| Morphine is the representative opiates of analgesics, which remains the primary drug treatment for severe pain, however repeated exposure to morphine and other opioids can lead to the development of tolerance, dependence or addiction. The challenging aspect of prevention of morphine tolerance, dependence or addiction had prompted a number of laboratory investigation, and had made fruitful achievements. In the past few decades, researches on morphine's mechanism of action focused mainly on the interaction between receptors and transmitters, and on intracellular signal transduction pathways. It is well documented that a novel family of neurosteroids, coined as steroids synthesized in situ by CNS, or metabolized from blood borne steroids by CNS, could act somewhat as the 4th genenration neurotransmitters, to widely influence the function of neurotransmitter receptors and ion channels in the CNS, and to regulate neuronal functions. It is reported as well that neurosteroids were effective in the prevention of morphine tolerance and dependence in animal experiments. Experimental results obtained by our lab showed that, in cases of morphine dependence, addiction and withdrawal, the levels of neurosteroids in rat brains changed dramatically, which indicated a possible relationship between neurosteroids and the development of morphine dependence, addiction or relapse. However, the underlying mechanism still waits to be identified. Therefore, the current research tried to explore the possible mechanism of effects of morphine dependence at cellular and molecular levels, and to provide new insights for the prevention and therapy of addictive disorders. Following methods were employed in our study: 1. Primary culture methods of cerebral cortical astrocytes and neurons: Primarily, cortical cells were obtained from the new borne rat cortices by mild digestion and mechanical dissection. The cells were plated on plastic culture plates pre-coated with poly-L-lysine or not, to facilitate the growth of neurons or glia. Cytosine arabinoside (Arc) were added to inhibit non-neuronal proliferation, and orbit shaker were used to purify the astrocytes. Typan blue resistant staining assay were employed to obtain a growth profile of astrocytes. We characterized the purified astrocytes by using immunocytochemistry with the antibody against GFAP, the specific protein marker of astrocytes. 2. Extraction and determination of the sulfated neurosteroids and un-conjugated neurosteroids in the culture media: The primary cultured astrocytes or neurons were randomly divided into several groups, and treated differently with morphine, opioid antagonists concomitant with morphine or opioid agonists alone. After 48h incubation, the culture media were collected and extracted using the method established by our lab to obtain the un-conjugated and sulfated neurosteroids. Using liquid chromatography-mass spectrometry (LC-MS) technique, we estimated the relative contents of several neurosteroids such as DHEA,PREG,AP,DS,PS, using MT and ES as the internal standards respectively, to observe whether drug treatment could affect the production of neurosteroids by the two types of cells or not. 3. Determination of the transcriptional levels of neurosteroidogenic key enzyme P450scc in the ASCs: We used Trizol reagents to extract the cellular total RNA,and amplified the gene fragment of P450scc and the internal standard β-actin by using One-step RT-PCR to observe the transcriptional regulation of opiates. 4. Determination of cellular p-CREB levels: The neuclear lysates were extracted and screened by using Western blot to observe the changes of p-CREB levels in astrocytes or neurons after drug treatment.Primary results are as follows: 1. Results of primary culture of rat cerebral cortical astrocytes and neurons: By means of mild digestion, we collected about 1.5 2.0×107 cells from a newborn SD rat brain,and the viability was 0.51.5×107 cells per brain. After orbit shaking, the cells were 97%±3% GFAP positive. Astrocytes remained their proliferative characters in vitro. The growth curve showed that a mass scale death 24h after cell seeding was followed by a recovery of cell proliferation 48h later. The growth of glial cells reached its climate at 6-7d, and showed a retardation due to the cell density and lack of space. Arc treated neurons remained their process of maturation, while the proliferation of the contaminant non-neuronal cells were actually hindered. Neurons of 56d showed bright and tight cell bodies with long and inter-connected axons and dendrites. The purity of neurons was about 90%, and were used for drug treatment. 2. Basal levels of PREG,DHEA and AP in primary cultured ASCs: The 48 h background production of DHEA, PREG and AP by astroyctes were 16.45±7.23, 39.89±22.57 and 190.77±77.85 ng·g-1 protein separately. When 1 μmol·L-1 cholesterol was added, the levels of DHEA, PREG and AP rose significantly to 152.43±38.73, 251.45±74.35 and 469.22±73.34 ng·g-1 protein respectively compared to the basal production(n=4,P<0.01). 3. Changes of neurosteroid levels in chronic drug treated ASCs: 3.1 Effects of chronic morphine treatment on the levels of neurosteroids in ASCs: Compared with the control group, after 48h-incubation with 1 μmol·L-1 morphine, the level of DHEA decreased by about 18.95% (n=12,P>0.05), the level of PREG and AP also dropped 41.46% and 26.35% respectively(n=12,P<0.01). The changes of sulfated steroids was quite different from the unconjugated ones. Compared with the control group, DS level dropped 48.26%(n=12, P<0.01), while PS elevated 88.00%(n=12,P<0.05).3.2 Effects of opioid receptor antagonists concomitant with morphine on the levels of neurosteroids in ASCs: Compared with the morphine treatment group, μ-opioid agonist CTAP(10 μmol ·L-1) significantly antagonized morphine's action by 43.88%( P<0.05 ) , 32.94%(P<0.05)and 19.18% for DHEA, PREG and AP respectively. However, the δ-and κ-antagonists ICI 174,864 and nor-BNI didn't reversed morphine's actions on DHEA and PREG. On the contrary, they differentially facilitated morphines action by 46.98% and 54.56% (P<0.01). CTAP didn't reversed morphine's inhibition of DS, but ICI-174,864 and nor-BNI significantly antagonized its action. As for PS, all three antagonists reversed morphine's stimulation of PS by 61.82%,53.39% and 86.43% respectively(P<0.01). 3.3 Effects of opioid agonists on the levels of neurosteroids of ASCs: Compared with the control group, the results showed a significant decrease of DHEA,PREG and AP by 47.49%, 37.48% and 32.62% (n=12, P<0.01) with the administration of DAMGO, the μ-agonist. DPDPE, the δ-agonist showed no influences on steroid levels, while U69593, the κ-agonist showed obvious inhibition of DHEA by 75.48%(P<0.01). DAMGO significantly inhibited the release of DS ( P<0.01 ), while DPDPE or U69593 alone showed no influence on DS. As for PS, DAMGO and DPDPE increased PS release, but showed no statistical differences. U69593 showed inhibitory effect on PS production when used alone (P<0.05). 3.4 Effects of orphanin FQ on the production of neurosteroids of ASCs: Comared with the control group, OFQ enhanced DHEA production by 33.74%(P<0.01)and decreased PREG level by 30.83% (P<0.01). AP was unaffected by OFQ (P>0.05). OFQ administration caused significant decrease of DS(P<0.01), and a increase of PS as well. 4. Chronic drug treatment on neurostroidogenesis of cortical neurons: 4.1 Effects of chronic morphine treatment on the levels of neurosteroids in cortical neurons:Compared with the control group, chronic morphine treatment caused 65.77%(P<0.01)decrease of PREG, while the same treatment significantly increased DHEA and AP by 58.88% and 110% respectively(P<0.01). 4.2 Effects of opioid-antagonists on the neursteroidogenesis of cortical neurons: Compared with the morphine group, the μ-antagonist CTAP reversed morphine's inhibition of PREG by about 593%(P<0.01),and decreased DHEA levels by 25.51% (P>0.05),while the AP level remained unaffected. ICI-174,864 and nor-BNI showed similar effects to CTAP against morphine. 4.3 Effects of opioid agonists on the neurosteroidogenesis of cortical neurons: Compared with the control group, DAMGO reduced the level of PREG by 44.02% ( P<0.01 ), and stimulated the production of DHEA and AP. Differently, DPDPE and U69593 showed stimulative effect to all three unconjugated neurosteoids. 4.4. Effects of OFQ on the neurosteroidogenesis of cortical neurons: Compared with the control group, OFQ alone enhanced the production of PREG, DHEA and AP by 68.05%, 95.23% and 213.76% respectively(P<0.01). Compared with the morphine group, the levels of all three unconjugated neurosteroids were higher in OFQ group than in the morphine group. 5. Transcriptional changes of ASCs P450scc gene with drug treatment: We amplified a 474bp fragment of P450scc from the total RNA of ASCs and adult male SD rat testis by using One-step RT-PCR. The transcripitional level of P450scc gene of the testis was much higher than that of the ASCs, and chronic morphine treatment dramatically decreased the level of P450scc mRNA of ASCs by 22.28% (P<0.01) compared with the control group. The results also showed that μ-and κ-antagonists CTAP and nor-BNI could reverse morphine's action , while δ-antagonist ICI174,864 failed to antagonize morphine's effect. 6. Effects of drugs on the level of p -CREB in cortical nerve cells: 6.1 Effects of acute morphine treatment on the p -CREB levels of ASCs:p-CREB antibody recognizes phosphorylated CREB,and shows across reaction towards p-ATF-1and CREM. Our results showed that morphine treatment(15min,60min and 24h)could significantly down regulate the level of p-CREB in ASCs by about 93.56%,74.33% and 67.05% respectively(n=4,P<0.01). κ-antagonist nor-BNI(10 μmol·L-1)concomitant with morphine fo 24h significantly reversed morphine's action alone, and increased p-CREB levels by about 15.69 times(n=4,P<0.01 vs morphine 24h)。6.2 Effects of chronic morphine treatment on the p-CREB levels of ASCs: The p-CREB levels increased significantly by 30% after 48h morphine treatment in ASCs compared with the control group(P<0.05). Compared with the morphine group, the opioid receptor antagonist CTAP, ICI 174,864 and nor-BNI differently reversed morphine's action significantly by about 33.12%,22.59% and 29.81% respectively(n=4,P<0.01). Compared with the control group, chronic treatment of μ-agonist DAMGO increased the level of p-CREB by 10%, which showed no statistic significance. DPDPE and U69593 showed little influence on the level of p-CREB. OFQ significantly increased the level of p-CREB in ASCs by 14.38%(P<0.05). 6.3 Effects of chronic morphine treatment on the p-CREB levels of neurons: Our results showed that chronic morphine treatment significantly increased the level of intracellular p-CREB by about 35%(P<0.01), while CTAP antagonized the increase of p-CREB by 10%(P<0.01,vs morphine). ICI174,864 and nor-BNI showed no obvious influences on the level of p-CREB levels. Otherwise, DAMGO increased p-CREB levels by about 23% compared to the control group ( P<0.01 ) , and U69,593 showed 14.76%(n=4,P<0.05) increase of p-CREB levels in neurons. OFQ showed no obvious influences on the p-CREB levels. To summarize, the present study established a primary culture method of rat cerebral cortical astrocytes and neurons. The results demonstrated for the first time that morphine chronic treatment could decrease the levels of several neurosteroids in ASCs and neurons. Opioid receptors were involved in the regulation of neurosteroids by morphine. Simultaneously, opioid receptors... |
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