丹参川芎嗪注射液抗脑缺血缺氧损伤的药效物质基础研究

目的从注射液、组分、成分3个层面,探究丹参川芎嗪注射液抗脑缺血缺氧损伤作用及其药效物质基础。方法首先采用SH-SY5Y神经细胞构建氧糖剥夺/复氧(oxygen-glucosedeprivation/reoxygenation,OGD/R)损伤模型,以细胞活力为指标,考察丹参川芎嗪注射液对神经细胞的保护作用。其次,采用制备液相色谱等技术制备丹参川芎嗪注射液标准组分,然后在SH-SY5Y神经细胞OGD/R模型上,系统筛选丹参川芎嗪注射液组分和成分中抗脑缺血缺氧损伤的药效物质,最后进一步通过LC-MS对有效组分进行分析,并对有效成分进行归属,初步明确丹参川芎嗪注射液抗脑缺血缺氧损伤的药效物质基础。结果与模型组相比,丹参川芎嗪注射液在2.5%~15%的稀释浓度下,具有显著的抗SH-SY5Y细胞OGD/R损伤的作用;组分I、J、L、M、O、P、Q以及丹酚酸C、丹酚酸A、丹参素和盐酸川芎嗪均具有显著的抗SH-SY5Y细胞OGD/R损伤的作用。LC-MS分析结果表明,组分O、P中均含有丹酚酸C,组分O中含有丹酚酸A、盐酸川芎嗪,组分P中含有丹参素。结论丹参川芎嗪注射液具有显著的抗脑缺血缺氧损伤作用,而丹酚酸C、丹酚酸A、丹参素和盐酸川芎嗪可能是其抗脑缺血缺氧损伤的主要药效物质。     

The protective effect of qiancao naomaitong mixture on neuronal damage and cerebral ischemia/reperfusion injury

Context Qiancao Naomaitong Mixture (QNM) is mainly used to treat ischemic stroke patients in the clinic.

Objective This study evaluates the protective effect of QNM on neuronal damage in vitro, and clarifies the underlying mechanism against cerebral ischemia-reperfusion (I/R) injury in vivo.

Materials and methods Activity assay of caspase 3 (C-3) and caspase 8 (C-8) were measured with microplate reader and cell apoptosis was investigated. Cerebral I/R injury was induced by MCAO model. QNM groups were given at 0.27, 0.54 and 1.08?mL/100?g body weight. The weight ratio of cerebral infarction tissue was obtained. The cytokine levels in serum and brain tissue were measured using ELISA.

Results Compared with the OGD group (C-3: 29.69?±?5.63, C-8: 74.05?±?6.86), 100?mg/mL QNM (C-3: 19.80?±?2.62, C-8: 48.94?±?6.41) and 200?mg/mL QNM (C-3: 16.28?±?4.55, C-8: 41.08?±?4.05) treatments decreased C-3 and C-8 activities significantly, and inhibited apoptosis of SH-SY5Y cells. The weight ratios of cerebral tissues in low, medium and high dose groups were 17.33?±?5.1%, 17.78?±?5.4% and 14.25?±?4.2%, respectively, significantly lower than in control group. QNM also improved the cytokine levels in serum and brain tissue. In addition, histological examination indicated that dense neuropil and largely surviving neurons were seen in treated rats.

Conclusion QNM exerted protective effect by inhibiting the cell apoptosis in vitro. The protective mechanisms of QNM were associated with its properties of anti-apoptosis and antioxidation as well as improved neuronal nutrition in I/R rats.     

Low concentration of formononetin stimulates the proliferation of nasopharyngeal carcinoma cell line CNE2 by upregulating bcl-2 and p-ERK1/2 expression

Context Formononetin is a typical phytoestrogen, which is a bioactive component found in red clover plants. Previous studies have shown that formononetin inhibits the proliferation of several types of cancer cells, including prostate cancer and osteosarcoma. However, how formononetin affects the proliferation of CNE2 is not clear.

Objective The objective of this study is to investigate the effects of formononetin on nasopharyngeal carcinoma cells in vitro, along with the underlying mechanism.

Materials and methods CNE2 cells were incubated with various concentrations of formononetin (0, 0.1, 0.2, 0.3 and 1?μM) for 48?h. Cell proliferation was measured by [3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay, while the rate of apoptosis was measured by flow cytometry. Bcl-2 and bax mRNA expression levels were determined by real time polymerase chain reaction (RT-PCR), while p-ERK1/2 and bcl-2 protein expression levels were quantified by Western blotting.

Results Formononetin promoted the proliferation of CNE2 cells at low concentrations (0, 0.05, 0.1, 0.2, 0.5, 1, 2 and 5?μM), OD values increased from 0.27?±?0.01 to 0.30?±?0.01, 0.30?±?0.01,0.36?±?0.01, 0.35?±?0.01, 0.34?±?0.01, 0.34?±?0.01 and 0.32?±?0.01, respectively. The percentage of late apoptosis declined from 6.77%?±?0.73% (0?μM group) to 6.2%?±?0.4% (0.1?μM group), 3.83%?±?0.71% (0.3?μM group) and 5.1%?±?0.52% (1M group). The mRNA levels of bax and bcl-2 were down- and upregulated, respectively, by formononetin. Bcl-2 and p-ERK1/2 protein levels were also upregulated.

Conclusions Formononetin stimulates CNE2 cell proliferation and has an inhibitory effect on CNE2 cells apoptosis, which is mediated by the activation of the ERK1/2 signaling pathways.     

Effects of Ginkgo biloba extract on the apoptosis of oxygen and glucose-deprived SH-SY5Y cells and its mechanism

Objective:The aim was to observe the effects of the extract of Ginkgo biloba (EGb761) on the apoptosis of oxygen and glucose-deprived (OGD) human neuroblastoma cells (SH-SY5Y) cells and explore its mechanism.Results:Cell viability was significantly lower in OGD group than in EGb761-pretreated groups, especially in moderate-concentration group (50 μg/ml) (P < 0.005). Apoptosis rate was significantly lower in EGb761-pretreated groups than in OGD group (P < 0.001). Immunofluorescent staining showed that there was AIF nuclear translocation in both EGb761-pretreated groups and OGD group, but AIF nuclear translocation was less in EGb761-pretreated groups than in OGD group.Conclusion:EGb761 can reduce the apoptosis of OGD SH-SY5Y cells probably through inhibiting AIF nuclear translocation. This study provides a theoretical basis for the application of EGb761 in clinical practice.KEY WORDS: Apoptosis, apoptosis-inducing factor, extract of Ginkgo biloba, oxygen and glucose-deprived model, SH-SY5Y     

Evaluation of the cytotoxic potential of a new pentacyclic triterpene from Rhododendron arboreum stem bark

Context: Traditionally, Rhododendron arboreum Sm. (Ericaceae) is a very important medicinal plant having oxytocic, estrogenic, anti-inflammatory, analgesic and hepatoprotective activities; it also inhibits the prostaglandin synthetase.

Objectives: This study determines the cytotoxic potential of 15-oxoursolic acid isolated from R. arboreum against selected human cancer cell lines.

Materials and methods: Extraction from stem bark (5?kg) of R. arboreum was performed with methanol, which was successively partitioned into hexane, dichloromethane and ethyl acetate fractions, respectively. The new antitumor agent [15-oxoursolic acid (1)] was isolated from ethyl acetate fraction through column chromatography. Structure elucidation of new compound was performed through extensive spectroscopy i.e., IR, MS and 1D and 2D NMR. Cytotoxicity of isolated compound was determined at doses 5–100?μM for a period of 72?h on specified human cancer cell lines [renal cell carcinoma (A498), non-small cell lung (NCI-H226), squamous cell carcinoma (H157) and human ovarian carcinoma (MDR-2780AD)].

Results: Structure of isolated compound was characterized as 15-oxoursolic acid on the basis of various extensive spectroscopic techniques. 15-Oxoursolic acid revealed considerable anticancer activity with IC₅₀ values of 2.3?±?0.1?μM, 4.9?±?0.2?μM, 9.2?±?0.2?μM and 10.3?±?0.1?μM against MDR 2780AD, Hep G2, H157 and NCI-H226, respectively, while in the case of A498, the activity was good (IC₅₀ 32.8?±?1.2?μM).

Conclusions: This study highlighted the potential of 15-oxoursolic acid to be further explored as a new lead compound for cancer chemotherapy.     

The effects of icariin on the expression of HIF-1α, HSP-60 and HSP-70 in PC12 cells suffered from oxygen–glucose deprivation-induced injury

Context: The effects of icariin, a chief constituent of ?avonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown.

Objective: To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability.

Materials and methods: PC12 cells were treated with icariin (10^?7, 10^?6 or 10^?5?mol/L) for 3?h (1?h before oxygen–glucose deprivation (OGD) plus 2?h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Results: After 2?h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3?±?1.9?ng/L, HSP-60: 199?±?16?ng/L, HSP-70: 195?±?17?ng/L, NSE: 1487?±?125?ng/L), and cell viability was significantly decreased (0.26?±?0.03), while icariin (10^?7, 10^?6, or 10^?5?mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1?±?1.4, 22.6?±?1.8, 15.7?±?2.1, HSP-60: 100?±?12, 89?±?6, 113?±?11, HSP-70: 139?±?9, 118?±?7, 95?±?9 and NSE: 1121?±?80, 1019?±?52, 731?±?88), and improved cell viability (0.36?±?0.03, 0.38?±?0.04, 0.37?±?0.03) in OGD-treated PC12 cells.

Discussion and conclusion: These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.     

Isolation of major phenolics from Launaea spinosa and their protective effect on HepG2 cells damaged with t-BHP

Context: Some Launaea species (Asteraceae) are used traditionally to treat liver oxidative stress.

Objective: The present study investigates the protective effects of isolated compounds from Launaea spinosa Sch. Bip. (Asteraceae) against oxidative stress on t-BHP-induced HepG2 cells.

Materials and methods: Major phenolic content from flowering aerial parts of L. spinosa was isolated and identified. The protective effects of isolated compounds (10 and 20?μM) against oxidative stress induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells were investigated through the measurement of aspartate aminotransferase (AST), alanine transaminase (ALT), and superoxide dismutase (SOD) levels.

Results: A new phenolic compound identified as 2,3-diferulyl R,R-(+) methyl tartrate (6), in addition to five known metabolites, esculetin (1), esculetin-7-O-d-glucoside (cichoriin) (2), fertaric acid (3), acacetin-7-O-d-glucoside (4), and acacetin-7-O-d-glucuronic acid (5), were isolated. Oxidant-induced damage by 200?μM t-BHP in HepG2 cells was inhibited by compounds 1, 4, and 5 (10 and 20?μM), or quercetin (10?μM; positive control). The protective effects of compounds 1, 4, and 5 were associated with decreasing in AST, ALT, and SOD levels. Compound 4 (20?μM) decreased the AST level from 128.5?±?13.9 to 7.9 ±1.8?U/mL. Meanwhile, compound 1 (20?μM) decreased ALT activity from 20.3?±?7.0 to 7.6?±?2.4?U/mL, while compound 5 decreased SOD levels from 41.6?±?9.0 to 28.3?±?3.4?mU/mg.

Conclusion: The major phenolic compounds isolated from L. spinosa displayed a significant cytoprotective effect against oxidative stress, leading to maintenance of the normal redox status of the cell.     

Membrane permeability-guided identification of neuroprotective components from Polygonum cuspidatun

Context: Polygonum cuspidatum Sieb et Zucc. (Polygonaceae) possesses various pharmacological activities and has been widely using as one of the most popular and valuable Chinese herbal medicines in clinics. Its usage has increasingly attracted much of our attention and urges investigation on its bioactive components.

Objective: To establish a rapid and valid approach for screening potential neuroprotective components from P. cuspidatum.

Materials and methods: Potential neuroprotective components from P. cuspidatum were screened utilizing liposome equilibrium dialysis followed by high-performance liquid chromatography (HPLC) analysis. Their neuroprotective effects on modulation of protein expression of α7 nAChR, α3 nAChR and synaptophysin (SPY) on SH-SY5Y human neuroblastoma cell line (SH-SY5Y) were evaluated by means of Western blotting.

Results: Two potential compounds, polydatin (C1) and emodin-8-O-β-d-glucoside (C2), were detected and identified in our study. The biological tests showed that both compounds C1 and C2, respectively, at concentrations of 0.1 and 0.25?mg/mL significantly increased protein expression of α7 and α3 nicotinic acetylcholine receptors (nAChRs) in SH-SY5Y cells. Moreover, C1 and C2 at 0.1?mg/mL significantly reversed the Aβ_1-42-induced decrease of α7 and α3 nAChRs protein expression in SH-SY5Y cells. In addition, C2 at 0.1?mg/mL significantly increased protein expression of SPY in SH-SY5Y cells and Aβ_1-42-induced SH-SY5Y cells whereas C1 did not provide any positive effects.

Discussion and conclusion: In conclusion, our approach utilizing liposome equilibrium dialysis combined with HPLC analysis and cell-based assays is a prompt and useful method for screening neuroprotective agents.     

Caffeic acid methyl and ethyl esters exert potential antidiabetic effects on glucose and lipid metabolism in cultured murine insulin-sensitive cells through mechanisms implicating activation of AMPK

Context: Caffeic acid methyl (CAME) and ethyl (CAEE) esters stimulate glucose uptake and AMP-activated protein kinase (AMPK) in C2C12 myocytes (ATCC^® CRL-1772^TM).

Objective: Effects of CAME and CAEE were now assessed on myocyte glucose transporter GLUT4 activity and expression, on hepatic gluconeogenesis and on adipogenesis as well as major underlying signaling pathways.

Materials and methods: GLUT4 protein translocation was studied in L6 GLUT4myc cells, glucose-6-phospatase (G6Pase) in H4IIE hepatocytes and adipogenesis in 3T3-L1 adipocytes. Key modulators were measured using western immunoblot. Cells were treated for 18?h with either CAME or CAEE at various concentrations (12.5–100?μM).

Results: Myocyte glucose uptake rose from 10.1?±?0.5 to 18.7?±?0.8 and 21.9?±?1.0?pmol/min/mg protein in DMSO-, CAME- and CAEE-stimulated cells, respectively, similar to insulin (17.7?±?1.2?pmol/min/mg protein), while GLUT4myc translocation increased significantly by 1.70?±?0.18, by 1.73?±?0.18- and by 1.95?±?0.30-fold (relative to DMSO), following insulin, CAME and CAEE stimulation, respectively. CAME and CAEE suppressed hepatocyte G6Pase by 62.0?±?6.9% and 62.7?±?6.0% with IC₅₀ of 45.93 and 22.64?μM, respectively, comparable to insulin (70.7?±?2.3% inhibition). Finally, CAME and CAEE almost abrogated adipogenesis (83.3?±?7.2% and 97.3?±?3.0% at 100?μM; IC₅₀ of 13.8 and 12.9?μM, respectively). The compounds inhibited adipogenic factors C/EBP-β and PPAR-γ and stimulated AMPK activity in the three cell-lines.

Discussion and conclusions: CAME and CAEE exerted antidiabetic activities in insulin-responsive cells through insulin-independent mechanisms involving AMPK and adipogenic factors.     

Modulation of chemotherapy-induced cytotoxicity in SH-SY5Y neuroblastoma cells by caffeine and chlorogenic acid

Chemotherapy is an important treatment modality for malignancy but is limited by significant toxicity and it susceptibility to numerous drug interactions. While the interacting effects with medications are well known, there is limited evidence on the interaction with commonly consumed food and natural products. The aim of this study was to evaluate the bioactive constituents of coffee (caffeine and chlorogenic acid) on the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in vitro. Pretreatment with caffeine (100?nM and 10?μM) sensitized SH-SY5Y cells to doxorubicin-induced toxicity and increased apoptosis and sensitized PC3 cells to gemcitabine-induced toxicity. Pretreatment with 10?μM caffeine decreased total cell reactive oxygen species (ROS) production but increased mitochondrial ROS production. In contrast, caffeine (10?nM and 10?μM) protected cells against gemcitabine-induced toxicity and apoptosis. Similarly, 1?μM and 10?μM caffeine protected cells against paclitaxel-induced toxicity and mitochondrial ROS production. Chlorogenic acid had no effect on chemotherapy-induced toxicity in SH-SY5Y cells. In conclusion, this study provides preliminary evidence that caffeine, not chlorogenic acid, modulates the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in SH-SY5Y cells via different mechanisms.     

Total flavones of Abelmoschus manihot improve diabetic nephropathy by inhibiting the iRhom2/TACE signalling pathway activity in rats

Context: Total flavones extracted from Abelmoschus manihot L. (Malvaceae) medic (TFA) have been proven clinically effective at improving renal inflammation and glomerular injury in chronic kidney disease (CKD).

Objective: This study evaluated the function of TFA as an inhibitor of iRhom2/TACE (tumour necrosis factor-α converting enzyme) signalling and investigated its anti-DN (diabetic nephropathy) effects in a DN rat model.

Materials and methods: In vitro, cells were treated with 200?μg/mL advanced glycation end products (AGEs), and then co-cultured with 20?μg/mL TFA for 24?h. Real time PCR, western blotting and co-immunoprecipitation assays were performed. In vivo, DN was induced in 8 week old male Sprague-Dawley rats via unilateral nephrectomy and intraperitoneal injection of streptozotocin, then TFA were administered to rats by gavage for 12 weeks at three different doses (300, 135 and 75?mg/kg/d). 4-Phenylbutanoic acid (2.5?mg/kg/d) was used as a positive control.

Results: IC₅₀ of TFA is 35.6?μM in HK2 and 39.6?μM in HRMC. TFA treatment (20?μM) inhibited the activation of iRhom2/TACE signalling in cultured cells induced by AGEs. LD₅₀>26?g/kg and ED₅₀=67?mg/kg of TFA in rat by gavage, TFA dose-dependently downregulated the expression of proinflammatory cytokines and exerted anti-inflammatory effects significantly though inhibiting the activation of iRhom2/TACE signalling.

Discussion and conclusions: Our results show that TFA could dose-dependently ameliorate renal inflammation by inhibiting the activation of iRhom2/TACE signalling and attenuating ER stress. These results suggest that TFA has potential therapeutic value for the treatment of DN in humans.     

The protective effect of dopamine against OGD/R injury-induced cell death in HT22 mouse hippocampal cells

Previous studies have shown that levo-dopamine (l-dopa) can improve the consciousness of certain patients with prolonged coma after cerebral ischemia–reperfusion injury, and promote cell growth in vivo. This study aimed to investigate whether l-dopa, which is used clinically to treat Parkinson's disease, might also ameliorate ischemia–reperfusion injury-induced cell death. The oxygen-glucose deprivation and re-oxygenation (OGD/R) model was used to mimic the ischemia–reperfusion pathological process in vitro. HT22 cells were treated with dopamine hydrochloride at different times (i.e., 2 h prior to OGD, during the period of OGD, during the period of R, and throughout the period of OGD/R) and at different concentrations (i.e., 25 μM, 50 μM, 100 μM). Lactate dehydrogenase (LDH) release, flow cytometry–annexin V, and propidium iodide staining with light microscopy showed that dopamine hydrochloride (added during re-oxygenation) promoted cell proliferation and facilitated maintenance of normal cell morphology. However, when present during oxygen-glucose deprivation for 18 h and present throughout OGD/R, dopamine hydrochloride increased cell damage as manifested by shrinkage, rounding up, and reduced viability. In conclusion, dopamine protected HT22 cells from OGD/R injury-induced cell death only at a particular point in time, suggesting that it may be useful for treating severe ischemia–reperfusion brain injury.     

The anti-inflammatory activity of several flavonoids isolated from Murraya paniculata on murine macrophage cell line and gastric epithelial cell (GES-1)

Context Murraya paniculata (L.) Jack (Rutaceae), Qianlixiang in Chinese, is distributed in China. As an important traditional Chinese medicine (TCM), it demonstrates many bioactivities, such as febrifuge, astringent, anti-dysenteric, and tonic.

Objective: The objective of this study is to evaluate the anti-inflammatory effect of three flavonoids isolated from M. paniculata in lipopolysaccharide (LPS)-activated murine macrophage cell line and ethanol-induced gastric damage on gastric epithelial cell (GES-1).

Materials and methods Three identified flavonoids were isolated from stems and leaves of M. paniculata using ultra performance liquid chromatography (UPLC). Cell viability was measured with MTT, mouse peritoneal macrophages and GES-1 cells were incubated with 0, 0.01, 0.1, 1, 10, and 100?μM P1, P3 and P8 for 24, 48, and 72?h. The inhibitory effect of pretreatment with various concentrations of 5,7,3′,4′,5′-pentamethoxyflavone (P1), 5,7,3′,4′-tetramethoxyflavone (P3), or 5-desmethylnobiletin 5-hydroxy-6,7,8,3′,4′-pentameth-oxyflavone (P8) ranging from 0.03 to 30?μM on nitric oxide (NO) secretion was quantified by the Griess assay for 24 and 48?h, while interleukin-6 (IL-6) was measured by ELISA for 24 and 48?h.

Results The effects of P1, P3, and P8 on mouse peritoneal macrophages and GES-1 cells were not attributable to cytotoxic effects at the doses of 0–10?μM. The IC₅₀ value of P1 is 53.40?μM, P3 is 120.98?μM, and P8 is 10.73?μM. The concentration of the three flavonoids had the best effects of anti-inflammation upon NO inhibition at the dose of 3?μM. P3 had the highest inhibition on IL-6 production. The GES-1 cells pretreated with three flavonoids showed a significant increase in the level of NO (P1: 7.94?±?0.0635?μM, P3: 8.81?±?0.0159?μM, and P8: 8.51?±?0.0522?μM) at 24?h and a more significant increase at 48?h (P1: 9.34?±?0.0975?μM, P3: 11.9?±?0.0672?μM, and P8: 9.34?±?0.0454?μM).

Discussion and conclusion The current results suggested that the anti-inflammatory activity of three flavonoids was mainly manifested in the reduction of production of NO and IL-6 production. Analysis of the structure–activity relationship indicated that the double bond at C₂–C₃ and the position of the B ring at C₂/C₃ seemed to be indispensable for the anti-inflammatory activity.     

咖啡酸对氧化应激诱导的PC12细胞5-脂氧酶激活的抑制作用

目的探讨咖啡酸是否通过抑制氧化应激诱导的5-脂氧酶(5-LOX)激活而减轻细胞损伤。方法稳定转染绿荧光蛋白(GFP)-5-LOX的PC12细胞,预先给予咖啡酸0.001~10μmol.L-1和对照药MK886,30min后观察缺氧缺糖/恢复(OGD/R)及过氧化氢(H2O2)160μmol.L-1处理后的变化。MTT法和碘化丙啶染色法分析细胞存活率和死亡率;荧光显微镜观察OGD 2 h/R2 h和H2O2处理40 min时5-LOX的核膜移位;ELISA法测定OGD 2 h/R 3 h时5-LOX代谢产物的生成;DCF法检测OGD 2 h/R 0.5 h细胞内活性氧(ROS)的产生。结果 OGD 2 h/R 24 h GFP-5-LOX转染和GFP-转染PC12细胞的存活率分别为(63.1±6.6)%和(70.7±6.9)%;H2O2处理24 h细胞存活率分别为(62.5±7.7)%和(75.7±9.5)%。在GFP-5-LOX转染的PC12细胞中,咖啡酸和对照药MK886可使OGD/R细胞存活率从(63.1±6.6)%分别增加到(87.3±2.0)%和(89.9±6.3)%,细胞坏死率从(31.4±1.5)%降低到(10.1±2.0)%和(11.7±1.3)%(P<0.01);使H2O2处理细胞存活率从(62.51±7.65)%增加到(92.59±4.02)%和(75.31±6.60)%;使OGD/R细胞CysLTs的生成从261.1±33.7降低到108.5±16.7和(90.6±19.5)ng.g-1蛋白(P<0.01)。此外,咖啡酸抑制OGD/R诱导PC12细胞的ROS产生,IC50值为8.021μmol.L-1;抑制OGD/R诱导的GFP-5-LOX转染的PC12细胞5-LOX核膜移位,IC50值为0.974μmol.L-1;抑制H2O2诱导的GFP-5-LOX转染的PC12细胞5-LOX核膜移位,IC50值为0.501μmol.L-1;MK886无上述作用。结论咖啡酸可抑制氧化应激诱导的PC12细胞5-LOX激活,对缺血损伤的PC12细胞具有保护作用。     

Bioactive glycosides from the roots of Ilex asprella

Context The roots of Ilex asprella (Hook. et Arn.) Champ. ex Benth. (Aquifoliaceae) are widely used in Chinese medicine to treat influenza, amygdalitis, pertussis, etc. Their mechanism of action is still unknown, which raises the need to identify new bioactive compounds in this plant.

Objective In this study, we isolated a novel saponin containing sulphonic groups, namely, asprellcoside A (1) and a known phenolic glycoside compound (2) from the roots of Ilex asprella and evaluated their bioactivities.

Materials and methods Molecular structures were elucidated by analysing their spectral and chemical properties. The viability of A549 cells was tested using a MTT assay. Ability of the compounds to inhibit viruses was determined using the neuraminidase activity assay. Their anti-inflammatory effects were tested using the IP-10 activity assay using various concentrations (compound 1: 0.6, 0.2, 0.6, 1.70, 5.00 and 15.00?μM; compound 2: 0.4, 1.2, 3.6, 11.0, 33.0 and 100?μM). Their inhibitory effect on platelet aggregation induced by adenosine diphosphate (ADP) in rabbit plasma was determined at 60 and 80?μM.

Results Both compounds inhibit influenza virus strain A/PuertoRico/8/1934 (H1N1) strongly with EC₅₀ values of 4.1 and 1.7?μM, respectively. Both compounds inhibit the secretion of IP-10 with EC₅₀ values of 6.6 and 2.5?μM, respectively. Compound 1 alone inhibited platelet aggregation significantly, with the rate of suppression being 47?±?8 and 38?±?3%, at 60 and 80?μM, respectively.

Conclusions The results suggest that both compounds may be valid therapeutics against influenza virus infection and that compound 1 may be a novel agent for treating thrombosis.     

Spirodela polyrhiza extract modulates the activation of atopic dermatitis-related ion channels,Orai1 and TRPV3, and inhibits mast cell degranulation

Context: Spirodela polyrhiza (L.) Schleid. (Lemnaceae), Spirodelae Herba (SH), has been known to relieve inflammation, urticaria and skin symptoms including pruritus, eczema and rash.

Objective: The effects of SH extract on two calcium ion channels, Orai1 and TRPV3, and their potential as novel therapeutics for atopic dermatitis (AD) were investigated. The regulatory role of Orai1 on mast cell degranulation was evaluated.

Materials and methods: The dried leaves of SH were extracted by 70% methanol. Effects of SH extract (100?μg/mL) in an HEK293T cell line overexpressing human Orai1 or TRPV3 were assessed. Ion channel modulation in transfected HEK293T cells was measured using a conventional whole-cell patch-clamp technique. IgE-antigen complex-stimulated mast cell degranulation was measured by β-hexosaminidase assay with morphological observation after treatment with 20, 50 and 100?μg/mL SH extract.

Results: SH extract (100?μg/mL) significantly inhibited Orai1 activity (63.8?±?0.97%) in Orai1-STIM1 co-overexpressed HEK293T cells. SH extract significantly increased TRPV3 activity (81.29?±?0.05% at ?100?mV) compared with the positive control 2-APB (100?μM), which induced full activation. SH extract inhibited degranulation in IgE-antigen complex-stimulated RBL-2H3 mast cells by decreasing β-hexosaminidase activity (3.14?±?0.03, 2.56?±?0.12 and 2.29?±?0.08?mU/mg, respectively).

Conclusion: Our results suggested that SH extract could treat abnormal skin barrier pathologies in AD through modulation of the activities of the calcium ion channels Orai1 and TRPV3 and inhibition of mast cell degranulation. This is the first report of an herbal effect on the modulation of ion channels associated with skin barrier disruption in AD pathogenesis.     

Antioxidant activities and molecular mechanisms of the ethanol extracts of Baccharis propolis and Eucalyptus propolis in RAW64.7 cells

Context Numerous studies have reported that propolis possesses strong antioxidant activities. However, their antioxidant molecular mechanisms are unclear.

Objective We utilize ethanol extracts of Chinese propolis (EECP) as a reference to compare ethanol extracts of Eucalyptus propolis (EEEP) with ethanol extracts of Baccharis propolis (EEBGP) based on their antioxidant capacities and underlying molecular mechanisms.

Materials and methods HPLC and chemical analysis are utilized to evaluate compositions and antioxidant activities. ROS-eliminating effects of EEBGP (20–75?μg/mL), EEEP (1.25–3.75?μg/mL) and EECP (1.25–5?μg/mL) are also determined by flow cytometry analysis. Moreover, we compared antioxidant capacities by determining their effects on expressions of antioxidant genes in RAW264.7 cells with qRT-PCR, western blot and confocal microscopy analysis.

Results EEBGP mainly contains chlorogenic acid (8.98?±?0.86?mg/g), kaempferide (11.18?±?8.31?mg/g) and artepillin C (107.70?±?10.86?mg/g), but EEEP contains 10 compositions, whereas EECP contains 17 compositions. Meantime, although EEEP shows DPPH (IC₅₀ 19.55?±?1.28), ABTS (IC₅₀ 20.0?±?0.31) and reducing power (2.70?±?0.08?mmol TE/g) better than EEBGP’s DPPH (IC₅₀ 43.85?±?0.54), ABTS (IC₅₀ 38.2?±?0.33) and reducing power (1.53?±?0.05?mmol TE/g), EEBGP exerts much higher ROS inhibition rate (40%) than EEEP (under 20%). Moreover, EEBGP strengthen antioxidant system by activating p38/p-p38 and Erk/p-Erk kinase via accelerating nucleus translocation of Nrf2. EEEP and EECP improve antioxidant gene expression only via Erk/p-Erk kinase-Nrf2 signalling pathway.

Discussion and conclusion EEBGP and EEEP exert antioxidant activities via different molecular mechanisms, which may depend on chemical compositions.     

Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells

Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson’s disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC).

Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated.

Materials and methods DMA (0–60?μM) or DFC (0–10?μM) was co-treated with 6-OHDA (200?μM, 24?h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression.

Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells.

Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.     

Effects of indirubin and isatin on cell viability,mutagenicity, genotoxicity and BAX/ERCC1 gene expression

Context: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood.

Objective: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes.

Materials and methods: HeLa and/or CHO-K1 cell lines were tested (3 or 24?h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72?h) tests after treatment with IRN (0.1 to 200?μM) or ISA (0.5 to 50?μM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24?h) by gavage (50, 100 and 150?mg/kg determined from the LD₅₀ – 1?g/kg b.w.) and submitted to comet assay in vivo.

Results: IRN reduced the viability of CHO-K1 (24?h; 5 to 200?μM) and HeLa cells (10 to 200?μM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10?μM; HeLa: 5 and 10?μM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24?h) at all doses tested. IRN (100 and 150?mg/kg) also induced genotoxicity in vivo (4?h).

Conclusion: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.