Effect Of NR2B-siRNA Mediated By Hydroxyapatite Nanoparticles On Inflammatory Pain Of Mice

Objective To observe the effect of NR2B-siRNA mediated by hydroxyapatite nanoparticles on formalin-induced inflammatory pain of mice and the expression of NR2B in spinal cord.To preliminarily investigate the feasibility of HA as siRNA carrier to transfer NR2B-siRNA in vivo.Methods Hydroxyapatite nanoparticles were prepared in low Ca/P ratio by a kind of electrodeposition-hydrothermal process.Agarose gel electrophoresis and ultraviolet spectrophotometer were used to analyse the ability of HA to bind NR2B-siRNA and the stability of the complex in normal sodium.Forty-eight male Kunming species of Mus musculus albus,weighing about 18-20g,were randomly divided into six groups. The first group(HN group,8 mice):NR2B-siRNA mediated by HA were administered into subarachnoid space of mice.The second group (PN group,8 mice):NR2B-siRNA were administered into subarachnoid space of mice via the same injection above when polyethylenimine(PEI) was used as siRNA carrier instead of HA.The third group(HG group,8 mice):as a negative control,green fluorescent protein(GFP)-siRNA mediated by HA were injected into mice in the same way.The fourth group(HA group,8 mice):isometric sterilized suspension of nanoparticles were intrathecally injected into mice.The fifth group(NS group,8 mice):isometric stroke-physiological saline solution were intrathecally injected into mice.The sixth group(BC group,8 mice):only intrathecal injection was performed,but nothing was injected into subarachnoid space of mice.On 7th day after intrathecal injection,the planta surface of the right hindpaw was injected with 20ul of a 1% formalin solution for the formalin test observed about 1 hour.The contralateral paw was not injected.Following formalin test,lumbar segments of spinal cords were dissected immediately in each group for use in immunohistochemical staining of spinal cord sections.Result Transmission electron microscope(TEM)analysis revealed that the diameter of hydroxyapatite nanoparticles was 40-50nm,and the nanoparticles were well-distributed like the little striga or rod.In condition of acidity and neutrality,HA was able to fully bind NR2B-siRNA when the ratio of HA quality to NR2B-siRNA quality was up to 35:1,even higher than the ratio.In contrast,the ability of HA to bind NR2B-siRNA was slightly weak in condition of alkalinity. Furthermore,the compound binding NR2B-siRNA could be stable in normal sodium,and not be dissociated.The nociceptive response in the acute phase was indistinguishable among all groups for nociception with regard to the flinching response(P＞0.05).HN group and PN group significantly decreased the nociceptive response in the tonic phase, compared to HG group,HA group,NS group and BC group(P＜0.05), while no significant difference between HN group and PN group was observed(P＞0.05).Neither was among the rest 4 groups(P＞0.05).The immunohistochemistry result of NR2B in spinal cord showed that the amount of cells expressing NR2B protein in HN group,PN group,HG group,HA group,NS group and BC group were 53.79±11.38,60.63±9.9, 99.04±16.39,91.42±14.39,101.21±16.82,96.38±10.27,respectively. Compared to the rest 4 groups,the amount of cells expressing NR2B protein in HN group and PN group were significantly reduced(P＜0.05). There was no significant difference between HN group and PN group(P＞0.05).Neither was among the rest 4 groups(P＞0.05).Conclusion 1.As a novel siRNA carrier,HA could effectively transfect siRNA via intrathecal injection,and this complex could significantly reduce formalin-induced nociception in the tonic phase of mice.2.HA/NR2B-siRNA complex injected intrathecally could effectively inhibit NR2B expression in spinal cord of mice.