Water Soluble Lipopolymer Can Efficient Deliver SiRNA Targeting NMDA Receptor1for Neuropathic Pain Rats

BackgroundPain is an unpleasant emotion experience accompanied with tissue damage or potential tissue damage, which defined by The International Association for The Study of Pain (IASP). This kind of pain often repeated attacks in a certain period (a few months to a few years). Prolonged suffering from pain, however, can become a serious burden for the affected persons and is one of the major reasons for people to seek medical care and pharmacological treatment. Despite the obvious need for pain medication and the great progress that has been made in recent years in elucidating the molecular and cellular mechanisms underlying nociception, pain drugs available to date are frequently unsatisfactory. Current pharmacotherapy of pain is still mainly based on two classes of drugs that have been established for a long time: non-steroidal anti-inflammatory drugs (NSAIDs) and opioids. Both classes of analgesic substances, however, have severe drawbacks, in that they frequently result in only partial and transient relief from pain. In addition, prolonged usage of these pain drugs produces severe side-effects in some patients, including gastric, kidney and liver toxicity in the case of NSAIDs and constipation, nausea, respiratory depression and sedation in the case of opioids. Thus, the need to develop new pharmacological strategies for pain relief.Chronic pain such as neuropathic pain is a performance of the nerve cells high reactive as a result of inflammation or nerve damage. Peripheral damage stimulation or tissue damage cause primary afferent nerve endings release excitability amino acid (EAAs) and some other neurotransmitters to the spinal cord. These excitability amino acid (EAA) such as N-methyl-D aspartate (NMDA) can through its receptor mediate exciting transfer on spinal cord and other parts of the postsynaptic. Yu and Salter found that NMDA receptors raised can cause sodium ion concentration increases in cells, cause synaptic transmission between excitability enhanced, thus produce pain central sensitization. Therefore, by blocking NMDA receptors then inhibition the effect of neurotransmitter of NMDA, It can block exciting transfer of pain central, then to treat chronic pain. However, at present, NMDA receptor blockers such as MK-801and APV can not Widely used in clinical because of side effects. So Garry put forward that to interfere with NMDA receptors genes by RNA interference to treat chronic pain.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 key problem is transduction tools that can carry siRNA in vivo safely and efficiently and then restrained the expression of genes effectively and appropriately. At present, the virus carrier is undoubtedly high efficiency gene transduction tools, however, the virus carrier is only used in vitro because of safety question. Recently, cholesterol has been used to modify low molecular weight PEI, water-soluble lipopolymer (WSLP) comprising of cholesterol and low molecular weight PEI exhibited good biocompatibility, high transfection efficiency and minimal toxicity To sum up, we hypothesizes that WSLP delivering siRNA targeting NR1mayinhibit NR1expression in the spinal dorsal horn,and may be used as a novel method for treating neuropathic pain. The first, designed and synthesised NR1-siRNA; The second, prepared WSLP,WSLP was connected with siRNA directly; The third, established the model of neuropathic pain and intrathecal injection of WSLP/siRNA according to previous methods; The last using real-time PCR technology and western-blot technology detected the change of NR1expression on the Spinal dorsal horns. 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 NR1in vivo and to determined whether intrathecal injection WSLP/NR1-siRNA complexes can be a new method for neuropathic pain treatment.MethodsThe research process is divided into four parts:(1) Synthesis WSLP/siRNA.①According to reference synthesis WSLP;②Selecting NR1-siRNA sequence according to reference, synthesis NR1-siRNA by guangzhou ruibo company;③Synthesis WSLP/siRNA according to reference and the results which gain from previous study, the quality ratio of WSLP and siRNA is5:1,after mixing, rest for30min at room temperature.(2) Water soluble lipopolymer can efficient delivery siRNA targeting spinal NR1expression in rat. Take adult SD rats(n=18) which catheterization of the Lumbar Spinal Subarachnoid Space, Divided into three groups randomly:NS group (intrathecal injection saline)、naked siRNA group (intrathecal injection naked NR1-siRNA), WSLP/siRNA group (intrathecal injection WSLP/NR1-siRNA), The amount of siRNA is1nmol. After three days, their spinal dorsal horns at L4-6were harvested on ice and divided equally for detection of NR1mRNA and protein expressions.①Take the spinal cord specimens, extraction total RNA, detection of NR1mRNA expression using RT-PCR ②Take the spinal cord specimens, detection of NR1protein expression using western blots(3) Water soluble lipopolymer can be efficient siRNA delivery targeting NR1for neuropathic pain rats. The model of neuropathic pain was established and intrathecal administration was performed according to previously described methods. Healthy Sprague-Dawley rats were randomly assigned to four groups (n=24):S group (n=6, only expose the sciatic nerve branch), C group (n=6, made neuropathic pain model and intrathecal injection saline), W group (n=6, made neuropathic pain model and intrathecal injection WSLP/siRNA, n=6) and L group (n=6, made neuropathic pain model and intrathecal injection WSLP/scRNA).Neuropathic pain model adopted the Spared nerve Injury model (SNI).After made neuropathic pain mode, the next day intrathecal injection saline, WSLP/siRNA, WSLP/scRNA respectively. After three days, their spinal dorsal horns at L4-6were harvested on ice and divided equally for detection of NR1mRNA and protein expressions.①Take the spinal cord specimens, extraction total RNA, detection of NR1mRNA expression using RT-PCR;②Take the spinal cord specimens, detection of NR1protein expression using western blots.(4) Water soluble lipopolymer delivery of NR1-siRNA relieves neuropathic pain in rats. The model of neuropathic pain was established and intrathecal administration was performed according to previously described methods. Healthy Sprague-Dawley rats were randomly assigned to four groups (n=24):S group (n=6, only expose the sciatic nerve branch), C group (n=6, made neuropathic pain model and intrathecal injection saline), W group (n=6, made neuropathic pain model and intrathecal injection WSLP/siRNA, n=6) and L group (n=6, made neuropathic pain model and intrathecal injection WSLP/scRNA).Neuropathic pain model adopted the Spared Nerve Injury model (SNI).After made neuropathic pain mode, the next day intrathecal injection saline, WSLP/siRNA, WSLP/scRNA respectively. Detection of pain behaviors according to previously described methods. The detection was performed on the1day before and1,3,5, and7days after model establishment and the detection was performed at09:00to15:00in a quiet environment.Results(1) Water soluble lipopolymer can efficient delivery siRNA targeting spinal NR1expression in rat.①Real-time RT-PCR results The differences of NR1mRNA expression level at spinal cord among NS group、The naked siRNA group and WSLP/siRNA group is significant statistically (F=1116.901, P=0.000). Compared with the NS group, WSLP/siRNA group NR1mRNA expression level decreased (P<0.01), but the naked siRNA groups NR1mRNA expression level remained unchanged in (P=0.441)②Western-Blot results The differences of NR1protein expression level among NS group、The naked siRNA group and WSLP/siRNA group is significant statistically (F=155.358, P<0.01).Compared with the NS group, The WSLP/siRNA group NR1protein expression level decreased (P<0.01), but the naked siRNA group remained unchanged (P=0.149).(2) Water soluble lipopolymer can be efficient siRNA delivery targeting NR1for neuropathic pain rats.①Real-time RT-PCR results The differences of NR1mRNA expression level at spinal cord of four groups is significant statistically (F=344.179, P<0.01). After3days after model establishment, Compared with the S group, the W group NR1mRNA levels were significantly increased (P<0.01), and the L group NR1mRNA levels were significantly increased (P<0.01). After3days after model establishment, Compared with the C group, the W group NR1mRNA level decreased (P<0.01), but the L group remained unchanged (P=0.201)②Western-Blot results The differences of NR1protein expression level at spinal cord of four groups is significant statistically (F=741.420, P<0.01). After3days after model establishment, Compared with the S group, the W group NR1protein were significantly increased (P<0.01), the L group NR1protein were significantly increased (P<0.01). After3days after model establishment, compared with the C group, the W group NR1protein level decreased(P<0.01), but the L group protein level remained unchanged (P=0.596)(3) Water soluble lipopolymer delivery siRNA targeting NR1relieves neuropathic pain in rats. The cumulative pain score was used to reflect the degree of pain. The cumulative pain score significantly decreased indicate less pain. The differences of cumulative pain score in four groups had significant statistically (F=458.347, P <0.01). The time (before and after surgery) had an influence on the cumulative pain score (F=749.112, P<0.01). These had cross effects between the time(before and after surgery)and intrathecal injection of WSLP/siRNA (F=86.405, P<0.001). After3days after model establishment, The differences of cumulative pain score in four groups had significant statistically (F=224.042, P<0.01), Compared with the C group, The cumulative pain scores of the W group were reduced (P<0.01), while the cumulative pain scores of the L group were remained unchanged (P=0.370)Conclusion(1)WSLP can effectively carry siRNA silence spinal cord NR1expression in Sprague-Dawley rats;(2)WSLP can effectively carry siRNA restrain NR1mRNA and protein excessive expression in neuropathic pain rats;(3) WSLP can carry siRNA effectively relieves neuropathic pain in rats.