Proteomic Analysis Of PKCγ Mediated Chronic Morphine Tolerance

Objective:To observe the effects of lentiviral vector-mediated short hairpin RNA of PKCy(LV-shPKCy) intrathecal on morphine analgesia effect in morphine tolerant rats, and its effects on the expression of PKCy mRNA and protein, to identificate PKCy related proteins in chronic morphine tolerant rats by proteomic approach, providing potential molecule target and theoretical exploration for preventing the morphine tolerance.Methods:1.We used a total of48male Sprague-Dawley (SD) rats weighting280-320mg. The rats were implanted with intrathecal catheter following the methods of Yaksh and randomly assigned to four groups: group Ⅰ was normal sodium(NS, n=12);groupⅡwas morphine tolerance (MT, n=12), group Ⅲ was MT+LV-GFP (n=12),group Ⅳ was MT+LV-shPKCy(n=12).In group Ⅰ, saline (20μl) was injected intrathecal (i.t.) twice a day for six days. Morphine tolerance was induced by i.t. morphine (10μg b.i.d.) for six consecutive days in groups Ⅱ, Ⅲ and Ⅳ. On the7th day, rats in groups Ⅲ and Ⅳ were given LV-GFP and LV-shPKCγ(10μl), respectively. We assessed the basic mechanical and thermal pain threshold two days before operation. After the operation, latencies of hind-limb withdrawal response to radiant heat and mechanical stimulation were measured30min after morphine injection per triduum for13days. On the1st,7th and13th days, only the thermal nociceptive assessment was performed before morphine administration and every60min after the morphine injection (for6h).2.48male SD rats weighting280-320g were implanted with intrathecal catheter following the methods of Yaksh and randomly assigned to four groups:group Ⅰ was normal sodium(n=6);group Ⅱ was morphine tolerance (n=24), group Ⅲ was MT+LV-GFP(n=9), and group Ⅳ was MT+LV-shPKCγ (n=9).In group Ⅰ, saline (20μl) was injected intrathecal (i.t.) twice a day for six days. Morphine tolerance was induced by i.t. morphine (10μg b.i.d.) for six consecutive days in groups Ⅱ, Ⅲ and Ⅳ. On the7th day, rats in groups Ⅲ and Ⅳ were given LV-GFP and LV-shPKCγ(10μl), respectively.36rats were euthanized via decapitation and the spinal cord of the lumbar enlargement was removed for reverse transcriptase-polymerase chain reaction(RT-PCR) and western bolt:6rats in group Ⅰ, Ⅲ and Ⅳ were killed on day14;in group Ⅱ,6rats were killed every day as such time point:one day before morphine administration,1st,7th and14th day. On14th day,3rats in group Ⅲand Ⅳ were perfused transaortally with paraformaldehyde and remove the spinal cord for frozen section examination.3.42male SD rats weighting280-320g,24rats were used for proteomic analysis, and18rats were used for immunohistochemical test. 24rats were implanted with intrathecal catheter and induced morphine tolerance as mentioned above. On the7th day, morphine tolerant rats were randomly assigned to two groups:control group (n=12) and PKCy RNA interference group(n=12), the rats were given LV-GFP or LV-shPKCγ10μl, respectively. Since2days before morphine injection, we assessed the thermal pain threshold every3days. On the14th day, proteins in the enlargement part of spinal cord were isolated, and identificated the proteomic difference by two dimensional gel electrophoresis and MALDI-TOF-MS, part of classic different proteins were authenticated by western blot.18rats were randomly divided into three groups as follows: I control group (n=6), Ⅱ MT+LV-NC group (n=6),ⅢMT+LV-shPKCy group (n=6). In control group, saline was intrathecally twice a day for7days. In group Ⅱ and Ⅲ, rats were treated as described above. On the13th day, all rats in three groups were killed and used for immunohistochemical test.Results:1.Pain threshold after morphine injection:in NS group, the thermal and mechanical pain threshold after morphine injection were significantly higher than the basic value (P<0.05), and there was no difference in morphine antinociceptive effects in different days(P>0.0.5); in group Ⅱ、Ⅲ and Ⅳ, injection of morphine made the pain threshold significantly higher than the basic value on1st and4th day(P<0.05), but fail to analgesia on7th day (P>0.0.5); on10th and13th day, injection of morphine made the pain threshold heighten in group Ⅳ, but still fail to analgesia in group Ⅱ and Ⅲ; there was no difference between the four groups in the basic pain threshold or the morphine analgesic effects on the1st day, and there was significant difference between NS group and rest groups on the other days. Time course for the effect of morphine on the thermal pain threshold:on the1st day, the peak effect of morphine was observed30-60min after morphine injection, and the analgesic effect decreased over time (it was still evident after6hours), there was no difference between four groups (P>0.0.5); in NS group, there was no difference between1st and other days in the peak effect of morphine (P>0.0.5), and the duration of analgesia effect was more than6hours; on the7th day, there was no difference between the pain threshold after morphine injection and basic value in group Ⅱ,Ⅲand Ⅳ (P>0.0.5),there was significant difference between the morphine exposure group and NS group in the corresponding pain threshold (P<0.05); on the13th day, there was no difference between group Ⅱand Ⅲ in the pain threshold (P>0.0.5), the peak analgesic effect in group Ⅳ was observed30min after morphine injection and duration more than6hours (P<0.05), there was difference between NS group and other groups in the corresponding pain threshold (P<0.05). There was linear correlation between thermal and mechanical pain threshold30min after morphine injection(r=0.972, r2=0.944, P<0.05), and between1st and13th day thermal pain threshold in 6hours in group Ⅳ (r=0.937, r2=0.878, P<0.05)。2.On the14th days, the fluorescence was observed in the spinal cord of the rats in the control and PKCy interfering group. In group Ⅱ, there was no difference between the1st day and basic value in the expression of PKCy mRNA (P>0.05), but up-regulated on the7th and14th day, the expression of PKCy protein was also up-regulated on the7th and14th day compared to the basic value (P<0.05). on the14th day, expression of PKCy was up-regulated in morphine tolerant group compared to control group, the injection of LV-shPKCy, but not LV-GFP reversed the morphine induced up-regulation of PKCy (P<0.05), there was no difference between group Ⅱ and Ⅲ (P>0.05). There was no difference in four groups in the expression of MOR on the14th day (P>0.05)3. Chronic morphine exposure (10μg, i.t, b.i.d) in rats lead to a stabilized antinociceptive tolerance, and LV-shPKCγ significant reversed morphine tolerance. Using PDQuest2-DE gels analysis software, approximately1100well-stained, clearly-delineated protein spots were detected, and most spots were distributed in range from pH5to pH8.25spots which showed remarkable change between saline and LV-shPKCγ groups had been observed, mass spectrometrical analysis resulted in identification of13distinct proteins. Glial fibrillary acidic protein (GFAP), fascin (FSCN1) and glial cell line-derived neurotrophic factor (GDNF) were taken for western blotting analysis and the results were agree with those from2D gel electrophoresis. Chronic morphine exposure up regulated the expression of fascin in the spinal cord, staining for fascin was more intensive in morphine treated groups than in the control group. In the LV-shPKCγ group, fascin stainng was significantly attenuated compared with group Ⅱ.Conclusions:1.Chronic exposure of the rats to morphine induced a time-dependent antinociceptive tolerance, the peak effect and duration of morphine were both changed in morphine tolerant rats, and LV-shPKCγ significant reversed morphine tolerance.2.The expression of PKCy was time dependently upregulated in the spinal cord of morphine tolerant rat, the injection of LV-shPKCy, but not LV-GFP, down-regulated the expression of PKCy in morphine tolerant rat; the expression of MOR was not mediated by PKCy in morphine tolerant rat.3.Proteins such as GFAP, FSCN1and GDNF were taken part in the development of morphine tolerance which was mediated by PKCy, and there were potential molecular targets for preventing morphine tolerance.