ABSTRACT
ObjectiveTo investigate the protective effect of microRNA-155 (miR-155) antisense oligonucleotid (ASO) on acute lung injury (ALI) mice by establishing a lentiviral expression vector of ASO of miRNA.Methods miR-155 antisense oligonucleotides amplified by polymerase chain reaction (PCR) from genomic, using BamH Ⅰ and Nhe Ⅰ double digestion, ligated into lentiviral expression vector. Sequence and virus titer were measured. According to the random number table method, 54 male BALB/c mice of 4-6 weeks old were divided into three groups. ALI animal models were prepared by intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS). The three groups were injected with 200μL phosphate buffered saline (PBS) containing 1×108/mL pmiR-155-ASO virus (pmiR-155-ASO group) or 200μL PBS containing 1×108/mL pSMPUW-miR-GFP empty virus (pmiR-cont group) or the same amount of PBS (PBS group) at 24 hours before the molding. Ten mice in each group were used to observe the 7-day survival rate. Blood samples and lung tissues of the remaining 8 mice were harvested after the model was established, and the levels of serum inflammatory cytokines were determined by enzyme linked immunosorbent assay (ELISA); the expression of miR-155 in lung tissue was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR); histopathological changes of lung and distribution of macrophages were observed under microscope.Results There was no significant difference in each index between pmiR-cont group and PBS group. The mature miR-155 expression in lung tissue in pmiR-155-ASO group was significantly lower than that in pmiR-cont group (2-ΔΔCt: 4.92±0.72 vs. 15.38±0.60,P < 0.05). Compared with pmiR-cont group, the injury degree of ALI mice after pretreatment with miR-155ASO was significantly improved, and the 7-day survival rate was significantly increased (72.1％ vs. 61.9％,P < 0.05 ); gross lung observation showed that congestion in lung tissue was significantly reduced, and the ratio of wet/dry weight (W/D) of lung was significantly decreased (4.50±0.13 vs. 5.64±0.61,P < 0.05);hematoxylin-eosin (HE) staining showed that inflammatory cell infiltration in lung tissue was decreased, while immunofluorescence assay showed that macrophage infiltration in lung tissue was significant decreased; the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL- 6) in serum were significantly decreased [TNF-α (ng/L):379.8±48.9 vs. 495.9±33.3, IL-6 (ng/L): 262.3±61.8 vs. 355.4±22.6, bothP < 0.05], but the level of IL-10 did not change significantly (ng/L: 143.6±32.5 vs. 140.4±22.3,P > 0.05).Conclusion miR-155 ASO has the effect of inhibiting LPS-induced inflammatory response and improving prognosis in ALI mice.
ABSTRACT
Purpose To explore the possibility and the effect of therapeutic bronchial asthma by antisense oligonucleotid. Methods Based on the IL-5 cDNA sequence of mouse,a segment of antisense oligonucleotid was designed and synthetized.5′-labeling of antisense oligonucleotid was signed by T4 PNK in order that the efficiency of stearylamine liposome in transfe-ting antisense oligonucleotid can be evaluated. Astham model was duplicated with ovalbumin (OVA) absorbed to aluminum hydroxide. T lymphocytes of mice were separated by nylon fiber method,then T lymphocytes transfected a different content of antisense oligonucleotid with stearylamine phys. positive liposome were cultured respectively in order to observe the effect of antisense oligonucleotid on IL-5 produced by T lymphocytes. IL-5 levels in the supernatants of T lymphocytes culture were determined by ELISA. Results Stearylamine liposome could markedly increase the efficiency of antisense oligonucleotid transfection. The efficiency of antisense oligonucleotid transfection was the best at 1∶15 m/m(antisense oligonucleotid and SA liposome) and it was increased approximately 12 times.In healthy and asthma Balb/c mice, IL-5 was not detected in the supernatants of T lymphocytes culture without challenge with OVA.However,IL-5 was increased markedly in the supernatants of T lymphocytes culture challenged with OVA. After transfecting a different concentration antisense oligonucleotid, IL-5 levels in the supernatants of T lymphocytes culture were significantly lower than those in control cells without antisense oligonucleotid transfection. IL-5 levels decreased from (44.60±6.23) to (30.70±7.362),(17.20±6.181) and(8.16±2.34)pg/ml respectively. And IL-5 synthesis was inhibited by 31.17% , 61.43% and 81.7% respectively. Conclutions IL-5 synthesis could be obviously inhibited by antisense oligonucleotid and showed a markedly relation between quantitative and effect. It is supported that the production of IL-5 be inhibited through preventing the transcription of IL-5 from T lymphocytes. The study provides foundation for antisense gene therapeutic asthma.