Immunomodulatory effects of Premna tomentosa extract against Cr (VI) induced toxicity in splenic lymphocytes--an in vitro study.

Premna tomentosa (L. Verbanacae) is a widely used medicinal plant. Our earlier studies show that the extract of P. tomentosa leaves prevents acetaminophen-induced hepatotoxicity owing to its antioxidant property. In the present study, we have investigated the immunomodulatory effects of P. tomentosa extract against Chromium (VI) induced immunosuppression in splenic lymphocytes. Chromium (Cr) addition at a concentration of 5 microg showed an increase in cytotoxicity, apoptosis and reactive oxygen species (ROS) and a decrease in lymphocyte proliferation and antioxidant levels, whereas pre-treatment of the cells with P. tomentosa extract (at 500 microg concentration) resulted in decreased cytotoxicity and ROS levels. Further, the drug treatment also maintained antioxidant levels and restored lymphocyte proliferation similar to that of control cells. The results indicated that the leaf extract of P. tomentosa has cytoprotective and immunomodulatory activities.     

Assessment of the protective potential of Premna tomentosa (L. Verbenaceae) extract on lipid profile and lipid-metabolizing enzymes in acetaminophen-intoxicated rats

OBJECTIVES: The liver is often damaged by environmental toxins, poor eating habits, alcohol and over-the-counter drug use that damage and weaken the liver, leading to important public health problems such as hepatitis, cirrhosis, and alcoholic liver diseases. It is cardinal to treat liver disorders, because it affects the biochemistry of the cell directly. Damage to the liver can be prevented by including a balanced diet that includes nutrients and herbs that support a healthy liver. Premna tomentosa (PT) is one such herbal drug used widely in India for the treatment of liver disorders, and we have already reported the hepatoprotective potential and antioxidant property of methanolic extract of PT leaves. Because injury to the liver can promote a variety of reactions with consequent effect on lipids, the present study was designed to elucidate the hypolipidemic effect of PT extract in acetaminophen (AA)-induced hepatotoxicity in rats. DESIGN AND SUBJECTS: Animals were pretreated with PT extract (750 mg/kg, orally) for 15 days and then induced with hepatotoxicity by AA (640 mg/kg, intraperitoneally). RESULTS: PT extract pretreatment significantly inhibited induced alterations in the levels of cholesterol, triglycerides, free fatty acids, phospholipids, serum lipoproteins, and lipid-metabolizing enzymes. CONCLUSIONS: The results indicate that PT extract improves lipid metabolism and has the potential for use in hepatic disorders.     

Comparative toxicity of fatty acids on a macrophage cell line (J774)

In the present study, the cytotoxicity of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on a macrophage cell line (J774) was investigated. The induction of toxicity was investigated by changes in cell size, granularity, membrane integrity, DNA fragmentation and phosphatidylserine externalization by using flow cytometry. Fluorescence microscopy was used to determine the type of cell death (Acridine Orange/ethidium bromide assay). The possible mechanisms involved were examined by measuring mitochondrial depolarization, lipid accumulation and PPARgamma (peroxisome-proliferator-activated receptor gamma) activation. The results demonstrate that fatty acids induce apoptosis and necrosis of J774 cells. At high concentrations, fatty acids cause macrophage death mainly by necrosis. The cytotoxicity of the fatty acids was not strictly related to the number of double bonds in the molecules: palmitic acid>docosahexaenoic acid>stearic acid=eicosapentaenoic acid=arachidonic acid>oleic acid>linoleic acid. The induction of cell death did not involve PPARgamma activation. The mechanisms of fatty acids to induce cell death involved changes in mitochondrial transmembrane potential and intracellular neutral lipid accumulation. Fatty acids poorly incorporated into triacylglycerol had the highest toxicity.     

Ultrasound-induced physiological effects and secondary metabolite (saponin) production in Panax ginseng cell cultures

This work was aimed at the effects of ultrasound (US) on the growth and secondary metabolite biosynthesis of cultured plant cells. Suspension cultures of Panax ginseng cells were exposed to US at power density below 82 mW/cm3 for short periods of time (1-4 min) in a US bath (38.5-kHz fixed frequency and 810 W maximum peak power). Under most exposure conditions, US stimulated the biosynthesis of secondary metabolites, the ginsenoside saponins of ginseng cells, increasing the total saponin content of the cell by up to 75%. The growth and viability of ginseng cells were usually depressed immediately after the exposure to US, but recovered gradually to levels similar to those of a normal culture in a few days, with virtually no net loss of biomass yield at the end of the culture period. At some lower US doses, sonicated cultures could even reach slightly higher biomass yields than that of normal cultures. The effects of US on cell growth and secondary metabolite yield showed a significant correlation with the total US energy emitted (i.e., the product of US power and exposure time). Mechanical stress and microstreaming induced by acoustic cavitation were considered as the most possible causes of the various physiological effects of US on ginseng cells. In particular, the stimulation of secondary metabolite production by US may be a result of US-induced plant cell defense response.     

Intracellular activity of zidovudine (3''-azido-3''-deoxythymidine, AZT) against Salmonella typhimurium in the macrophage cell line J774-2.

The antibacterial effect of zidovudine (AZT) has been demonstrated both in vitro and in vivo with experimental models of gram-negative bacterial infections. It has been associated with the absence or low occurrence of nontyphoid Salmonella infections in AIDS patients treated with AZT. Using the macrophage cell line J774-2, we demonstrate the inhibition of intracellular growth of Salmonella typhimurium by AZT. This effect is obtained with one-half of the MIC (1 microgram/ml) of AZT for S. typhimurium. Inhibition of intracellular growth is observed after 4 h of incubation and persists at 24 h. Maximal inhibition is shown at a concentration of 128 micrograms/ml, and no further effect is observed with higher concentrations. When the inhibitory effect of AZT is compared with that of pefloxacin or that of ceftriaxone at half their MICs (0.2 and 0.02 microgram/ml, respectively), AZT and pefloxacin give better results than ceftriaxone. In this study, using an intracellular model, we show that AZT is able to inhibit the intracellular multiplication of S. typhimurium at a minimal effective concentration lower than the MIC, indicating its potential for antibacterial accumulation in the macrophages.     

Dynorphin-A(1-17) decreases nitric oxide release and cytotoxicity induced with lipopolysaccharide plus interferon-gamma in murine macrophage cell line J774.

Nitric oxide (NO) is an important mediator of cytotoxicity caused by macrophages or by their resident counterpart in brain-glial cells. Modulation of NO release by both activated macrophages and glial cells has been reported in the presence of endogenous (peptide) and synthetic (non-peptide) agonists with kappa opioid-receptors (KOR) selectivity. The data obtained with macrophages and glial cells are contradictory: enhanced NO release by mouse macrophages was reported in the presence of synthetic agonist of KOR selectivity (Neuropeptides 32 (1998) 287), and decreased NO release by glial cells, in the presence of dynorphin-A((1-8)), endogenous opioid peptide with KOR selectivity (J. Biomed. Sci. 7 (2000) 241). In this study, we used a murine cell line J774 of macrophage origin and examined the effect of dynorphin-A((1-17)), endogenous opioid peptide with selectivity for KOR, on NO release induced with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Dynorphin-A((1-17)) was chosen since in comparison to dynorphin-A((1-13)), it is more resistant to biodegradation (Peptides 17 (1996) 983), and its effects during prolonged treatment of cells could be more pronounced. The effect of dynorphin-A((1-17)) on NO release was compared to its effect on cytotoxicity, induced with LPS plus IFN-gamma. The data obtained have shown that activation-induced NO release by J774 cells is decreased in the presence of dynorphin-A((1-17)). This was associated with deceased LPS and IFN-gamma-induced cytotoxicity of J774 cells, suggesting their causal relationship. Neither of the observed effects of dynorphin-A((1-17)) could be prevented with the KOR selective antagonist, norbinaltorphimine, suggesting that they are mediated via non-opioid mechanism. By diminishing NO release dynorphin-A((1-17)) may affect cytotoxic ability of macrophages, but may also beneficially influence inflammation-induced damage of local tissue.     

Use of Mono Mac 6 human monocytic cell line and J774 murine macrophage cell line in parallel antimycobacterial drug studies.

The Mono Mac 6 (MM6) human monocytic cell line was evaluated with the established J774 murine macrophage cell line to ascertain its effectiveness in determining the intracellular activities of antimycobacterial drugs. Cells were infected with Mycobacterium tuberculosis H37Ra and treated with drug concentrations corresponding to the MICs, as well as to threefold higher than and threefold less than the MICs. Changes in CFU were compared after 7 days to determine significant differences between treated and nontreated groups. The results suggest that MM6 will make a useful model for testing the intracellular activities of antituberculosis drugs.     

Crocus sativus L. (Saffron) extract and its active constituents (crocin and safranal) on ischemia-reperfusion in rat skeletal muscle

Saffron and its constituents have been shown to decrease ischemia-reperfusion (I/R) injury in kidney or brain tissues. In this study, the effects of saffron ethanolic extract and its constituents, crocin and safranal, were evaluated in skeletal muscle during I/R injury. Hind limb ischemia was induced using clamping the common femoral artery and vein. After 2 h ischemia, the clamp of the femoral vessels of animals was taken off and the animal underwent 1h reperfusion. Muscle injuries were evaluated by recording of the electromyographic (EMG) potentials and performing some biochemical analysis including thiobarbituric acid reactive substances (TBARS), total sulfhydryl (SH) groups and antioxidant capacity of muscle (using FRAP assay). The ethanolic extract of saffron (5, 20 and 80 mg kg⁻¹), crocin (50, 200 and 400 mg kg⁻¹), safranal (0.1, 0.25 and 0.5 ml kg⁻¹) and normal saline (10 ml kg⁻¹) were administered intraperitoneally 1 h prior reperfusion. The average peak-to-peak amplitude during I/R was significantly increased in extract, crocin and safranal groups in comparison with control-ischemic group. Following saffron, crocin and safranal administration, the total SH contents and antioxidant capacity were elevated in muscle flap. The MDA level was declined significantly in test groups. It is concluded that saffron extract and its constituents show a protective effect against lower limb I/R in rat.     

Inhibitory effect of phenolic extract from squirting cucumber (Ecballium elaterium (L.) A. Rich) seed oil on integrin-mediated cell adhesion,migration and angiogenesis

Integrin targeted therapies by natural bioactive compounds have attracted attention in the field of oncology and cancer treatment. This study evaluates the potential of phenolic extract from the medicinal herb Ecballium elaterium L. seed oil (PEO) to inhibit the adhesion and migration of the highly invasive human fibrosarcoma cell line HT1080. At safe concentrations (up to 40 μg mL⁻¹), results show that PEO dose-dependently inhibits adhesion and migration of HT1080 to fibronectin (IC₅₀ = 18 μg mL⁻¹) and fibrinogen (IC₅₀ = 12.86 μg mL⁻¹). These observations were associated with the reduction of cell motility and migration velocity as revealed in the Boyden chamber and random motility using two-dimensional assays, respectively. Additional experiments using integrin blocking antibodies showed that PEO at the highest safe concentration (40 μg mL⁻¹) competitively inhibited the attachment of HT1080 cell to anti-αvβ3 (>98%), anti-α5β1 (>86%), and to a lesser extent anti-α2 (>50%) immobilized antibodies, suggesting that αvβ3 and α5β1 integrins were selectively targeted by PEO. Moreover, PEO specifically targeted these integrins in human microvascular endothelial cells (HMEC-1) and dose-dependently blocked the in vitro tubulogenesis. In the CAM model, PEO inhibited the VEGF-induced neoangiogenesis confirming its anti-angiogenic effect. Collectively, these results indicate that PEO holds promise for the development of natural integrin-targeted therapies against fibrosarcoma.

Phenolic extract from Ecballium elaterium inhibits integrin-mediated adhesion and migration, and hinders VGEF-induced angiogenesis.     

脑缺血再灌注对小鼠学习记忆的损伤及山茶花提取物的保护作用

摘要 目的：探讨山茶花提取物（ECJ）对缺血性记忆障碍的保护作用及其与炎症和血脑屏障损伤的关系。方法：小鼠分为假手术组、模型组、阳性药物对照组 （银杏叶提取物，EGB，35 mg·kg-1，ip）和山茶花提取物治疗组（ECJ，50，15，5 mg·kg-1，ip）。 山茶花提取物治疗组和阳性药物对照组分别注射山茶花提取物和银杏叶提取物，连续给药9d。给药后第7d，采用双侧颈总动脉结扎法建立重复前脑缺血再灌注模型。术后第7d用水迷宫检测小鼠学习记忆能力；用紫外分光光度计检测脑组织髓过氧化物酶（MPO）活性和伊文思蓝（EB）含量；同时测定脑组织的含水量。结果：模型组小鼠的学习记忆能力显著下降（P＜0.01)，同时伴随脑内MPO活性上升（P＜0.01），脑组织EB及水分量升高（P＜0.01）。ECJ预防和治疗给药可提高缺血小鼠的学习记忆能力；并使脑组织EB含量下降（P＜0.05和P＜0.01），脑水分含量减少（P＜0.05），血脑屏障损伤程度有所减轻；同时伴随MPO活性下降（P＜0.05和P＜0.01），炎症反应减弱。结论：ECJ能减轻缺血再灌注引起的脑损伤，改善小鼠学习记忆能力，该作用可能与其提高脑组织抗血脑屏障损伤和抗炎症反应能力有关。 关键词 山茶花提取物；脑缺血再灌注；学习记忆；血脑屏障；炎症反应 中图分类号：R965，R743 文献标识码：A 文章编号：1001-1242(2008)-03-0245-03     

Cytotoxic and antioxidative potentials of ethanolic extract of Eugenia uniflora L. (Myrtaceae) leaves on human blood cells

Eugenia uniflora is used in the Brazilian folk medicine to treat intestinal disorders and hypertension. However, scanty information exist on its potential toxicity to human, and little is known on its antioxidant activity in biological system. Hence, we investigated for the first time the potential toxic effects of ethanolic extract (EtOH) of E. uniflora (EEEU) in human leukocytes and erythrocytes, as well as its influence on membrane erythrocytes osmotic fragility. In addition, EEEU was chemically characterized and its antioxidant capacity was evaluated. We found that EEEU (1–480 μg/mL) caused neither cytotoxicity nor DNA damage evaluated by Trypan blue and Comet assay, respectively. EEEU (1–480 μg/mL) did not have any effect on membrane erythrocytes fragility. In addition, EEEU inhibited Fe²⁺-induced lipid peroxidation in rat brain and liver homogenates, and scavenged the DPPH radical. EEEU presented some polyphenolic compounds with high content such as quercetin, quercitrin, isoquercitrin, luteolin and ellagic acid, which may be at least in part responsible for its beneficial effects. Our results suggest that consumption of EEEU at relatively higher concentrations may not result in toxicity. However, further in vitro and in vivo studies should be conducted to ascertain its safety.     

Enhanced activity of streptomycin and chloramphenicol against intracellular Escherichia coli in the J774 macrophage cell line mediated by liposome delivery.

AT-2266 (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-1, 8-naphthyridine-3-carboxylic acid) showed marked activity in vivo when administered orally to mice bearing systemic, pulmonary, dermal, or urinary tract infections due to variety of organisms. The activity of AT-2266 was uniformly higher than those of norfloxacin, pipemidic acid, and nalidixic acid against all of the infections. The activity of AT-2266 administered orally was almost comparable to that of gentamicin administered subcutaneously against urinary tract infections due to gram-negative organisms but was generally lower against other infections. AT-2266 exhibited significant activity against infections due to gentamicin-resistant and nalidixic acid-resistant organisms.     

The role of antigen mobility in anti-Rh0(D)-induced agglutination.

Intact human erythrocytes were cross-linked with glutaraldehyde (GA) or dimethyladipimidate (DMA) and tested for their ability to bind [125I]-IgG anti-Rh0(D) and to undergo antibody-mediated hemagglutination. There was no decrease in antibody binding after treatment with GA concentrations up to 1.25% and DMA concentrations up to 1%. Red cells treated with these concentrations of GA and DMA did not agglutinate. The techniques employed to induce agglutination of the cross-linked red cells involved "incomplete" IgG anti-Rho (D) in albumin, "complete" IgM anti-D Rho (D) in saline, and the antiglobulin (Coombs) reaction. The agglutinability of the chemically modified red cells was inversely correlated with the extent of fixation. The dissociation of antibody binding from agglutinability in cross-linked erythrocytes suggests that Rho (D) antigen mobility is required for red cell agglutination. Antigen mobility was manifested by the transition from a relatively monodisperse distribution pattern of Rho (D) antigen sites to one of large aggregates or clusters when agglutination was induced by IgM anti-Rho (D), IgG anti-Rho (D) agglutination of protease modified red cells, and by anti-IgG agglutination of IgG anti-Rho (D)-coated red cells. Antigen clustering was not as prominent in red cells agglutinated by IgG anti-Rho (D) in the presence of albumin. Even though antigen mobility is a prerequisite for antibody-mediated hemagglutination, clustering does not appear to be an absolute requirement. The degree of antigen clustering differs with varying types of agglutination.     

Selective immunosuppression by administration of major histocompatibility complex (MHC) class II-binding peptides. I. Evidence for in vivo MHC blockade preventing T cell activation.

Draining lymph node cells (LNC) from mice immunized with hen egg white lysozyme (HEL) display at their surface antigen-MHC complexes able to stimulate, in the absence of any further antigen addition, HEL peptide-specific, class II-restricted T cell hybridomas. Chloroquine addition to these LNC cultures fails to inhibit antigen presentation, indicating that antigenic complexes of class II molecules and HEL peptides are formed in vivo. MHC class II restriction of antigen presentation by LNC from HEL-primed mice was verified by the use of anti-class II monoclonal antibodies. Coinjection of HEL and the I-Ak-binding peptide HEL 112-129 in mice of H-2k haplotype inhibits the ability of LNC to stimulate I-Ak-restricted, HEL 46-61-specific T cell hybridomas. Similar results are obtained in mice coinjected with the HEL peptides 46-61 and 112-129. Inhibition of T hybridoma activation can also be observed using as antigen-presenting cells irradiated, T cell-depleted LNC from mice coinjected with HEL 46-61 and HEL 112-129, ruling out the possible role of either specific or nonspecific suppressor T cells. Inhibition of T cell proliferation is associated with MHC-specific inhibition of antigen presentation and with occupancy by the competitor of class II binding sites, as measured by activation of peptide-specific T cell hybridomas. These results demonstrate that administration of MHC class II binding peptide competitors selectively inhibits antigen presentation to class II-restricted T cells, indicating competitive blockade of class II molecules in vivo.     

Selection of L cell sublines resistant to (E)-5-(2-bromovinyl)-2'-deoxyuridine.

A mouse thymidine kinase (TK) deficient L cell subline and L cell sublines biochemically transformed by herpes simplex virus TK were cultured in the presence of increasing concentrations of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) which inhibited the growth of all sublines, and resistant sublines were isolated. Their growth properties were dependent on the medium condition used for selection. One subline had lost viral TK activity, while another retained viral TK activity with altered sensitivity of TK to BVDU in comparison with that of the original subline. Growth characteristics and TK activity of sublines are discussed.