BACKGROUND： Studies have demonstrated that exogenous neurosteroid treatment prevents the development of morphine tolerance and dependence, and attenuates abstinence behavior in mice. However, there are few studies on...BACKGROUND： Studies have demonstrated that exogenous neurosteroid treatment prevents the development of morphine tolerance and dependence, and attenuates abstinence behavior in mice. However, there are few studies on whether the levels of endogenous neurosteroids can be changed by morphine dependence and withdrawal. OBJECTIVE： To investigate the levels of various neurosteroids in rat brain following morphine dependence and withdrawal. To evaluate the expressions of steroidogenic enzyme mRNAs and proteins. To identify the relationship between neurosteroids and morphine dependence at the whole animal behavior, neural biochemistry, and molecular levels. DESIGN, TIME AND SETTING： A randomized, controlled study. Experiments were performed at the Department of Pharmacology of Hebei Medical University and Department of Pharmacology of Beathune International Peace Hospital, China, from June 2004 to October 2007. MATERIALS： Morphine hydrochloride injection （Shenyang First Pharmaceutical Factory, China）, naloxone hydrochloride （Hunan Yiqiao Pharmaceutical Co., China） and a gas chromatography-mass spectrometry system （Agilent, CA, USA） were used in this study. METHODS： Healthy adult Sprague Dawley rats were randomly divided into three groups： a morphine dependence group, morphine withdrawal group and control group （n = 20）. The rats in the morphine dependence and morphine withdrawal groups were given increasing doses of morphine （5, 10, 15, 20, 30, 40 and 50 mg/kg, intraperitoneal） to create morphine dependence. The rats in the morphine withdrawal group were injected with 2 mg/kg naloxone to precipitate withdrawal 1 hour after the last morphine injection. Rats in the control group were treated with an equal volume of saline. MAIN OUTCOME MEASURES： Following morphine dependence and withdrawal, brain levels of the neurosteroids pregnenolone, progesterone and allopregnanolone were analyzed using gas chromatography-mass spectrometry. The mRNA expression of two key steroidogenic enzymes, P450 side-chain cleavage enzyme （P450scc） and 3[B-hydroxysteroid dehydrogenase （313-HSD）, were determined in rat brain regions using reverse transcription-polymerase chain reaction. The distribution and expression of P450scc protein were visualized in brain regions associated with addiction by immunohistochemistry. RESULTS： In brain tissue from the morphine dependence group, the levels of pregnenolone and progesterone were decreased by 62% （P 〈 0.01） and 92% （P 〈 0.01 ） respectively, compared with the control group. In the morphine dependence group, the key steroidogenic enzyme P450scc mRNA was decreased in striatum （P 〈 0.05）, while 3-HSD mRNA was decreased in amygdala （P 〈 0.05）, striatum （P 〈 0.05） and frontal cortex （P 〈 0.05） compared with the control group. Morphine withdrawal induced a significant increase in the neurosteroid levels compared with the control group （P 〈 0.01）. However, there was no significant difference in the expressions of P450scc and 36-HSD mRNAs between the morphine withdrawal and control groups （P 〉 0.05）. CONCLUSION： The neurosteroid levels and expressions of steroidogenic enzymes changed similarly in morphine dependent rats, suggesting that the morphine dependence-induced decrease in neurosteroids might depend on local expression of steroidogenic enzymes in the central nervous system. However, the changes in neurosteroids in morphine withdrawal rats were not in accordance with the changes in the expression of steroidogenic enzymes, suggesting that the effects of morphine withdrawal on brain neurosteroid levels may not depend primarily on the local expression of steroidogenic enzymes in the central nervous system.展开更多
Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances However, s...Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances However, some compounds do not show enough sensitivity in LC/MS and soft ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information This report presents an overview of the derivatization methods in the LC/MS analysis of neurosteroids or neuroactive neurosteroids, which are synthesized and accumulated in the nervous system The derivatization of pregnenolone 3 sulfate, one of these steroids, with 4 ( N,N dimethylaminosulfonyl) 7 hydrazino 2,1,3 benzoxadiazole gave a satisfactory sensitivity during the quantitative analysis using LC/ESI MS The obtained results are much lower than those previously obtained using gas chromatography/mass spectrometry or radioimmunoassay On the other hand, the derivatization to acetate was useful for the treatment of labile catechol estrogens in rat brains and gave enough structural information in LC/APCI MS, which confirmed the existence of catechol estrogens in mammalian展开更多
The etiological factors contributing to depression and other neuropsychiatric disorders are largely undefined. Endoplasmic reticulum stress pathways and autophagy are well-defined mechanisms that play critical functio...The etiological factors contributing to depression and other neuropsychiatric disorders are largely undefined. Endoplasmic reticulum stress pathways and autophagy are well-defined mechanisms that play critical functions in recognizing and resolving cellular stress and are possible targets for the pathophysiology and treatment of psychiatric and neurologic illnesses. An increasing number of studies indicate the involvement of endoplasmic reticulum stress and autophagy in the control of neuroinflammation, a contributing factor to multiple neuropsychiatric illnesses. Initial inflammatory triggers induce endoplasmic reticulum stress, leading to neuroinflammatory responses. Subsequently, induction of autophagy by neurosteroids and other signaling pathways that converge on autophagy induction are thought to participate in resolving neuroinflammation. The aim of this review is to summarize our current understanding of the molecular mechanisms governing the induction of endoplasmic reticulum stress, autophagy, and neuroinflammation in the central nervous system. Studies focused on innate immune factors, including neurosteroids with anti-inflammatory roles will be reviewed. In the context of depression, animal models that led to our current understanding of molecular mechanisms underlying depression will be highlighted, including the roles of sigma 1 receptors and pharmacological agents that dampen endoplasmic reticulum stress and associated neuroinflammation.展开更多
Neurosteroids are synthesized in the nervous system from cholesterol or steroidal precursors imported from peripheral sources. These compounds are important allosteric modulators of γ-aminobutyric acid A receptors （...Neurosteroids are synthesized in the nervous system from cholesterol or steroidal precursors imported from peripheral sources. These compounds are important allosteric modulators of γ-aminobutyric acid A receptors （GABAARs）, which play a vital role in pain modulation in the lateral thalamus, a main gate where somatosensory information enters the cerebral cortex. Using high-perfor mance liquid chromatography/tandem mass spectrometry, we found increased levels of neurosteroids （pregnenolone, progesterone, deoxycorticosterone, allopregnanolone, and tetrahydrodeoxycorticosterone） in the chronic stage of neuropathic pain （28 days after spared nerve injury） in rats.The expression of the translocator protein TSPO, the upstream steroidogenesis rate-limiting enzyme, increased at the same time. In vivo stereotaxic microinjection of neurosteroids or the TSPO activator AC-5216 into the lateral thalamus （AP -3.0 mm, ML 4-3.0 mm, DV 6.0 mm） alleviated the mechanical allodynia in neuropathic pain, while the TSPO inhibitor PK 11195 exacerbated it. The analgesic effects of AC-5216 and neurosteroids were sig- nificantly attenuated by the GABAAR antagonist bicuculline. These results suggested that elevated neurosteroids in the lateral thalamus play a protective role in the chronic stage of neuropathic pain.展开更多
基金the National Natural Science Foundation of China, No. 30772082the Natural Science Foundation of Hebei Province of China, No. C2005000834
文摘BACKGROUND： Studies have demonstrated that exogenous neurosteroid treatment prevents the development of morphine tolerance and dependence, and attenuates abstinence behavior in mice. However, there are few studies on whether the levels of endogenous neurosteroids can be changed by morphine dependence and withdrawal. OBJECTIVE： To investigate the levels of various neurosteroids in rat brain following morphine dependence and withdrawal. To evaluate the expressions of steroidogenic enzyme mRNAs and proteins. To identify the relationship between neurosteroids and morphine dependence at the whole animal behavior, neural biochemistry, and molecular levels. DESIGN, TIME AND SETTING： A randomized, controlled study. Experiments were performed at the Department of Pharmacology of Hebei Medical University and Department of Pharmacology of Beathune International Peace Hospital, China, from June 2004 to October 2007. MATERIALS： Morphine hydrochloride injection （Shenyang First Pharmaceutical Factory, China）, naloxone hydrochloride （Hunan Yiqiao Pharmaceutical Co., China） and a gas chromatography-mass spectrometry system （Agilent, CA, USA） were used in this study. METHODS： Healthy adult Sprague Dawley rats were randomly divided into three groups： a morphine dependence group, morphine withdrawal group and control group （n = 20）. The rats in the morphine dependence and morphine withdrawal groups were given increasing doses of morphine （5, 10, 15, 20, 30, 40 and 50 mg/kg, intraperitoneal） to create morphine dependence. The rats in the morphine withdrawal group were injected with 2 mg/kg naloxone to precipitate withdrawal 1 hour after the last morphine injection. Rats in the control group were treated with an equal volume of saline. MAIN OUTCOME MEASURES： Following morphine dependence and withdrawal, brain levels of the neurosteroids pregnenolone, progesterone and allopregnanolone were analyzed using gas chromatography-mass spectrometry. The mRNA expression of two key steroidogenic enzymes, P450 side-chain cleavage enzyme （P450scc） and 3[B-hydroxysteroid dehydrogenase （313-HSD）, were determined in rat brain regions using reverse transcription-polymerase chain reaction. The distribution and expression of P450scc protein were visualized in brain regions associated with addiction by immunohistochemistry. RESULTS： In brain tissue from the morphine dependence group, the levels of pregnenolone and progesterone were decreased by 62% （P 〈 0.01） and 92% （P 〈 0.01 ） respectively, compared with the control group. In the morphine dependence group, the key steroidogenic enzyme P450scc mRNA was decreased in striatum （P 〈 0.05）, while 3-HSD mRNA was decreased in amygdala （P 〈 0.05）, striatum （P 〈 0.05） and frontal cortex （P 〈 0.05） compared with the control group. Morphine withdrawal induced a significant increase in the neurosteroid levels compared with the control group （P 〈 0.01）. However, there was no significant difference in the expressions of P450scc and 36-HSD mRNAs between the morphine withdrawal and control groups （P 〉 0.05）. CONCLUSION： The neurosteroid levels and expressions of steroidogenic enzymes changed similarly in morphine dependent rats, suggesting that the morphine dependence-induced decrease in neurosteroids might depend on local expression of steroidogenic enzymes in the central nervous system. However, the changes in neurosteroids in morphine withdrawal rats were not in accordance with the changes in the expression of steroidogenic enzymes, suggesting that the effects of morphine withdrawal on brain neurosteroid levels may not depend primarily on the local expression of steroidogenic enzymes in the central nervous system.
文摘Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances However, some compounds do not show enough sensitivity in LC/MS and soft ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information This report presents an overview of the derivatization methods in the LC/MS analysis of neurosteroids or neuroactive neurosteroids, which are synthesized and accumulated in the nervous system The derivatization of pregnenolone 3 sulfate, one of these steroids, with 4 ( N,N dimethylaminosulfonyl) 7 hydrazino 2,1,3 benzoxadiazole gave a satisfactory sensitivity during the quantitative analysis using LC/ESI MS The obtained results are much lower than those previously obtained using gas chromatography/mass spectrometry or radioimmunoassay On the other hand, the derivatization to acetate was useful for the treatment of labile catechol estrogens in rat brains and gave enough structural information in LC/APCI MS, which confirmed the existence of catechol estrogens in mammalian
基金funded by the Taylor Family Institute for Innovative Psychiatric Researchthe Bantly FoundationMH122379 from the National Institute of Mental Health (to YI)。
文摘The etiological factors contributing to depression and other neuropsychiatric disorders are largely undefined. Endoplasmic reticulum stress pathways and autophagy are well-defined mechanisms that play critical functions in recognizing and resolving cellular stress and are possible targets for the pathophysiology and treatment of psychiatric and neurologic illnesses. An increasing number of studies indicate the involvement of endoplasmic reticulum stress and autophagy in the control of neuroinflammation, a contributing factor to multiple neuropsychiatric illnesses. Initial inflammatory triggers induce endoplasmic reticulum stress, leading to neuroinflammatory responses. Subsequently, induction of autophagy by neurosteroids and other signaling pathways that converge on autophagy induction are thought to participate in resolving neuroinflammation. The aim of this review is to summarize our current understanding of the molecular mechanisms governing the induction of endoplasmic reticulum stress, autophagy, and neuroinflammation in the central nervous system. Studies focused on innate immune factors, including neurosteroids with anti-inflammatory roles will be reviewed. In the context of depression, animal models that led to our current understanding of molecular mechanisms underlying depression will be highlighted, including the roles of sigma 1 receptors and pharmacological agents that dampen endoplasmic reticulum stress and associated neuroinflammation.
基金supported by grants from the National Basic Research Development Program of China(2013CB531905,2014CB548200,and 2015CB554503)the National Natural Science Foundation of China(81230023,81221002,31200835,81571067,and 21305005)+1 种基金a Key Project of the Ministry of Education of China(109003)the ‘‘111’’ Project of the Ministry of Education of China(B07001)
文摘Neurosteroids are synthesized in the nervous system from cholesterol or steroidal precursors imported from peripheral sources. These compounds are important allosteric modulators of γ-aminobutyric acid A receptors （GABAARs）, which play a vital role in pain modulation in the lateral thalamus, a main gate where somatosensory information enters the cerebral cortex. Using high-perfor mance liquid chromatography/tandem mass spectrometry, we found increased levels of neurosteroids （pregnenolone, progesterone, deoxycorticosterone, allopregnanolone, and tetrahydrodeoxycorticosterone） in the chronic stage of neuropathic pain （28 days after spared nerve injury） in rats.The expression of the translocator protein TSPO, the upstream steroidogenesis rate-limiting enzyme, increased at the same time. In vivo stereotaxic microinjection of neurosteroids or the TSPO activator AC-5216 into the lateral thalamus （AP -3.0 mm, ML 4-3.0 mm, DV 6.0 mm） alleviated the mechanical allodynia in neuropathic pain, while the TSPO inhibitor PK 11195 exacerbated it. The analgesic effects of AC-5216 and neurosteroids were sig- nificantly attenuated by the GABAAR antagonist bicuculline. These results suggested that elevated neurosteroids in the lateral thalamus play a protective role in the chronic stage of neuropathic pain.
