Wnt/β-catenin Signaling Pathway Involves In The Establishment Of Morphine Induced Analgesia Tolerance And Hyperalgesia In Rats

Background Wnt signaling pathway widely exists in multicellular eukaryotes, and is highly conserved in the process of evolution. During the development and adult life, Wnt signaling cascade controls a number of pivotal biological phenomena. As accepted by most researchers, Wnt signaling pathways involved in regulating cell proliferation, differentiation, polarity formation, migration, apoptosis and other physiological and pathological processes. Recent studies have shown that Wnt signal dysfunction is closely relevant to the pathogenesis of various diseases. For example, mutation of APC, β-catenin, Axin, etc, the components of Wnt signaling pathways, caused tumorigenesis(colorectal cancer, melanoma, adenomatous polyposis, etc.).Wnt signaling pathway disruption can cause a variety of central nervous system diseases and metabolic abnormalities disease.3types of Wnt pathways are discovered by now.The Wnt/β-catenin pathway, Ca2+ pathway and PCP pathway.Among them,Wnt/β-catenin pathway is better understood.Morphine is separated from opium alkaloid for the first time by German chemist F.W.A. in 1806. Its derivatives morphine hydrochloride is still commonly used in clinical analgesic, such as used in severe trauma, surgery, burns, and severe pain caused by advanced cancer. However, long time use of morphine is easy to cause weaken of morphine analgesia, namely morphine tolerance. And after discontinuation it also can cause the symptom of hyperalgesia(slight touch which is not of damage can cause severe pain). There is still no effective means to control these side effects which are greatly influenced the clinical application of morphine. Therefore, the key to solve this problem is to understand the molecular mechanism of morphine tolerance and hyperalgesia.The mechanism of morphine tolerance and hyperalgesia is not yet clear. Study has implied that there is the same molecular and cellular basis undering in morphine tolerance and neuropathicpain, including the activation of central nervous system excitatory amino acid receptors, and subsequent the protein kinase C translocation and activation, nitric oxide production, etc. The recent study has already been reported that the Wnt/β-catenin signaling pathway is involved in the development and maintainent of neuropathic pain, but whether it play a role in the molecular mechanism of morphine tolerance and hyperalgesia still needs further study.Objective This experiment is to determine the role of Wnt/β-catenin signaling in morphine induced analgesis tolerance and subsequent hyperalgesia in rat, by the observation of the expression changes of Wnt/β-catenin signaling molecules of spinal cord dorsal horn and dorsal root ganglion in rats of morphine induced analgesis tolerance and subsequent hyperalgesia, and bocking the Wnt/β-catenin signaling pathway for the interference of morphine induced analgesis tolerance and subsequent hyperalgesia in rats.Methods 1. Morphine tolerance and hyperalgesia animal model was induced by intrathecal injection of low-dose morphine repeatedly. Intrathecal(i.t.) catheter implantation was performed using SD rats weighting about 250 g under isoflurane inhalation anesthesia, with PE10 tube open end in the lumbar intumescence and the other side was pull out of the neck skin. The animal accepted adaptative training for 3 to 5 days during surgery recovery period. Then rats were injected i.t. with 10 μg/10 μl morphine twice daily for 6 consecutive days; at the same time, rats receiving i.t. saline(10 μl) twice daily for 6 days as control group. 2. Intrathecal injection of Wnt inhibitor IWR to observe the effect on morphine tolerance and hyperalgesia.Experimental groups inducing:(1) saline + DMSO grou;(2) morphine + DMSO;(3) high dose IWR 50 μM/10 μl + morphine;(4) low-dose IWR 25 μM/10 μl + morphine.To specify, morphine was injected twice a day, each time 10 μg/10 μl; IWR was once a day 30 min before the 1th injection of morphine each day. 3. Establishment and reverse of morphine tolerance and hyperalgesia in rats was confirmed with pain behavior tests. We habituated animals in the testing environment for 3 days and carried out behavioral testing(tail-flick latency test,mechanical paw-withdrawal threshold,thermal paw-withdrawal latency) in a blinded manner.The tail-flick latency(TFL) was taken before morphine injection(0 d)(baseline) or at the 1,3,5,7,9,11 days of intrathecal injection of morphine;while the paw-withdrawal mechanical threshold(PWMT) and paw-withdrawal thermal latency(PWTL) were taken after discontinuation of Intrathecal injection of morphine. Accordingly, the values of TFL were calculated using the following equator. The percentage of maximal possible analgesic effect(%MPAE) was: %MPAE = [(test latency-baseline latency)/(10-baseline latency)]*100%. 10 was the cut-off point in TFL test. 4. In spinal cord and DRG the protein level of Wnt3 a, the ligand of Wnt/β-catenin signaling pathway, was tested by Western blot. 5. Immunofluorescent staining was performed to test the expression of Wnt3 a in spinal cord and DRG; and fluorescent costaining with IB4, GFAP, NF-200, Neu N, OX-42, etc. to decide the specific cell type of Wnt3 a cells. 6. Change of gene expression of Wnt/β-catenin pathway components was tested with q PCR..Total RNA animal tissue of spinal cord and DRG(L4, L5)was extracted from 3 groups:(1)normal saline + DMSO;(2) morphine + DMSO;(3)high dose IWR 50 μM/10 μl + morphine. The concentration and purity of total RNA was determined by Nanodrop.The the same amount of total RNA was reverse transcribed into c DNA.With Thermo Scientific SYBR Green q PCR kit, gene expression of Wnt/β-catenin pathway targets Axin2,β-catenin,c-Myc was detected. And Wnt ligands Wnt3 a and Wnt5 a, Wnt receptors Fzd1 and Fzd8 were tested as well. 7. Statistics analysis: All the tests above were repeated at least three times. The statistics were carried out software SPSS21.0. Comparison of experimental group and control was perfoemed by Single factor analysis of variance.Data were presented as mean ± standard difference( x±s), and α=0.05 as the significant standard of test, P <0.05 was considered statistically significant. Results 1. Morphine tolerance model was stable establishedduring in our experiments.Analgesic effect began to decline 3 days after intrathecally morphine injection,and the analgesic effect of morphine almost disappeared on the 7th day, which was determined by pain behavior test. 2. Protein level of Wnt3 a rapidly increased and kept high level in the spinal cord and DRG after morphine intrathecal injection. 3. Wnt3 a expression was positive in the spinal cord and DRG in rats of morphine tolerance, and Wnt3 a mainly distributed in astrocytes and microglia in the spinal cord, or in small cell and secondary cells in DRG, while the satellite glial cell contained few Wnt3 a. 4. When received i.t. injections of the Wnt/β-catenin signaling pathway inhibitor IWR(25 μM/10 μl or 50 μM/10 μl), the tail flick shows a significant increase in %MPAE compared with the control group.Meanwhile PMWT(Paw withdrawal mechanical threshold)and PWTL(Paw withdrawal thermal latency)were also significantly increased. 5. The results of real time quantitative PCR showed that in the morphine tolerance animal model, Wnt/β–catenin signaling pathway was activated in Spinal cord, the expression of the target genes Axin2,c-Myc, and β–catenin were increased.And the gene of Fzd1 and Fzd8 in Spinal cord and DRG, as well as the gene of Wnt3 a and Wnt5 a in DRG were also increased.After the IWR blocking Wnt/β–catenin signaling pathway, the expression of target genes c-Myc and β–catenin wree significantly decreased in the spinal cord. Conclusion The Wnt/β-catenin signaling pathway was involved in morphine tolerance and hyperalgesia in rats.