| Objective: to investigate the effects of mild hypothermia on mitochondrial apoptotic pathways in mice with acute lung injury(ALI)induced by lipopolysaccharide(LPS)inhalation,and explore the protective effect and mechanism of mild hypothermia on ALI.Methods: 36 male Kunming mice were divided into 3 groups acaccording to the random number table method: the control group(group C,n=12),the mild hypothermia group(group H,n=12),and the normal temperature group(group N,n=12).Under the guidance of a light source,tracheal intubation was performed with a microsampler.The model group(H,N)was instilled with 50 ul of lipopolysaccharide at a dose of 3mg/kg to establish a mouse ALI model,while the control group(C)was infused with an equal volume of normal saline.After1 h of administration,the model group began to perform physical temperature control.The hypothermia group controlled the anal temperature at 33-34℃ and the normal temperature group was controlled at 36-37℃,while the control group did not undergo temperature intervention.After continuous temperature intervention for 4h and 8h,the mice were sacrificed in batches,and the lungs of the mice were removed by thoracotomy.The right lung of the mouse was isolated and stored in 10% paraformaldehyde and stored at room temperature for later paraffin section.After hematoxylin-eosin staining(HE),the pathological changes of the tissue were observed by light microscopy and semi-quantitative scoring was performed.After washing the blood stains,a small portion of the left lung(50 mg)was quickly cut out,and then immediately cut into pieces and digested in a collagenase solution to prepare a single cell suspension.After staining with Annexin V-FITC and Propidium iodide(PI),the apoptosis rate of lung tissue cells was detected by flow cytometry.The remaining lungs are stored in a-80℃ refrigerator for later extraction of total RNA and total protein from lung tissue.Real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-qPCR)was used to detect the expression of cysteine aspartic protease 3(Caspase-3)mRNA,and Western blot was used to detect the proto-oncogene protein c-bcl-2(Bcl-2),bcl-2 related X protein(Bax),Cleaved caspase-3 proteins expression.Results: 1.Pathological changes of lung tissue:No or only slight pathological damage was observed in the control group under light microscope,and obvious pathological damage was seen in the mild hypothermia group and the normal temperature group.Through semi-quantitative scoring,the score of the module was significantly increased(the gold standard of the ALI model),and at the same time,the mild hypothermia group was significantly lower than the normal temperature group(4h: 0.83±0.41 vs.5.67±0.82 vs.83±0.75;8h: 1.17±0.41 vs.8.33±1.03 vs.11.83±0.98).Comparing the different periods in the same group,the control group had no significant changes,but the mild hypothermia group and the normal temperature group scored significantly higher at 8h than4 h.2.Caspase-3 mRNA expression level in lung tissue: Compared with the control group,the expression of Caspase-3 mRNA in the mild hypothermia group and the normal temperature group was significantly increased,and at the same time,the mild hypothermia group was significantly lower than the normal temperature group(4h: 1.00±0.08 vs.1.96±0.27 vs.4.75±0.37;8h: 1.07±0.14 vs.2.40±0.32 vs.4.88±0.29).Comparing the different periods in the same group,there was no significant change in the control group and the normal temperature group,while the expression of the hypothermia group increased with time.3.Bcl-2,Bax,Cleaved caspase3 protein expression in lung tissue: Bcl-2: Compared with the control group,the mild hypothermia group was significantly increased,and the normal temperature group was significantly increased at 4h,but it was significantly reduced after 8h of intervention;in addition,the mild hypothermia group was significantly higher than the normal temperature group at the same time(4h: 0.35±0.05 vs.0.94±0.06 vs.0.83±0.08;8h: 0.36±0.03 vs.0.73±0.09 vs.0.26±0.05).Comparing the different periods in the same group,There was no significant change in the control group;the expression level of the hypothermia group and the normal temperature group decreased significantly with time.Bax: Compared with the control group,the mild hypothermia group and the normal temperature group increased significantly.The 4h in the mild hypothermia group was significantly lower than the 4h in the normal temperature group,but the 8h in the mild hypothermia group was not significantly different from the 8h in the normal temperature group(4h: 0.53±0.07 vs.0.77±0.06 vs.0.86±0.06;8h: 0.74±0.04 vs.0.92±0.05 vs.0.96±0.07).Comparing the different periods in the same group,the expression of Bax in each group increased significantly with time.Bcl-2/Bax ratio: Compared with the control group,the mild hypothermia group was significantly increased,and the normal temperature group was significantly increased at 4h,but it was significantly reduced after 8h of intervention;in addition,the mild hypothermia group was significantly higher than the normal temperature group at the same time(4h:0.66±0.03 vs.1.23±0.09 vs.0.97±0.07;8h: 0.49±0.04 vs.0.79±0.11 vs.0.27±0.06).Comparing the different periods in the same group,the ratio in each group decreased significantly with time.Cleaved caspase3: Compared with the control group,the mild hypothermia group and the normal temperature group were significantly increased,and at the same time,the mild hypothermia group was significantly lower than the normal temperature group(4h: 0.42±0.08 vs.0.59±0.05 vs.0.75±0.09;8h: 0.58±0.04 vs.0.89±0.09 vs.0.08±0.06).8h and 4h in each group were significantly higher.Comparing the different periods in the same group,the ratio in each group increased significantly with time.4.Lung tissue cell apoptosis rate: Compared with the control group,the mild hypothermia group and the normal temperature group were significantly increased,and at the same time,the mild hypothermia group was significantly lower than the normal temperature group(4h: 22.12±1.46 vs.33.07±2.25 vs.39.90±3.50;8h:23.03±1.92 vs.41.20±3.05 vs.54.60±4.97).Comparing the different periods in the same group,there was no significant change in the control group,the hypothermia group and the normal temperature group increased significantly with time.Conclusion: Mild hypothermia can down-regulate the expression of Bcl-2,Bax,and Caspase-3 in the mitochondrial apoptotic pathway of lung tissue in mice with LPS-induced acute lung injury,thereby reducing the rate of apoptosis and reducing the severity of lung injury. |
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