| BackgroundChronic pain had been estimated to affect one-third of the population in world. People quality of life have seriously affected by neuropathic pain. The current conventional therapy for neuropathic pain is not satisfactory due to the long duration, complex etiology, the mechanism of neuropathic pain not clear, including the nerve block therapy, surgical therapy, physiotherapy etc. Neuropathic pain is defined pain caued by a lesion or disease of the somatosensory system by IASP in 2011. It has already become the research hotspot to develop a safe and effective therapy for neuropathic pain.Recent a series of evidence have demonstrated nNOS up-regulation in spinal cord and brain are considered to play a key role in generating and development of neuropathic pain. Peripheral nerve injury can lead to overexpression of nNOS in spinal cord dorsal horn, accompanied by hyperalgesia and pain disorders. Systemic or intrathecal injection of non-specific NOS inhibitors or selective nNOS inhibitors can relieve thermal hypersensitivity of neuropathic pain induced by chronic constriction injury of the sciatic nerve or spinal nerve ligation. Mice that were lacking in the nNOS gene do not exhibit nerve injury-induced mechanical hyperalgesia. After nerve damage, Noxious stimuli signal delivered to the spinal cord, excessive excitatory amino acids generated in combination with NMDA receptor, NMDA receptor activation, promoting Ca2+ influx, then to activate nNOS.Neuronal nitric oxide synthase (nNOS) mediates many neurophysiological and neuropathological processes, through not only catalyzing that NO is synthesized from the terminal guanidino nitrogen of arginine, but also acted on interaction of nNOS protein domain with NMDA receptors and postsynaptic protein PSD and other substances, including participation in synaptic transmission, plasticity regulation, long-term potentiation in the hippocampus, neuronal apoptosis and regeneration, mediating nerve damage and neurodegenerative process and adjusting various stages of neural differentiation. Eventually, The pathology of primary sensory neurons would lead to the primary central sensitization and synaptic plasticity, until functional changes in the cerebral cortex of the senior center. It could be an effective approach for the treatment of neuropathic pain that siRNA downregulate expression of nNOS.However, the development of RNAi-based therapeutics faces amount of challenge for in vivo application as follow:safe and efficient accomplish a therapeutic effect after accurate delivery into the target tissue. Due to lack of reliable gene vector, the application of siRNA gene therapy is still severely restrict in clinical. In recent years, cell-penetrating peptides (CPPs) as a gene delivery tool were got extensive attention, CPPs can traverse deliver siRNA into intracellular through non-receptor-dependent, then give play to biological activity.One of the most studied and most promising CPPs is the HIV-1 transactivator of transcription (Tat) peptide. Tat with penetrating activity of CPPs is rich in Polypeptide fragments of basic amino acids. It can quickly and efficiently mediated the gene fragment into the intracellular, and does not affect the normal structure and function of cells. There are advantage of high transfection efficiency, low toxicity and stability in plasma.The amino acids 49-57 of the Tat protein is the base sequence for derivery various molecules. Alain Pluen have found that it can optimize transfection efficiency by amino acids 49-57 of the Tat peptide conducted to combine to cationic membrane active peptide LK15 (KLLKLLLKLLLKLLK), and more importantly without considerable cytotoxicity. Therefore, we speculate that Tat-LK15 would be an available gene vector in biological applications of the nervous system. Tat-LK15 delivery siRNA targeting nNOS would be a prospective strategy for the treatment of pathological pain diseases.Firstly in vitro, compared with LipofectamineTM RNAiMAX(Lipo), siRNA marked with FAM (Carboxyfluorescein, FAM-siRNA) as the reporter gene, Tat-LK15 as transfection reagents mediated siRNA into 293T cells and RGC-5 cells respectively. The positive cells were counted by fluorescence microscopy after transfection 24h and the cell viability was calculated by CCK-8 assay in both cells. Secondly, Prepare the model of RGC-5 cell overexpression of nNOS protein. To investigates the potential application of Tat-LK15 as gene carrier for delivering siRNA targeting nNOS in vitro. Finally, Intrathecal administration of Tat-LK15 nNOSsiRNA complex in SNL rat,50% Paw withdrawal mechaical threshold (50% PWMT) and paw withdrawal thermal latency (PWTL) were measured and the expression of nNOS mRNA and protein in the spinal cord were detected. To investigates the potential treatment of neuropathic pain through Tat-LK15 delivery siRNA targeting nNOS to interfere expression of nNOS in rat spinal dorsal horn.ObjectiveTo investigates the transfection efficiency, cellar toxicity and Tat-LK15 as gene carrier for delivering siRNA targeting nNOS in vitro, which provides evidence of Tat-LK15 as gene carrier for gene therapy. Fartherly, to investigates the potential treatment of neuropathic pain through cell penetrating peptide Tat-LK15 delivery siRNA targeting nNOS to interfere expression of nNOS in rat spinal dorsal horn. It would offer a new idea and a new strategy in gene treatment of neurological pan.MethodsThe research process is divided into three parts:1. Transfection Efficiency and Cytotoxicity Studies of siRNA Transfected by Cell-Penetrating Peptides Tat-LK15. According to Alain Pluen, Tat-LK15 (RKKRRQRRRGGGKLLKLLLKLLLKLLK) was synthesized by BoXin Ximen Chain and was >86.59% pure. Compared with LipofectamineTM RNAiMAX(Lipo), to evaluate transfection efficiency and cytotoxicity of siRNA into 293T cells and RGC-5 cell by CPPs Tat-LK15. ①. Tat-LK15 siRNA complex were prepared at weight ratio from 1:3,1:2,1:1,2:1 and 3:1, then electrophoresed on 20% nondenaturing polyacrylamide gel to analyze the best ratio of Tat-LK15 and siRNA. siRNA marked with FAM(Carboxyfluorescein) as the reporter gene, Tat-LK15 siRNA complex at weight ratio from 1:2,3:4,1:1,4:3,2:1 (μg/μg), and Lipo siRNA at ratio of 1:1,2:1, 3:1,4:1,5:1 (μL/μg) transfected into 293T cells and RGC-5 cells respectively. After transfection 24h, the positive cells were counted by fluorescence microscopy. Calculate the rate of positive cells through each hole looking for three representative vision of the observer 100 cells. Then the cell viability was calculated by CCK-8 assay in both cells.2. Delivery of therapeutic siRNA by Tat-LK15 Targeting nNOS fusion gene in RGC-5 cells in vitro. ①. Compared with LipofectamineTM RNAiMAX (Lipo), to evaluate transfection efficiency of Tat-LK15 siRNA complex prepared at weight ratio 3:4,1:1 and 1:2 transfected into RGC-5 cells by Flow Cytometry. The apoptosis ratio of RGC-5 was identified by Flow Cytometry 24h after treated with the different doses of Tat-LK15 (1 μg,2.5 μg,5 μg,10 μg and 20 μg). ②. Prepare the model of RGC-5 cell overexpression of nNOS protein by OGD and high NaCl (145 mmol·L-1). RGC-5 cells were randomly divided into 5 groups:control group,model group, Tat-S group (Tat-LK15 mediate nNOS/siRNA transfection model cell), Lipo-S group (LipofectamineTM RNAiMAX mediate nNOS/siRNA transfection model cell) and Tat-N group (Tat-LK15 mediate NCsiRNA transfection model cell). Real-time Quantitative polymerase chain reaction(Q-PCR) and western blotting were used to evaluate nNOS expression level assay.3. Study of siRNA targeting nNOS mediated by cell penetrating peptides for treatment of neuropathic pain in rats. ①. As transfection reagents, Tat-LK15 mediated siRNA marked with FAM (FAM-siRNA) into Rat Neurons-dorsal spinal cord cells (RN-dsc), and then observed by inverted fluorescence microscopy after transfection 24h. ②. Fifty healthy male SD rats were randomly divided into 5 groups (n= 10 each):control group (group C), Sham operation group (group Sham), neuropathic pain group (group SNL), Tat-LK15-nNOS siRNA group (group TS) and Tat-LK15 NCsiRNA group (group TN). Neuropathic pain was induced by spinal nerve ligation (SNL), rats of group C without operation and rats spinal nerve just being exposed in group S. Group SNL, TS and TN induced by SNL and implanted intrathecal catheter. Intrathecal administration was performed from the 7 day after model establishment. Normal saline, Tat-LK15-nNOSsiRNA complex and Tat-LK15-NCsiRNA 10μL (including 5 μg of siRNA) were injected intrathecal ly each day for 7 days. Paw withdrawal mechaical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at 1 day before (baseline) and 3,7,10 and 14 days after model establishment. Then animals were sacrificed on the 14th day after operation and the lumbar segment (L4～6-) of the spinal cord was removed to detect the expression of nNOS mRNA and protein using Q-PCR and Western blot analysis.Results1. ①. It indicated that the Tat-LK15/siRNA complex completely formed at weight ratio of 2:1 (μg/μg) by Gel retardation. ②. The transfection efficiency was increased along with the increasing dosage of Lipo and Tat-LK15. The highest transfection efficiency of Tat-LK15(2:1) was (87.3±4.5)% for RGC-5 cells, which showed significantly lower than the Lipo (96.2±3.2)% when the ratio of Lipo/siRNA (μL/μg) is 5:1 (P<0.05).There was no significant difference of the highest transfection efficiency between Tat-LK15 and Lipo for 293T cells(P>0.05).But the cytotoxicity of Lipo was increased along with the increasing dosage of Lipo,than the cytotoxicity of Tat-LK15 was not significant change. When transfection efficiency was the highest, The cell viability of 293T cells and RGC-5 deliveried by Lipo were (77.8±4.1)% and (73.4±7.7)%, which were significantly lower than Tat-LK15(2:1) (91.0±3.7)% and (90.0±6.1)% in same cells. The cytotoxicity of Tat-LK15 was the lower (P<0.05).2. ①.The result of measurement by flow cytometry indicated that the transfection efficiency was (84.4±3.9)% of Tat-LK15/siRNA complex at weight ratio of 2:1 (μg/μg), which showed significantly lower than the Lipo at 5 μL (95.6±2.4)% (P<0.05). It caused cotytoxicity when Tat-LK15 dose was 20 μg (6.1 μmol·L-1), the apoptosis rate more than control group[(10.3±1.1)% vs (7.4±0.9)%, P<0.05]. ②. The nNOS protein level of RGC-5 cell was significantly elevated after modeling using OGD and high NaCl. While there is no difference increase of nNOS protein level of RGC-5 cell in OGD. Therefore OGD and high NaCl can induced the model of RGC-5 cell overexpression of nNOS protein. Compared with that of model group, Tat-LK15/siRNA efficiently inhibited the expression of nNOS at mRNA level (63.1%) nor protein level(58.9%) in Tat-S group that Tat-LK15/siRNA transfection model cell (P<0.05). But there was no significantly different of the efficiency inhibited between Tat-S group and Lipo-S group.3. ①. After detection of microtubule-associated protein 2 (MAP 2) which is the special character of these neuronal cells and DAPI using immunofluorescence techniques, it indicated Tat-LK15 can effectively mediate FAM-siRNA into RN-dsc cells that high purity. ②. Effect of 50% Paw withdrawal mechaical threshold (50%PWMT) and paw withdrawal thermal latency (PWTL) after injected intrathecally of Tat-LK15 nNOS/siRNA in neuropathic pain rats. Before modeled, there were no significantly difference about the base values of 50%PWMT and PWTL in 5 groups. Compared with group Sham, spinal nerve ligation significantly decreased 50%PWMT and PWTL in group SNL, TS and TN at 3d and 7d after SNL (P<0.01), but there were no significantly difference in group C (P>0.05). Intrathecal injection after SNL 7d, compared with group SNL, Tat-LK15-nNOS siRNA complex significantly increased 50%PWMT and PWTL in group TS at 10 ～14 d (P<0.01); There were no significantly difference between group TN after intrathecal injection of Tat-LK15 NCsiRNA and group SNL. Effect of nNOS expression in spinal cord after injected intrathecally of Tat-LK15 nNOS/siRNA in neuropathic pain rats. Compared with group Sham, SNL significantly increased expression of nNOS mRNA and protein in group SNL (P<0.01), but there were no significantly difference in group C (P>0.05). After intrathecal injection, Tat-LK15 nNOS/siRNA complex significantly down-regulated nNOS mRNA and protein expression in group TS compared with SNL (P<0.01); There were no significantly difference between group TN and group SNL (P>0.05).Conclusion(1) In vivo, cell penetrating peptide Tat-LK15 can mediate nNOS siRNA successful transfection into various cells. It indicated that the Tat-LK15/siRNA complex has high efficiency when the best ratio at weight ratio of 2:1 (μg/μg). Tat-LK15’advantage is the biosafety cause low cytotoxicity. The Tat-LK15 can efficiently deliver nNOS siRNA interfere expression of nNOS in RGC-5 cells. Therefore, Tat-LK15 would be the available gene vector for derivery siRNA into neuronal cell. And this project also demonstrated that Tat-LK15 can effectively mediate siRNA into primary culture Rat Neurons-dorsal spinal cord cells.(2)There are significantly decrease in 50% Paw withdrawal mechaical threshold (50%PWMT) and paw withdrawal thermal latency (PWTL) of neuropathic pain rats. Intrathecal injection Tat-LK15-nNOS siRNA complex after SNL, there is significantly increased 50%PWMT and PWTL. SNL rats have significantly increased expression of nNOS mRNA and protein. After intrathecal injection, Tat-LK15-nNOS siRNA complex significantly inhibited nNOS mRNA and protein expression.(3)The results showed that:in vivo, Tat-LK15 not only can mediate nNOS siRNA successful transfection and inhibits the expression of nNOS into rat neurons-dorsal spinal cord, but also effectively relieve spinal nerve ligation-induced neuropathic pain in rats. |
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