[Effects of antisense oligonucleotide to nuclear factor-kappaB on the development of bleomycin-induced pulmonary fibrosis and IL-4 expression therein: experiment with mice].
OBJECTIVE
To investigate the expression of nuclear factor-kappaB (NF-kappaB) in the cells in the bronchoalveolar lavage fluid (BALF) of idiopathic pulmonary fibrosis (IPF) and its effects on IL-4 expression therein, and to investigate the therapeutic role of antisense oligonucleotide (ASON) to NF-kappaB on IPF.


METHODS
C57BL/6 mice were randomly divided into 4 groups: bleomycin (BLM) group (n = 35, injected with BLM through caudal vein), control group [n = 20, injected with normal saline (NS) via caudal vein], ASON group (n = 35, injected with ASON to p65, a subunit of NF-kappaB, at the dose of 900 microg), and SON group (n = 35, injected with sense oligonucleotide to p65 subunit). Six hours after intravenous injection, the BLM, ASON, and SON groups were treated with BLM-A5 (5 mg/kg dissolved in 20 microl NS) by intratracheal installation, and the control group was treated with NS (20 microl). 0.5, 1, 3, 7, 14, and 28 days following intratracheal instillation of BLM or 0.5, 1, 14 days following intratracheal instillation of NS, 5 mice of every group were sacrificed and bronchoalveolar lavage was performed. The BALF was collected and assayed with ELISA for IL-4. Immunohistochemistry (IHC) and microscope image analysis were completed to detect the expression of p65 and IL-4 in the bronchoalveolar lavage cells. Another 5 mice from each group were sacrificed 28 days after intratracheal instillation with their total right lungs taken out to undergo pathohistological examination. The content of hydroxyproline in the left lung was detected by high performance liquid chromatography and ELISA.


RESULTS
(1) Twenty-eight days after intratracheal instillation, the BLM and the SON groups showed consolidation of the lung parenchyma with loss of the alveolar architecture and increased cellularity, while the ASON and control groups showed no significant pulmonary consolidation or fibrosis. (2) Twenty-eight days after intratracheal instillation, the hydroxyproline content of the BLM group was 876.8 +/- 91.1 nmol/lung, significantly higher than that of the control group (347.6 +/- 53.9 nmol/lung, t = -9.833, P < 0.001); the hydroxyproline content of the ASON group was 505.6 +/- 34.8 nmol/lung, significantly lower than that of the BLM group (t = -9.862, P < 0.001); however, the hydroxyproline content of the SON group was 775.2 +/- 68.9 nmol/lung, not significantly different from that of the BLM group (t = 2.118, P = 0.102). (3) One day after the intratracheal instillation of BLM, the value of average integral optical density of p65 in the bronchoalveolar lavage cells of the BLM, SON, and ASON groups were 275 +/- 13, 233 +/- 60, 233 +/- 60, and 126 +/- 34 respectively, all significantly higher that of the control group (38 +/- 18, t = 27.350, 8.039, and 6.107, P < 0.001, = 0.001, and = 0.004), that of the ASON group being significantly lower than those of the BLM and SON groups (t = 7.664 and -3.407, P = 0.002 and 0.027). (4) IHC showed that 1 day after the intratracheal instillation, the value of average integral optical density of IL-4 of the BLM, SON, and ASON groups were 134 +/- 16, 128 +/- 2, and 80 +/- 9 respectively, all significantly higher than that of the control group (33 +/- 12, t = 10.346, -5.927, and 5.313, P < 0.001, = 0.004, = 0.006), that of the ASON group being significantly lower than those of the BLM and SON groups (t = 6.967 and -3.591, P = 0.002 and 0.023). (5) ELISA showed that 1 day after the intratracheal instillation the IL-4 level BLM, SON, and ASON groups were (20.8 +/- 7.2) ng/L, (21.4 +/- 8.0) ng/L, and (9.7 +/- 1.4) ng/L respectively, all significantly higher than that of the control group [(1.6 +/- 3.6) ng/L, t = 6.494, 4.143, and 4.331, P = 0.003, 0.014, and 0.012], that of the ASON group being significantly lower than those of the BLM and SON groups (t = -3.553 and -3.577, P = 0.024 and 0.023) (6) Correlation analysis showed that 1 day after intratracheal instillation the expression of p65 was positively correlated with IL-4 expression in the bronchoalveolar lavage cells in the treatment group (r = 0.890, P < 0.05) and the ASON group (r = 0.909, P < 0.05).


CONCLUSIONS
The expression of NF-kappaB is significantly increased and augments the expression of IL-4 indirectly in the BALF cells during the process of BLM-induced lung fibrosis. ASON significantly inhibits the NF-kappaB activation and the IL-4 expression, and may be useful in gene therapy for pulmonary fibrosis.
Zhang
Xiao-ye
XY
Department of Respiratory Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, China.
Zhou
Yan
Y
Liu
Wei-qing
WQ
Zhao
Li
L
Li
Sheng-qi
SQ
D000818:Animals; D001761:Bleomycin / Q000031:analogs & derivatives; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry / Q000166:cytology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007150:Immunohistochemistry; D015847:Interleukin-4 / Q000096:biosynthesis*; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D011897:Random Allocation
D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D001761:Bleomycin; D015847:Interleukin-4; C025703:bleomycetin