Intervention Study Of Interleukin-35 On Endothelial Cells Associated With Transfusion-related Acute Lung Injury

Background:Transfusion-related acute lung injury(TRALI)is the main cause of death related to transfusion.At present,the pathophysiology mechanism of TRALI has not been fully elucidated.The clinical measures taken are mainly supportive measures,and there is no specific treatment for TRALI.Intervention and treatment measures studied at home and abroad were mainly target to neutrophils,macrophages,monocyte and inflammation reduction and so on.However,the activation of endothelial cells also plays a vital role in the occurrence and development of TRALI.At present,no intervention measures for endothelial cells in TRALI have been reported,so this research was focused on this point.Objective:To provide new insights for the prophylactic treatment and further study on the mechanisms of occurrence and development in TRALI,we used the "two hit" TRALI mouse model and human microvascular endothelial cells(HMVECs)in vitro to explore the interventional treatment of interleukin-35(IL-35)on endothelial cells in mice with TRALI in this study.Methods:1.Animal experiments["two hit"(LPS+MHC-I mAb)model]:32 male BALB/c mice aged 8-10 weeks were selected and randomly divided into 4 groups(8 in each group),which were Naive group,LPS group,TRALI group and rIL-35 group.Mice in the TRALI group were injected intraperitoneally with a low dose of LPS(0.1 mg/kg),and 24 hours later,MHC-I mAb(2mg/kg)was injected into the tail vein.In the rIL-35 intervention group,rIL-35(100μg/kg)was injected intravenously for two consecutive days before LPS injection on the third day,and rIL-35 was injected again before 34-1-2s antibody injection on the fourth day.In the LPS group,the same dose of LPS was given.Naive group and LPS group were used as controls.Samples were taken after the death or observation for 2h of the mice,and the wet-dry weight ratio(W/D)of lung tissues and pathological sections of lung tissues(HE staining)were analyzed.The supernatants of lung tissue homogenate was collected to detect protein concentration,myeloperoxidase(MPO)activity,and cytokines IL-6,IFN-γ,TNF,E-selectin,P-selectin and ICAM-1 levels.Blood was collected for detection of E-selectin,P-selectin,and ICAM-1 levels.2.HMVECs experiment:Human microvascular endothelial cells(HMVECs)were cultured in vitro.When the cells grew to 90%-95%,they were divided into three groups:Naive group,LPS intervention group and rIL-35 intervention group.LPS intervention group:LPS(0.5μg/mL)stimulation for 6h.HMVECs were activated fully;rIL-35 intervention group:rIL-35(2μg/mL)pretreatment for 1 8h and LPS(0.5μg/mL)stimulation for 6h.The expression levels of E-selectin,P-selectin and ICAM-1 were detected by flow cytometry.Results:1.Animal experiment:Compared with the control groups,pathological sections of lung tissue of TRALI mice showed a large number of infiltration of inflammatory cells,destruction of the alveolar structure,a significant increase in exudates in the alveolar cavity,and diffuse bleeding,indicating a successful TRALI model.Compared with the TRALI group,pathological sections of mice in the rIL-35 intervention group showed a decrease in red blood cells and inflammatory cells in the alveolar cavity,a significant reduction in pulmonary edema,and a decrease in lung W/D(4.158±0.257 vs 5.987±0.745,P<0.05);Protein concentration(1787.50±363.89 vs 2449.98±372.34,P<0.05),MPO activity(208.70±48.82 vs 528.58±95.38,P<0.05),IL-6(11.84±3.37 vs 294.80±105.31,P<0.05),IFN-γ(4.74±0.60 vs 6.88±1.57,P<0.05),and TNF(9.70±2.94 vs 14.76±1.97,P<0.05)were reduced in the supernatant of lung homogenate,and inhibited neutrophil aggregation in the lung;The levels of E-selectin(21.66±1.42 vs 24.40±3.27),P-selectin(29.61±3.62 vs 34.02±2.38)and ICAM-1(54.16±6.32 vs 62.72±5.51)in the supernatant of lung tissue homogenate were significantly reduced,and the difference was statistically significant(P<0.05);In addition,the levels of E-selectin(18.52±3.63 vs 22.66±3.17),P-selectin(21.14±3.43 vs 25.64±3.04),and ICAM-1(52.85±7.70 vs 62.72±5.51)were significantly reduced in blood,and the differences were statistically significant(P<0.05)and the 2h survival rate increased from 37.5%to 100%.2.HMVECs experiment:Compared with the LPS intervention group,E-selectin(1.24±0.45 vs 3.23±0.45),P-selectin(0.15±0.04 vs 0.29±0.06),and ICAM-1(52.92±0.42 vs 59.37±1.81)were significantly reduced in the rIL-35 intervention group,and the difference was statistically significant(P<0.05).Conclusions:This study showed that the mechanism of TRALI was related to the activation of endothelial cells.Antibody-mediated murine TRALI can be prevented by rIL-35 and rIL-35 appears to work by inhibiting the activation of lung endothelial cells.This could provide new insights for further studies on the treatment and mechanism of TRALI.