ABSTRACT
Objective To investigate the therapeutic effect of cell penetrating peptide Tat-LK15 mediating small interfering RNA (siRNA) interference with the expression of neuronal nitric oxide synthase (nNOS) in rat spinal dorsal horn on neuropathic pain. Methods The transfection reagent, Tat-LK15, was used to mediate the transfection of rat spinal dorsal horn (SDH) neuronal cells with carboxyfluorescein (FAM), and then the transfection effect was observed under inverted fluorescence microscope. Fifty healthy male SD rats were randomly divided into 5 groups (n=10): control group, sham operation group (sham group), neuropathic pain group (SNL group), Tat-LK15-nNOS siRNA group (TS group) and Tat-LK15-NC siRNA group (TN group). Neuropathic pain was induced by spinal nerve ligation (SNL), rats in control group did not receive operation and only the spinal nerve was exposed in sham group. Groups SNL, TS and TN were made into the models by SNL and implanted intrathecal catheter, intrathecal administration was performed from the 7th day after model establishment, and 10μl normal saline, 10μl TS complex (including 5μg siRNA) and 10μl TN (including 5μg siRNA) were injected intrathecally each day for 7 days. Paw withdrawal mechanical 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 the operation and the lumbar segment (L4-6) of the spinal cord was removed to detect the expressions of nNOS mRNA and protein using q-PCR and Western blotting analysis. Results Tat-LK15 effectively mediated FAM-siRNA into SDH neuronal cells. Compared with sham group, SNL significantly decreased PWMT and PWTL and increased expressions of nNOS mRNA and protein from the 3rd day (P<0.01), but there was no significant difference between the sham and control group. Tat-LK15-nNOS siRNA complex significantly increased PWMT and PWTL and down-regulated nNOS mRNA and protein expressions in TS group compared with SNL group on the days 10 and 14. There was no significant difference between TN and SNL group. Conclusion Tat-LK15 not only can mediate successful nNOS siRNA transfection and inhibit the expression of nNOS, but also effectively relieve SNL-induced neuropathic pain in rats.
ABSTRACT
Objective We used peptide array technique to construct a peptide FynP inhibiting the interac-tion between Fyn and PSD95 in vitro therefor with a potential for inbiting NR2B phosphorylation level(p-NR2B). This experiment was designed to examine whether FynP(deliverd with TAT-LK15)can inhibit interaction between Fyn and PSD95 in inflammatory pain rats,and therefore inhibit NR2B phosphorylationin in vivo. Methods TAT-LK15 was complexed with FynP(cell-penetrating peptide Tat-LK15/FynP)or scrambled control FynP(Tat-LK15/mFynP). Changes of p-NR2B were detected using western-blot in SCDH of chronic inflammatory pain rats following intraperitoneal injection of Tat-LK15/FynP,meanwhile,the effect of Tat-LK15/FynP on the interaction between Fyn and PSD-95 was tested by co-immunoprecipitation. Pain control efficacy was evaluated by changes of mechani-cal withdrawal threshold(MWT)and thermal withdrawal duration(TWL)in these rats. Results Interaction be-tween Fyn and PSD-95 was efficiently inhibited by intraperitoneal injection of TAT-LK15/FynP complexes while in-jection of FynP or TAT-LK15/mFynP complexes did not show this inhibitory effect. NR2B phosphorylation level was also inhibited by injection of TAT-LK15/FynP,and the changes of p-NR2B levels were reduced by 52%compared to chronic inflammatory pain rats without treatment. FynP or TAT-LK15/mFynP did not show this effect. Moreover, injection of TAT-LK15/FynP complexes significantly reduced MWT and increased TWL of chronic inflammatory pain rats accordingly. Conclusion FynP delivered by Tat-LK15 can perturb Fyn and PSD95 interaction and then inhibit NR2B phosphorylation activation therfor relieve chronic inflammatory pain.
ABSTRACT
Objective We used peptide array technique to construct a peptide FynP inhibiting the interac-tion between Fyn and PSD95 in vitro therefor with a potential for inbiting NR2B phosphorylation level(p-NR2B). This experiment was designed to examine whether FynP(deliverd with TAT-LK15)can inhibit interaction between Fyn and PSD95 in inflammatory pain rats,and therefore inhibit NR2B phosphorylationin in vivo. Methods TAT-LK15 was complexed with FynP(cell-penetrating peptide Tat-LK15/FynP)or scrambled control FynP(Tat-LK15/mFynP). Changes of p-NR2B were detected using western-blot in SCDH of chronic inflammatory pain rats following intraperitoneal injection of Tat-LK15/FynP,meanwhile,the effect of Tat-LK15/FynP on the interaction between Fyn and PSD-95 was tested by co-immunoprecipitation. Pain control efficacy was evaluated by changes of mechani-cal withdrawal threshold(MWT)and thermal withdrawal duration(TWL)in these rats. Results Interaction be-tween Fyn and PSD-95 was efficiently inhibited by intraperitoneal injection of TAT-LK15/FynP complexes while in-jection of FynP or TAT-LK15/mFynP complexes did not show this inhibitory effect. NR2B phosphorylation level was also inhibited by injection of TAT-LK15/FynP,and the changes of p-NR2B levels were reduced by 52%compared to chronic inflammatory pain rats without treatment. FynP or TAT-LK15/mFynP did not show this effect. Moreover, injection of TAT-LK15/FynP complexes significantly reduced MWT and increased TWL of chronic inflammatory pain rats accordingly. Conclusion FynP delivered by Tat-LK15 can perturb Fyn and PSD95 interaction and then inhibit NR2B phosphorylation activation therfor relieve chronic inflammatory pain.
ABSTRACT
Aim To investigate the potential applica-tion of a non-viral gene carrier Tat-LK15 for delivering siRNA targeting nNOS in vitro, which provides evi-dence of Tat-LK15 mediating siRNA targeting nNOS in vivo for treatment of neuropathic pain. Methods 1. Tat-LK15 was mixed with siRNA, then the mixture was analyzed the best ratio by Gel retardation. The trans-fection efficiency of FAM-siRNA mediated by Tat-LK15 on RGC-5 cells was examined by Flow Cytome-try. The apoptosis ratio of RGC-5 was identified by Flow Cytometry 24 h after treated with the different do-ses of Tat-LK15 (1, 2. 5, 5, 10 and 20 μg). 2. The model of RGC-5 cell overexpression of nNOS protein was prepared. 3. 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 me-diate nNOS/siRNA transfection model cell) and Tat-N group ( Tat-LK15 mediate NCsiRNA transfection model cell) . Real-time Quantitative polymerase chain reac-tion( Q-PCR) and Western blot were used to evaluate nNOS expression level assay. Results It indicated that the Tat-LK15/siRNA complex completely formed at the weight ratio of 2∶ 1 (μg/μg) , and the transfec-tion efficiency was (84. 4 ± 3. 9)%. It caused cotytox-icity when Tat-LK15 dose was 20 μg ( 6. 1 μmol · L-1 ) , and the apoptosis rate more than control group [(10. 3 ± 1. 1)% vs (7. 4 ± 0. 9)%, P<0. 05]. The nNOS protein level of RGC-5 cells was significantly el-evated after modeling. Compared with that of model group, Tat-LK15/siRNA efficiently inhibited the ex-pression of nNOS at transcriptional level or protein leve1 of Tat-S group ( P <0. 05 ) , and there was no significant difference of the efficiency inhibited between Tat-S group and Lipo-S group. Conclusions Tat-LK15’ advantage is with high efficiency, low cytotox-icity. The Tat-LK15 can deliver siRNA targeting nNOS in vitro efficiently and safely.