Effect Of Toll-Like Receptor 4 Gene Knockout On Lung Injury In Mice Treated With Mild Hypothermia

Objective To investigate the protective effect of mild hypothermia on acute lung injury(ALI)in mice and its mechanism,and the effect of Toll-like receptor 4 gene knockout on ALI in mice treated with mild hypothermia.Methods 48 male normal mice(C57BL / 10J)and 48 male TLR 4knockout mice(C57BL / 10ScNJNJU)were randomly divided into normal mice control group(A,n = 16),normal mice mild hypothermia model group(B,n = 16),normal mice normal temperature model group(C,n = 16),TLR4 knockout mice control group(D,n = 16)and TLR 4 knockout mice mild hypothermia model group.(E,n = 16),TLR 4 knockout mice normal temperature model group(F,n = 16).The ALI model of mice was replicated by intratracheal drip of Lipopolysaccharide(LPS)10 mg / kg in the model group,while saline of the same volume was injected into the control group A and D.The model group was given alcohol and animal thermostat temperature control intervention after 1 hour of administration.The body temperature was continuously measured.The Anal temperature was controlled at 33-35?(mild hypothermia model group)and 36-37?(normal temperature model group),while the control group was not given temperature intervention.After 4 and 8hours of temperature intervention,mice were sacrificed in batches,and blood and lung tissue samples were collected.(1)Hematoxylin-eosin staining(H E)was used to observe the pathological changes of lung tissue in mice,and semi-quantitatively evaluate the degree of pathological damage of lung tissue;(2)Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of tumor necrosis factor-alpha(TNF-alpha)in serum of mice in each group;(3)Western Blot was used to detect the expression of TLR 4 and NF-kappa B proteins in lung tissues of mice in each group.(4)RNA was extracted from lung tissues by TRIzol,and the transcription levels of MyD88 and i NOS in lung tissues of mice in each group were detected by real-time fluorescence quantitative PCR(RT-PCR).The data were analyzed by SPSS 22.0 software.Results1.Normal mice(1)Lung injury score : At the same time,there was significant difference between the control group,mild hypothermia group and normal temperature group,and there was significant difference between mild hypothermia group and normal temperature group(P < 0.05)(4 h : 0.67±0.52 vs.6.67±1.03 vs.8.67±1.21;8 h : 1.33±0.52 vs.8.67±1.21 vs.11.66±0.82).(2)Serum TNF-alpha expression : At the same time,there was significant difference between the control group,mild hypothermia group and normal temperature group,and there was significant difference between mild hypothermia group and normal temperature group(P < 0.05)(4 h : 13.68±1.44 vs.53.44±4.61 vs.87.60±28.09;8h : 13.89±1.64 vs.81.01±22.54 vs.155.64 ±24.08).(3)The expression levels of TLR 4 and NF-?B protein in lung tissue : TLR 4 : At the same time,there were significant differences between normal temperature group and control group and mild hypothermia group(P < 0.05)(4 h :0.87±0.07 vs.0.39±0.04 vs.0.59±0.08;8h : 0.90 ±0.08 vs.0.47±0.12 vs.0.66±0.02).NF-?B:There was significant difference between the normal temperature group and the control group and mild hypothermia group at 4 h and8 h(P < 0.05)(4 h :0.84±0.05 vs.0.60±0.16 vs.0.64±0.10;8h :0.90±0.08 vs.0.76±0.06 vs.0.72±0.03).(4)The transcription levels of MyD88 and i NOS in lung tissue : MyD88 : 4 h in control group was significantly different from mild hypothermia group and normal temperature group(P < 0.05)(0.88±0.20 vs.1.80±0.48 vs.2.05±0.54);8 h in control group was significantly different from mild hypothermia group and normal temperature group(P < 0.05)(0.84±0.22 vs.2.91±0.51 vs.4.59±0.39).i NOS mRNA : At the same time,there was significant difference between the control group,mild hypothermia group and normal temperature group,and there was significant difference between mild hypothermia group and normal temperature group(P < 0.05)(4h : 1.00±0.22 vs.6.06±2.03 vs.13.11±2.62;8 h : 3.24±0.76 vs.10.87±1.50 vs.18.35±2.74).2.TLR4 knockout mice(1)Lung injury score : At the same time,there was significant difference between control group and mild hypothermia group and normal temperature group,and there was significant difference between mild hypothermia group and normal temperature group(P < 0.05)(4 h : 0.83±0.41 vs.2.50±0.55 vs.3.83±1.47;8 h : 1.17±0.40 vs.3.00±0.90 vs.4.17±0.98).Comparison with ordinary mice: there was no statistically significant difference between the control groups,There were statistically significant differences between the mild hypothermia group and the normal temperature group(P < 0.05).Factorial analysis showed that mild hypothermia therapy and TLR 4 gene knockout had an interactive effect on reducing lung injury in mice(P < 0.05).(2)Serum TNF-alpha expression : At the same time,there was significant difference between control group and mild hypothermia group and normal temperature group,and there was significant difference between mild hypothermia group and normal temperature group(P < 0.05)(4 h : 14.55±0.17 vs.31.00 ±2.17 vs.41.59±3.49;8 h : 15.04±0.17 vs.33.08±4.74 vs.45.87±10.75).Comparison with ordinary mice : there was no statistically significant difference between the control groups,There were statistically significant differences between the mild hypothermia group and the normal temperature group(P<0.05).Factorial analysis showed that mild hypothermia therapy and TLR 4 gene knockout had an interactive effect on reducing the expression of TNF-alpha in serum of mice(P<0.05).(3)The expression levels of TLR 4 and NF-?B in lung tissue : TLR4 gene knockout mice had no expression of TLR 4 protein in lung tissue.NF-?B: There was no significant difference among the control group,mild hypothermia group and normal temperature group(4 h : 0.47±0.18 vs.0.75±0.03 vs.0.65 ±0.16;8 h : 0.40±0.14 vs.0.56±0.12 vs.0.64±0.12).(4)MyD88 and i NOS transcription levels in lung tissues : MyD88 mRNA : At the same time,there were significant differences between the control group,mild hypothermia group and normal temperature group(P < 0.05),but there was no significant difference between mild hypothermia group and normal temperature group(4 h : 0.86±0.17 vs.1.43±0.26 vs.1.67±0.24;8 h : 0.89±0.26 vs.1.79±0.64 vs.2.11±0.55).Compared with normal mice : 4 h : there was no statistically significant difference between the control group,mild hypothermia group and normal temperature group.8 h : no statistically significant difference was found in the control group.The difference between mild hypothermia group and normal temperature group was statistically significant(P < 0.05).i NOS mRNA : At the same time,there was significantdifference between control group and mild hypothermia group and normal temperature group,and there was significant difference between mild hypothermia group and normal temperature group(P < 0.05)(4 h : 0.95±0.18 vs.1.67±0.29 vs.3.53±0.75;8 h : 1.10±0.17 vs.2.20±0.25 vs.3.07 ±0.97).Compared with normal mice,except the control group for 4 hours,there were significant differences in other groups at different time points(P < 0.05).Factorial analysis showed that mild hypothermia treatment and TLR 4 gene knockout had an interaction effect on the reduction of i NOS transcription level in lung tissue of mice(P < 0.05).Conclusion Mild hypothermia can protect LPS-induced ALI in mice,at least partly by down-regulating the transcription and expression of related signaling molecules in TLR 4 / NF-kappa B signaling pathway,thus inhibiting the release of inflammatory factors and reducing the degree of lung injury.In addition,TLR 4 signaling pathway blockade has synergistic effect on mild hypothermia treatment of ALI in mice.