transcription(ssDNA and 2LTR),likely acting at the viral entry stage,yet showed no affinity for the PKC-δprotein.These results position compound S11 as a potential candidate for further preclinical investigation and for studies aimed at elucidating the pharmacological mechanism underlying its anti-HIV-1 activity.

Yuanhuacin A Is a Selective Antagonist of Phorbol Ester Receptor in Protein Kinase C

Protein kinase C with a molecular weight of 82 kD has been purified to electrophoresis homogenous from rat brain through a series of chromatography cohmns including DE-52, Sepharose G-200 and phenyl-Sepharose. The enzyme possessed autophosphorylation activity. Yuanhuacin A inhibited the ~3H-phorbol-12, 13-dibutyrate (~3H-PdBu) binding of PKC with an IC_(50) value of 1.48±0.287×10^(-8) mol/L when the concentration of ~3H-PdBu was 1.5×10^(-9) mol/L (K_i=1.2×10^(-8) mol/L). Yuanhuacin A inhibited the PdBu-stimulated PKC activity in the catalysis of the phosphorylation of Histone Ⅲ-S with an IC_(50) of 2.82±0.37×10^(-9) mol/L (PdBu=10^(-6) mol/L), while it had no effect on the basal and Ca^(2+)-stimulated PKC activity in the same assay system. This result suggests that Yuanhuacin A is a selective antagonist of the phorbol ester receptor in protein kinase C.

Two novel phorbol esters from Croton tiglium L.

To investigate the phytochemical constituents of aboveground parts of Croton tiglium L. （family Euphorbiaceae）, its ingredients were isolated by repeated chromatography on silica gel, Sephadex LH-20, ODS and preparative HPLC. Their structures were identified based on 1D, 2D NMR and mass spectral analysis. A total of 10 phorbol esters were obtained. Among them, compound 1 （12-O-（2-methyl）butyryl-4ct-deoxyphorbol-β-isobutyrate） and compound 10 （20-formyl-4a-deoxyphorbol- β-acetate） were two new compounds, and the other eight were known compounds.

Forskolin and Phorbol 12-myristate 13-acetate modulates the expression pattern of AP-1 factors and cell cycle regulators in estrogen-responsive MCF-7 cells

Activator protein-1(AP-1)transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to the variety of stimulus.The phorbol 12-myristate 13-acetate and Forskolin(Fo)are well-known kinase activators/stimulators of Protein Kinase C(PKC)and Protein Kinase A(PKA)respectively.Importantly,these kinases are found to be present in transitional points of many cell signaling pathways,especially those involved in proliferation.The stimulating effect of PKC and PKA on the expression of AP-1 factors in MCF-7 breast cell proliferation is not well characterized.Hence,the role of PKC by PMA treatment and the role of PKA by using Fo in MCF-7 cells is investigated.Where,cells treated with PMA showed increased cell proliferation,while Fo had no effect,but inhibited the PMA induced proliferation.The RT-PCR results showed the PMA induced c-Jun,c-Fos and Fra-1 expressions compared to control and Fo.However,Fo in combination with PMA,inhibit the PMA induced above mRNA expressions where Fo alone has no effect.Western blot studies validated the c-Jun expressions in PMA treated MCF-7 cells.Further,PMA increases the mRNA expression of Cyclin-E1,Cyclin-D1,and CDK-4,whereas Fo decreases their expressions.Thus,mitogenic effect of PMA and inhibitory action of Fo on MCF-7 cells is probably enhanced via activation of AP-1 factors and concomitant action of cell cycle regulators in the downstream singling cascade.

细胞因子释放综合征的体外试验探索性研究

目的建立细胞因子释放综合征(CRS)的体外评价方法,对试验体系细胞密度、阳性药种类及浓度进行探索。方法分别用CD3抗体(OKT3)(0.1~5μg)、OKT3(0.1~1μg)+CD28抗体(anti-CD28)(1、2μg·mL^(-1))、佛波酯(PMA)(10~100 ng·mL^(-1))、PMA(10~100 ng·mL^(-1))+离子霉素(ION)(1、5μg·mL^(-1))刺激外周血单个核细胞(PBMC)48 h,用CCK-8法检测细胞增殖情况来筛选PBMC的最佳密度及药物的最佳浓度。实验分为7组:对照组、OKT30.1μg+anti-CD281μg·mL^(-1)组、OKT30.5μg+anti-CD281μg·mL^(-1)组、OKT31μg+anti-CD281μg·mL^(-1)组、PMA 10 ng·mL^(-1)+ION 1μg·mL^(-1)组、PMA 25 ng·mL^(-1)+ION 1μg·mL^(-1)组、PMA 50 ng·mL^(-1)+ION 1μg·mL^(-1)组,分别与PBMC细胞暴露48 h,收集细胞上清,通过酶联免疫吸附试验(ELISA)检测药物作用下PBMC释放细胞因子白介素-6(IL-6)、干扰素γ(IFN-γ)情况。结果与对照组相比,OKT3和PMA均引起中密度、高密度PBMC显著增殖,各剂量OKT3和PMA均引起细胞增殖。与阳性药单独使用相比,药物联合使用产生更强的细胞活化效应。与对照组相比,在OKT3(0.1~1μg)+anti-CD28(1μg·mL^(-1))作用下,PBMC释放IL-6、IFN-γ显著增加;在PMA(10~50 ng·mL^(-1))+ION(1μg·mL^(-1))作用下,PBMC分泌IFN-γ与对照组相比显著增加,在PMA(25~50 ng·mL^(-1))+ION(1μg·mL^(-1))作用下,PBMC分泌IL-6与对照组相比显著增加。结论确定了PBMC体外CRS风险评价方法的细胞密度、阳性药物种类及浓度、药物联合使用浓度。

佛波酯诱导THP-1单核细胞分化为巨噬细胞的条件与机制研究进展

人类白血病单核细胞系THP-1细胞,已被广泛运用于研究单核细胞/巨噬细胞的生物学功能、作用机制和巨噬细胞参与炎症性疾病及肿瘤微环境的模型中。然而,影响THP-1分化为巨噬细胞的因素较多,如佛波酯(PMA)浓度、戒断期长度、PMA刺激持续时间、细胞的基线状态、细胞密度以及环境氧张力等。优化PMA诱导THP-1的分化方案不仅可以助力于实验研究,还可减少实验室之间的结果差异性。本综述旨在总结PMA诱导THP-1单核细胞分化条件的影响因素、优化方案,探讨其作用机制,为单核细胞分化为巨噬细胞的研究提供参考。

佛波双酯对照品的制备研究

目的研究佛波双酯(TPA)对照品的制备方法及质量标准,为佛波双酯原料和相关制剂的质量控制提供依据。方法利用硅胶柱制备色谱和反相制备色谱对巴豆油的甲醇-水提取物进行分离纯化,分别以高效液相色谱法(HPLC)和气相色谱对佛波双酯对照品进行纯度检查和含量测定,用紫外光谱(UV)、红外图谱(IR)、质谱(MS)、核磁共振氢谱(1H-NMR)、碳-13核磁共振(13C-NMR)等方法进行图谱分析及结构确证,确认分子式为C36H56O8。结果纯化制备得到的佛波双酯对照品,紫外最大吸收波长为232 nm,MS结果为高分辨电喷雾质谱(HRESI-MS)m/z 615.4120(M-H)-,HPLC检查纯度>99.5%。结论本制备方法科学有效,制备的对照品纯度符合中药化学对照品的相关要求,可作为佛波双酯原料及相关制剂质量控制的化学对照品。

千年桐根的化学成分研究

研究千年桐Vernicia montana根的化学成分。采用各种现代色谱手段对其化学成分进行分离,测定各种波谱数据并结合文献研究以鉴定化合物的结构。分离到5化合物,分别鉴定为二萜12-O-palmityl-13-O-acetyl phorbol（1）,三萜maslinic acid（2）,木脂素syringaresinol（3）,棕榈酸palmitic acid（4）和单棕榈酸甘油酯glycerol monopalmitate（5）。五个化合物（1~5）均为首次从该植物中分得。

不同产地麻疯树种仁的含油量及脱油种仁的佛波酯含量

选用国内4种不同产地的麻疯树种仁,对其含油量以及脱油后的佛波酯含量进行了研究.采用GB/T 5512-85粮食、油料检验粗脂肪测定法进行麻疯树种仁含油量的测定;脱油后种仁经二氯甲烷超声提取佛波酯,采用高效液相色谱(HPLC)对其含量进行检测.结果表明,小金河、攀枝花、仁里、片角乡麻疯树种仁的含油量分别为58.29%、60.84%、58.83%和56.12%,脱油后种仁中佛波酯含量分别为2.43mgg-1、2.25mgg-1、2.03mgg-1和1.21mgg-1.不同产地的麻疯树种仁含油量均有差异,在深入开发麻疯树资源时,种仁含油量的高低可作为其选种依据之一.4种不同产地的麻疯树种仁脱油后的佛波酯含量均高于无毒麻疯树品种,要利用麻疯树的脱油种仁这一潜在饲料资源,对其进行进一步脱毒处理是必要的.

肝素增强佛波酯对人鼻咽癌CNE2细胞的作用 :上调c-jun ,p53 ,p21的表达(英文)

目的 :观察肝素联合佛波酯对人鼻咽癌细胞的增殖与凋亡的影响及其可能的分子机制。方法 :用细胞记数法、流式细胞术观察肝素联合佛波酯对人鼻咽癌细胞的增殖及细胞周期的影响 ;而用TUNEL、琼脂糖凝胶电泳、Westernblot等方法观察肝素与佛波酯联合应用对人鼻咽癌细胞凋亡的作用。结果 :肝素与佛波酯联合应用后对鼻咽癌细胞的生长抑制及其凋亡具有显著增强的作用 ,同佛波酯单独应用于诱导细胞凋亡相比 ,低剂量的肝素与佛波酯联合应用后发现 :TUNEL阳性细胞明显增多 ;G1 期细胞阻止 ,S期细胞明显减少 ,凋亡率增加 ;DNA“梯形”变化更加明显 ;c-jun ,p53 ,p2 1基因表达明显升高 ,而c-fos在整个用药过程中没有改变。结论 :肝素增强佛波酯对人鼻咽癌细胞的抗增殖及促凋亡 ,这可能与c-jun ,p53 ,p2