Effect of miltefosine on erythrocytes

BackgroundMiltefosine, an alkylphosphocholine drug with antiparasite, antibacterial, antifungal and antineoplastic potency, is the only oral drug that can be used to treat visceral and cutaneous leishmaniasis. The effect of miltefosine is at least partially due to triggering of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca²⁺-level ([Ca²⁺]_i). The present study explored, whether miltefosine elicits eryptosis.MethodsCell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence.ResultsA 48 h exposure to miltefosine (?4.9 μM) was followed by significant decrease of forward scatter and significant increase of annexin-V-binding. The effect was paralleled by significant increase of [Ca²⁺]_i. The annexin-V-binding following miltefosine treatment was significantly blunted in the nominal absence of extracellular Ca²⁺.ConclusionMiltefosine stimulates eryptosis, an effect at least partially due to stimulation of Ca²⁺ entry.     

Induction of apoptotic erythrocyte death by rotenone

The pesticide rotenone stimulates apoptosis and rotenone intoxication has been considered a cause of Parkinson's disease. Rotenone further sensitizes tumor cells to cytotoxic drugs. The apoptotic effect of rotenone is at least partially due to mitochondrial injury. Even though lacking mitochondria and nuclei, erythrocytes may undergo eryptosis, an apoptosis-like suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and enhanced ceramide formation. The present study explored, whether rotenone elicits eryptosis. To this end, [Ca(2+)](i) was estimated utilizing Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, ceramide utilizing fluorescence antibodies and hemolysis from hemoglobin release. A 48 h exposure to rotenone significantly increased Fluo3-fluorescence(i) (≥1 μM), increased ceramide abundance (10 μM), decreased forward scatter (≥2.5 μM) and increased annexin-V-binding (≥ 1 μM). Rotenone exposure was further followed by slight but significant hemolysis. Rotenone-induced cell membrane scrambling was significantly blunted, but not completely abrogated by removal of extracellular Ca(2+). The present observations disclose a novel effect of rotenone, i.e. triggering of erythrocyte shrinkage and cell membrane scrambling, an effect paralleled by and partially dependent on Ca(2+)-entry.     

Suicidal erythrocyte death triggered by cisplatin

Cisplatin, a cytotoxic drug for the treatment of cancer, induces suicidal death or apoptosis of nucleated cells. Side effects of cisplatin include anemia, which, at least in theory, could similarly result from suicidal cell death. Erythrocyte suicidal death or eryptosis is characterized by cell shrinkage and cell membrane scrambling, the latter leading to exposure of phosphatidylserine (PS) at the cell surface. PS-exposing cells are rapidly cleared from circulating blood. The present experiments explored whether cisplatin could trigger eryptosis. According to forward scatter in FACS analysis, a 48 h exposure to cisplatin (≥1 μM) indeed decreased cell volume and, according to annexin V-binding, cisplatin (≥1 μM, 48 h) indeed increased PS exposure at the cell surface. Cisplatin did not induce hemolysis. According to Fluo3 fluorescence, cisplatin increased cytosolic Ca²⁺ activity, a known stimulator of eryptosis. In the absence of extracellular Ca²⁺, the effect of cisplatin on annexin V-binding was blunted. Cisplatin did not significantly modify the formation of ceramide, another stimulator of eryptosis. Cisplatin moderately decreased the cellular concentration of ATP, which is known to favour eryptosis. In conclusion, cisplatin triggers suicidal erythrocyte death at least partially by increasing cytosolic Ca²⁺ activity. The effect contributes to or even accounts for the development of anemia during cisplatin treatment.     

Beauvericin induced erythrocyte cell membrane scrambling

Beauvericin is a mycotoxin with antiviral, antibacterial, nematicidal, insecticidal, cytotoxic, and apoptotic activity. Similar to nucleated cells erythrocytes may undergo suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by energy depletion leading to increase of cytosolic Ca²⁺ activity. The present study thus explored whether beauvericin is able to trigger eryptosis and influence eryptosis following energy depletion. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in FACS analysis, cytosolic Ca²⁺ concentration from Fluo3 fluorescence, cytosolic ATP concentration from a luciferase-assay and ion channel activity with whole cell patch clamp. Exposure to beauvericin (≥5 μM) significantly decreased erythrocyte ATP concentration and increased cytosolic Ca²⁺ concentration as well as annexin V-binding. The effect of beauvericin on annexin V binding was significantly blunted by removal of extracellular Ca²⁺. Glucose depletion (48 h) was followed by, increase of Fluo3 fluorescence, decrease of forward scatter and increase of annexin V-binding. Beauvericin (≥1 μM) augmented the effect of glucose withdrawal on Fluo3 fluorescence and annexin V-binding, but significantly blunted the effect of glucose withdrawal on forward scatter, an effect paralleled by inhibition of Ca²⁺ activated K⁺ channels. The present observations disclose novel effects of beauvericin, i.e. stimulation of Ca²⁺ entry with subsequent cell membrane scrambling and inhibition of Ca²⁺ activated K⁺ channels with blunting of cell shrinkage.     

Arsenic-induced suicidal erythrocyte death

Environmental exposure to arsenic has been associated with anemia, which could result from suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase in cytosolic Ca²⁺ concentration, ceramide and energy depletion. The present experiments explored, whether arsenic stimulates eryptosis. According to annexin V-binding, arsenic trioxide (7 μM) within 48 h significantly increased phosphatidylserine exposure of human erythrocytes without inducing hemolysis. According to forward scatter, arsenic trioxide (7 μM) significantly decreased cell volume. Moreover, Fluo3-fluorescence showed that arsenic (10 μM) significantly increased cytosolic Ca²⁺ concentration. According to binding of respective fluorescent antibodies, arsenic trioxide (10 μM) significantly increased ceramide formation. Arsenic (10 μM) further lowered the intracellular ATP concentration. Removal of extracellular Ca²⁺ or inhibition of the Ca²⁺-permeable cation channels with amiloride blunted the effects of arsenic on annexin V-binding and cell shrinkage. In conclusion, arsenic triggers suicidal erythrocyte death by increasing cytosolic Ca²⁺ concentration, by stimulating the formation of ceramide and by decreasing ATP availability.     

Inhibition of erythrocyte “apoptosis” by catecholamines

Osmotic shock, oxidative stress and Cl⁻ removal activate a non-selective Ca²⁺-permeable cation conductance in human erythrocytes. The entry of Ca²⁺ leads to activation of a scramblase with subsequent exposure of phosphatidylserine at the cell surface. Phosphatidylserine mediates binding to phosphatidylserine receptors on macrophages which engulf and degrade phosphatidylserine exposing cells. Moreover, phosphatidylserine exposure may lead to adherence of erythrocytes to the vascular wall. In the present study, we explored whether activation of the non-selective cation conductance and subsequent phosphatidylserine exposure might be influenced by catecholamines. Phosphatidylserine exposure has been determined by FITC-annexin V binding while cell volume was estimated from forward scatter in FACS analysis. Removal of Cl⁻ enhanced annexin binding and decreased forward scatter, an effect significantly blunted by the β agonist isoproterenol (IC₅₀ approx. 1 μM). Fluo-3 fluorescence measurements revealed an increase of cytosolic Ca²⁺ activity following Cl⁻ removal, an effect again significantly blunted by isoproterenol exposure (10 μM). Whole-cell patch-clamp experiments performed in Cl⁻ free bath solution indeed disclosed a time-dependent inactivation of a non-selective cation conductance following isoproterenol exposure (10 μM). Phenylephrine (IC₅₀<10 μM), dobutamine (IC₅₀ approx. 1 μM) and dopamine (IC₅₀ approx. 3 μM) similarly inhibited the effect of Cl⁻ removal on annexin binding and forward scatter. In conclusion, several catecholamines inhibit the Cl⁻ removal-activated Ca²⁺ entry into erythrocytes, thus preventing increase of cytosolic Ca²⁺ activity, subsequent cell shrinkage and activation of erythrocyte scramblase. The catecholamines thus counteract erythrocyte phosphatidylserine exposure and subsequent clearance of erythrocytes from circulating blood.     

和厚朴酚神经保护作用的研究进展

和厚朴酚作为中药厚朴的主要活性成分之一,是一种多酚类化合物,具有抗炎、抗菌、抗肿瘤、抗氧化等多种作用.近年来发现,和厚朴酚对神经系统具有明显的保护作用.和厚朴酚易于通过血脑屏障,具有发展为广谱抗神经系统疾病药物的潜力.本文主要概述了目前和厚朴酚在神经保护方面的作用及机制,以期为后续的深入研究提供参考.     

Specific oxidation of honokiol by Cunninghamella echinulata

Among 37 species of microbial strains, Cunninghamella echinulata AS 3.3400 were found to possess the ability to transform honokiol to (R)-magnolignan C (1) and (S)-magnolignan C (2) by regio-specific oxidation. Among them, 1 was a new compound. The structures of two compounds were determined by the analyses of CD, MS and NMR spectroscopic data.     

Acute and sub-chronic toxicity studies of honokiol microemulsion

The purpose of this study was to investigate the acute and sub-chronic toxicity of honokiol microemulsion. In the acute toxicity tests, the mice were intravenously injected graded doses of honokiol microemulsion and were observed for toxic symptoms and mortality daily for 14 days. In the sub-chronic toxicity study, rats were injected honokiol microemulsion at doses of 100, 500, 2500 μg/kg body weight (BW) for 30 days. After 30 days treatment and 14 days recovery, the rats were sacrificed for hematological, biochemical and histological examination. In the acute toxicity tests, the estimated median lethal dosage (LD₅₀) was 50.5 mg/kg body weight in mice. In the sub-chronic toxicity tests, the non-toxic reaction dose was 500 μg/kg body weight. In each treatment group, degeneration or/and necrosis in vascular endothelial cells and structure change of vessel wall can be observed in the injection site (cauda vein) of a few animals while there were no changes in the vessels of other organs. The overall findings of this study indicate that the honokiol microemulsion is non-toxic up to 500 μg/kg body weight, and it has irritation to the vascular of the injection site which should be paid attention to in clinical medication.     

HPLC法测定胃健宁胶囊中和厚朴酚及厚朴酚的含量

目的建立胃健宁胶囊中和厚朴酚及厚朴酚的检测方法。方法色谱条件为C18色谱柱;流动相:乙腈-水-冰醋酸(60∶40∶1)(V∶V∶V);流速:1.000 mL·min-1;检测波长:294 nm。结果胃健宁胶囊中和厚朴酚在0.02⁰.32μg(r=0.999 6)范围内线性关系良好,平均回收率为96.58%,RSD为1.78%(n=9);厚朴酚在0.019 60.32μg(r=0.999 6)范围内线性关系良好,平均回收率为96.58%,RSD为1.78%(n=9);厚朴酚在0.019 6⁰.313 6μg(r=0.999 8)范围内线性关系良好,厚朴酚的平均回收率为97.29%,RSD为1.39%(n=9)。结论该方法简便,准确度高,可有效控制胃健宁胶囊的质量。     

高效液相色谱法测定加味藿香正气丸中厚朴酚与和厚朴酚的含量

目的 建立加味藿香正气丸中厚朴酚与和厚朴酚含量测定的高效液相色谱（HPLC）分析方法.方法 采用 Agilent ZORBAX SB-C18（4.6 mm×150.0 mm,5 μm）色谱柱,流动相为甲醇-水（70∶30）,检测波长为294 nm,流速为1.0 mL/min,进样量10 μL,柱温25 ℃.结果 厚朴酚与和厚朴酚的线性范围分别为0.113 0～1.130 0 μg[相关系数（r）=0.999 99,n=6]和0.061 5～0.615 0 μg（r=0.999 98,n=6）;平均回收率分别为98.4%[相对标准偏差（RSD）=0.98%,n=9]和98.8%（RSD=0.54%,n=9）.结论 HPLC法测定加味藿香正气丸中厚朴酚与和厚朴酚的含量,操作简便、准确、重现性好,可作为该药品质量控制的方法.     

HPLC法测定藿香正气口服液中橙皮苷、和厚朴酚、厚朴酚的含量

目的用HPLC法进行藿香正气口服液中几种有效成分的含量测定方法。方法采用DiamonsilTMC18柱（250×4.6mm,5μm）,以甲醇-水梯度洗脱,检测波长283nm,流速1mL.min-1,柱温25℃。结果橙皮苷、和厚朴酚、厚朴酚线性范围分别在0.1311～2.622μg、0.1728～3.4568μg、0.1751～3.5020μg。结论该测定方法简便可行,可用于藿香正气口服液中几种有效成分的含量测定。     

和厚朴酚对小鼠背根神经节细胞河豚毒素不敏感钠电流的影响

目的 通过观察和厚朴酚（honokiol, Hon）对河豚毒素不敏感（TTX-R）钠电流的作用,探讨它可能的镇痛机制。方法 应用酶解法急性分离小鼠背根神经节细胞,全细胞膜片钳技术记录河豚毒素不敏感钠电流。结果 和厚朴酚对河豚毒素不敏感钠电流的抑制呈现浓度依赖性,半抑制浓度（IC₅₀）为28.1 μmol·L^-1。和厚朴酚（30 μmol·L^-1）使河豚毒素不敏感钠电流密度下降48.1%,稳态激活和失活曲线分别向右和左偏移约7 mV和11.1 mV,但不影响通道的恢复时间。结论 和厚朴酚明显抑制河豚毒素不敏感钠电流,其作用可能与它的镇痛机制有关。     

Antifungal activity of magnolol and honokiol

Two neolignan compounds, magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl, 1) and honokiol (5,5'-diallyl-2,4'-dihydroxybiphenyl, 2), were isolated from the stem bark of Magnolia obovata and evaluated for antifungal activity against various human pathogenic fungi. Compound 1 and 2 showed significant inhibitory activities against Trichophyton mentagrophytes, Microsporium gypseum, Epidermophyton floccosum, Aspergillus niger, Cryptococcus neoformans, and Candida albicans with minimum inhibitory concentrations (MIC) in a range of 25-100 microg/ml. Therefore, compound 1 and 2 could be used as lead compounds for the development of novel antifungal agents.     

Protective effects of orally administered honokiol on cerebral ischemia reperfusion in rats and on stroke in SHRsp

Honokiol is a protective agent for cerebral ischemia injury when administered intravenously. In the present study, we aimed to investigate the oral effect of honokiol microemulsion on cerebral ischemia-reperfusion (I-R) injury in rats and stroke in SHRsp. Both tMCAO and SHRsp models in rats were used to evaluate the efficacy of the microemulsion. Rat aortic segment contraction test, primary rat aortic endothelial cells and primary brain microvascular endothelial cells (BMECs) injured by OGD-R were used to explore its potential action mechanism. Oral honokiol microemulsion significantly reduced infarct volume, neurological score and brain water content in tMCAO model, and it evidently reduced neurological score and increased the survival rate of SHRsp. Moreover, honokiol significantly inhibited aortic contraction induced by KCl and phenylephrine, and L-NAME suppressed these inhibitory effects. On the other side, honokiol increased NO and p-eNOS levels in rat endothelial cells. In addition, it also protects BMECs against OGD-R injury and increased eNOS expression in BMECs. In conclusion, oral honokiol administration has protective effects in tMCAO and in SHRsp rats, and its action mechanism is likely to be associated with its vasodilative effect produced by eNOS activation and with its protective effect on BMECs.     

HPLC测定拈痛丸中厚朴酚与和厚朴酚的含量

目的 建立拈痛丸中厚朴酚与和厚朴酚含量的测定方法.方法 采用RP-HPLC法,色谱柱为DiamonsidTM-C18柱,甲醇-水-磷酸(68:32:0.1)为流动相;流速1.0 ml·min-1;检测波长294 nm;柱温30℃.结果 厚朴酚的平均回收率为97.7%,RSD=1.78%(n=6);和厚朴酚的平均回收率为100.9%,RSD=1.97%(n=6).结论 所建方法可用于拈痛丸中厚朴酚与和厚朴酚的含量测定.     

Anti-tumor effect of honokiol alone and in combination with other anti-cancer agents in breast cancer

Honokiol, an active component isolated and purified from Chinese traditional herb magnolia, was demonstrated to inhibit growth and induce apoptosis of different cancer cell lines such as human leukaemia, colon, and lung cancer cell lines; to attenuate the angiogenic activities of human endothelial cells in vitro; and to efficiently suppress the growth of angiosarcoma in nude mice. In this study, we have demonstrated that treatment of different human breast cancer cell lines with honokiol resulted in a time- and concentration-dependent growth inhibition in both estrogen receptor-positive and -negative breast cancer cell lines, as well as in drug-resistant breast cancer cell lines such as adriamycin-resistant and tamoxifen-resistant cell lines. The inhibition of growth was associated with a G1-phase cell cycle arrest and induction of caspase-dependent apoptosis. The effects of honokiol might be reversely related to the expression level of human epidermal growth receptor 2, (HER-2, also known as erbB2, c-erbB2) since knockdown of her-2 expression by siRNA significantly enhanced the sensitivity of the her-2 over-expressed BT-474 cells to the honokiol-induced apoptosis. Furthermore, inhibition of HER-2 signalling by specific human epidermal growth receptor 1/HER-2 (EGFR/HER-2) kinase inhibitor lapatinib synergistically enhanced the anti-cancer effects of honokiol in her-2 over-expressed breast cancer cells. Finally, we showed that honokiol was able to attenuate the PI3K/Akt/mTOR (Phosphoinositide 3-kinases/Akt/mammalian target of rapamycin) signalling by down-regulation of Akt phosphorylation and upregulation of PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) expression. Combination of honokiol with the mTOR inhibitor rapamycin presented synergistic effects on induction of apoptosis of breast cancer cells. In conclusion, honokiol, either alone or in combination with other therapeutics, could serve as a new, promising approach for breast cancer treatment.     

Inhibitory effects of honokiol on lipopolysaccharide-induced cellular responses and signaling events in human renal mesangial cells

Honokiol has been shown to possess a lot of pharmacologic benefits, including antioxidative, antiangiogenic and antineoplastic effects. In the present study, we investigated the anti-inflammatory effects of honokiol and the signaling mechanisms involved in lipopolysaccharide (LPS)-induced conditions in human renal mesangial cells (HRMCs). Honokiol did not significantly change HRMC viability when used at a concentration of < 20 μmol/l but markedly altered cell viability at concentrations of > 40 μmol/l. In this study, LPS treatment led to a marked upregulation of the levels of IL-1β, IL-18, TNF-α, TGF-β1, CCL2, CCL3, and CCL5 in HRMCs. The expression of COX-2, iNOS, and their products PGE₂ and NO also increased. The upregulation of these molecules was significantly abolished by honokiol in a dose-dependent manner. Moreover, honokiol almost completely reversed IL-1β, CCL3, and NO expression at 10 μmol/l, and IL-18, TNF-α, TGF-β1, and COX-2 expression at 20 μmol/l. In addition, phospho-NF-κB p65 at Ser536, phospho-Akt, and phospho-p42/44 were dramatically suppressed by honokiol in LPS-treated HRMCs. These results indicate that honokiol can inhibit the LPS-induced expression of inflammatory cytokines and mediators in HRMCs. The anti-inflammatory mechanisms of honokiol are partly due to the suppression of the phospho-NF-κB p65, phospho-Akt and phospho-p42/44 pathways.     

Quantification and structural identification of related phenolic compounds in the raw medicinal material honokiol

High-performance liquid chromatography (HPLC) was used to quantify magnaldehyde B (6), magnaldehyde E (4) and 8',9'-dihydroxyhonokiol (7) simultaneously in the raw Chinese medicinal material honokiol. The separation was performed on a reversed-phase C₁₈ column by using a gradient elution with mobile phases of water (A) and methanol (B). The mobile phase gradient was run from 40% B to 56.5% B in 55 min, 55-67 min from 56.5% to 51.5%, 67-80 min from 51.5% to 70%, 80-170 min at 70%. The elution was carried out at a flow rate of 1.0 mL/min at the column temperature of 35 ^оC with the UV detection wavelength at 256 nm. Magnaldehyde B, magnaldehyde E and 8',9'-dihydroxyhonokiol showed good linear relationships with peak areas in the range of 0.00864 to 0.07776 mg/mL, 0.01488 to 0.13392 mg/mL and 0.01568 to 0.10976 mg/mL, respectively. Their corresponding average recoveries were 100.30%, 99.63% and 98.29%, respectively. Our results showed that the established method is simple, rapid, and accurate with good reproducibility for evaluating the quality of raw Chinese medicinal material honokiol. Moreover, another five phenolic compounds, namely erythro-7-O-methylhonokitriol (1), threo-7-O-methylhonokitriol (2), 7-O-ethylhonokitriol (3), magnaldehyde C (5), honokiol (8), together with compounds 4, 6 and 7, were isolated and purified from the remaining substance in the process of preparing the raw material honokiol by silica gel column and semi-preparative HPLC. Their structures were characterized by 1D and 2D NMR spectroscopy. Among them, compounds 1 and 2 were reported to have common planar structures and their relative configurations were identified for the first time. Compounds 3 and 7 were not only obtained from the raw medicinal material for the first time but also novel compounds.     

Cordycepin induced eryptosis in mouse erythrocytes through a Ca<Superscript>2+</Superscript>-dependent pathway without caspase-3 activation

Cordyceps sinensis is a prized traditional Chinese medicine and its major component cordycepin is found to have anti-leukemia activities. However, its cytotoxicity in erythrocytes was unclear. To examine the effect of cordycepin on the induction of eryptosis (an apoptosis-like process in enucleated erythrocytes), flow cytometric assays based on membrane integrity and asymmetry were employed. For comparison, analyses were performed in parallel with two other anti-leukemia agents, indirubin 3′-monoxime (IDM) and As₂O₃. We found that at the IC₅₀ against leukemia HL-60, cordycepin elicited eryptosis while IDM and As₂O₃ showed no erythrotoxicity in mouse erythrocytes. Mechanistically, cordycepin increased the [Ca²⁺]_i and activated μ-calpain protease in a dose-dependent manner. Yet, no caspase-3 activation was observed in the cordycepin-treated erythrocytes. When extracellular Ca²⁺ was depleted, both the cordycepin-induced eryptosis and μ-calpain cleavage were suppressed. Our study therefore demonstrated for the first time that cordycepin induces eryptosis through a calcium-dependent pathway in the absence of mitochondria and caspase-3 activation.