The Mechanism Of The Effect Of（BTK）Bruton Tyrosine Kinase On Hyperoxia Induced Acute Lung Injury In Mice

Objective:1.To explore the relationship between lung injury and Btk by establishment of hyperoxia induced lung injury in mice model, understand the expression and activation level of Btk in hyperoxia induced lung injury2.To investigate the interactions of Btk and NF kappa Bsignaling pathway which in hyperoxia induced lung inflammation responses and in apoptosis regulation role, to further explore the Btk in hyperoxic injury in the pathogenesis in molecular mechanism.3.To explore regulatory direction of the signaling pathway and understanding its role in pathological mechanism under oxidative stress by further study the relationship between Btk and inflammatory cytokines.Methods:Part one:60 healthy Kunming mice were selected and randomly divided to control group and hyperoxia exposed group(H Group), the hyperoxia exposure group respectively according to the 1,2,3,7 days time points divided into four subgroups, with 12 mice in each group. The control group was breathed indoor air; H group animals were placed in the oxygen tank and maintain oxygen concentration in 95%. Modeling is completed then observation lung tissue of the eye view, determination of W / D ratio, the value of TP in BALF and HE staining were measured to evaluate the model of lung injury severity.Moreover,by Western blot determination Btk and p-Btk expression levels in each time the mouse lung tissue.Part two:According to the results of the first part of the experiment, the experiment was divided into four groups: control group, hyperoxia exposure 3 days(H3d group), the inhibitor group and hyperoxia exposure 3 days + inhibitor group(H3d+I group). Control group breathed room air; H3 d group were exposed to oxygen cabin inhaled high concentration oxygen and oxygen concentration was maintained at 95%; inhibitor group given intraperitoneal injection of Btk specific inhibitor LFM-A13(50mg / kg), per 12 h after repeated dosing time. H3d+I group also give the two interventions above. Modeling is completed after through the determination of W / D ratio in each group, the value of TP in bronchoalveolar lavage fluid(BALF) and HE staining to evaluate the lung injury severity.In addition,to determination Btk, p-Btk, p-NF- kappa Bp65 and 14-3-3 protein expression levels by Western blot in mice lung tissue eaach groups.Then using RT-q PCR to quantitate TNF-a, IL-6 m RNA levels and enzyme-linked immunosorbent assay to measure the expression of MCP-1 in serum.Results:Part one:View of lung, W / D ratio, TP content in BALF and HE staining showed H7 d group was the most significant injury.Western blot analysis showed that Btk and p-Btk increased gradually with the extension of time and the difference is statistically significant(P < 0.05), but H7 d group and H3 d group expression difference no significant(P > 0.05).Part two:The experimental results show W / D ratio, TP content in BALF and HE staining pathologic evaluation score in H3d+I group is significantly lower than H3 d group.Western blot analysis showed that Btk,p-Btk and p-NF- kappa Bp65 in the H3 d group expression was higher than those in H3d+I group, control group and inhibitor group,the difference is statistically significant(P < 0.05). In addition, Western blot of14-3-3 protein quantitative results suggest that H3 d only a barely expression.The H3 d group compared with control group, inhibitor group and H3d+I group are reduce significantly(P < 0.01).RT- q PCR results showed the TNF-a and IL-6 expression in H3 d group was significantly higher than those in control group, inhibitor group and H3d+I group(P < 0.05).ELISA detection of serum MCP-1 expression show although H3d+I group expression higher than that of the control group but significantly lower than that in H3 d group.Conclusion:Part one:With prolongation of the hyperoxia exposure time, lung injury severity and protein Btk and p-Btk expression level increased gradually.The both proteins’ expression in H3 d group are peak, suggest that Btk in hyperoxic lung injury pathological formation of early play a role.Part two:Inhibition of Btk can effectively improve lung injury severity, this effect may be related to the down-regulation of NF kappa B and up-regulated 14-3-3 protein expression and inhibition of inflammatory factor activation in the lung.These results suggestion that Btk via regulation of NF kappa B promote inflammatory cytokines TNF-a,IL-6 activation and chemokines MCP-1 release and mediate of 14-3-3 protein to lead the formation of lung injury in oxidative stress.