The mechanism of Xuebijing injection in preventing and treating lung injury induced by cardiopulmonary bypass by regulating the apoptosis of alveolar polymorphonuclear neutrophil
Objective To investigate the protective effect of Xuebijing injection on acute lung injury(ALI)associated with cardiopulmonary bypass(CPB)by regulating the apoptosis of polymorphonuclear neutrophils(PMN).Methods Thirty male Sprague-Dawley(SD)rats were randomly divided into sham operation group(Sham group),CPB model group(CPB group)and Xuebijing pretreatment group(XBJ group)according to the random number table method,with 10 rats in each group.Rats in the CPB group and XBJ group undergoing CPB procedures for 60 minutes.Rats in the Sham group did not undergo CPB.Rats in the XBJ group received intraperitoneal injection of 4 mL/kg Xuebijing injection 2 hours before CPB.Rats in the Sham group and CPB group were injected with an equal amount of normal saline.4 hours after CPB,arterial blood was collected for blood gas analysis to calculate respiratory index(RI),and lung tissue of rats was collected for determination of lung index(LI)and pulmonary water containing rate.PMN in bronchoalveolar lavage fluid(BALF)were collected and the activity of caspase-3 was detected.The apoptosis rate was detected by flow cytometry.The expressions of microRNA-142-3p(miR-142-3p)and FoxO1 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The protein expression of FoxO1 was detected by Western blotting.In addition,HL-60 cells were divided into control oligonucleotide transfection group,miR-142-3p mimics transfection group,and miR-142-3p inhibitor transfection group.After 48 hours of transfection,the activity of miR-142-3p binding to FoxO1 was detected using dual luciferase reporter genes.Results Compared with Sham group,RI,LI and pulmonary water containing rate were significantly increased in CPB group.The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly decreased,the expression of miR-142-3p was decreased,and the expression of FoxO1 protein was increased.However,compared with CPB group,RI,LI and pulmonary water containing rate were significantly decreased in XBJ group[RI:0.281±0.066 vs.0.379±0.071,LI:4.50±0.26 vs.5.71±0.42,pulmonary water containing rate:(80.31±32.50)%vs.(84.59±3.41)%,all P<0.01].The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly increased[caspase-3 activity:0.350±0.021 vs.0.210±0.014,apoptosis rate:(15.490±1.382)%vs.(8.700±0.701)%,both P<0.01],the expression of miR-142-3p was significantly up-regulated(2-ΔΔCt:2.61±0.17 vs.0.62±0.05,P<0.01),and the protein expression of FoxO1 was decreased[FoxO1/GAPDH(relative expression level):0.81±0.04 vs.1.22±0.06,P<0.01].However,there was no statistically significant difference in FoxO1 mRNA expression among the three groups.The bioinformatics analysis results showed that miR-142-3p can bind to the FoxO1 3'untranslated region(3'UTR).In HL-60 cells,compared with control oligonucleotide transfection group,the transfection of miR-142-3p mimics could reduce the expression of FoxO1 protein[FoxO1/GAPDH(relative expression level):0.48±0.06 vs.1.00±0.05,P<0.01],however,the transfection of miR-142-3p inhibitor increased the expression of FoxO1 protein[FoxO1/GAPDH(relative expression level):1.37±0.21 vs.1.00±0.05,P<0.05].But,transfection with miR-142-3p mimics or inhibitor had no effect on FoxO1 mRNA expression.The luciferase reporter gene showed that miR-142-3p could bind to the FoxO1 3'UTR to inhibit FoxO1 expression.Conclusion Xuebijing injection may promote the apoptosis of pulmonary alveolar PMN through the miR-142-3p/FoxO1 axis,and play a role in the prevention and treatment of CPB-induced ALI.
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