Protective Role Of The Cholinergic Anti-Inflammatory Pathway In A Mouse Model Of Acute Lung Injury

Objective To explore whether the cholinergic anti-inflammatory pathway has a protective effect on a mouse model of acute lung injury(ALI)through the study of the expression of marker protein I?B?p-I?B?NF-?B p65 in the pathway.To determine whether nicotine plays a protective role in ALI through the cholinergic anti-inflammatory pathway.Methods The male BALB/c mice of grade SPF were selected and the weight was 20-22g.Mice were randomly divided into four groups(n= 10 per group):control(group C),lipopolsaccharide(LPS)(group L),LPS+nicotine(group LN),LPS+methyllycaconitine(group LM).An ALI model was established through the intraperitoneal injection of LPS.Mice in group L were only injected with LPS(10 mg/kg)intraperitoneally.Nicotine(1.2 mg/kg)and methyllycaconitine(2.4 mg/kg)were administered intraperitoneally 30 min prior to the LPS challenge.By contrast,the control group only received the same volume of normal saline intraperitoneally.All mice were killed at 6h after LPS injection.4%chloral hydrate(1 ml/100 g)was injected intraperitoneally,and the supernatant was obtained by centrifuging the blood obtained from the eyeball.The expression of IL-6 and TNF-a,the main inflammatory factor in serum was detected by Elisa.Then the mice were exposed to open the chest,heart and lung tissue,ligation of hilum of left lung to take fresh lung tissue.One part was used to measure the degree of pulmonary edema by weighing the wet weight and dry weight to calculate of dry and wet ratio;the other part was used to detect the expression of marker protein I?B.p-IKB?NF-?B p65 in the cholinergic anti-inflammatory pathway in ALI by Western Blot.The right lung was perfused with 4 ? polyformaldehyde(4%),and paraffin was embedded and sectioned.The histopathological changes of lung tissue were observed by HE staining.The nuclear transfer of NF-?B p65 protein in cholinergic anti-inflammatory pathway was observed by immunofluorescence.Results Elisa data showed that the expression of IL-6 and TNF-? had the same trend.Compared with group C,the expression of IL-6 and TNF-a increased significantly in group L and group LM(p<0.01),and that in group LN significantly decreased compared with that in L group(p<0.01).The results of dry and wet ratio showed that,the numerical value was significantly increased in group L(p<0.05)and in group LM(p<0.05)compared with group C,which indicated serious edema.Compared with the group L,the W/D in group LN was significantly decreased(p<0.05),and the degree of edema was obviously improved.Western blot showed that in total protein,compared with group C,the expression of p-I?B increased significantly in group L and LM(p<0.05).Compared with group L,the expression of p-I?B in group LN decreased significantly(p<0.05).The expression of I?B in the total protein showed the opposite trend of that of p-I?B.Compared with group C,the nuclear expression of NF-?B p65 increased significantly in group L and group LM(p<0.05);compared with group L,the expression of NF-?B p65 in group LN decreased significantly.In cytoplasm,compared with group C,the expression of NF-?B p65 in group L and group LM was significantly down-regulated(p<0.05);compared with group L,the expression of NF-?B p65 in group LN was significantly increased(p<0.05).HE staining showed that group C had normal lung tissue structure.Compared with group C,the structure of lung tissue in group L and group LM was significantly damaged,and edema in lung tissue was severe,and there was a large number of inflammatory cells infiltration.Compared with group L,the destruction degree of lung tissue in group LN was significantly improved.Immunofluorescence results showed that the lung tissue of group C was normal,karyoplasms is clearly visible;compared with group C,NF-?B p65 fluorescent antibody and a large number of nuclei obviously overlap of group L and group LM and structure of lung tissue was destroyed.Compared with group L,NF-?B p65 fluorescent antibody and nuclei overlap almost disappeared in group LN.The nucleoplasm is relatively clear.Conclusion Under the conditions of this experiment,we can draw the following conclusions:Firstly,the cholinergic anti-inflammatory pathway protects the ALI induced by LPS in the ALI mouse model.Secondly,the cholinergic anti-inflammatory pathway play the protective role by reducing the phosphorylation of the downstream protein I?B and nuclear transfer of NF-?B p65.