The Effects And Mechanisms Of Toll-Like Receptors In Morphine-Induced Central Nervous Systerm Apoptosis

Objective:Toll-like receptors(TLRs) are special type I transmembrane receptors and pathogen associated molecular pattern recognition receptors in innate immune system,participating in acute inflammation,cell signaling transduction and apoptosis. Although TLRs and their functions have been established in immune cells,the functional role of TRLs in the central nervous systerm(CNS) remains unclear.TLR2 and TLR9 express in CNS and play pivotal roles in brain injuries and functional deficits.Morphine has been widely applied in clinics as one of the most potent pain relievers,however, chronic use of opioids as clinical therapies produces benefits as well as severe side effects.Morphine causes cell death and apoptosis in various systems.We have previously reported that morphine promotes apoptosis both in vitro and in vivo.In CNS,morphine induced neuronal apoptosis.Whether TLRs plays a role in morphine-mediated apoptosis in neurons and microglia are unknown.Methods:There are three parts in this study.(1) We used TLR2 deficient and wild type primary neurons to study the consequence of TLR2 deficiency to the response of the neurons to morphine.Quantitative real time RT-PCR analysis and immunostaining were performed to examine the mRNA and protein level of TLR2.PI staining and TUNEL assay determined the apoptotic cells in wild type and TLR2 knockout neurons. Western blot and immunostaining examined the protein expression of cleaved-caspase-3 and GSK3β.(2) We treated HEK293 and HEK293 cells stably transfected with TLR2 (TLR2/HEK293) with new compound resveratrol aliphatic acid R6A at different concentrations for 24 hours and then subjected to serum deprivation for 12 hours and 24 hours.The apoptotic cells were determined by TUNEL assay.Protein expression of TLR2,phospho-Akt and phospho-GSK3βwere evaluated by western blot.(3) We used TLR9 deficient,μreceptor deficient and wild type primary microglia to study the mechanisms of TLR9 in morphine-induced apoptosis in microglia.The mRNA expression of TLR9 and microglia cell marker CD11b were studied by RT-PCR to determined the time dependent and dose dependent in wild type microglia.The apoptotic cells were measured by TUNEL assay in wild type and TLR9 knockout microglia.Immunostaining were performed to investigate the cleaved-caspase-3 after morphine treatment in wild type,TLR9 knockout andμreceptor knockout microglia.Proinflammation cytokine tumor necrosis factor-alpha(TNFα),interlukin-1β(IL-1β) and interlukin-6(IL-6) were analyzed by RT-PCR.Western blot were used to determine the phospho-GSK3βand phospho-P38.Results:(1) Cortical neurons from wild type mice were treated with morphine sulfate at 15μM for different periods of time and the expression of TLR2 was examined by quantitative real time RT-PCR and immunohistochemistry in primary culture.TLR2 expression in mRNA level was significantly increased following 15μM morphine treatment for 1 days.Morphine-induced TLR2 expression was also detected at the protein level.We evaluated the effect of TLR2 on morphine-induced primary neuronal apoptosis using PI staining and TUNEL technique.A significant number of cells in the wild type neurons after morphine treatment were undergoing apoptosis,whereas only a few apoptotic cells were detected in the TLR2 deficient neurons following morphine treatment.Morphine causes caspase-3 activation in wild type neurons but not in TLR2 deficient neuron.(2)New compound resveratrol aliphatic acid R6A inhibited the TLR2 protein expression on 100μM after 12hours and 24hours serum free.TUNEL assay displayed that R6A significantly decreased the apoptotic cell in TLR2/HEK293 cell.The mechanism of R6A inhibited TLR2-induced apoptosis involvment increasing the protein level of phospho-Akt and phospho-GSK3β.(3)In primary microglia cell,morphine induced the TLR9 mRNA increased in dose-dependent and time-dependent.The inhibitor of opioid receptor naloxone can block the effect of morphine and the agonist ofμreceptor DAMGO also induced the expression of TLR9.But inμreceptor knockout microglia,there was no change of the expression of TLR9.TUNEL assay showed that the apoptotic cells increased significantly after morphine treatment in wild type microglia but not in TLR9 knockout microglia.So we investigated next apoptosis index cleaved caspase-3.The results revealed that both TLR9 deficiency andμreceptor deficiency are resistant to the morphine-induced activity of caspase-3.CD11b is a marker of actived microglia.The evidences of CD11b indicate morphine induces the CD11b increased significantly on time-dependent and dose-dependent in wild type microglia,not in TLR9 knockout andμreceptor knockout microglia.That is coincident with TLR9 expression.That means morphine induces apoptosis throughμreceptor which active the TLR9 next.Microglia cells are immune cells in CNS and they can induce the expression of inflammatory cytokine.We examined the proinflammatory cytokine by RT-PCR and found that TLR9 mediates the increasing of TNFα,IL-1βand IL-6.GSK3βand P38 plays a pivotal role in regulating many cellular functions,including cell survival and apoptosis.We investigated the effect of TLR9 on phosphorylation of GSK3βand P38 with or without morphine treatment using western blot.The results showed morphine decreased the levels of GSK3βserine-9 phosphorylation in the wild type microglia,but not TLR9 knockout microglia.Since phosphorylation of serine-9 is inhibitory for GSK3βactivity, these data demonstrated that morphine decreases GSK3βactivity through a TLR9-dependent signaling pathway.On the other hand morphine increased the levels of P38 phosphorylation in the wild type microglia,but not TLR9 knockout microglia.Conclusion:(1)The present studies showed that TLR2 is required for morphine-induced neuronal apoptosis and an involvement of GSK3β.Further understanding the mechanisms mediated by TLRs may provide insights into potential therapeutic interventions morphine-related neurotoxicity.(2)Our findings revealed that the new compound R6A could effectively inhibit the expression of TLR2.Moreover,our results showed that R6A blocks TLR2-mediated apoptosis and an involvement of Akt/GSK3βpathway.Therefore,our data suggest that R6A is a novel and potent TLR2 inhibitor.(3) Morphine induces the microglia apoptosis throughμreceptor and TLR9 mediates it by GSK3β,P38,caspase-3 pathway.At the same time,TLR9 regulates the expression of proinflammation cytokines which induce apoptosis.The present study provided evidence of the important effect of TLR2 in morphine-induced apoptosis in neuron for the first time.We provide some basic materials for novel TLR2 inhibitor resveratrol aliphatic acid R6A.Also for the first time,we reported that TLR9 mediates morphine-induced apoptosis in primary microglia.All of them provided further evidence for morphine-induced neurotoxicity and produced a trend toward reduced neurotoxicity effects of morphine perform as attention deficits and cognitive capability in pain patients.