| According to the World Health Organization released the report on morphine, the abuse of morphine has increased greatly in recent years, and has led to a series of undesirable effects. Morphine are known to cause varying degrees of harm to each system of the body. The pathological damage to the heart and lung, and the direct inhibition to the central nervous system and respiratory center by morphine, causing acute or chronic ischemic brain and hypoxia, further induce pathological damage in nerve cells and fibers. The mesencephalic dopamine system, one of the key brain regions of drug addiction, participates almost all the rewarding effects of dependency drugs,and is thought to be the final pathway for the reward system. The mesencephalic dopaminergic neurons and their target structures are critically involved in the neural circuit modifications that underlie a variety of adaptive changes, including mental disorders and the development and maintenance of addiction.Multiple adaptive changes in molecular and cellular function as well as structure remodeling of nerve cells in the mesolimbic dopaminergic system have been shown after morphine dependence, which are thought to be linked to the persistent craving and relapse of the addicts with losing resistance.Among transcription factors involved in development and physiological function of mesencephalic dopaminergic neurons, Nurr1(nuclear receptor related factor 1) and Pitx3(pituitary homeobox 3) may play critical role for determining the dopamine transmitter identity and neurotransmission, as well as for the survival and maintenance of dopaminergic neurons. Nurr1 is an orphan member of the nuclear receptor superfamily of transcription factors,which is critical for the differentiation, migration, maturity and survival of thedopaminergic neurons in ventral tegmental area(VTA) and substantia nigra(SN) as well as non-dopaminergic neurons. It is essential for transcription of tyrosine hydroxylase(TH) involved in dopaminergic neurons metabolism in the VTA and SN. Another critical transcription factor for the development and for the survival and maintenance of dopaminergic neurons is the homeobox protein Pitx3. The gene encoding for Pitx3 is expressed exclusively in mesencephalic dopaminergic neurons and activates the transcription of genes directly involved in the differentiation and maintenance of dopaminergic neurons. Previous studies indicated that Nurr1 and Pitx3 significant decreased in mice after chronic cocaine administration, suggesting that these transcription factors could mediate the neuroadaptive processes leading to alteration in dopamine circuits and remodeling of neural struction, which may finally result in more serious damage.Endoplasmic reticulum stress is a self protection mechanism for the stress cells, but excessive endoplasmic reticulum stress could result in pathological changes of cells. Recent studies have shown that endoplasmic reticulum stress was not only critical for the pathogenesis and nerve cells death induced by neurodegenerative diseases, but also involved in the development and maintenance of addiction. But the detailed report is lack. When the endoplasmic reticulum stress, glucose-regulated protein 78(GRP78),endoplasmic reticulum chaperon, binds with unfolded or misfolded protein,thus makes it dissociation with the endoplasmic reticulum transmembrance protein receptors such as PKR-like endoplasmic reticulum kinase(PERK),inositol requirement 1(IRE1) and artificial transcription factor 6(ATF6),which promoting the correct folding and finally protecting those cells through activating the three pathways. However, if the function of endoplasmic reticulum is damaged due to over strong or long time of endoplasmic reticulum stress, PERK and ATF6 will promote the action of CHOP and increase its expression. The high level of CHOP suggests the endoplasmic reticulum stress reach its peak and then induce apoptosis. Caspase 12, a key molecule of apoptosis induced by endoplasmic reticulum stress, was onlyactivated in the endoplasmic reticulum stress and not activated in the process of apoptosis induced by the mitochondrial and death receptor. Caspase 12, an inactive zymogen, was induced by recombination. Activation of caspase 12 could cut and activate caspase 9 and then start the caspase 3 classic apoptotic pathway, which finally lead to apoptosis. Therefore, caspase 12 is thought to be a marker of endoplasmic reticulum stress, and it plays critical role in the apoptotic pathway.The present studies indicated that chronic morphine dependence may result in the neurons damage. However, it is not clear the pathological changes of mesencephalic dopaminergic neurons in VTA and SN and the mechanism of the related regulatory proteins involved in the injury induced by morphine.Therefore, in order to mimic human with long-term morphine exposure, the rat model with different duration of chronic morphine dependence was established. The effect of chronic morphine dependence on dopaminergic neurons in VTA and SN was observed from the perspective of pathological morphology. Meanwhile, the dynamic changes and the relationship of related proteins associated with the dopaminergic neurons, Nurr1, Pitx3 and TH, and endoplasmic reticulum stress proteins, GRP78, P-e IF2α, ATF6 and CHOP, as well as apoptotic proteins, caspase 12 and caspase 3, induced by morphine dependence was explored. It will provide a new approach for the further study of dopaminergic neuron injury and the search for new drug targets.Part one Establishment of different durations of chronic morphine dependent rats and pathological observationObjective: Modes of different durations of chronic morphine dependent rats were established. Using conventional histological staining, special histochemical staining and Fluoro-Jade B fluorescence staining, the study investigated the effect of different durations of morphine dependence on dopaminergic neurons in VTA and SN.Methods:1 The model of morphine was established by increasing subcutaneous injections of morphine hydrochloride. Withdrawal symptoms were induced by subcutaneous injection of naloxone hydrochloride, and the scores were given.Models of different durations of chronic morphine dependent rats were established after morphine dependence.2 The weight of rats in each group were detected, and the effect of different durations of chronic morphine exposure on the weight of rats was observed.3 HE staining was used to observe the pathological changes of VTA and SN in chronic morphine dependent rats.4 Thionine staining was used to observe the changes of Nissl bodies of dopaminergic neurons in VTA and SN after different durations of chronic morphine dependence.5 Fluoro-Jade B staining was used to detected degeneration and death of dopaminergic neurons in VTA and SN after different durations of chronic morphine dependence.Results:1 Compared with the control group, the scores of withdrawal symptoms were obviously different in morphine dependent group(P < 0.05), suggesting that the models of morphine dependent rats were successfully established.2 With prolonged time, the weight of control group was obviously increased. The weight of morphine dependent rats was slightly decreased in a week, but after that time the weight also increased. However, compared with control group, the weight of morphine dependent group was lower and had significant difference(P < 0.05).3 HE staining showed that with prolonged of chronic morphine dependence, tissue was edema and neuronophagia, glial cells were proliferation, and finally the nerve cells were shrinking.4 Thionine staining indicated that with prolonged of chronic morphine dependence, tissue structure was not clear, Nissl bodies were disappeared,nerve cells were pycnosis and deep dyeing.5 Fluoro-Jade B staining indicated that dopaminergic neurons in VTA and SN were degeneration and death.Summary: The study has been successfully established the model of different durations of morphine dependent rats. After observing by HE staining, thionine staining and Fluoro-Jade B staining, we get the conclusion as follows: with prolonged of morphine dependence, dopaminergic neurons in VTA and SN were edema and neuronophagia, glial cells were proliferation,Nissl bodies were disappeared, and nerve cells were death. All results suggested that dopaminergic neurons in VTA and SN were degeneration and death induced by chronic morphine dependence.Part two Effect on the expression of mesencephalic dopaminergic regulatory proteins Nurr1, Pitx3 and TH induced by different durations of chronic morphine dependenceObjective: Through systematic observation the changes of Nurr1, Pitx3 and TH as well as the relationship between each other, the study was to detect whether Nurr1, Pitx3 and TH were involved in nerve injury induced by chronic morphine dependence, which could provide morphological basis for studying the mechanism of nerve injury associated with chronic morphine dependence, and a new approach for the treatment of nerve injury induced by morphine.Methods:1 Immunohistochemistry was used to observe the effect of different durations of chronic morphine dependence on dopaminergic neuron marker TH in VTA and SN.2 Immuofluorescence double staining was used to detect the effect of different durations of chronic morphine dependence on Nurr1 and Pitx3 as well as the relationship between these and TH in dopaminergic neurons of VTA and SN.3 Western blot was used to detect the effect of different durations of chronic morphine dependence on protein content of Nurr1, Pitx3 and TH in dopaminergic neurons of VTA and SN.Results:1 TH immunohistochemical results in VTA and SN: with prolonged of chronic morphine exposure, the number of TH positive cells was decreased.2 Double immunofluorescence results in VTA and TH: with prolonged of chronic morphine exposure, the number of TH positive cells expressing Nurr1/Pitx3 in VTA and SN was decreased.3 Western blot analysis of TH expression in VTA and SN: there was no significant difference between the 1 week morphine dependent group and the control group(P >0.05), but the relative TH expression of the 3 weeks and 6weeks morphine dependent groups decreased markedly(P <0.05 and P <0.01,respectively).4 Western blot analysis of Nurr1 and Pitx3 expression in VTA and SN:the relative level of Nurr1/ Pitx3 in the VTA and SN after 1 week morphine exposure was increased compared with the control group(P <0.05). However,with prolonged morphine exposure, the relative Nurr1/ Pitx3 expression of the3 week and 6 week morphine dependent groups decreased markedly(P<0.05).Summary:Long duration of chronic morphine dependence could decrease the expression of dopaminergic regulatory proteins of Nurr1 and Pitx3, and then could not effectively regulate TH. Thus, the survival of dopaminergic neurons was affected and appeared significant damage. All the results suggested that the decreased expression of Nurr1, Pitx3 and TH caused by chronic morphine dependence may be the one of dopaminergic neurons injury ways.Part three The effect on endoplasmic reticulum stress proteins induced by different durations of chronic morphine dependenceObjective: Through investigated changes of endoplasmic reticulum stress proteins GRP78, P-e IF2α, ATF6 and CHOP, as well as apoptotic proteins, caspase 12 and caspase 3, the study was to investigate whether endoplasmic reticulum stress proteins and apoptotic proteins were involved in the damage of dopaminergic neurons induced by chronic morphine dependence, which provided a scientific basis for prevention and treatment of nerve injury caused by drug dependence.Methods:1 Multiple fluorescent labeling and confocal microscopy were used to investigate changes of endoplasmic reticulum stress proteins GRP78, P-e IF2α,ATF6, CHOP as well as apoptotic proteins caspase-12 and caspase-3 in dopaminergic neurons of VTA and SN induced by different chronic morphine dependence.2 Western blot was used to detect the effect of different durations of chronic morphine dependence on protein content of endoplasmic reticulum stress marker proteins GRP78 and CHOP in dopaminergic neurons of VTA and SN.Results:1 Multiple immunofluorescence staining With confocal microscopy, the colocalization of GRP78, P-e IF2α, ATF6,CHOP, caspase-12 and caspsae-3 with TH in dopaminergic neurons of VTA and SN were investigated. The result was got as follows: with prolonged of morphine dependence, the number of GRP78, P-e IF2α, ATF6 and CHOP colocalized with TH was slightly decreased after significantly increase, and the number of caspase-12 and caspsae-3 was increased sustainability.2 Western blot analysis With prolonged of morphine dependence, protein of GRP78 and CHOP was slightly decreased after significantly increase(P <0.05), which consistent with the result of immunofluorescence.Summary: The endoplasmic reticulum stress proteins GRP78, P-e IF2α,ATF6, CHOP as well as apoptotic proteins caspase-12 and caspsae-3 in dopaminergic neurons could changes after chronic morphine dependence. The endoplasmic reticulum stress marker protein CHOP was increased induced by the activation of PERK and ATF6 signaling pathways. Meanwhile, caspase-12 and caspsae-3 were increased. All the results suggested that the activation of endoplasmic reticulum stress could induce apoptosis, which may be involved in the damage of dopaminergic neurons induced by chronic morphine dependence.Part four The effect of different durations of morphine stimulation on endoplasmic reticulum stress proteins in MN9 D cellsObjective: The study was to investigate the changes of endoplasmic reticulum stress proteins GRP78, P-e IF2α, ATF6, CHOP as well as apoptotic proteins caspase-12 and caspsae-3 after different durations of morphine stimulation, which verified the overall experiment.Methods:1 Immunofluorescence was used to investigate the expression of TH in MN9 D cells and SH-SY5 Y cells, and the number of TH was compared with each other.2 MTT was used to detect the survival rate of MN9 D cells after different concentrations of morphine stimulation.3 Immunofluorescence was used to investigate changes of endoplasmic reticulum stress proteins GRP78, P-e IF2α, ATF6, CHOP as well as apoptotic proteins caspase-12 and caspsae-3 in MN9 D cells induced by different durations of morphine stimulation.4 TUNEL staining was used to investigate the apoptosis of MN9 D cells.Results:1 TH-positive cells were expressed in MN9 D cells and SH-SY5 Y cells,and the rate of TH in MN9 D cells was significantly higher than that in SH-SY5 Y cells(P <0.05).2 MTT result indicated that the survival of MN9 D cells was significantly decreased after the concentration of 1μM morphine effected on MN9 D cells for 24 hours(P <0.05), and the survival rate of MN9 D cells was decreased with the raise of morphine concentration.3 Immunofluorescence staining suggested that endoplasmic reticulum stress proteins GRP78, P-e IF2α, ATF6, CHOP and apoptotic proteins caspase-12, caspsae-3 were increased with prolonged duration of morphine stimulation.4 TUNEL result indicated that apoptotic cells were not found in the control and the 24 h of morphine stimulation, apoptotic cells were found randomly after 48 h of morphine stimulation. A few scattered apoptotic cells were observed after 72 h of morphine stimulation.Summary:1 The number of TH positive cells in MN9 D cells is significantly more than that in SH-SY5 Y cells, suggesting that it is suitable for studying the mechanism of dopaminergic addiction.2 With prolonged duration of morphine stimulation, endoplasmic reticulum stress proteins GRP78, P-e IF2α, ATF6, CHOP as well as apoptotic proteins caspase-12 and caspase-3 were increased, which may be involved in apoptosis.Conclusions:In the study, we studied the effect of morphine on VTA and SN dopaminergic neurions as well as MN9 D cells, based on successfully established the model of different durations of morphine dependent rats and the model of different durations of morphine stimulation on MN9 D cells, and got conclusions as follows:1 Chronic morphine dependence could result in the damage of dopaminergic neurons.2 The reduction of Nurr1, Pitx3 and TH could affect the survival of cells,which may be involved in the damage of dopaminergic neurons induced by chronic morphine dependence.3 The activation of endoplasmic reticulum stress and its downstream apoptosis may be involved in the damage of dopaminergic neurons induced by chronic morphine.In summary, chronic morphine could lead to the damage of dopaminergic neurons in VTA and SN, one possible reason is that dysregulation of dopaminergic regulatory factors affects the survival of dopaminergic neurons,and another reason is that the upregulation of endoplasmic reticulum stress proteins and apoptotic proteins may induce the apoptosis. |
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