| Background:Acute limb ischemia(ALI)is one of the most common ischemic diseases in clinical practice,it has a sharp attack and a poor clinical prognosis,which is often caused by sudden interruption of blood flow in the distal limb artery.The most common causes are Arterial thromboembolism(AE),ectopic emboli(heart or aorta),Arterial compression,Arterial discontinuity,etc.One of the most common phenomenon is arterial thrombosis.ALI generally has a course of no more than 2 weeks,it is generally believed that acute ischemia lasts from 6 to 8 hours and irreversible histopathological damage may occur in the limb.With the continuous progress of drug thrombolysis,thrombectomy,fragmentation and catheter technology,the clinical efficacy has been significantly improved,but the amputation rate is still up to 10-30%within a month,and the mortality rate reaches up to 15%.After clinical intervention,the pathogenic factors were relieved,the blood flow was restored,and the limb injury was further aggravated on the basis of Ischemia,which led to Ischemia reperfusion(I/R)injury.The prognosis of ALI was related to the duration of ischemic symptoms,due to the severity of ischemia,the number of collateral circulation,whether the thrombus affected the important collateral circulation,and the time of medical treatment.Therefore,further understanding of the pathogenesis of ALI,correct understanding and treatment of ALI-related diseases and prognosis is essential.When limb muscle,fat,blood vessels and other soft tissue are damaged,striated muscle will dissolve,the fat will necrosis,and a large amount of toxic substances are released in cells,and the ion will disorder,of which will often lead to one or more distant organ dysfunction,failure,toxic shock and even death.In clinical work,many patients with ALI are suffering from hypertension,coronary heart disease and other basic diseases at the same time.After the onset of ALL,the body can not tolerate reoperation of blood circulation within a short time.How to prolong the surgical treatment time window and protect the structure and function of the vessel walls as much as possible has become an outstanding issue to be solved.After limb ischemia and ischemia reperfusion,tissue and organs are damaged,which leads to the burst of inflammatory response,and a large number of inflammatory mediators are released,including Tumor necrosis factor induction(TNF-a induction),Intercellular adhesion molecule(ICAM),Vascular celladhesion molecule(VCAM),etc.Inflammatory mediators chemokine white blood cells,resulting in vascular endothelial damage,middle elastic fiber board and smooth muscle destruction,pipe wall edema,eventually leading to vascular wall contraction dysfunction,vascular remodeling,tube stenosis,microthrombosis,these will aggravate tissue ischemia and hypoxia.After the cause of ischemia is removed,the arterial blood flow will restore,and the high-pressure blood flow will open the damaged intercellular connections,increase vascular wall permeability,inflammatory mediators in the tissues and blood infiltrated into the vascular wall,the vascular wall edema and stenosis are aggravated,further aggravating the limb ischemia.Many factors are involved in the process of acute limb ischemia and post-ischemia reperfusion injury,but the release of inflammatory factors and inflammatory mediators are throughout the entire I/R injury process.Therefore,eliminating the inflammatory factors and inhibiting the release of inflammatory mediators can play a protective role in vascular walls function.In the relationship between High mobility group box protein 1(HMGB1)and vascular endothelial cells,tumor necrosis factor activation(tnf-activation),interleukin(IL)and lipopolysaccharide can stimulate endothelial cells to secrete HMGB1,while the co-incubation of HMGB1 with endothelial cells will induce the expression of intercellular adhesion molecule(ICAM),Vascular celladhesion molecule(VCAM)and e-selectin.Exogenous high mobility group protein B1 also promotes release of TNF-a,McP-1,IL-8 and G-CSF.Therefore,HMGB1 plays an important role in regulating and amplifying inflammatory reactions.Our preliminary study also found that heparin reduced the release of inflammatory mediators,maintained the stability of the intramural environment,and protected the structure and function of the vessel walls during the acute I/R/process,but the specific mechanism is still unclear.Interleukin 11(IL-11)is a member of the interleukin 6(IL-6)family,which is mainly secreted by bone marrow stromal cells and stromal cells(being mainly bone marrow fibroblasts).Human body is widely divided,with more substantial viscera and less cavity viscera,whose effect is almost opposite to IL-6(the main effect of IL-6,which is the inflammatory IL-11,is anti-inflammatory).In hematopoietic system diseases,IL-11 mainly cooperates with IL-3 to stimulate the proliferation,differentiation,maturity of bone marrow megakaryocytes and multilineage progenitor cells,colony formation,increase peripheral blood platelet count,simultaneously promote the proliferation of erythrocytes,inhibit the differentiation of adipocytes,and participate in the occurrence and development of various clinical diseases.Du et al.reported that IL-11 pretreatment in the animal model of small intestine preadaptation and intestinal I/R injury,it can protect the intestinal mucosa from damage,increase the intestinal absorption function,reduce the occurrence of intestinal necrosis and lower the mortality of mice.In the animal model experiments of the myocardial infarction,Fujio Y,Maeda m.et al.reported that the pre-treatment of IL-11 reduced the area of myocardial infarction in rats and promoted the formation of collateral circulation after myocardial infarction in 2011.After femoral artery ligation,pretreatment with IL-11 increased the number of new blood vessels and improved the repair ability of endothelial cells after injury.In the theoretical study of ischemia reperfusion diseases,hemodynamic changes,free radical production,intracellular calcium overload,inflammatory cytokine formation,autophagy,and cell apoptosis were all involved in the pathophysiological process of I/R,but the specific mechanism is not very clear.Many evidences show that IL-11 has important physiological functions in reducing the release of inflammatory factors,regulating leukocyte chemotaxis,platelet adhesion,anticoagulation/pro-coagulation,and reducing apoptosis.However,its effect on vascular walls damage in the course of ALI is not unclear,and the mechanism remains to be furtherly studied.Purpose:We constructed an ischemia/reperfusion model of the lower extremity in rats by clipping the femoral artery and evaluated the effect of the model.By means of histomorphology,molecular biology,biochemistry,etc.,from the perspective of inflammatory factors,cell apoptosis and oxidative stress,the study was carried out on the injury of lower extremity ischemia/reperfusion vessel wall,and the influence of IL-11 pretreatment on the vascular wall damage in the process of lower extremity ischemia/reperfusion was clarified,and its mechanism was expounded as acute.It provided an important theoretical basis for the acute ischemic/reperfusion diseases of lowering extremities and exploring possible therapeutic targets.Method:1.Animal groups:six weeks old,healthy,adult,male,and Wistar rats were selected,with a weight of about 200±20 g;Randomly divided them into 3 groups:(1)control group(S group);(2)ischemia reperfusion group(I/R group);(3):IL-11 pretreatment group(IL-11 + I/R group);Each group has eight rats.2.Establish the rat model of lower limb ischemia/reperfusion by using the microarterial clamping method to clamp the right femoral artery of the rat;3.HE staining and pathological analysis:morphological changes of the femoral artery after right leg ischemia/reperfusion in rats were observed,including the damage condition of the intima,the middle elastic fiber board and the smooth muscle layer,and the thickness of each layer of pipe wall and the change of lumen area were all measured.4.Immunohistochemical detection:the expression of inflammatory factors HMGB1,TNF-a,IL-6 and JAK2/STAT3 in the right femoral artery wall of rats were observed.5.TUNEL method was used to detect apoptosis in femoral artery walls of rats in each group.6.RT-PCR method was used to detect the expression level of JAK2/STAT3 mRNA in IL-11 related pathway.7.Western blot detection:detect the expression level of HMGB1,TNF-a,IL-6 and JAK2/STAT3 protein.8.Biochemical methods:the changes of SOD and MDA,the oxidative stress products in the femoral artery tissue of the rats.Results:1.Endothelial injury,cell edema.neutrophil infiltration,collagen fibre degradation and elastic plate destruction can be seen in the right femoral artery wall of rats in the I/R group and the IL-11+I/R group under the light microscope.Compared with the I/R group,the IL-11+I/R group showed the right femoral artery wall was relieved.There was no obvious injury of arterial wall in group S.2.Compared with the IL-11+I/R group,the thickness of the intima,media,and exterior module of the tube wall in IR group increased in varying degrees,and the lumen area decreased(P<0.05),and occasionally there were incidental wall microthrombus formation.3.Significant expressions of HMGB1,TNF-a,IL-6,JAK2/STAT3 were detected in the femoral artery walls of both the IL-11+I/R group and the I/R group,while few were detected in the S group.Compared with the I/R group,the release of HMGB1,TNF-a,and IL-6 was significantly reduced in the IL-11+I/R group(P<0.05),while the expression of JAK2/STAT3 was increased(P<0.05).4.Apoptosis was detected in the femoral artery wall of rats in each group,there are more apoptotic cells in the IL-11+I/R group and I/R group and less apoptotic cells in the S group.The apoptosis index of femoral artery cells in the IL-11+I/R group was significantly lower than that in the I/R group(p<0.01).5.Compared with the I/R group,the content of SOD in femoral artery wall increased significantly,while the content of MDA decreased significantly(p<0.01).Conclusions:1.The model of ischemia/reperfusion in the lower thigh of rats was successfully established by the method of microarterial clamping for femoral artery occlusion.The method was simple and easy,and the success rate of the model was high.2.IL-11 may protect the femoral artery wall from ischemia/reperfusion injury by inhibiting the release of inflammatory factors,reducing the apoptosis of vascular wall cells and improving the oxidative stress state of femoral artery.3.The protective effect of IL-11 on vascular wall may be achieved through the molecular signaling pathway of JAK2/STAT3. |
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