Construction and mechanism of Mindin gene macrophage-specific knockout mice in lung ischemia-reperfusion injury / 中华器官移植杂志

ABSTRACT

Objective:To explore the construction and mechanism of Mindin gene specific macrophage knockout mice in acute lung injury induced by lung ischemia-reperfusion injury(IRI).Methods:Mindin gene knockout mice were constructed by CRE-LOP system, Mice were divided into four groups of C57/B6 wild-type mice sham operation(n=10), C57/B6 mice operation(n=10), Mindin-/-macrophage-specific knockout mice operation(n=10)and C57/B6 mice operation + Mindin recombinant protein intervention(n=10). And lung ischemia-reperfusion injury model was established by clamping pulmonary portal.The effects of Mindin gene knockout and recombinant protein intervention on acute lung injury were observed in vivo and in vitro.t-test and ANOVA test were employed for data processing.Results:Mindin gene macrophage specific knockout mice was successfully constructed.Surgery(Mindin-/-)group significantly reduced pulmonary edema, release of inflammatory factors(IL1β 2.73±0.19 vs. 5.81±0.61; IL-18 6.52±0.63 vs. 11.03±0.34; TNF-α 2.18±0.14 vs. 4.76±0.20; HMGB1 4.57±0.33 vs. 8.76±0.87), expression of NLRP3(2.07±0.27 vs. 4.91±0.22)and secretion of GSDMD(2.78±0.37 vs. 5.78±0.29)as compared with surgery group in vivo.In surgery(WT)+ Mindin group, the expression of lung IRI, inflammatory factors and cell pyroptosis were opposite, And the results were consistent in vitro and in vivo.As compared with surgery group, the above parameters were up-regulated in surgery(WT)+ Mindin protein group.And inter-group differences were statistically significant(all P<0.05). In vitro, the expressions of NLRP3(1.00±0.36, 0.41±0.06, 4.13±0.23), GSDMD(1.00±0.17, 0.34±0.16, 6.32±0.46)and integrin β4(1.00±0.11, 0.28±0.07, 3.53±0.17)were detected in different groups including hypoxia-recovery oxygen(HR), HR+ Mindin siRNA and HR+ Mindin protein groups in macrophage cell line(J774A); As compared with HR group, the above parameters were up-regulated in HR+ Mindin protein group and down-regulated in HR+ Mindin siRNA group.And the differences were statistically significant( P<0.05). The expressions of NLRP3(1.00±0.07, 1.13±0.11, 0.51±0.14)and GSDMD(1.00±0.09, 0.87±0.16, 0.37±0.12)were detected in Mindin, Mindin protein+ vehicle and Mindin protein+ integrin β4 knockout groups.The above parameters were down-regulated in Mindin protein+ integrin β4 knockout group as compared with Mindin protein and Mindin protein + vehicle groups.And the inter-group differences were statistically significant(all P<0.05). Conclusions:During pulmonary IRI, Mindin knockdown can alleviate pulmonary IRI.Mindin gene may promote the expression of inflammatory factors, NLRP3 and GSDMD protein by activating integrin β4 and aggravate cell pyroptosis to promote the development of pulmonary IRI.