The Effect Of Rosuvastatin On Morphine Analgesic Effect In Rats With Morphine Tolerance

Pain is one of most common clinical symptoms. It is not only a protective response that motivated by damaging situations, but also could be a symptom of certain disease. Acute pain always accompany with endocrine or immune alterations while chronic pain could lead to both physiological and psychological changes. Hence treatment is needed timely. Morphine is a class of the most effective analgesics for treating many forms of acute and chronic pain, especially for neuropathic pain and cancer pain. However, its effectiveness gradually subsides following repetitive administration, leading to the diminished analgesic effect in the clinical setting. Besides the known side effects such as respiratory depression, nausea and vomiting, the clinical utility of morphine is often hampered by the development of analgesic tolerance that necessitates dose escalation regardless of the disease progression.Abundant research had been carried out in recent decades. The mechanisms of morphine tolerance are not fully understood, but appear to be comprised of two types of plasticity or counter-adaptation at the cellular level and through neuronal circuits. Besides, it has been reported that opioid receptors are phosphorylated by many different kinases, such as cAMP-dependent protein kinase (PKA), protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase, G protein-coupled receptor kinases (GRKs), and mitogen-activated protein kinase (MAPK), which are responsible for morphine tolerance.Glial cells, components of nervous system, wide spread in central and peripheral nervous system, which number are10to50times of neuron and had same volume as neuron in the nervous system. Traditionally, it was considered that the signal modulation of morphine tolerance in spinal cord level were related to neuron and its mediators, glial cells were viewed as only structural supports for neurons and took no part in the modulation. Glial cells had been long overlooked since Song reported that chronic morphine treatment enhanced GFAP immunoreactivity in lumbar spinal cord, PCg and hippocampus of rats. The following research further revealed various kinds of glial cells inhibitors such as, propentofylline, minocycline, AV411attenuated morphine tolerance. Blocking up P2X4, P2X7receptors on the microglia and p38MAPK signal pathway which activate microglia can prevent morphine tolerance. These findings indicated that the functions of microglia and astrocytes were not only limited in maintaining nervous system homeostasis, they can produce and release a variety of neuroexcitatory substances, such as excitatory amino acids to regulate neural plasticity. Hence, modulation of the activity of spinal glial could be an effective strategy to prevent or even cure morphine tolerance.Statins, known as3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors, are commonly used as cholesterol-lowering drugs. Recent experimental and clinical evidence suggested that statins exert pleiotropic effects independent of lowering cholesterol, and a significant proportion of these effects may be considered to be anti-inflammatory. For instance, Lovastatin inhibit the induction of cytokines in rat primary astrocytes, microglia. Simvastatin attenuates microglial cells and astrocyte activation and decreases IL-1β level after traumatic brain injury. Rosuvastatins alleviate experimental nerve injury-induced neuropathic pain through attenuates microglial cells and astrocyte activation and decreases IL-1β level.Though statins can regulate the inflammation in central nervous system, its effect on morphine tolerance is still unknown. Thus, we designed this research to explore whether rosuvastatin can attenuated morphine tolerance through the similar way and analyzed its possible molecular mechanism.Part I Rosuvastatin prevented the development of morphine analgesic tolerance in normal ratsAim To explore the effect of rosuvastatin on the analgesic effect of morphine in normal rats and investigate possible underlying mechanisms.Methods Forty-eight male SD rats were randomly divided into six groups (n=8):saline control group (group C), morphine tolerance group (group MT),10mg/Kg rosuvastatin control group (group RC),0.4mg/kg rosuvastatin treatment group (group R1),2mg/kg rosuvastatin treatment group (group R2) and10mg/kg rosuvastatin treatment group (group R3). Basic Paw withdrawal thermal latency (PWTL) was first assessed on day1, after that morphine (4mg/kg) was injected via tail vein to determine the percentage of maximal possible effect (%MPE)30min after the injection. Rats were treated with subcutaneous injections of either saline (group C,RC) or10mg/kg morphine (group MT,R1,R2,R3) on day2. The injections were given twice daily at8:00and16:00for5d. The oral gavage of either Saline (group C, MT) or rosuvastatin (10,0.4,2,10mg/kg respectively for group RC, R1,R2,R3) was performed30min before the injections and lasted for5consecutive days. Behavioral tests repeated on day7as same as day1. Lumbar5spinal cord was obtained immediately after the behavioral tests to determine the expression of ERK and p-ERK by western bolt while IL-1β and TNF-a by immunofluorescence.Results On day1, the basic PWL and%MPE of six groups had no significant difference. On day7, the basic PWL was similar among six groups. Compared with the group C, group MT and R1showed a significant difference decrease of%MPE at30min while group RC, R2, R3revealed no marked difference. Compared with%MPE at30min of group MT, group RC, R2and R3showed a significant difference increase while group R1revealed no marked difference. The expression of ERK in L5spinal cord showed no significant difference among six groups. However, compared with group C, the expression of p-ERK, IL-1β and TNF-ain group MT and Rl were obviously increased. Compared with group MT, the expression of p-ERK, IL-1β and TNF-a in group RC,R2and R3were obviously decreased while group R1showed no marked difference.Conclusion Rosuvastatin can prevent the development of morphine tolerance in normal rats by inhibition of the phosphorylation of ERK and proinflammatory cytokine IL-1β and TNF-a expression.Part II Rosuvastatin partially restored the analgesic effect of morphine in rats with morphine toleranceAim To explore the effect of rosuvastatin on the analgesic effect of morphine in rats with morphine tolerance and investigate possible underlying mechanisms.Methods Forty-eight male SD rats were randomly divided into six groups (n=8):saline control group (group C), morphine tolerance group (group MT),10mg/kg rosuvastatin control group (group RC),0.4mg/kg rosuvastatin treatment group (group R1),2mg/kg rosuvastatin treatment group (group R2) and10mg/kg rosuvastatin treatment group (group R3). Rats were treated with subcutaneous injections of either saline (group C,RC) or10mg/kg morphine (group MT,R1,R2,R3). The injections were given twice daily at8:00and16:00for10days. The oral gavage of either Saline (group C, MT) or rosuvastatin (10,0.4,2,10mg/kg respectively for group RC, R1,R2,R3) began on day6and lasted for5consecutive days. Basic Paw withdrawal thermal latency (PWTL) was assessed on day6and11, after that morphine (4mg/kg) was injected via tail vein to determine the percentage of maximal possible effect (%MPE)30min after the injection. Lumbar5spinal cord was obtained immediately after the behavioral tests to determine the expression of ERK and p-ERK.Results On day6, the basic PWTL was similar among six groups. Compared with group C, group MT,R1,R2and R3showed significant decrease of%MPE at30min. On day11, there was no significant difference of the basic PWTL among six groups. Compared with the group C, group MT and Rl showed a significant difference decrease of%MPE at30min. Compared with%MPE at30min of group MT, group RC,R2and R3showed a significant difference increase. The expression of ERK in L5spinal cord showed no significant difference among six groups. However, compared with group C, the expression of p-ERK in group MT and R1were obviously increased. Compared with group MT, the expression of p-ERK in group RC,R2and R3were obviously decreased. Compared with group C, The expression of GFAP in group MT were obviously increased, Compared with group MT, the expression of GFAP in group R3were obviously decreased.Conclusion Rosuvastatin partially restored the analgesic effect of morphine in rats with morphine tolerance possibly through inhibiting the phosphorylation of ERK and activation of astrocytes in the spinal cord.