| Background and Objective:TAO,also known as Buerger’s disease,is a Non-atheromatous diseases,which mainly invades the small and middle arteries of the extremities in human body.However,the etiology and pathogenesis of TAO are still not fully understood.PGI2 and TXA2 are important bioactive substances that are synthesized and secreted by vascular endothelial cells and closely related to coagulation function in vivo.PGI2 is metabolized from AA by cPLA2/COX/PGIS pathway,and TXA2 is catalyzesd by cPLA2/COX/TXAS pathway.TXA2/PGI2 is usually in a dynamic equilibrium under physiological conditions,when the ratio is imbalance,thrombosis and tissue ischemia may be emerged.Our previous studies and other literatures showed that the levels of TXB2 and COX-2 were significantly increased in the TAO rat model,while the level of 6-K-PGF1α was decreased.However,the role of COX-1,PGI2 and cPLA2 in the pathogenesis of TAO has not yet been reported.LTs,a potent proinflammatory factor,are a metabolite of AA.5-LO is a key enzyme in the biosynthesis of LTs.LTB4 enhances adhesion of vascular endothelium to other cells and increases vascular permeability;CysLTs increase capillary permeability and contractility.It has been reported that 5-LO and LTB4 are highly expressed in the carotid artery,aorta,abdominal aorta and coronary artery plaque.However,the role of LTs in the pathogenesis of TAO has not yet been reported.NO is catalyzed by three NOS enzymes and is a paracrine factor that affects vascular tone and platelet function,preventing leukocyte adhesion and reducing intimal hyperplasia.Studies have showed that the level of iNOS and eNOS were increased in cardiac I/R,pulmonary hypertension,peripheral arterial disease,cerebrovascular disease,and coronary artery disease;there are a few literatures showed that NOS activity was increased in the pathological mechanism of TAO.Modern pharmacological studies have showed that SFI can significantly increase vascular perfusion,scavenge oxygen free radicals,improve microcirculation,protect endothelial cells and reduce inflammatory response,as well as have anti-apoptoticand anti-inflammatory effects.It is typically used for the treatment of cardiovascular diseases.However,few studies have applied SFI to TAO.Therefore,this project will further explore the protective effect and mechanism of SFI for TAO from the pathways of TXA2/PGI2 on the basis of previous experiments.Methods:1.TAO rat model: According to the previous method reported by our research group,the femoral artery of male SD rats were injected with lauric acid to make TAO animal model,while sham group rats were injected with saline,and SFI group rats were given SFI on the third day after TAO model achieved and administered daily for15 days.The drug dosage was calculated according to the body surface area.The rats in sham and TAO groups were administered with the same dosage of saline as vehicle daily for 15 days.2.Main symptoms: body weight of rats in each experimental group was recorded daily.Skin temperature,colors,arterial pulse,limb swelling,the scope of gangrene and mummification of the rat hind limb in all groups were checked daily after the operation.3.Main pathological signs: The obtained femoral artery was sectioned and observed the pathological changes of the femoral artery through HE staining.4.Blood coagulation indexes and hemorheological indexes were measured in the Second Affiliated Hospital of Nanchang University.5.Content of TXB2 and 6-K-PGF1α in plasma were determined by radioimmunoassay measuring.6.Content of LTB4 and LTC4 in plasma were determined by ELISA.7.Content of NO in plasma was determined by Griess method.8.The mRNA expressions of TXAS,PGIS,COX-1,COX-2,cPLA2,5-LO,LTA4 H,LTC4S,iNOS and eNOS in femoral artery were examinated by quantitative real-time RT-PCR.9.The protein levels of TXAS,PGIS,COX-1,COX-2,cPLA2,5-LO,LTA4 H,iNOS and eNOS in femoral artery were measured by western blot.10.The distributions of TXAS,PGIS,COX-1,COX-2,cPLA2 and LTC4 S in femoral artery were evaluated by immunohistochemistry.Result:1.About 10-15 minutes after the femoral artery of male SD rats were injected5% sodium laurate solution,the hind paw and the knee joint appeared to be pale,then the hind paw became cyanosis;the local skin temperature decreased,the foot swelled,and the pulse of the dorsal foot artery weakened or disappeared.On the second day the hind limbs of the TAO rats showed varying degrees of ischemic symptoms,the fingers of the affected paw became dark and the dark area extended to the whole upper paw,then the paw was gangrened and then mummified with time elapsed.There were pain,limp and drag-line phenomenon in the affected limb of some animals.15 days later typical symptoms of TAO appeared.2.From the second day,food intake and activity of rats from TAO model group and SFI group began to decline.Compared with the sham group,the weight of rats in TAO group was significantly lower(p<0.05).Body weight of rats in all SFI groups increased from the sixth day compared to that in TAO group(p<0.05),it is especially marked in medium and high-dose groups.3.The sham group had no gangrene symptoms in the limbs,model group and all SFI groups all have different degrees of gangrene symptoms,while the most serious gangrene was appeared in the model group in which III,IV,and V were 3,3,2respectively.The gangrene degree of rats in TAO model group was significantly increased when compared with the sham group(p<0.001).Compared to model group,gangrene degree of rats in medium and high dose SFI groups markedly decreased(p<0.01).4.HE staining showed that in sham group,thefemoral arterial had smooth intima and intact internal elastic lamina,no exfoliation and shedding,endothelial cells and smooth muscle cells were arranged in order,and no inflammatory cell infiltration existed in the blood vessel.In TAO group,the thin internal elastic layer of the femoral artery was basically intact;Inflammatory cell infiltration was observed along the elastic layer,fresh thrombus and tissue thrombus were seen in the lumen;The degree of occlusion of the vascular cavity and inflammatory cell infiltration was reduced in all SFI group with minimum occlusion inthe femoral artery of middle dose group.5.FIB was elevated(P<0.05)in TAO model group compared with the shamgroup.The level of FIB was significantly reduced in all SFI groups compare to TAO model group(P<0.05).Reversely,compared with sham group,PT,APTT,and TT were all significantly declined in TAO group(P<0.01),while SFI was significantly reversed these changes(p<0.05).6.INR and prothrombin activity were markedly lower in TAO model group than those in the sham group(p<0.01).INR and prothrombin activity were increased in all SFI dose group when compared with TAO model group(p<0.05).However,D-dimer,ERI,EAI,hematocrit,whole blood reduced viscosity(low cut and high cut),whole blood viscosity(1/S,30/S and 200/S)and plasma viscosity were all significantly increased in TAO rats compared to sham group(p<0.01),while D-dimer,ERI,EAI,hematocrit,whole blood reduced viscosity(low cut and high cut),whole blood viscosity(1/S,30/S,and 200/S)and plasma viscosity were all slightly decreased in all dose SFI group when compared with TAO model group(p<0.05).7.Compared with sham group,TXB2 was significantly increased in TAO model group(P<0.01),while SFI significantly reversed TXB2 level increase in TAO rats in dose-dependent manner(p<0.05).In TAO model group plasma 6-K-PGF1α were significantly lower than that in the sham group(p<0.001),while SFI especially in medium-dose groups significantly increased plasma 6-K-PGF1α level when compared with TAO model group(p <0.05)8.The expression of LTB4 and LTC4 in TAO model group plasma were significantly increased when compared with the sham group(P<0.05),and SFI can significantly decrease the expression of LTB4 in TAO plasma when compared with the sham group(P<0.05),while LTC4 was insignificant(P>0.05).9.Compared with sham group,NO was significantly increased in TAO model group(P<0.01),while SFI significantly reversed NO level increase in TAO rats(p<0.05).10.The protein and mRNA expressions of TXAS,COX-1,COX-2,cPLA2,5-LO,LTA4 H,LTC4S and iNOS of femoral arteries in the TAO model and all SFI groups were increased markedly when compared with the sham group(P<0.05),and the protein and mRNA expressions of TXAS,COX-1,COX-2,cPLA2,5-LO,LTA4 H,LTC4S and iNOS were decreased in all SFI group compared to TAO model group(p<0.05).The protein and mRNA expressions of PGIS and eNOS in femoral arteries in TAO rats were decreased when compared to sham group,while all dose SFI treatment increased significantly the protein and mRNA expressions of PGIS and eNOS when compared to TAO model group.Conclusion:1.TAO rat model was successfully established.After sodium laurate solution injected,the limb of SD rat showed typical TAO symptoms after 15 days2.SFI can increase the weight of TAO rats,reduce the degree of gangrene and occlusion of the uterine cavity,indicating that SFI has a therapeutic effect on TAO rats.3.SFI can positively affect four coagulation indexes and hematology to produce a protective effect on the coagulation function and blood flow of TAO rats.4.SFI can produce a therapeutic effect on TAO rats by keeping the balance of TXA2/PGI2,its underlying mechanism may be involved in its effects on the signaling pathways of cPLA2 /COX/TXAS or PGIS.5.SFI has a therapeutic effect on TAO rats by decreasing LTB4 level,which may involve in its reducing the expressions of 5-LO and LTA4 H.6.SFI has a therapeutic effect on TAO rats by maintaining the appropriate concentration of plasma NO,which may be involved its reducing the expression of iNOS and increasing the expression of eNOS. |
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