The Effects And Mechanisms Of Opioid Induced Microglia Apoptosis

Chronic use of addictive drugs, such as opioids and alcohol, can lead to toxic encephalopathy, which mainly displays addiction (reward, dependence and tolerance) and damage of central nervous system. The patients gradually appear weakening of memory, slow response and personality change. Some patients also appear perception disorder, headache and cerebrovascular accident. The persons suffer from this disease increase enormously with the development of the Chinese economy, but the contradiction between the neglect of society and medicine and the status has become more and more acute.Morphine is extensively applied in clinics as one of the most effective pain relievers for centuries, but triggers apoptosis in various systems. In the central nervous system (CNS), morphine induces neuronal apoptosis. Glia, once thought to be merely affiliated cells of the CNS, are now recognized to play an important role in the formation and maintenance of opioid tolerance and dependence. Microglial cells represent the resident immune host defense and are considered the principal immune inflammatory cells of the CNS. Opioid receptors play important roles in the processes of opioids-induced effects. All of three opioid receptor types,?,8 and?have been indentified on microglial cells. Recent studies have shown that chronic morphine treatment induces apoptosis in microglia. Naloxone (an opioid-receptor antagonist) blocked this effect, but the molecular mechanism of this effect is still unknown.Serine/threonine kinase Akt regulates cellular activation, apoptosis and inflammatory response. Activated Akt can phosphorylate a lot of proteins involved in apoptotic and survival signaling pathways resulting in diminished cell apoptosis. Activated Akt phosphorylates some downstream targets of the PI3K signaling including GSK3?. GSK3?is a constitutively active enzyme which can be inactivated by Akt through phosphorylation of serine 9. GSK3?is an important regulator of cell apoptosis and survival. Generous evidence supports that GSK3 regulates important steps in both of the two major apoptotic pathways (the death receptor-mediated extrinsic and the mitochondrial intrinsic apoptotic apoptotic pathways). Recently we have revealed that the Akt/GSK3?signaling plays a critical role in morphine-induced apoptosis in breast cancer and neuronal cells. However, the specific role of the Akt/GSK3?signaling in microglia is still unknown. One of three major members of MAPKs, p38 MAPK exerts pro-apoptotic role. Chronic morphine administration increases the level of phosphor-p38 in dorsal root ganglion neurons. Inhibition of p38 MAPK by p38 specific inhibitor SB203580 notably attenuated morphine-induced tolerance to analgesia. P38 MAPK can sensitize cells to apoptosis by up-regulating Bax, the pro-apoptotic member of Bcl-2 family. But the precise role of p38 MAPK in morphine-induced microglia apoptosis is still unknown.?-arrestin 2, the member of arrestins family, is a multifunctional adaptor/scaffold protein that plays an important role in G-protein-coupled receptor (GPCR) regulation.?-arrestin 2 functions in anti-apoptotic pathway through regulating the activity of interacted kinases. Recently,?-arrestin 2 has been shown to influence growth factor receptor-mediated cell proliferation through phosphorylation of Akt. But the role of?-arrestins in microglia apoptosis induced by morphine is unknown.In this study, we explored the molecular mechanisms by which morphine induced microglia apoptosis. Specifically, we determined the involvement of the Akt/GSK3?and?-arrestin 2 signaling pathways. Thus, our results provide a significative insight into the mechanism of microglia apoptosis induced by morphine.PART?The effects and mechanisms of morphine induced microglia apoptosis ObjectiveTo investigate the activation and apoptosis of microglia induced by morphine in brain microglia as well as in the microglial cell line BV-2, and to define the effect of morphine on microglia. Methods1. Cell culture and morphine treatmentBrain microglia and microglial cell line BV-2 were randomly divided into 3 groups for treatment:1. control, morphine treatment (2,5,10,20?M); 2. control, morphine treatment (6,12,24h); 3. control, morphine treatment (10?M), naloxone treatment (10?M), morphine and naloxone treatment. Cultures were incubated at 37?and 5% in a fully humidified incubator.2. Cell viability assayThe experimental cells were seeded in 96-well pates at an initial cell density of 1 X 104/well. Cell viability was assessed by the reduction of MTT. Briefly,20?1 MTT was added to each well, and plates were incubated at 37?for 4h. The color formation was quantified by means of an ELISA plate reader at 570nm wavelength using 100?1 solubilization solution.3. Quantification of apoptosis by TUNEL assayThe experimental cells were treated with morphine in the presence or absence of naloxone for 24h. Apoptotic cells were determined by terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling (TUNEL) assay using in situ cell death detection kit.4. Western blot analysisWestern blot was performed to detect the protein levels of cleaved caspase-3, caspase-3 in microglia. Briefly, the cellular proteins were separated by SDS-PAGE and transferred on to nitrocellulose membrane. The membrane was then incubated at room temperature in a blocking solution composed of 5% skim milk powder for 1h. The membrane was then incubated with the blocking solution containing the first antibody overnight at 4?. The blot was then incubated with a second antibody for 1h at room temperature. Band intensity was quantified by densitometric analyses using a densitometer.5. ImmunohistochemistryPrimary microglial cells were fixed and immunostained to detect the protein levels of cleaved caspase-3 and caspase-3 after morphine treatment. Briefly, microglial cells were fixed in acetone/methanol for 5 min at room temperature, and then immunostained using specific primary and secondary antibodies. Fluorescent images were captured by a fluorescent microscope, and confocal images were captured by a Leica TCS SP2 Laser Scanning Confocal Microscope.6. RT-PCRThe RT-PCR detection technique was performed to detect the mRNA level of CDllb, Fas, FasL, TNF-a, IL-1?and IL-6 in microglia after morphine treatment. Briefly, Total RNA was extracted from the cells, first-strand cDNA was synthesized from l?g of total RNA. After incubation at 70?for 3 min and cooling down to 37?for 10 min, RT cocktail was added to the annealing mixture and further incubated at 37?for 60 min.Results1. Morphine induces the expression of CDllb in an opioid-receptor dependent manner in microgliaThe mRNA level of CDllb in microglia increased in a dose-dependent and time-dependent manner after morphine treatment, and this process could be blocked by naloxone, an opioid-receptor antagonist.2. Morphine induces microglia apoptosis in an opioid-receptor dependent manner in microgliaMorphine caused significant decreases in cell viability in a dose-dependent manner and this process could be inhibited by naloxone. Apoptotic cells were determined using TUNEL assay. We found that morphine significantly induces cell apoptosis both in BV-2 and primary mouse microglia. In addition, opioid-receptor antagonist naloxone blocked morphine-primed apoptosis. We examined caspase-3 activation in microglia, and found BV-2 and mouse primary microglia have a significantly higher level of cleaved caspase-3 than control cells after exposure to morphine for 24h. Moreover, morphine-induced activation of caspase-3 can be blocked by naloxone in both BV-2 and mouse primary microglia.3. Morphine increases the expression of proinflammatory cytokine in an opioid-receptor dependent manner in microglia We examined the proinflammatory cytokine by RT-PCR, and found that morphine induced the expression of TNFa, IL-1?IL-6 in microglia. Naloxone could block this process.Conclusions1. Morphine activates microglia and induces the expression of proinflammatory cytokines in microglia in an opioid-receptor dependent manner2. Morphine induces microglia apoptosis in an opioid-receptor dependent mannerPART IIThe role of GSK3 signaling in morphine induced microglia apoptosis ObjectiveTo investigate the effects and mechanisms of GSK3?signaling in morphine-induced microglia apoptosis in brain microglia as well as in the microglial cell line BV-2.Methods1. Cell culture and morphine treatmentBrain microglia and microglial cell line BV-2 were randomly divided into 4 groups for treatment:1. control, morphine treatment (2,5,10,20?M); 2. control, morphine treatment (6,12,24h); 3. control, morphine treatment (10?M), naloxone treatment (10?M), morphine and naloxone treatment.4. control, morphine treatment (10?M), SB216763 (10?M), morphine and SB216763 treatment. Cultures were incubated at 37?and 5% in a fully humidified incubator.2. Western blot analysisWestern blot was performed to detect the protein levels of phosphor-Akt, phosphor-GSK3?, cleaved caspase-3, cleaved caspase-8, phosphor-p38, Bax, Bcl-2 in microglia.3. Quantification of apoptosis by TUNEL assay Apoptotic cells were determined by terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling (TUNEL) assay using in situ cell death detection kit.4. RT-PCR The RT-PCR detection technique was performed to detect the mRNA level of Bax and Bcl-2 in microglia after morphine treatment.Results1. Morphine decreases the levels of microglia phosphor-Akt and phosphor-GSK3?through an opioid receptorWe examined the levels of phosphor-Akt and phosphor-GSK3?in BV-2 cells in the presence or absence of morphine. The levels of phosphor-Akt and phosphor-GSK3?in morphine treated cells were significantly lower compared to control cells in a dose-and time-dependent manner. Furthermore, pretreatment of naloxone to BV-2 cells blocked morphine decreased the levels of phosphor-Akt and phosphor-GSK3?.2. GSK3?inhibition increases morphine-induced microglia apoptosisBV-2 and mouse primary microglial cells were pretreated with the GSK3?inhibitor SB216763, and then treated with morphine for 24h. We found that treatment with SB216763 significantly increased morphine-induced apoptosis in both BV-2 and mouse primary microglia. Significantly greater activation of caspase-3 and caspase-8 was observed when BV-2 cells were exposed to both SB216763 and morphine than they were exposed to SB216763 or morphine alone.3. Effect of GSK3?on the levels of Bcl-2/Bax and p38 MAPK in microglia following morphine treatmentTreatment of morphine with SB216763 significantly decreased the levels of Bcl-2 and increased the levels of Bax. SB216763 alone did not alter the expression of Bcl-2 and Bax. To investigate whether GSK3?regulate the levels of Bax and Bcl-2 at transcription or post-transcription, we determined the levels of Bax and Bcl-2 by RT-PCR. We found that inhibition of GSK3?by SB216763 increased morphine-induced changes of Bax and Bcl-2. These results are consistent with the Western blot analysis. BV-2 cells were pretreated with SB216763 and then treated with morphine. Morphine alone significantly increased the level of phosphor-p38 MAPK. Of great significance, combination of SB216763 and morphine treatment has a significant synergistic effect on the level of phosphor-p38 MAPK. Moreover, naloxone significantly attenuated morphine-induced the level of phosphor-p38 MAPK.Conclusions1. Morphine decreases the levels of phosphor-Akt and phosphor-GSK3? in an opioid-receptor dependent manner in microglia2. Inhibition of GSK3?increases morphine-induced apoptosis in microglia3. Inhibition of GSK3p increases morphine-induced changes of Bax and Bcl-24. GSK3?regulates the levels of Bax and Bcl-2 at transcription level5. Inhibition of GSK3?increases morphine-induced changes of phosphor-p38 in microgliaPART IIIThe role of p38 MAPK in morphine induced microglia apoptosisObjectiveTo investigate the effects and mechanisms of p38 MAPK signaling in morphine-induced microglia apoptosis in brain microglia as well as in the microglial cell line BV-2.Methods1. Cell culture and morphine treatmentBrain microglia and microglial cell line BV-2 were randomly divided into 2 groups for treatment:1. control, morphine treatment (10?M), naloxone treatment (10?M), morphine and naloxone treatment.2. control, morphine treatment (10?M), SB203580 (10?M), morphine and SB203580 treatment. Cultures were incubated at 37?and 5%in a fully humidified incubator.2. Western blot analysisWestern blot was performed to detect the protein levels of phosphor-p38, cleaved caspase-3, cleaved caspase-8, Bax, Bcl-2 in microglia.3. Quantification of apoptosis by TUNEL assayApoptotic cells were determined by terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling (TUNEL) assay using in situ cell death detection kit.4. RT-PCRThe RT-PCR detection technique was performed to detect the mRNA level of Bax and Bcl-2 in microglia after morphine treatment.Results1. Morphine increases the levels of microglia phosphor-p38 through an opioid receptorWe examined the levels of phosphor-p38 in microglia in the presence or absence of morphine. The levels of phosphor-p38 in morphine treated cells were significantly higher compared to control cells. Furthermore, pretreatment of naloxone to BV-2 cells blocked morphine-increased the levels of phosphor-p38.2. P38 MAPK inhibition attenuates morphine-induced microglia apoptosisWe pretreated BV-2 and mouse primary microglial cells with the specific p38 MAPK inhibitor SB203580 and then treated the cells in the presence or absence of morphine. Apoptotic cells were examined by TUNEL assay. We found that SB203580 significantly blocked morphine-induced apoptosis in both BV-2 and mouse primary microglial cells. In addition, inhibition of p38 by SB203580 dramatically inhibited morphine-induced activation of caspase-3 and caspase-8.3. Effect of p38 MAPK on the levels of Bcl-2/Bax in microglia following morphine treatmentBV-2 cells were treated with SB203580 for 1h and then treated with or without morphine for 24h, and found that inhibition of p38 MAPK by SB203580 significantly inhibited morphine-induced changes of Bcl-2 and Bax compared with the morphine treatment alone. We determined the levels of Bax and Bcl-2 by RT-PCR. We found that inhibition of p38 MAPK by SB203580 attenuated morphine-induced changes of Bax and Bcl-2. These results are consistent with the Western blot analysis. Conclusions 1. Morphine increases the levels of phosphor-p38 in an opioid-receptor dependent manner in microglia2. Inhibition of p38 MAPK attenuates morphine-induced microglia apoptosis3. Inhibition of p38 MAPK attenuates morphine-induced changes of Bax and Bcl-24. P38 regulates the levels of Bax and Bcl-2 at transcriptionPART IVThe role of?-arrestin 2 signaling in morphine induced microglia apoptosisObjectiveTo investigate the effects and mechanisms of?-arrestin 2 signaling in morphine-induced microglia apoptosis in brain microglia as well as in the microglial cell line BV-2.Methods1. Cell culture and morphine treatmentBrain microglia and microglial cell line BV-2 were randomly divided into 2 group for treatment:1.control, morphine treatment (2,10?M); 2.control, morphine treatment (10?M), naloxone treatment (10?M), morphine and naloxone treatment. Cultures were incubated at 37?and 5%in a fully humidified incubator.2. Gene transfectionThe experimental cells were transfected with either?-arrestin2 full length plasmid or?-arrestin 2 RNA interfering (RNAi) plasmid and control vector. Forty-eight hours after transfection, the medium was changed with fresh DMEM for further treatment with different concentrations of morphine.3. Cell viability assayCell viability was assessed by the reduction of MTT. Briefly,20?1 MTT was added to each well, and plates were incubated at 37?for 4h. The color formation was quantified by means of an ELISA plate reader at 570nm wavelength using 100?1 solubilization solution. 4. Quantification of apoptosis by TUNEL assayApoptotic cells were determined by terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling (TUNEL) assay using in situ cell death detection kit.5. Western blot analysisWestern blot was performed to detect the protein levels of phosphor-Akt, cleaved caspase-3 in microglia.Results1. Morphine decreases the levels of microglia?-arrestin2 through an opioid receptormicroglia cells were treated with morphine at indicated concentrations for 24h and the expression of P-arrestin 1 and?-arrestin 2 were determined by Western blotting. We found that morphine significantly decreased the expression of?-arrestin 2 but had no effects on p-arrestin 1.2. Effect of P-arrestin 2 on morphine-induced cell death in microgliaWe transfected BV-2 microglia cells with?-arrestin 2 full length plasmid and control vector plasmid, after 48h, the cells were incubated with morphine at different concentrations for 24h. We found that overexpression of?-arrestin 2 significantly attenuated morphine-induced cell death in microglia compared to the control cells.3. Effect of p-arrestin 2 on morphine-induced apoptosis in microgliaApoptotic cells were examined by TUNEL assay. It showed that overexpression of P-arrestin 2 significantly decreased apoptotic cells in microglia following morphine treatment compared with control cells. Morphine significantly induced activation of caspase-3 in microglia, and overexpression of?-arrestin 2 significantly inhibited morphine-induced activation of caspase-3.4. Effect of P-arrestin 2 on the levels of phosphor-Akt in microglia following morphine treatmentWe found that morphine decreased the levels of phosphor-Akt, and overexpression of P-arrestin 2 could significantly inhibited morphine-induced changes of phosphor-Akt in microglia following morphine treatment. Conclusions1. Morphine decreases the level of?-arrestin 2 in microglia in an opioid-receptor dependent manner.2. Overexpression of?-arrestin 2 attenuates morphine-induced apoptosis in microglia.3. p-arrestin 2 protects microglia from apoptosis through activation of Akt.