Effect Of Cold Exposure On TLR4/NF-κB Signaling Pathway Of Acute Lung Injury Induced By Endotoxin In Mice

[Objective]This study established two models of acute lung injury induced by LPS in vivo and in vitro.Through various experiments in vivo and in vitro to study the effect of cold exposure on LPS-induced acute lung injury.To explore the relationship between LPS-induced acute lung injury exacerbated by cold exposure and toll like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway,and to provide a theoretical basis for the prevention and treatment of acute lung injury caused by infection in cold regions.[Methods]1.In vivo experiment:40 clear grade BALB/c healthy male mice were randomly divided into 4 groups,10 mice in each group,control(Con)group,cold exposure(Cold)group,LPS group,cold exposure+LPS(Cold+LPS)group,where mice in LPS group and Cold+LPS group nasal drops LPS 5mg/kg body weight,Con group and Cold group nasal drops of equal volume of sterile PBS(0.01 M,PBS as solvent)solution,after drops the nose,the Con group and LPS group mice were placed at room temperature,and the Cold group and Cold+LPS group mice were placed in 4℃ ventilated cold room.After 6 hours,the survival status and Survival rate,determination of total protein concentration in alveolar lavage fluid(BALF),white blood cell count;detection of IL-6 and TNF-α content in BALF by ELISA method;retention of lung tissue,weighing to calculate lung wet/dry Specific gravity,hematoxylin-eosin(HE)staining was used to observe lung tissue changes;western blotting method was used to determine the expression levels of TLR4 and NF-κB protein in lung tissue.Explore the effects of cold exposure on lung injury in mice.2.In vitro experiment:A549 cells were divided into control(Con)group,cold exposure(Cold)group,LPS group,LPS+cold exposure(Cold+LPS)group,of which LPS group,Cold+LPS group adds 1 μg/ml LPS to the cells of the group,then put the Con group and the LPS group at room temperature,and the Cold group and the Cold+LPS group in a 4℃ temperature-controlled incubator.After 6 hours,test each group by ELISA Inflammatory factors IL-6,TNF-α in the cell supernatant,and Western blotting were used to determine the expression levels of TLR4 and NF-κB protein in the cells.Explore the effect of cold exposure on LPS-induced A549 cell damage model.[Results]1.Survival status and survival rate of mice:the mice in Con group and Cold group had normal diet,good activity and slow breathing;the mice in LPS group had relatively poor survival state,reduced diet,reduced activity and shortness of breath;Cold+LPS group had the worst condition,little dietary activity and respiratory distress.2.The protein expression levels of TLR4 and NF-κB(p65)in lung tissue of mice:there was no significant difference between Cold group and Con group(P>0.05);the protein expression levels of TLR4 and NF-κB(p65)in LPS group and Cold+LPS group were all increased(P<0.05);Compared with the LPS group,the protein expression levels of TLR4 and NF-κB(p65)in Cold+LPS group were all increased(P<0.05).3.The contents of TNF-α and IL-6 in BALF of mice:there was no significant difference between Cold group and Con group(P>0.05);the contents of TNF-α and IL-6 in LPS group and Cold+LPS group were all increased(P<0.05);Compared with the LPS group,the contents of TNF-α and IL-6 in Cold+LPS group were all increased(P<0.05).4.Total protein concentration and white blood cell count in BALF of mice:the results showed that there was no significant difference between Cold group and Con group(P>0.05);the contents of total protein and white blood cell count in LPS group and Cold+LPS group were all increased(P<0.05);Compared with the LPS group,the contents of total protein and white blood cell count in Cold+LPS group were all increased(P<0.05).5.Lung wet dry specific gravity(W/D):there was no significant difference between cold group and con group(P>0.05);in LPS group and Cold+LPS group were all increased(P<0.05);Compared with the LPS group,the wet dry specific gravity of lung tissue in Cold+LPS group was increased(P<0.05).6.Pathological changes of lung tissue:HE staining showed that there were no obvious pathological changes in the lung tissue of Cold group and Con group,but inflammatory cell infiltration,pulmonary edema,alveolar hemorrhage,alveolar septum thickening and alveolar structure damage occurred in LPS group and Cold+LPS group,and the histopathological changes of Cold+LPS group were more obvious than those of LPS group.7.The protein expression levels of TLR4 and NF-κB(p65)in A549 cells:there was no significant difference between Cold group and Con group(P>0.05),the protein expression levels of TLR4 and NF-κB(p65)in LPS group and Cold+LPS group were all increased(P<0.05);Compared with the LPS group,the protein expression levels of TLR4 and NF-κB(p65)in Cold+LPS group were all increased(P<0.05).8.The contents of TNF-α and IL-6 in A549 cell supernatant:there was no significant difference between Cold group and Con group(P>0.05),the contents of TNF-α and IL-6 in LPS group and Cold+LPS group were all increased(P<0.05);Compared with the LPS group,the contents of TNF-α and IL-6 in Cold+LPS group were all increased(P<0.05).[Conclusions]Cold exposure could not induce acute lung injury,but may aggravate LPS-induced acute lung injury by up-regulating TLR4/NF-κB signaling pathway.