Intervention Study Of FTY720 In Mice With Aged Platelet Mediated Transfusion-related Acute Lung Injury

Background:The transfusion reaction known as transfusion-related acute lung injury(TRALI)is serious and potentially fatal.Transfusion of aged platelets(PLT)can result in the development of TRALI compared to fresh platelets,albeit the precise mechanism is still unknown.Sphingolipids ceramide(Cer)and sphingosine-1-phosphate(S1P)are biologically active sphingolipids that either degrade or enhance the endothelial barrier depending on how they affect the expression of zonula occludens-1(ZO-1)and vascular endothelial cadherin(VE-cadherin)in endothelial cells.The "sphingolipid rheostat"—a dynamic equilibrium between the relative concentrations of Cer and S1P—is crucial for maintaining healthy endothelial barrier function.According to lipidomic research on human platelets,aged platelets had much greater Cer concentrations and significantly lower SIP concentrations than fresh platelets.In various forms of acute lung injury,fingolimod(FTY720),a structural counterpart of S1P,may enhance pulmonary endothelial function and protect lung function in a dose-dependent manner.It is unknown if FTY720 could prevent TRALI by restoring the "sphingolipid rheostat" to balance and protecting the endothelium barrier.Objective:1.To analyze the alteration in Cer and S1P levels in mouse platelet concentrates,and to build a mouse model of TRALI mediated by aged platelets using a "two-hit" model;2.To examine the effects of various doses of FTY720 on TRALI and on the expression of endothelial junction proteins VE-cadherin and ZO-1.Methods:1.Mouse platelet concentrates were isolated and prepared,and samples were taken at d0(i.e.,the day of preparation),d1,d3,and d5 of storage to detect hematological indices,CD62P activation rate,and the concentration of Cer and S1P;d1PLT was taken for bacterial culture.2.Establishment of the TRALI mouse model with aged platelets:35 mice were divided into five groups at random:normal group,lipopolysaccharide(LPS)control group,d1PLT group,d3PLT group,and d5PLT group.On the test day,LPS was administered intraperitoneally to the last three groups at a dose of 2 mg/kg,and two hours later,stored platelets were administered through tail vein at a dose of 10 mL/kg for d1,d3,d5 respectively.At the appropriate times,the normal group and the LPS control group received injections of the appropriate quantities of saline and/or LPS.The mice were put to death and samples were obtained six hours following LPS administration.We investigated the myeloperoxidase(MPO)activity,protein concentration in lung tissue homogenates,and wet-to-dry weight ratio(W/D ratio).Lung histopathological sections were prepared and scored using the lung histopathological damage scoring criteria.3.Following the TRALI model’s successful creation,42 mice were split into seven groups at random:the d5PLT group,the LPS control group,the normal group,and the groups receiving 1 mg/kg FTY720,0.5 mg/kg FTY720,0.2 mg/kg FTY720,and 0.1 mg/kg FTY720.Following the injection of LPS,the FTY720 group was given at doses of 1,0.5,0.2,and 0.1 mg/kg FTY720,respectively,and 2 hours later,d5PLT was pumped into the tail vein.As mentioned above,sampling and testing were done.4.The expression levels of VE-cadherin and ZO-1 in mouse lung tissues were determined using Western blot(WB).Results:1.Establishment of aged platelet mediated TRALI mouse model1.1 Assessment of mouse platelet concentratesWith increasing storage time,PLT counts(d0 vs d5:965.75±54.36 vs 579.12±51.38109/L,P<0.0001)gradually decreased,and the mean platelet volume(d0 vs d5:5.82±0.89 vs 10.32±0.41 fL,P<0.0001),platelet distribution width(d0 vs d5:5.21 ±0.14 vs 14.46± 1.24 fL,P<0.0001),and CD62P activation rate(d0 vs d5:30.01 ±2.13 vs 54.98±2.06%,P<0.0001)gradually increased.The bacterial culture test was negative.1.2 Changes of Cer and S1P concentration in mouse platelet concentratesWith increasing storage time,the concentration of Cer in mouse concentrated platelets(d0 vs d5:43.68±3.00 vs 58.37±5.69 umol/L,P<0.0001)gradually increased and the concentration of S1P(d0 vs d5:201.85±9.92 vs 149.81 ±4.86 nmol/L,P<0.0001)gradually decreased,and the ratio of Cer to S1P was gradually imbalanced.1.3 Establishment of TRALI mouse modelCompared with the normal group,the protein concentration in the lung tissue homogenates(d3PLT group vs d5PLT group:5 21 1.27±461.60 vs 6 546.38±409.50 ug/mL,P<0.0001),W/D ratio(d3PLT group vs d5PLT group:4.50±0.18 vs 4.79±0.21,P<0.05),MPO activity(d3PLT group vs d5PLT group:28.01±7.81 vs 49.38±4.43 U/L,P<0.001),and lung injury score(d3PLT group vs d5PLT group:6.00±0.18 vs 7.24±0.38,P<0.01)in the LPS control,d1PLT,d3PLT and d5PLT group gradually increased.The alveolar walls of the mice in the d5PLT group had significantly thicker walls,and there was severe alveolar congestion and a significant infiltration of inflammatory cells in the interstitial lung,according to histopathological lung sections.According to these findings,infusion of d5PLT following the first injection of LPS might produce the TRALI mouse model as opposed to d1PLT and d3PLT.2.The intervention effects of different doses of FTY720 on TRALI1 mg/kg FTY720 did not prevent TRALI in "two-hits" mice:compared with the normal group,the protein concentration in lung tissue homogenates(4,700.24±335.28 vs.6,170.26±545.50 ug/mL,P<0.0001),W/D ratio(4.41 ±0.30 vs 4.88 ± 0.25,P<0.01),MPO activity(19.88±7.54 vs 45.97±4.79 U/L,P<0.0001),and lung injury score(4.08±0.71 vs 7.92±0.65,P<0.0001)of the 1 mg/kg FTY720 group were significantly higher,and the differences with d5PLT group were not statistically significant(P>0.05).Lung histopathological sections also showed severe alveolar congestion and significant thickening of the alveolar wall in the 1 mg/kg FTY720 group.Low-dose(0.5,0.2,0.1 mg/kg)FTY720 could prevent the occurrence of TRALI:the assessment indexes in all groups of low-dose FTY720 were significantly lower than those in the d5PLT group(P<0.05),and the differences with the normal group were not statistically significant(P>0.05),and there were fewer inflammatory cells in the interstitial infiltrate of the lung and less alveolar congestion.3.Effect of different doses of FTY720 on the expression levels of VE-cadherin,ZO-1The expression levels of VE-cadherin and ZO-1 were significantly lower in the d5PLT group compared with the normal group(P<0.05).After prophylactic administration of FTY720,the expression levels of VE-cadherin in the 1 mg/kg FTY720 group were still significantly lower than those in the normal group(P<0.05),and the expression levels of ZO-1 were increased,but the differences with the normal and d5PLT groups were not statistically significant(P<0.05).The differences between the normal and d5PLT groups were not statistically significant(P>0.05).Compared with the d5PLT group,the expression levels of VE-cadherin and ZO-1 in all groups of low-dose FTY720 were significantly higher(P<0.05)and tended to be normal.Conclusions:1.By transfusing aged platelets(d5)with an imbalanced "sphingolipid rheostat" based on LPS pre-stimulation,a TRALI animal model can be created.2.FTY720 has a dose-dependent effect on TRALI.Low dosages of FTY720(0.5,0.2,and 0.1 mg/kg)were able to prevent TRALI in "two-hits" mice by improving endothelial barrier function,lowering permeability,and reducing inflammatory cell infiltration.High doses of FTY720(1 mg/kg)were unable to do so.3.A low dose of FTY720 may improve the endothelium barrier’s ability to prevent TRALI by promoting the upregulation of VE-cadherin and ZO-1.