Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were alpha-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 mug/ml against promastigotes, respectively. These values were 11.6 and 40.8 mug/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 mug/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were alpha-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 mug/ml against promastigotes, respectively. These values were 11.6 and 40.8 mug/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 mug/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

Susceptibilidad in vitro a infección por Leishmania y sensibilidad a medicamentos difiere según tipo de macrófagos / The susceptibility to infection by Leishmania panamensis and the sensitivity to drugs differ according to the macrophage type

Introducción: Los sistemas in vitro son útiles en la evaluación de compuestos con actividad biológica, permitiendo por ejemplo, determinar el potencial citotóxico y leishmanicida de un compuesto. Objetivo: Identificar el tipo de célula mamífera que permita una óptima infección in vitro por Leishmania y que constituya el sistema adecuado para el tamizaje in vitro de compuestos con actividad anti-Leishmania. Métodos: La susceptibilidad de las células a infección por L. panamensis se evalúo según la Concentración Infectiva 50 determinada por microscopía de luz y citometría de flujo; la supervivencia intracelular de los amastigotes se evaluó por microscopía de fluorescencia y la sensibilidad de las células a anfotericina B y antimoniato de meglumina se evalúo por espectrofotometría. Resultados: Los cultivos primarios son más susceptibles a la infección por L. panamensis in vitro. Sin embargo, la supervivencia intracelular del parásito fue mejor en U-937. Por su parte, la sensibilidad de las células a anfotericina B y antimoniato de meglumina vario según el tipo de célula. Conclusiones: Las células U-937 son las adecuadas para la infección por Leishmania porque: a) presentan crecimiento ilimitado y se les puede inducir transformación a macrófagos. b) la susceptibilidad a la infección por Leishmania es similar a la observada en cultivos primarios de macrófagos y c) permiten mayor supervivencia de los amastigotes luego de la infección. Adicionalmente, las células U-937 son menos sensibles a la acción de los fármacos comúnmente utilizados como control en la detección de compuestos con actividad leishmanicida.

Introduction: In vitro systems are useful in the evaluation of compounds with biological activity determining the cytotoxic and leishmanicidal activity of the candidates. Objective: To identify the mammalian cell that allows the optimal in vitro infection by Leishmania and therefore, identify the suitable system for the in vitro evaluation of leishmanicidal activity of drugs. Methodology: The susceptibility to the infection by L. panamensis was evaluated according to the Infective Concentration 50 tested by light microscopy and flow citometry; the intracellular survival of amastigotes was determined by fluorescence microscopy and the sensitivity to amphotericine B and meglumine antimoniate was evaluated by spectrophotometry. Results: The primary culture cells were more susceptible to the in vitro infection by L. panamensis because they did require fewer parasites per cell ratio to achieve the 50% infection rate whereas the intracellular survival of parasites was better in the U-937 cells. All cells showed differential sensitivity to amphotericine B and meglumine antimoniate. Conclusion: The U-937 cells are the most suitable model for the in vitro infection by L. panamensis because: a) they are a cell line with unlimited growth where transformation into macrophages can be induced. b) The susceptibility to infection by L. panamensis is similar to that observed in primary cultures macrophages and, c) They allow the intracellular survival of amastigotes after the infection process. In addition the U-937 cells are less sensitive to the action of the commonly drugs used as a control in the screening of compounds with leishmanicidal activity.

Study on expression of NO and iNOS in J774A. 1 cells induced by rVvhA / 中华微生物学和免疫学杂志

Objective To investigate the effect of recombinant Vibrio vulnificus hemolysin (rVvhA) on the expression of nitric oxide(NO) and induced nitric oxide synthase(iNOS) in J774A. 1 cells.Methods The inhibitory effect of rVvhA on J774A. 1 proliferation was measured by MTF colorimetry technique. The content of nitrite in culture medium was determined by Griess reagent. The expression of iNOS protein and mRNA were measured by immunofluorescence and RT-PCR, respectively. Results The viability of J774A. 1 cells was apparently inhibited when exposed to 0.8 HU/ml rVvhA and up. With the help of IFN-γ, the expression of NO and the activity of iNOS in J774A. 1 cells were remarkably increased when exposed to 0.4 HU/ml rVvhA. Conclusion rVvhA can increase the expression of NO and iNOS, it may play an important role in the pathogenic mechanism of Vibrio vulnificus.

Effects of cell cycles and their regulating genes on apoptosis of mononuclear-macrophages induced by Leptospira interrogans / 中华微生物学和免疫学杂志

Objective To investigate the effects of different cell cycles and their regulating genes on apoptosis of mononuclear-macrophages induced by Leptospira interrogans. Methods The diversity and alteration of cell cycles of murine mononuclear-macrophage line(J774A. 1 ) and human monocyte line(THP-1 ) before and after infected with L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai were detected using Cell Cycle Stain Kit plus flow cytometer. The cell cycle synchronized J774A. 1 and THP-1 cells were generated and then identified by using different cell cycle blocking agents and flow cytometer. By using Annexin V/PI Detection Kit combined with flow cytometer, the rates of early-apoptosis and late-apoptosis/necrosis in the synchronized and non-synchronized J774A. 1 and THP-1 cells after infection with L. interrogans strain Lai were determined. Several real-time fluorescence quantitative RT-PCRs were performed to the changes of mRNAs levels of p21, p27, p53, c-myc and cycA genes that associated with cell cycle and apoptosis in J774A. 1 and THP-1 cells before and after infected with L. interrogans strain Lai. Results There were G1, S and G2/M phases in both the non-infected normal J774A. 1 and THP-1 cells. On the contrast,the majority of infected J774A. 1 and THP-1 cells were stagnated at G1 phase, but the amount of S phase THP-1 cells was elevated while that of S phase J774A. 1 cells was not(P ＜0.05). No remarkable early-apoptosis in both the infected G1 phase J774A. 1 and THP-1 cells was found, whereas the rates of early-apoptosis and late-apoptosis/necrosis in the infected M phase J774A. 1 and THP-1 cells were significantly increased (P ＜0.05 ). Additionally, late-apoptosis / necrosis rate in the infected G1 phase THP-1 cells (P ＜ 0.05 )that not found in the infected G1 phase J774A. 1 cells. Compared to the non-infected cells, the p21 mRNA levels in the infected J774A. 1 and THP-1 cells were significantly elevated(P ＜0.05), and the c-myc and p27 mRNA levels in the infected J774A. 1 cells and the cycA mRNA level in the infected THP-1 cells were also higher than those in both the non-infected cells ( P ＜ 0.05 ). Conclusion Different cell cycles and their regulating genes have a role to affect the apoptosis of human and murine mononuclear-macrophages caused by L. interrogans with a diversity of cell line origins.

Effect of apoptosis of rVvhA on J774A.1 and the related mechanism / 中华微生物学和免疫学杂志

Objective To investigate the activity of recombinant Vibrio vulnificus hemolysin (rVvhA) on the apoptosis of J774A.1 cells and the related mechanism. Methods The cytotoxic effect of rVvhA on the growth of J774A.1 cells was identified by MTT, celluar and mitochondrial morphology were observed by transmission electron microscopy, apoptosis or necrosis and mitochondrial membrane potential in J774A.1 cells were measured by flow cytometry, activities of caspase-3 ,-8,-9 were detected by spectrophotometry. Results The viability of J774A.1 cells exposed to rVvhA was inhibited, and it is dependent on dose. Celluar and mitochondrial uhrastructure both occurred to change obviously observed by transmission electron microscopy in J774A.1 treated by 2.0 HU/ml and 3.0 HU/ml rVvhA after 8 hours; and 3.0 HU/ml rVvhA group had a better cytotoxic effect on J774A.1 than that of 3.0 HU/ml rVvhA group. The percentage of apoptosis is (7.80±0.62)%, (12.33±0.12)%, respectively. Besides, the mitochondriai membrane potential also reduced, because the rate of fluorescence which is green increase 1.0% (normal) to 9.8% (2.0 HU/ml rVvhA) and 39.2% (3.0 HU/ml rVvhA). At the same time, the caspase-3, -9 activity increased gradually, but caspase-8 remained unchanging. In J774A.1 cells treated by 3.0 HU/ml rV-vhA + caspase-3 inhibitor(Ac-DEVD-FMK) or caspase-9 inhibitor(Ac-LEHD-FMK), The apoptosis of was reduced to(6.23±3.95)% ,(9.60±3.14)%, and the activity of caspase-3, -9 reduced, too. Conclusion The rVvhA has cytotoxic effect on J774A.1. Mitochondria-mediated apoptosis pathway which is dependent on caspase may be related to apoptosis induced by rVvhA in J774A.1.

Variance and resource of intracellular free calcium and extracellular arachidonic acid of host cells during the invasion of Toxoplasma gondii / 中华传染病杂志

Objective To explore variance and resource of intracellular free Ca2+ and extracellular arachidonic acid (AA) in different types of passage cells during the invasion of T. gondii. Methods The variance and resource of extracellular AA and intraceUular free Caz+ of Vero and J774A. 1 cells during the invasion of T. gondii were detected by multi-purpose scintillation counter and laser scanning confocal microscope. Data were analyzed using Chi-square test and t test. Results The intracellular free Ca2+ levels in J774A. 1 and Vero cells were both increased after T. gondii infection. The maximal changes of fluorescence intensity were (1 219.7±58.4)% (P＜0.01) and (356.3±23.6)% (P＜0.05), respectively.The increase of intracellular Ca2+ level in Vero cell was mostly from the release of intracellular Ca2+ store.And the Ca2+ increase in J774A. 1 cell was from both the release of intracellular Ca2+ store and extracellular Ca2+ influx. Extracellular AA levels were significantly increased in both Vero and J774A. 1 cells after T.gondii infection 5.02 and 8. 44 times respectively (t= 3. 124, t = 3. 852, P＜0.01). The AA elevations could be significantly inhibited by phospholipase A2 (PLA2) inhibitor pretreating T. gondii. Conclusions The phospholipase C of phagocytic host cell and PLA2 of T. gondii are activated by T. gondii infection,which results in the increase of intracellular free Ca2+ and extraeellular AA level. Combined actions of Ca2+and AA play a major role in the invasion of T. gondii to host cell. While only PLA2 of T. gondii may be activated in nonphagoeytic host cell.

Change of Cytoskeleton and Variance of Ca~(2+) in Cultured Cells During the Invasion of Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To explore the change of cytoskeleton and the variance of Ca2+ in cultured cells during the invasion of Toxoplasma gondii. Methods Tachyzoites suspensions were gathered by routine method and used to infect phagocytic cells(J774A.1)and non-phagocytic cells (HUVEC). The ability of T. gondii invading into the cells and the influence of cytoskeleton inhibitor,colchicine and cytochalasin D,were observed by microscopy. The rearrangement of cytoskeleton of cells was observed by fluoromicroscopy. By using laser scanning confocal microscope,the variance of Ca2+ in J774A.1 and HUVEC was detected. Results Ca2+ increased greatly in J774A.1 during the invasion of T.gondii(P0.05). The microfilaments of J774A.1 were agglomerated during the invasion of T. gondii. Cytoskeleton inhibitor,cytochalasin D(P0.05)during the invasion and cytoskeleton was not changed. Cytochalasin D and colchicine showed little effect on the infection rate of HUVEC. Conclusion The concentration of Ca2+ increases greatly and cytoskeleton(mainly the microfilament)has been rearranged in phagocytic cell during the invasion of T. gondii,while both of them show no significant change in non-phagocytic cell.