| BackgroundChronic pain(Classified As inflammatory pain, neuropathic pain and cancer pain) is characterized by hyperalgesia and allodynia. Central sensitization caused by the alteration of synaptic plasticity is involved in the generation of chronic pain. The glutamate N-methyl-d-aspartate receptors (NMDARs), especially those located in the dorsal horn of the spinal cord, are critically involved in nociceptive transmission and synaptic plasticity. Because small interfering RNA (siRNA) can inhibit NR2B expression, siRNA may provide a novel approach to treat neuropathic pain and possibly nerve injury. RNA interference (RNAi)is a kind of evolutionary conservative defense mechanism that against genetically modified or foreign virus defense, exogenous and endogenous double-stranded RNA(dsRNA)existing homologous complementary sequence with target genes transcription product mRNA,then the mRNA was degraded in cells, so that the specific gene was effectively closed, which is specific gene sequences post-transcriptional gene silencing(PTGS). The clinical success of gene therapy depends on the development of suitable efficient and targeted gene transfer system. At present, the virus vector is undoubtedly high efficiency gene transduction tools. However, the virus vector is only used in vitro because of safety consideration. Non-viral vector, as its low immunogenicity and power of carrying high molecular weight of DNA, is more suitable for in vivo gene delivery. Recently, Kim modified low molecular weight PEI with cholesterol, generated a new non-viral gene delivery vector-water-soluble lipopolymer(WSLP).The preliminary study showed that WSLP has high transgene efficieney and low toxicity. Besides, WSLP readily formed nano-sized complexes (～50nm) with siRNA, indicated that WSLP can permeate the blood-brain barrier. Previous research showed that intrathecal injection of WSLP/NR2B-siRNA can inhibit the NR2B expressing in spinal cord and hyperpathia in neuropathic pain rats. However.intrathecal injection have the potential of inducing nervous system side effect. NR2B-siRNA packaged by WSLP can avoid from enzyme and become capable to pass the BBB(Blood-brain barrier), thus intraperitoneal injection of WSLP/NR2B-siRNA can also inhibit spinal cord NR2B expressing, further more,to treat chronic pain by inhibiting the hyperpathia in neuropathic pain rats.To sum up, we hypothesizes that WSLP delivering siRNA targeting NR2B may inhibit NR2B expression in the spinal dorsal horn,and may be used as a novel method for treating neuropathic pain. The first, designed and synthesised NR2B-siRNA and prepared WSLP,WSLP was connected with siRNA directly,then established the model of neuropathic pain and intraperitoneal injection of WSLP/siRNA; The second, using Q-PCR technology and western-blot technology detected the change of NR2B expression on the Spinal dorsal horns.50%mechanical withdrawal threshold was measured by von Frey hair through Up-Down method, and thermal withdrawal latency was measured by Plantar Test instrument. This study may be provides a novel approach or choice for treating neuropathic pain. ObjectiveTo examine the potential application of a non-viral gene carrier, water-soluble lipopolymer (WSLP) delivering siRNA targeting NR2B in vivo and to determined whether intraperitoneal injection WSLP/NR2B-siRNA complexes instead of intrathecal injection, can be a new method for neuropathic pain treatment.MethodsThe research process is divided into two parts:(1) Water-soluble lipopolymer can efficient delivery siRNA targeting spinal NR2B expression in neuropathic pain rat. Synthesis WSLP according to reference and previous study, then synthesis WSLP/NR2B-siRNA,(NR2B-siRNA was supplied by guangzhou ruibo company). Take adult SD rats(n=28), Divided into groups randomly:control group:C group (blank control group, no treatment, n=4)、 NP group (neuropathic pain induced by ligating L5spinal nerve+intraperitoneal injection of0.9%NS, n=4) and sham group(expose L5spinal nerve, not ligate, n=4); neuropathic pain treatment group:siWSLP group(model+intraperitoneal injection of WSLP/NR2B-siRNA, n=4), PEI group (model+intraperitoneal injection of PEI/NR2B-siRNA, n=4), ncWSLP group(model+intraperitoneal injection of WSLP/negative control siRNA, n=4) and WSLP group(model+intraperitoneal injection of WSLP, n=4). The drugs were injected at8days following neuropathic pain model establishment, and the amount of siRNA(or ncRNA) is10nmol. Three days after intraperitoneal injection, their spinal dorsal horns at L4-L6were harvested on ice and divided equally for detection of NR2B mRNA and protein expressions.①Take the spinal cord specimens, extraction total RNA, detection of NR2B mRNA expression using Q-PCR②Take the spinal cord specimens, detection of NR2B protein expression using western blots.(2) Water-soluble lipopolymer delivery of NR2B-siRNA relieves neuropathic pain in rats. The model of neuropathic pain was established and intraperitoneal administration was performed according to previously described methods. Healthy Sprague-Dawley rats were randomly assigned into groups (n=42):control group:C group (blank control group, no treatment. n=6), NP group (neuropathic pain induced by ligating L5spinal nerve+intraperitoneal injection of0.9%NS, n=6) and sham group(expose L5spinal nerve, not ligate, n=6); neuropathic pain treatment group:PEI group (model+intraperitoneal injection of PEI/NR2B-siRNA, n=6), siWSLP group(model+intraperitoneal injection of WSLP/NR2B-siRNA, n=6), ncWSLP group(model+intraperitoneal injection of WSLP/negative control siRNA, n=6) and WSLP group(model+intraperitoneal injection of WSLP, n=6). The drugs were injected at8days following neuropathic pain model establishment, and the amount of siRNA(or ncRNA) is10nmol.50%mechanical withdrawal threshold was measured by von Frey hair through Up-Down method, and thermal withdrawal latency was measured by Plantar Test instrument. The detection was performed on the1day before,7day after model establishment;3,7,14and21days after intraperitoneal injection, and the detection was performed at09:00to18:00in a quiet environment.Results(1)The effect of intraperitoneal injection of WSLP/NR2B-siRNA on spinal NR2B expression in neuropathic pain rat.①Q-PCR results:The differences of NR2B mRNA expression level at spinal cord of seven groups is significant statistically (P<0.01).3days after intraperitoneal injection, compared with the blank control group, NR2B mRNA levels were significantly increased in the neuropathic pain treatment group, PEI/NR2B-siRNA group, WSLP/NR2B-siRNA, WSLP/ncRNA group, WSLP group and sham group (P<0.01). NR2B mRNA levels in WSLP/NR2B-siRNA group were significantly decreased by38.45%compared with the neuropathic pain treatment group(P<0.01), while the PEI/NR2B-siRNA group, WSLP/ncRNA group and WSLP group remained unchanged (P>0.05).3days after intraperitoneal injection of WSLP/NR2B-siRNA, NR2B mRNA levels was similar between WSLP/NR2B-siRNA group and sham group (P>0.05)②Western blot results:3days after intraperitoneal injection, the NR2B protein level were significantly increased in the neuropathic pain treatment group, PEI/NR2B-siRNA group, WSLP/ncRNA group, and WSLP group(P<0.01), compared with the blank control group. Compared with the neuropathic pain treatment group, the NR2B protein levels in WSLP/NR2B-siRNA group were significantly decreased by44.09%(P<0.01), while the PEI/NR2B-siRNA group, WSLP/ncRNA group and WSLP group remained unchanged (P>0.05)(2)Effect of intraperitoneal injection of WSLP/NR2B-siRNA on relieving neuropathic pain in rats.Before the neuropathic pain model establishment, the50%mechanical withdrawal threshold and thermal withdrawal latency were similar in the seven groups.7days after the model establishment, compared with the the basic level before neuropathic pain model establishment and the control group, the50%mechanical withdrawal threshold and thermal withdrawal latency were significantly decreased in neuropathic pain treatment group, PEI/NR2B-siRNA group, WSLP/NR2B-siRNA group, WSLP/ncRNA group, WSLP group and sham group.3days after intraperitoneal injection, the50%mechanical withdrawal threshold and thermal withdrawal latency in WSLP/NR2B-siRNA group were significantly increased (P<0.01) compared with the level before intraperitoneal injection, while PEI group, ncWSLP group and WSLP group remains unchanged (P>0.05) Comparisons between groups shows:3days after intraperitoneal injection, the 50%mechanical withdrawal threshold and thermal withdrawal latency in WSLP/NR2B-siRNA group were significantly increased (P<0.01) compared with neuropathic pain treatment group, while PEI/NR2B-siRNA group, WSLP/ncRNA group and WSLP group were similar to the neuropathic pain treatment group (P>0.05). Intraperitoneal injection of WSLP/NR2B-siRNA can significantly increased the50%mechanical withdrawal threshold and thermal withdrawal latency of the neuropathic pain rat, and the changes were maintained for7days.ConclusionIntraperitoneal injection of WSLP/NR2B-siRNA can effectively decrease NR2B mRNA and protein level which over-expressed in neuropathic pain rats.Intraperitoneal injection of WSLP/NR2B-siRNA can effectively increase the50%mechanical withdrawal threshold and thermal withdrawal latency of neuropathic pain rats.WSLP can effectively deliver NR2B-siRNA and treat neuropathic pain. |
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