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.

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.

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.

Objective To evaluate the CT features of small lung invasive adenocarcinoma with air?containing space and its relationship with pathological types, and to explore the pathological basis of air?containing space. Methods CT and pathological data of fifty patients with surgically proven lung invasive adenocarcinoma with air?containing space in our hospital from January 2012 to December 2017 were retrospectively reviewed. CT image analysis included image features of tumor and air?containing space. Pathological analysis included pathological subtype, differentiation degree. CT features of tumor and air?containing space were compared with regard to pathological types using chi?square test or Fisher exact text. Analysis of variance was used to compare quantitative data satisfying normal distribution, while those data not satisfying normal distribution were compared with Kruskal?Wallis test. In addition, Spearman correlation was used to analyze the correlation between nodule density and pathological types. Results (1) CT features of tumors: Tumors were predominantly located in peripheral lungs (46/50). Most of the tumors were subsolid nodules (37/50). Tumor?lung interface was generally clear (46/50). Tumors are often accompanied by malignant signs such as lobulation (37/50), spicule sign (27/50), air bronchogram sign (43/50), and pleural indentation (36/50). The mean diameter of nodules ranged from 7.50 mm to 18.12 mm, with an average of (12.91±2.85)mm. The nodule density ranged from-657.00 to 73.00 HU with an average of (-213.88±206.16) HU. (2) CT features of air?containing space:Air?containing spaces were commonly solitary (37/50) and were found to be mainly in an eccentric distribution (29/37). The maximum diameter of air?containing space ranged from 1.00 to 16.00mm, and the average diameter was (4.23±3.14)mm. Air?containing spaces less than 5mm were found in 35 cases (70%), and air?containing spaces more than 5mm were found in 15 cases. (3) Comparison of CT features and corresponding pathological types: Nodule density, number of air?containing space, and type of nodule density in different pathological types were statistically different (P<0.05). There was a correlation between nodule density and pathological subtypes (r=0.371, P=0.008). Differences of nodule density, short?dimension of nodule, type of nodule density, spicule sign, pleural indentation among different tumor differentiation degrees were significant (P<0.05). The degree of tumor differentiation was negatively correlated with nodule density (r=-0.451, P=0.001). Conclusion The detection rate of air?containing space in lung invasive adenocarcinomas is 12.7%. Most small lung invasive adenocarcinomas with air?containing space are presented as peripheral subsolid nodule, and there is a certain correlation between their CT features and pathological types. The pathological basis of air?containing space was supposed to be dilated distal bronchiole induced by check?valve mechanism and destruction of alveolar structure by tumor.

Objective: To evaluate the CT features of small lung invasive adenocarcinoma with air-containing space and its relationship with pathological types, and to explore the pathological basis of air-containing space. Methods: CT and pathological data of fifty patients with surgically proven lung invasive adenocarcinoma with air-containing space in our hospital from January 2012 to December 2017 were retrospectively reviewed. CT image analysis included image features of tumor and air-containing space. Pathological analysis included pathological subtype, differentiation degree. CT features of tumor and air-containing space were compared with regard to pathological types using chi-square test or Fisher exact text. Analysis of variance was used to compare quantitative data satisfying normal distribution, while those data not satisfying normal distribution were compared with Kruskal-Wallis test. In addition, Spearman correlation was used to analyze the correlation between nodule density and pathological types. Results: (1) CT features of tumors: Tumors were predominantly located in peripheral lungs (46/50). Most of the tumors were subsolid nodules (37/50). Tumor-lung interface was generally clear (46/50). Tumors are often accompanied by malignant signs such as lobulation (37/50), spicule sign (27/50), air bronchogram sign (43/50), and pleural indentation (36/50). The mean diameter of nodules ranged from 7.50 mm to 18.12 mm, with an average of (12.91±2.85)mm. The nodule density ranged from-657.00 to 73.00 HU with an average of (-213.88±206.16) HU. (2) CT features of air-containing space: Air-containing spaces were commonly solitary (37/50) and were found to be mainly in an eccentric distribution (29/37). The maximum diameter of air-containing space ranged from 1.00 to 16.00mm, and the average diameter was (4.23±3.14)mm. Air-containing spaces less than 5mm were found in 35 cases (70%), and air-containing spaces more than 5mm were found in 15 cases. (3) Comparison of CT features and corresponding pathological types: Nodule density, number of air-containing space, and type of nodule density in different pathological types were statistically different (P<0.05). There was a correlation between nodule density and pathological subtypes (r=0.371, P=0.008). Differences of nodule density, short-dimension of nodule, type of nodule density, spicule sign, pleural indentation among different tumor differentiation degrees were significant (P<0.05). The degree of tumor differentiation was negatively correlated with nodule density (r=-0.451, P=0.001). Conclusion The detection rate of air-containing space in lung invasive adenocarcinomas is 12.7%. Most small lung invasive adenocarcinomas with air-containing space are presented as peripheral subsolid nodule, and there is a certain correlation between their CT features and pathological types. The pathological basis of air-containing space was supposed to be dilated distal bronchiole induced by check-valve mechanism and destruction of alveolar structure by tumor.