Tempol decreases the levels of reactive oxygen species in human neutrophils and impairs their response to

SUMMARY Background and aims: Mycobacterium tuberculosis (Mtb), the main causative agent of human tuberculosis (TB), remains as a serious public health problem. The innate immune response following Mtb infection plays a crucial role in preventing the onset of active TB and limiting its spread. Since phagocytes-derived reactive oxygen/nitrogen species (ROS/RNS) during the oxidative burst can fight Mtb, we hypothesized that the use of antioxidants could increase the host's susceptibility to Mtb/TB. In that way, we investigated the effects of the nitroxide Tempol, an antioxidant with superoxide dismutase-like activity, on the response of neutrophils against Mtb. Methods: Human blood-derived neutrophils were isolated from healthy volunteers and incubated with Mtb (H37Ra) at different multiplicities of infection (MOIs), in the absence or presence of Tempol. The levels of ROS in neutrophils were evaluated using the cytochrome C reduction assay (extracellular O2 •-) and luminol-(total intracellular and extracellular ROS) and isoluminol-(extracellular ROS) amplified chemiluminescence assay. The killing assay (two-step protocol) checked the mycobactericidal capacity of neutrophils, as calculated the phagocytosis (K p ) and intracellular killing (Kk) rates. Results: The levels of ROS and killing capacity in Mtb-stimulated neutrophils were significantly decreased by 450 µM Tempol (p < 0.05). Interestingly, Tempol decreased the k k of neutrophils, but did not affect their kp, demonstrating that a putative diminution in ROS levels, ultimately, affected the intracellular killing of Mtb. Conclusion: This study provides insights regarding the role of antioxidants on the neutrophil response toward Mtb, so that our findings deserve to be considered regarding further studies and clinical implications.

Contextualización y objetivos: Mycobacterium tuberculosis (Mtb), el principal agente causal de la tuberculosis humana (TB), es un grave problema de salud pública. La respuesta inmune innata desencadenada en la infección por Mtb juega un papel crucial en la prevención de la aparición de la tuberculosis activa y en la limitación de su propagación. Dado que las especies reactivas de oxígeno/nitrógeno derivadas de los fagocitos (ERO/ERN) durante el burst oxidativo pueden combatir la infección pulmonar por Mtb, planteamos la hipótesis de que el uso de antioxidantes podría aumentar la susceptibilidad del huésped humano hacia Mtb/TB. De esa manera, investigamos los efectos del nitróxido Tempol, un antioxidante con actividad similar a la superóxido dismutasa, sobre la respuesta de los neutrófilos contra Mtb. Métodos: se aislaron neutrófilos derivados de sangre humana de voluntarios sanos y se incubaron con Mtb (H37Ra) en diferentes multiplicidades de infección (MOI), en ausencia o presencia de Tempol. Los niveles de ERO en neutrófilos se evaluaron mediante el ensayo de la reducción del citocromo C (O2'- extracelular) y el ensayo de quimioluminiscencia, amplificada mediante el uso de luminol (ERO total, intracelular y extracelular) e isoluminol (ERO extracelular). La prueba de actividad microbicida (protocolo de dos pasos) verificó la capacidad micobactericida de los neutrófilos, calculando las tasas de fagocitosis (Kp) y muerte intracelular (Kk). Resultados: los niveles de ERO y la capacidad micobactericida en neutrófilos estimulados con Mtb disminuyeron significativamente en los grupos tratados con 450 µM de Tempol (p < 0,05). Curiosamente, Tempol disminuyó la tasa de muerte intracelular (Kk) en neutrófilos, pero no tuvo ningún efecto sobre la tasa de fagocitosis (Kp), lo que demuestra que una supuesta disminución en los niveles de ERO, en última instancia, afectó la destrucción intracelular de Mtb. Conclusión: este estudio proporciona información sobre el papel de los antioxidantes en la respuesta de los neutrófilos hacia Mtb, por lo que nuestros hallazgos merecen ser considerados con respecto a más estudios e implicaciones clínicas.

Contextualização e objetivos: Mycobacterium tuberculosis (Mtb), principal agente causal da tuberculose humana (TB), é um grave problema de saúde pública. A resposta imune inata desencadeada pela infecção por Mtb desempenha um papel crucial na prevenção do aparecimento da tuberculose ativa e na limitação de sua disseminação. Como as espécies reativas de oxigênio/nitrogênio derivadas de fagó-citos (ERO/ERN) durante a burst oxidativa podem combater a infecção pulmonar por Mtb, hipotetizamos que o uso de antioxidantes poderia aumentar a suscetibilidade do hospedeiro humano ao Mtb/TB. Assim, investigamos os efeitos do nitróxido de Tempol, um antioxidante com atividade semelhante a superóxido dismutase, na resposta de neutrófilos contra o Mtb. Métodos: neutrófilos derivados de sangue humano foram isolados de voluntários saudáveis e incubados com Mtb(H37Ra) em diferentes multiplicidades de infecção (MOI), na ausência ou presença de Tempol. Os níveis de ERO em neutrófilos foram avaliados por ensaio de depleção de citocromo C (O2•- extracelular) e ensaio de quimioluminescência, amplificado com luminol (ERO total, intracelular e extracelular) e isoluminol (ERO extracelular). O teste de atividade microbicida (protocolo de duas etapas) verificou a capacidade micobactericida dos neutrófilos, calculando as taxas de fagocitose (Kp) e morte intracelular (Kk). Resultados: os níveis de ERO e a capacidade micobactericida em neutrófilos estimulados por Mtb diminuíram significativamente nos grupos tratados com Tempol 450 µM (p < 0,05). Curiosamente, Tempol diminuiu a taxa de morte intracelular (Kk) em neutrófilos, mas não teve efeito sobre a taxa de fagocitose (Kp), mostrando que uma diminuição putativa nos níveis de ERO afetou a morte intracelular de Mtb. Conclusão: este estudo fornece informações sobre o papel dos antioxidantes na resposta dos neutrófilos ao Mtb, portanto, nossos achados merecem consideração para estudos adicionais e implicações clínicas.

Effects of nitronyl nitroxide on myocardial hypoxia and apoptosis-associated proteins in hypobaric hypoxia mice / 中国药学杂志

OBJECTIVE: To investigate the effects of nitronyl nitroxide(HPN) on the myocardial hypoxia- and the expression of apoptosis-associated proteins in hypobaric hypoxia condition mice. METHODS: Sixty BALB/c mice were divided into normal control group, hypoxia model group, acetazolamide group and HPN group randomly. After single intraperitoneal injection of HPN for 30 min, the mice were exposed to a simulated high altitude of 8 000 m for 12 h. After hypoxic exposure, mice were sacrificed and the content of lactate(LD) and lactate dehydrogenase(LDH) activity in heart were determined. HIF-1, VEGF, caspase-3, Bax and Bcl-2 were detected by immunohistochemistry. RESULTS: Compared with normal control group, the LD level and LDH activity in hypoxia model group increased significantly. In addition, the expression of HIF-1α, VEGF, caspase-3 and Bax were increased, Bcl-2 and Bc1-2/Bax ratio was decreased. Pre-treated with HNP the LD level and LDH activity, the expression of HIF-1α, VEGF, caspase-3 and Bax were decreased effectively, the expression of Bcl-2 and Bc1-2/Bax ratio was increased. CONCLUSION: HPN has protective effect on heart injury induced by hypobaric hypoxia in mice. Its mechanism may be related to the amelioration of energy metabolism, alleviation of oxidative stress as well as inhibition of myocardial apoptosis.

EFFECTS OF PODOPHYLLIC ACID PIPERIDYL HYDR-AZONE NITROXIDE RADICAL ON THE PROLIFERATION, MITOTIC INDEX AND DNA SYNTHESIS OF SOME CANCER CELLS IN VITRO / 中国药理学通报

It was found that the proliferation of mice L7712 leukemic cells in vitro was markedly inhibited by 5 mg/L GP1 and VP10 The inhib itory rate increased with the incubation time. At a concentration of 0 .04-20mg/L, the mitotic index ( MI ) of GP1 group increased, but the MI of VP16 group decreased. After L7712 cells were treated with GP, 5 mg/L for 12 h the MI reached the highest point which was 8 times as high as that of the control, at the same time, the MI of VP16 ( 5 mg/L) group was about one-third of that of the control. The result of the combination of GP1 with VP16 showed that VP16 could antagonize the effect of GP on MI of L7712 cells. After being treated by GP1 and VP18 for 24 h, serious damage of L7712 cells could be observed. Both drugs inhibited the incorporation of (3H) TdR into DNA of S180, ascitic hepatoma (AH), L1210 and L7712 cells incubated for 24 h. It was further observed that S180 and L7712 cells were more sensitive than other cells to both drugs.