Objective: To investigate the effect of hypothermia on the expression Toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88), nuclear factor-ΚBp65 (NF-ΚBp65), plasminogen activator inhibitor-1 (PAI-1) in the TLR2/MyD88 pathway in rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS) inhalation.
Methods: Ninety male Sprague-Dawley (SD) rats were randomly divided into control group (n=18), hypothermia group (n=24), temperature controlled group (n=24), and temperature-uncontrolled group (n=24). The ALI model was reproduced by 0.5 mL/kg LPS intratracheal instillation, while only normal saline was instilled intratracheally for control group. Arterial blood was collected and physical cooling was started 1 hour after instillation. The body temperature was lowered to 32-34 centigradein hypothermia group and 36-37 centigradein temperature controlled group, and no intervention was used for temperature-uncontrolled group and control group. The arterial blood gas was determined in all the groups before and 1 hour after instillation of saline or LPS and 1, 6, 12 hours after intervention. Rats were sacrificed respectively at 1, 6 and 12 hours after temperature control therapy, the morphological changes in lung tissue were observed under light microscope. The protein expression of PAI-1 in bronchoalveolar lavage fluid (BALF) was determined by enzyme linked immunosorbent assay (ELISA). TLR2 mRNA and MyD88 mRNA transcriptional level were determined by reverse transcription-polymeras chain reaction (RT-PCR). NF-ΚBp65 protein level was determined by Western Blot.
Results: After instillation of LPS, the oxygenation index (PaO₂/FiO₂) of each group was decreased obviously, the damage of lung tissues was aggravating, the lung injury score was increased significantly, PAI-1 protein in BALF and the expressions of TLR2 mRNA, MyD88 mRNA, NF-ΚBp65 protein in lung tissues were increased obviously. Each index was improved by therapeutic Hypothermia, the effect of which was best in using a cooling period in the 1-6 hours, while might be benefit at 6-12 hours. Compared with temperature controlled group, PaO2/FiO2 (mmHg, 1 mmHg=0.133 kPa) at 1 hour and 6 hours of hypothermia group was improved(1 hour: 402.49 ± 38.61 vs. 324.36 ± 28.93, 6 hours: 349.72 ± 98.20 vs. 284.35 ± 13.68, both P<0.01), the lung injury score at 1, 6 and 12 hours were significantly decreased (1 hour: 6.04 ± 0.74 vs. 7.96 ± 0.65, 6 hours: 9.09 ± 0.80 vs. 13.13 ± 1.02, 12 hours: 10.79 ± 1.42 vs. 13.42 ± 0.68, all P<0.01), the PAI-1 protein (ng/L) in BALF at 1, 6 and 12 hours were significantly decreased(1 hour: 121.36 ± 4.62 vs. 197.74 ± 9.42, 6 hours: 230.53 ± 10.76 vs. 294.06 ± 16.60, 12 hours: 270.48 ± 13.20 vs. 319.40 ± 10.24, all P<0.01), TLR2 mRNA and MyD88 mRNA expressions(2 (-Δ ΔCt)) in the lung tissues at 1, 6 and 12 hours were significantly decreased(TLR2 mRNA 1 hour: 2.18 ± 0.26 vs. 3.04 ± 0.39, 6 hours: 4.09 ± 0.29 vs. 4.90 ± 0.35, 12 hours: 6.02 ± 0.43 vs. 7.10 ± 0.54; MyD88 mRNA 1 hour: 2.25 ± 0.41 vs. 3.04 ± 0.30, 6 hours: 5.67 ± 0.55 vs. 7.01 ± 0.76, 12 hours: 7.14 ± 0.60 vs. 8.87 ± 0.54, all P<0.01), NF-ΚBp65 protein expression (A value) at 6 hours and 12 hours was significantly decreased(6 hours: 0.31 ± 0.08 vs. 0.53 ± 0.12, 12 hours: 1.05 ± 0.17 vs. 1.76 ± 0.35, both P<0.01). There was no difference in each index between temperature controlled group and temperature-uncontrolled group.
Conclusions: Hypothermia can down-regulate the expression of TLR2 mRNA, MyD88 mRNA, NF-ΚBp65 protein and PAI-1 in the TLR2/MyD88 pathway to protect lung tissue of rats with ALI induced by LPS inhalation from injury.