Objective: To investigate the impact of hypothermia on Toll-like receptor 4 (TLR4) mRNA transcription and downstream inflammatory balance of macrophage induced by lipopolysaccharide (LPS).
Methods: RAW264.7 mouse macrophage cell line was collected and cultivated in incubator at 37 centigrade or 32 centigrade. The cells in stimulation group were incubated with 10 ng/mL LPS, while normal saline was used instead in control group. The cells culture supernatant was collected at 2, 8, 24 hours to determine the interleukin (IL-1β and IL-10) levels. Total RNA was collected with TRIzol, and TLR4 mRNA transcriptional level was determined by reverse transcription-polymerase chain reaction (RT-PCR).
Results: Under the intervention of LPS, TLR4 mRNA expression in normal temperature group and hypothermia group was increased first followed by a decreasing trend (both P<0.001), and at the same time point, TLR4 mRNA expression (2(-ΔΔCt)) in hypothermia group was lower than that in normal temperature group (2 hours: 9.19 ± 0.57 vs. 10.08 ± 1.02, t=-2.285, P=0.036; 8 hours: 13.58 ± 1.57 vs. 15.24 ± 1.63, t=-2.201, P=0.042; 24 hours: 6.85 ± 1.17 vs. 8.17 ± 1.21, t=2.353, P=0.032). After the administration of LPS, IL-1β and IL-10 levels in both normal and hypothermia group were gradually increased (all P<0.001), and at the same time point, the level of IL-1β in hypothermia group was lower than that in normal temperature group (2 hours: 87.08 ± 29.35 ng/L vs. 110.53 ± 26.58 ng/L, t=1.777, P=0.047; 8 hours: 119.19 ± 49.14 ng/L vs. 173.25 ± 37.21 ng/L, t=2.631, P=0.018; 24 hours: 208.66 ± 53.83 ng/L vs. 346.56 ± 64.30 ng/L, t=4.933, P<0.001), whereas the level of IL-10 of hypothermia group was higher than that in normal temperature group (8 hours: 170.01 ±2 4.90 ng/L vs. 140.02 ± 15.08 ng/L, t=-3.091, P=0.007; 24 hours: 423.10 ± 52.40 ng/L vs. 165.06 ± 31.97 ng/L, t=-12.611, P<0.001). The IL-1β/IL-10 ratio in normal temperature group was gradually increased (F=20.003, P<0.001), and it was decreased gradually in hypothermia group (F=1.811, P=0.185). At the same time point, IL-1β/IL-10 ratio in hypothermia group was lower than that in normal temperature group (2 hours: 0.740 ± 0.214 vs. 0.993 ± 0.256, t=2.275, P=0.037; 8 hours: 0.701 ± 0.363 vs. 1.237 ± 0.455, t=2.763, P=0.014; 24 hours: 0.493 ± 0.292 vs. 2.099 ± 0.428, t=9.299, P<0.001).
Conclusions: Hypothermia can reduce TLR4 mRNA transcription level of macrophage after the challenge of LPS, and it can relatively lower the level of IL-1β, but a higher level of IL-10 is maintained, thus creating a tendency of anti-inflammation.