Research Progress On FTY720 Combined With HAMSCs In The Treatment Of Smoke Induced Acute Lung Injury In Rats

Objective:Acute lung injury（ALI）is a kind of serious lung injury that can cause infiltration of double pneumonia and acute respiratory distress syndrome（ARDS）in severe cases,with a fatality rate of 30%-40%.At present,the supportive treatment of ALI has certain limitations and is prone to secondary injury and infection.It is urgent to explore new diagnosis and treatment schemes.At present,the supportive treatment of ALI has certain limitations and is prone to secondary injury and infection.It is urgent to explore new diagnosis and treatment plans.In recent years,mesenchymal stem cells（MSCs）have shown therapeutic advantages in hematopoiesis,immune regulation and tissue cell repair due to their self-replication,multidirectional differentiation potential and the ability to secrete a variety of biological active factors,which can be used as a potential strategy for ALI treatment.Human amniotic mesenchymal stem cells（h AMSCs）have attracted more and more attention due to their wide source,unrestricted ethics and easy acquisition.However,there are still many uncertainties in their application.As an important metabolite in sphinolipid metabolism,sphingosine-1-phosphate（S1P）is an important regulator of vascular endothelial cell permeability and body fluid balance in vivo,which can reduce lung injury.As an S1P receptor agonist,FTY720has been widely used in the repair of lung injury,which can limit the destruction of vascular endothelial monolayer in pulmonary microcirculation and inhibit the secretion of relevant inflammatory factors,providing a new research idea for the combined treatment of ALI.In this study,rats with smoke injury were used to establish ALI model to observe the treatment of ALI with h AMSCs and FTY720 and the changes of S1P,and to explore the effect and possible mechanism of the combined treatment of ALI with FTY720 and h AMSCs.Methods:（1）Establishment of ALI model in rats with smoke injury:SPF SD rats aged 8-10 weeks were used in this study.Using 30g pine sawdust as smoking material,and a self-made"multi-functional smoke injury experimental equipment"was used to smoke.ALI model of rat was established by smoking for 5min/3 times.（2）Isolation,culture and identification of h AMSCs:The healthy placentas were obtained from cesarean delivery women at term.Amniotic membrane was bluntly separated and digested with 0.25%trypsin and 0.1%type IV collagenase.After filtration and centrifugation,the cell precipitates were obtained,and the cells were re-suspended in complete culture medium.The surface markers CD73,CD90 and CD105 were identified by parallel flow cytometry.（3）Study on the treatment of ALI in rats with h AMSCs:SD rats were divided into normal group,smoke injury group and h AMSCs treatment group.The rats in the normal group breathed air freely in the injury equipment.Rats in the injury group were injected with 0.5ml normal saline through tail vein after injured by the above methods;In the h AMSCs treatment group,1×10~6h AMSCs were injected via tail vein（injection volume:0.5 m L）after injury.After 1d,3d and 7d,the lung tissue was collected,and the abdominal aortic blood was taken for blood gas analysis.The wet/dry weight ratio of lung tissue was measured in the right apical lobe.The septum of the right lung was stained with HE staining.The expression of IL-6,TNF-αand IL-10 in lung tissue were detected by ELISA.The m RNA expression levels of S1P,Sph K1 and S1PR1 in lung tissue were detected by RT-PCR.The protein expression level of S1P,Sph K1 and S1PR1 were detected by WB.（4）Study on the proliferative activity of S1P receptor agonist FTY720 on h AMSCs:HAMSCs were treated with FTY720 at different concentration gradients,and the cell viability was detected by CCK8 at different treatment time points.（5）Study on the combination of FTY720 and h AMSCs in the treatment of ALI rats:The rats were randomly divided into normal group,injury group,h AMSCs treatment group,FTY720 treatment group,and FTY720+h AMSCs combined treatment group.The rats in the FTY720 treatment group and the combined treatment group were injected with 0.1mg/kg FTY720 solution by intraperitoneal injection.The rats in the h AMSCs treatment group and the combined treatment group were injected with 1×10~6 h AMSCs through tail vein after injury.fter 1d,3d and 7d,the lung tissue was collected,and the abdominal aortic blood was taken for blood gas analysis.The wet/dry weight ratio of lung tissue was measured in the right apical lobe.The septum of the right lung was stained with HE staining.The expression of IL-6,TNF-αand IL-10 in lung tissue were detected by ELISA.The m RNA expression levels of S1P,Sph K1 and S1PR1 in lung tissue were detected by RT-PCR.The protein expression level of S1P,Sph K1 and S1PR1 were detected by WB.（6）Statistical analysis:Experimental data were analyzed by using SPSS 20.0 software,and single-factor LSD analysis was performed for the comparison between multiple groups at a single time point.The statistical results are expressed as“Standard Deviation,Mean±SD”,and P<0.05indicates statistically significant difference.Results:（1）Chapter 2:Compared with the normal group,the arterial oxygen partial pressure,oxygenation index and the level of anti-inflammatory factor IL-10 decreased significantly at 1,3 and 7 days after smoke injury（P<0.05）,carbon dioxide partial pressure,wet/dry weight ratio of lung tissue and IL-6 and TNF-a in lung tissue、S1P,Sph K1 and S1PR1 were significantly increased（P<0.05）.There were also obvious bleeding,edema and inflammatory cell infiltration in lung tissue,and the pathological score was significantly increased（P<0.05）.HAMSCs in isolation and culture grew adherently and grew in spindle and spindle shape.As identified by flow cytometry,the cells mainly expressed up to CD90,CD105 and CD73,but did not express CD34,CD45,CD14,CD19 and HLA-DR.They consistent with the immunophenotype of MSCs.Compared with the injury group,the oxygen partial pressure of rats in h AMSCs treatment group increased significantly（P<0.05）,and the wet/dry weight ratio of lung tissue decreased significantly（P<0.05）.The pathological changes of lung tissue were improved,the expression of IL-10 in lung tissue increased significantly（P<0.05）,and the levels of IL-6 and TNF-αin lung tissue were increased.The expressions of S1P,Sph K1and S1PR1 decreased significantly（P<0.05）.（2）Chapter 3:CCK8 results showed that FTY720 10μM significantly promoted the proliferation of h AMSCs at 12-24h（P<0.05）.FTY720 combined with h AMSCs can significantly improved the arterial partial pressure of oxygen and oxygenation index,reduced pulmonary edema and alveolar structure,and significantly reduced inflammatory factors IL-6、TNF-αand the expression of S1P,its kinase and receptor in lung tissue decreased significantly,and its therapeutic effect was significantly better than that in h AMSCs group.Conclusions:1.ALI model of smog-induced injury rats was established by self-made smog-induced injury device.ALI induced a decrease in oxygen partial pressure and oxygenation index,severe pulmonary edema and bleeding,and up-regulated expression of sphingolipid metabolite S1P and its receptor.2.HAMSCs can effectively improve the injury of ALI lung tissue in rats,reduce the inflammatory response,and reduce the expression of S1P,Sph K1 and S1PR1.3.S1P receptor agonist FTY720 can significantly promote the proliferation of h AMSCs.4.The combined treatment of FTY720 and h AMSCs can effectively improve lung tissue injury,increase oxygen partial pressure and oxygenation index,improve pulmonary edema,reduce lung tissue inflammation,and reduce the expression of S1P,Sph K1 and S1PR1.The combined treatment effect is better than h AMSCs single treatment.