Study On Polymorphonuclear Leukocytes Chemotaxis Mediated By JNK1/2/AP-1 Signaling Pathway During Acute Lung Injury In Mice

Objective: Under the intervention of JNK1/2 specific inhibitor SP600125,the JNK1/2/AP-1 signaling pathway in PMNs aggregation in the early stage of ALI was explored by observing lung tissue samples,lung tissue wet/dry ratio,serum NO,TNF-? and MPO and other inflammatory changes.Methods: Adopt an approach to random sampling.C57BL/6 mice were randomly divided into three groups according to simple random sampling.Control group received intranasal drip of 50?l PBS solution + intraperitoneal injection of 2 ml PBS solution,the LPS group received intranasal drip of 50?l LPS(40mg/ml)intraperitoneal injection of 2 ml PBS solution,and the LPS + SP600125 group received intranasal drip of 50?l LPS(40mg/ml)intraperitoneal injection of 2ml SP600125 solution.After modeling,mice were observed for general behavior at 12 h,24h,48 h,and 72 h.Lung tissue injury,wet/dry lung tissues were evaluated;ICAM-1and c-Jun level in lung tissues were measured by immunohistochemistry;ELISA measured TNF-? expression in the blood;NO and MPO contents in lung tissues were measured by kit;RT-PCR measured i NOS m RNA transcription levels in the lung,and the Western Blot was used to detect the JNK1,JNK 2 and p-JNK1/2 protein expression levels.Results: 1.The mice's general behavior: The lips of the Control group were rosy,breathing was stable,and the appearance of the lung tissue was pink after dissection.The mice of the LPS group showed erect hair,shortness of breath,and cyanosis of the lips.The escape action was slow during grasping,and the volume of the lungs increased after dissection.Red hemorrhages;SP600125 intervention reduced the shortness of breath of the mice,and the mental weakness improved but did not return to normal.2.Pathological injury of lung tissue: H&E staining showed that the lung tissue in the Control group was complete,with bright alveolar spaces,no alveolar wall rupture,no alveolar and interstitial congestion,and infiltration of inflammatory cells.In the LPS group,alveolar walls were ruptured,and inflammatory cells,red blood cells,and the protein-containing slurry exuded,the pathological damage worsened with the prolonged modeling time.Lung injury was reduced in the SP600125 group compared with the LPS group.3.The wet/dry weight ratio(W/D)of lung tissue in mice: Compared with group Control,the lung tissue W/D value of LPS group increased(P<0.05),and the W/D value of lung tissue after SP600125 intervention was lower than that of LPS group(P<0.05).4.Expression of TNF-? in serum: The concentration of serum TNF-? in LPS group was higher than that in group Control at different time points(P<0.01),and the serum TNF-? concentration after SP600125 was lower than that in LPS group(P<0.05),but it was still higher than that in Control group.5.The production of NO in serum: The serum NO level in group LPS increased compared with that in group Control(P<0.01).Compared with LPS,the serum NO level decreased at different time points(P<0.05)after SP600125 intervention.6.MPO activity in lung tissue: Compared with group Control,the MPO operation in lung tissue of group LPS increased(P<0.01).MPO activity in lung tissue after SP600125 intervention was lower than in the LPS group(P<0.05).7.Immunohistochemical detection of ICAM-1 and c-Jun expression in lung tissues: Positive expression was mainly observed in the cytoplasm,and was visualized as yellowish-brown or yellow granular staining.The appearance of ICAM-1 and c-Jun in group Control was small.The positive expression of ICAM-1 and c-Jun in the LPS group was higher than that in the Control group at all-time points.After SP600125 intervention,ICAM-1 and c-Jun positive expression cells decreased to varying degrees.8.RT-PCR determined the expression intensity of i NOS m RNA in mice's lung tissue: The expression of i NOS m RNA in lung tissue of the LPS group was higher than that of the Control group at each time point(P<0.01).After SP600125 intervention,the expression levels of i NOS m RNA in lung tissue were lower than those in LPS group at 24 h,48h,and the 72h(P<0.05),and there was no significant difference between i NOS m RNA in lung tissue at 12 h compared with LPS group(P>0.05).9.The expression of JNK1,JNK2,and p-JNK1/2 in lung tissue were measured by Western Blot: The protein expressions of JNK1,JNK2,and p-JNK1/2 in the LPS group increased at different time points,compared with the Control group(P<0.05).After the intervention,SP600125 JNK1 expression of mouse lung tissue compared with the LPS group(P<0.05)at each time point.At 24 h and 48 h,the expression intensity of the JNK2 protein was lower than that of the LPS group(P<0.01).At 12 h and 72 h,there was no significant difference in the expression intensity of JNK2 compared with the LPS group(P>0.05).The expression intensity of p-JNK1 / 2 in lung tissue was lower than in the LPS group at each time point(P<0.05).Conclusion: 1.SP600125 inhibits the activation of JNK1/2/AP-1,alleviates pulmonary edema in LPS induced ALI process,reduces inflammatory cytokines NO and TNF-?,and reduces PMNs lung tissue aggregation,ultimately reduces ALI inflammatory response.2.In LPS-induced ALI of mice,JNK1/2 in the cytoplasm is activated to produce phosphorylated JNK1/2(p-JNK1/2).p-JNK1/2 enters the nucleus and interacts with AP-1 to promote cytokines such as TNF-?,i NOS m RNA,ICAM-1,and so on.Eventually,a large number of PMNs migrated to the lungs to accumulate,activate,and release cytotoxic substances,thus increasing the corresponding biological effects.