The Influence Of Mild Hypothermia On The TLR4/MyD88 MRNA-NF-κB Protein Signaling Pathway In LPS-Induced Alveolar Macrophages In Vitro

Objective: To study the influence of sub-hypothermia(34℃)on the expressions of Toll-like receptor 4(TLR4)mRNA and myeliod differentiation factor 88(MyD88)mRNA,nuclear transcription factor-κB(NF-κB)and inflammatory factors TNF-α in lung macrophages of rats that infected with lipopolysaccharide(LPS)in vitro.To investigate the effect of mild hypothermia on TLR4/MyD88mRNA-NF-κB protein signal transduction pathway of lps-infected rat pulmonary macrophages in vitro.Methods: The lungs of adult male Sprague-Dawley rats were bronchially occluded with 0.9% saline.The lavage fluid was collected,and the lung macrophages were purified by filtration,centrifugation and adherence.Identification of extracted cells was determined by the immunofluorescence of differentiation antigen 68(CD68),the trypan blue cell reactive dye was used to detect cell survival,the cell counting kit(CCK8)was used to detect macrophage proliferation,and the Diff-quick kit was used to detect cell purity.The identifiedlung macrophages were divided into sub-hypothermia group(34℃)and normal temperature group(37℃).Each group was further divided into lipopolysaccharide(LPS)intervention group and control group,and the control group was added with equal dose of PBS,then cells of four groups were cultured at the same time,and the number of samples each group was ten.Cell and cell supernatants of each group were collected at 1h,4h,8h,16 h,and 24 h after infected,TLR4 mRNA and MyD88 mRNA were detected by real-time fluorescence reverse-polymerase-PCR(RT-PCR).The level of NF-κB was detected by Western Blot,and the expression of TNF-α was detected by enzyme-linked immunosorbent assay(Elisa).Results: Having sorted and purified the cells,results of trypan blue and CD68 suggested that more than 90% cells were macrophages,results that by using CCK8 kit suggested that cell proliferation reached the highest on the third day,and Diff-quick detected a cell purity greater than 95%.Having been infected of LPS,the transcriptional levels of TLR4 mRNA and MyD88 mRNA in the hypothermia group and the normal group increased gradually and then decreased.At the same time point,the transcription levels of TLR4 mRNA and MyD88 mRNA in the hypothermia group were lower than those of the normal temperature group(TLR4 mRNA 1h: 13.72 ± 0.56 vs 18.86 ± 0.09,P < 0.01;4h:14.59 ± 0.88 vs 21.14 ± 0.16,P < 0.01;8h: 13.86 ± 0.58 vs 25.63 ± 0.27,P <0.01;16h: 20.64 ± 1.01 31.19 ± 1.14,P < 0.01;24h: 18.08 ± 0.92 vs 26.81 ±1.17,P < 0.01.MyD88 mRNA 1 h: 12.68 ± 0.41 vs 17.12 ± 0.08,P < 0.01;4 h:20.07 ± 0.88 vs 23.41 ± 0.13,P < 0.01;8 h: 21.85 ± 0.12 vs 26.37 ± 0.20,P <0.01;16 h: 28.41 ± 0.76 is 32.83 ± 0.52,P < 0.01;24 h : 23.60 ± 0.34 is 27.39 ±0.91,P < 0.01).Having been infected of LPS,the NF-κB increased gradually within 24 hours of the normal temperature group and the hypothermia group.Atthe same time point,the level of NF-κB in the hypothermia group was significantly lower than that of the normal temperature group(1 h: 0.34 ± 0.03 vs.0.46 ± 0.06,P < 0.05;4 h: 0.51 ± 0.03 vs 0.61 ± 0.01,P < 0.01;8 h: 0.64 ±0.03 vs 0.93 ± 0.07,P < 0.01;16 h: 0.62 ± 0.04 is 1.06 ± 0.04,P < 0.01;24 h:0.71 ± 0.03 is 1.16 ± 0.07,P < 0.01).Infected of LPS,TNF-a increased first and then decreased in the normal temperature group and the hypothermia group.At the same time point,the level of TNF-a in the hypothermia group was significantly lower than that of the normal temperature group(1 h : 125.00 ±38.00 pg/ml ratio 577.43 ± 43.70 pg/ml,P < 0.01;4 h : 250.00 ± 32.00 pg/ml ratio 628.07 ± 55.84 pg/ml,P < 0.01;8 h : 500.75 ± 43.39 pg/ Mil ratio 682.57 ±39.50 pg/ml,P<0.01;16 h: 980.19 ± 30.38 pg/ml ratio 734.21 ± 48.92 pg/ml,P<0.01;24 h: 756.14 ± 49.47 pg/ml ratio 453.21 ± 32.81 pg/ml,P<0.01)Conclusion: Mild hypothermia may down-regulate the expression levels of cellular proteins NF-κB and TLR4 mRNA and MyD88 mRNA through the inflammatory signal transduction pathway of TLR4/MyD88 mRNA-NF-κB protein,and inhibit the expression level of inflammatory factor TNF-α in vitro.LPS-infected rat lung macrophages have protective effects within 24 hours.