The Role Of Spinal Cord CX-30 In The Development Of Morphine Tolerance In Rats

ObjectiveTo investigate the effect of intrathecal injection of si RNA targeting spinal cord gap junction protein-30(CX30) on the development of morphine-induced tolerance in the standard rats and its related potential molecular mechanism.MethodForty-eight male Sprague-Dawley rats under the weight range of 180-220 g were stochastically divided into 4 groups(n=12): blank control groupⅠ; morphine-tolerance groupⅡ; mismatch si RNA control groupⅢ; CX30 si RNA treatment groupⅣ. All rats were treated with intrathecal catheters which were confirmed whether in spinal or not in several days’ recovery after the surgery and record as day zero. At 8:00-10:00 of the first day, Basic PWTL(P1) was tested at a quiet indoor environment. Then all groups of rats were subcutaneous injected with morphine 5 mg/kg and PWTL(P2) was tested 30 minute later. After that we reckoned the percentage of maximal analgesic possible effect(%MPE) of 30 minute. On the day of 2-8, rats were injected with either DEPC menstruum 15 μl(groupⅠandⅡ) or mismatch si RNA 15 μl(groupⅢ)or CX30 si RNA 15 μl(groupⅣ) through intrathecal catheters at 8:30. After that all groups of rats were subcutaneous injected with morphine 10 mg/kg(groupⅡ、ⅢandⅣ) or the same volume of saline(groupⅠ) at 9:00 and 17:00.We repeated behavioral tests and calculations of %MPE on the ninth day as the same as day 1. Then Lumbar spinal cord 4-6 was harvested immediately after rats were killed and the expression of CX30 or ERK and p-ERK were assessed by western bolt while GFAP by immunofluorescence.Result1. Behavior Test: On day1, the P1、P2 and %MPE of 4 groups had no conspicuous statistical discrepancy(P>0.05). On day 9, the P1 had no evident statistical difference among 4 grous(P>0.05), but contrasted with groupⅠ, the P2 and %MPE of other three groups obviously reduced(P<0.05); contrasted with groupⅡ, the P2 and %MPE of group Ⅲ had no conspicuous statistical discrepancy(P>0.05), but obviously ascended in groupⅣ(P<0.05). 2. Western bolt: The expression of ERK in lumbar spinal cord showed no conspicuous statistical discrepancy among 4 groups(P>0.05); Nevertheless, contrastedwith groupⅠ, the expression of p-ERK and CX30 in groupⅡ、Ⅲ and Ⅳ were conspicuous enhanced(P<0.05); Contrasted with groupⅡ, the expression of p-ERK and CX30 in group Ⅲ had no conspicuous statistical discrepancy(P>0.05), but reduced in groupⅣ(P<0.05). 3. Immunofluorescence: Contrasted with groupⅠ, the expression of GFAP in groupⅡ,Ⅲ and Ⅳ were obviously enhanced(P<0.05); compared with groupⅡ, the expression of GFAP in group Ⅲ had no conspicuous statistical discrepancy(P>0.05), but reduced in groupⅣ(P<0.05).ConclusionIntrathecal injection of siRNA targeting spinal cord CX-30 can partially alleviate the development of morphine-induced tolerance in the standard rats probably by retardation of the expression of CX30 between astrocytes and diminution the phosphorylation of ERK and reduction the activation of astrocytes in the spinal cord and dorsal root ganglion.