La expresión del canal iónico TRPM7 es modulada por estrés oxidativo generado en neuronas expuestas a lipopolisacárido
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2011
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Las infecciones bacterianas del sistema nervioso se mantienen como una causa común de mortalidad debido a la ineficacia de las terapias farmacológicas actuales. Entre las infecciones causadas por bacterias Gram negativo, es ampliamente aceptado que las consecuencias fisiopatológicas son producidas principalmente por la acción del lipopolisácarido (LPS) en el sistema nervioso.
En el sistema nervioso, es tradicionalmente aceptado que el efecto neurotóxico mediado por el LPS es debido a la previa activación de células microgliales, sin embargo, evidencia reciente sugiere que el LPS podría ejercer un efecto neurotóxico de manera directa, es decir, sin la necesidad de una previa activación de células gliales.
Para evaluar los mecanismos implicados en la muerte neurona1 inducida por la exposición a LPS en ausencia de células gliales, se utilizaron dos modelos de estudio: 1- células PC12 diferenciadas y 2- Hippocampal primary neurons (HPNs) deprivadas de glia. Los resultados muestran que el LPS incrementa la generación de especies reactivas de oxígeno (ROS) y aumenta la expresión del canal iónico TRPM7, proteína con un rol crítico y que está involucrada en la neurotoxicidad provocada por isquemia cerebral. Adicionalmente, utilizando concentraciones moderadas de peróxido de hidrógeno, células PC12 diferenciadas también
aumentan la expresión de TRPM7.
Para determinar si el aumento de las especies reactivas de oxígeno gatilla el aumento de la expresión de TRPM7, células PC12 diferenciadas fueron expuestas a DTT, un agente altamente reductor. El efecto reductor del DTT impidió el incremento de la expresión de TRPM7 por parte del LPS.
Finalmente, células PC12 diferenciadas se expusieron a los inhibidores farmacológicos DPI, inhibidor no selectivo de la NAD(P)H y cheleritrina, inhibidor de la PKC para determinar las fuentes de ROS relacionadas con el aumento de la expresión de TRPM7. El efecto de estos inhibidores impidió el incremento de la expresión de TRPM7 mediada por LPS.
Bacterial nervous system infections remain a common cause of mortality due to the unsuccessful current drug therapies. Among the infections caused by gram negative bacteria, is widely accepted that the pathophysiological consequences are mainly produced by the action of LPS in the nervous system. In the nervous system, it is traditionally accepted that the neurotoxic effects mediated by LPS is due to prior activation of microglial cells, however, recent evidence suggests that LPS may exert a direct neurotoxic effect, without glial cells activation. To evaluate the mechanisms involved in neuronal cell death induced by LPS exposition without glial cells presence, two study models were used: 1- differentiated PC12 cells and 2- HPNs. The results show that LPS increases TRPM7 expression, an ion channel critically involved in neurotoxicity caused by brain ischemia. When differentiated PC12 cells were exposed to LPS, intracellular reactive oxygen species (ROS) levels were increased. Additionally, using moderate-dose hydrogen peroxide, differentiated PC12 cells also overexpress TRPM7. To determine whether increased superoxide is required for TRPM7 overexpression, differentiated PC12 cells were exposed to DTT, a highly reducing agent. DTT prevented TRPM7 overexpression after LPS exposition. Finally, to determine the ROS sources related with TRPM7 overexpression, differentiated PC12 cells were exposed to a NADP(H) oxidase pharmacological inhibitor, DPI, and cheleritrine, a PKC inhibitor. The effect of these inhibitors prevented the TRPM7 overexpression promoted by LPS.
Bacterial nervous system infections remain a common cause of mortality due to the unsuccessful current drug therapies. Among the infections caused by gram negative bacteria, is widely accepted that the pathophysiological consequences are mainly produced by the action of LPS in the nervous system. In the nervous system, it is traditionally accepted that the neurotoxic effects mediated by LPS is due to prior activation of microglial cells, however, recent evidence suggests that LPS may exert a direct neurotoxic effect, without glial cells activation. To evaluate the mechanisms involved in neuronal cell death induced by LPS exposition without glial cells presence, two study models were used: 1- differentiated PC12 cells and 2- HPNs. The results show that LPS increases TRPM7 expression, an ion channel critically involved in neurotoxicity caused by brain ischemia. When differentiated PC12 cells were exposed to LPS, intracellular reactive oxygen species (ROS) levels were increased. Additionally, using moderate-dose hydrogen peroxide, differentiated PC12 cells also overexpress TRPM7. To determine whether increased superoxide is required for TRPM7 overexpression, differentiated PC12 cells were exposed to DTT, a highly reducing agent. DTT prevented TRPM7 overexpression after LPS exposition. Finally, to determine the ROS sources related with TRPM7 overexpression, differentiated PC12 cells were exposed to a NADP(H) oxidase pharmacological inhibitor, DPI, and cheleritrine, a PKC inhibitor. The effect of these inhibitors prevented the TRPM7 overexpression promoted by LPS.
Notas
Tesis (Bioquímico, Magíster en Bioquímica)
Palabras clave
Canales Iónicos, Estrés Oxidativo, Lipopolisacáridos