The Mechanism Of Apoptosis In Cortical Neuron Induced By TCDD Via Microglial Pro-inflammation

ObjectiveTo investigate the mechanism of TCDD induces microglial pro-inflammation and subsequent rat primary cortical neuron apoptosis.Methods1. In this study, HAPI microglia cells were used to analyze the mechanism of inflammatory activation induced by TCDD in microglia.(1) The time dependent(0.5, 1, 3, 4, 6, 12 h) manner were respectively detected by RT-PCR and ELISA for the transcription and secretion of TNF-α and IL-1β induced by 10 n M TCDD induced HAPI cells.(2) The m RNA expressions of c PLA2 and COX-2,which were induced by TCDD in “non-genomic pathway”, were determined by RT-PCR.(3) Western blot analysis was used to determine the levels of phospho-IкBα, phospho-p65 and IкBαprotein induced by TCDD in HAPI cells. To further investigate the activation of NF-κB signaling pathway, the representative levels of NF-κB p65 in nuclear and cytosolic fractions were analyzed byWestern blot. The transportation of p65 from cytosolic to nuclear induced by TCDD was detected by Immunofluorescence staining.2. In order to investigate the effects of activated microglia on neuron, we chose NO, which was harmful to neuron, to determine the effects of activated HAPI cells on rat primary cortical neuron.(1)TCDD induced HAPI cells expression of i NOS in a dose-(0.01,0.1,1,10,50,100 n M) and time-dependent(0.5, 1, 3, 4, 6, 12 h)manner were analyzed by RT-PCR and Western blot. The release of NO was detected with Griess method. The protein levels of MAPKs(p38, JNK, ERK), which were the key signaling pathway promoting the release of NO, were analyzed by Western blot.(2) After treatment rat primary cortical neuron with media conditioned(CM),which was obtained from TCDD treated HAPI cells for different times(0.5, 1, 3, 4, 6, 12 h), we used CCK-8 kit and LDH release kit to determine the cell viability and cell toxicity, respectively. Then,we used TUNEL staining to detect the apoptosis effect of CM on rat primary cortical neuron.(3) Western blot and Griess method was used to detect the protein level of MAPKs and the secretion level of NO induced by TCDD after pretreatment HAPI cells with the inhibitors of MAPKs signaling pathway. Together, RT-PCR and Western blot was used to analyze the expression of i NOS, the main kinase promote synthesis of NO. Next, the cell viability and cell damage of rat primary cortical neuron induced by CM were determined by CCK-8 kit and LDH released kit. Further, the apoptosis effect of CM(pretreatment with MAPKs inhibitors) on rat primary cortical neuron was detected by TUNEL staining.Results1.(1) The results of RT-PCR and ELISA showed that 10 n M TCDD could induce the secretion of TNF-α in a time dependent manner compared with control groups(DMSO) in HAPI cells. The levels of TNF-α were increased from 3 h(P<0.05), and peaking at 4h. The m RNA expression and secretion of IL-1β were also increased.(2) RT-PCR showed that 10 n M TCDD could up-regulate c PLA2 and COX-2, which were two kinases of “non-genomic pathway”, at0.5 h and 1 h, respectively.(3) Western blot analysis showed 10 n M TCDD up-regulated the levels of phospho-IкBα, phospho-p65 and IкBα protein. Together, Nuclear slurry separation indicated that p65 was increased in nuclear and decreased in cytosolic. Furthermore,Immunofluorescence staining showed that treatment with 10 n M TCDD induced nuclear distribution of NF-кB p65.2.(1) RT-PCR and Western blot showed that the expression of i NOS induced by 10 n M TCDD was increased in dose- and time-dependent manner in HAPI cells. Griess method showed that TCDD increase the release of NO were in dose- and time-dependent manner in HAPI cells, and continue increased with time. Western blot showed the protein levels of p38 MAPK and JNK were increased, but ERK not changed.(2) The result of CCK-8 and LDH release showed that CM, which obtained from TCDD induced HAPI cells, can decrease rat primary cortical neuron cell viability and increase LDH release. Together, TUNEL staining showed the CM can significantly induce apoptosis of rat primary cortical neuron.(3)Pretreatment HAPI cells with SB202190 and SP600125(which were inhibitors of p38 MAPK and JNK, respectively) before treatment with TCDD, Western blot showed that phosphorylation of p38 MAPK and JNK were inhibited. The results of RT-PCR and Western blot showed that the expression of i NOS were attenuated,the increased of NO was also inhibited in HAPI cells. Further, CM induced decrease of cell viability and increase of LDH release were inhibited analyzed by CCK-8 kit and LDH release kit. Moreover,CM induced TUNEL-positive cells was decreased determined by TUNEL staining.Conclusions1.(1) TCDD can induce pro-inflammatory activation of HAPI microglial cells, and release inflammatory cytokine, such as TNF-α,IL-1β, and kinases, like i NOS.(2) TCDD rapid activated non-genomic pathway and up-regulated c PLA2 and COX-2.(3)Further, NF-κB signaling pathway is activated by TCDD in HAPI cells.2.(1) TCDD can induce activation of p38 MAPK and JNK signaling pathway, up-regulate i NOS expression and the synthesis of NO in HAPI cells.(2) The increasing of NO, which is induced by activated HAPI cells significantly decreases cell viability and apoptosis of rat primary cortical neurons.(3) Inhibition of p38 MAPK and JNK attenuate the release of NO, further inhibite activated microglia induced damage and apoptosis of primary cortical neuron.