The Role And Mechanism Of SphK-1/S1P Signaling Pathway Mediated By Endoplasmic Reticulum Stress In Lung Injury Induced By Limb Ischemia/Reperfusion

Part? Establishment and Evaluation of a Model of Lung Injury Induced by Limb Ischemia/ Reperfusion in RatsObjective To establish the model of lung injury induced by limb ischemia/reperfusion in rats by clamping the femoral artery in the proximal inguinal ligament and to evaluate the the severity of lung injury induced by ischemia / reperfusion in rats.Methods Forty SD rats were randomly divided into the following four groups with ten animals per group :ischemia 2h and then reperfusion 3h(A group)?ischemia 3h and then reperfusion 4h(B group)?ischemia 4h and then reperfusion 5h(C group)and control group(D group).The SD rats were anesthetized via the intraperitoneal administration of10% chloral hydrate(3ml/kg body weight),then were fixed and preserved skin.The auricular vein was cannulated for fluid administration(Ringer's lactate,8-10ml/h)to maintain normal physiological needs.ECG monitoring used RM6240 BD biological signal acquisition and processing system to monitor ECG.The arterial blood pressure was monitored continuously with the catheterization of the artery.After isolated femoral artery,micro artery clamp was used to clamp femoral artery in the proximal inguinal ligament to cause limbs ischemia,then suture the incision,a rubber tourniquet was used to bind the double hind legs root to prevent collateral circulation in order to make absence of dorsalis pedis artery pulse and the double foot become dark and cool as a sign of the success of ischemia.Afterischemia 2h,opened the original incision,the clamp and tourniquet were removed and the rats underwent 3h of reperfusion,we considered that the recovery of arterial pulse and the gradual warming of the double foot were successful(A group ischemia 2h and then reperfusion 3h?B group ischemia 3h and then reperfusion 4h?C group ischemia 4h and then reperfusion 5h)?Control group(D group)only incised the rightfemoral triangle skin,isolated and lined femoral artery without ligation,then sutured wound.We monitored continuously blood oxygen saturation ? invasive arterial blood pressure and ECG,collected blood samples for arterial blood gas analysis and stored lung tissue samples for pathology assessment after ischemia /reperfusion.Results Clamping the femoral arteryin the proximal inguinal ligament to induce limb ischemia 3h and then reperfusion 4h in rats was the best way to establish the model of lung injury induced by limb ischemia /reperfusion.Conclusion A model of lung injury induced by limbischemia /reperfusion in rats was established successfully.Part? The Role of Sph K-1/S1 P Signaling Pathway Mediated by Endoplasmic Reticulum Stress in Lung Injury Induced by Limb Ischemia/ReperfusionObjective To explore the role of Sph K-1/S1 P signaling pathway mediated by endoplasmic reticulum stress in lung injury induced by limb ischemia /reperfusion.Methods Fifty SD rats were randomly divided into the following six groups with ten animals per group :sham operation group(A group);normal saline via intragastric administration for a week + I/R(B group);FTY720 3mg(3 mg/kg/d)via intragastric administration for a week + I/R(C group);FTY720 5mg(5 mg/kg/d)via intragastric administration for a week + I/R(D group);FTY720 10mg(10 mg/kg/d)via intragastric administration for a week + I/R(E group).LIRLI model was prepared by SD rats in each group after the pretreatment throughlimb ischemia 3h and then reperfusion 4h.All the mice in sham operation group were fed with standard rat chow,and the other mice were treated with FTY720 via intragastric administration for a week,specific operations are as follows:pinched the mouse tail by right hand,pinched the neck skin by left thumb and forefinger to make mouse's head,neck and trunk in a straight line,then used a 1ml syringe with gavage needle gently push into the mouth after pressed the tongue against the palate,then injected FTY720 after the needle was comfirmed into the esophagus,the gavage volume of each mouse was no more than 1ml,at the end,made the mouse's head keep up for 3 minutes to prevent liquid reflux.The lung tissue homogenate was stored for measurement the levels of inflammatory factor by euzyme-linked immunosorbent assay,detection the expressionof S1P?Sph K-1 by Realtime quantitative PCR technique.We detected apoptotic cells in lung tissue by flow cytometry and evaluated pathology of lung tissue.Results Compared to normal saline pretreatment group,the levels of IL-6?IL-10?TNF-??IL-1? in lung tissue homogenate decreased significantly in FTY720 pretreatment group.The normal structure of lung tissue was observed in sham operation group while I/R group showed severe pathological changes of lung tissue.This injury was significantly reduced with the increase of the dose of FTY720 pretreatment.There were a large number of apoptotic cells in lung tissue induced by limb ischemia 3h and then reperfusion 4hin rats,while it was improved in different degree in FTY720 pretreatment group.Conclusion FTY720 could inhibit the cell apoptosis caused by a large number of inflammatory factors by upregulation the expression of S1 P and Sphk-1 m RNA,thereby improving lung injury.Part ? The Mechanism of Sph K-1/S1 P Signaling Pathway Mediated by Endoplasmic Reticulum Stress in Lung Injury Induced by Limb Ischemia/ReperfusionObjective To investigate the role of Sph K-1/S1 P signaling pathway mediated by endoplasmic reticulum stress in the pathogenesis of lung injury induced by limb ischemia/reperfusion.Methods Thirty adult healthy SD rats and thirty CHOP gene knockout rats were randomly divided into six groups.The control group was treated with saline via intraperitoneal injection,the others were pretreated with intraperitoneal injection of Sph K-1 activator PMA or inhibitor DMS.All the experimental animals were divided into six groups: group SD: SD rats +I/R;group SD+PMA: SD rats +I/R+PMA5ml(100nmol/L);group SD+DMS: SD rats+I/R+DMS5ml(50 ? mol/L);group CHOP: CHOP gene knockout rat +I/R;group CHOP+PMA: CHOP gene knockout rat +I/R+PMA5ml(100nmol/L);group CHOP+DMS: CHOP gene knockout rat +I/R+DMS5ml(50?mol/L).24 h later,we established the model through clamping the femoral artery in the proximal inguinal ligament to induce limb ischemia 3h and reperfusion 4h in rats.We measured the levels of IL-1?,IL-6,TNF-?,ICAM-1,MIF and IL-10 in blood plasma by euzyme-linkedimmunosorbent assay,detected protein expression by Western Blot,detected apoptotic cells by TUNEL staining and evaluated pathology of lung tissue.Results Compared to SD group,the levels of IL-1?,IL-6,TNF-?,ICAM-1,MIF and IL-10 increased significantly,the expression of apoptosis protein BAX,P53 and Caspase-3increased and the expression of antiapoptotic protein Bcl-2 decreased in SD+PMA group.Compared to SD group,the levels of inflammatory factor decreased and apoptosis of lung tissue was not obvious in CHOP group.Conclusion PMA pretreatment group could aggravate lung injury induced by I/R while DMS pretreatment group could attenuate lung injury.Between CHOP gene knockout rats after I/R,lung injury was not obvious in either PMA or DMS pretreatment group.