| BACKGROUND AND OBJECTIVEThromboangiitis obliterans (TAO) or Buerger’s disease is a segmental occlusive inflammatory disorder that involves primarily small and medium arteries, veins, and nerves in upper and lower extremities, which cause limb ischemic. It accounts for0.5-5%of patients hospitalized for arterial occlusive disease in Europe and up to16%of such patients in Japan. TAO typically occurs in young male smokers, the course of the disease is progressive, and patients with Buerger’s disease have lower quality of life because of intermittent claudication, rest pain, ulcers, several episodes of lower extremity superficial thrombophlebitis, and even face the risk of amputation. Recent studies showed the expression of intercellular adhesion molecule1(ICAM-1) and vascular cell adhesion molecule1(VCAM-1), E-selectin and tumor necrosis factor (TNF)-alpha increased in endothelium and some inflammatory cells in the thickened intima in all TAO patients. A great deal of research has been conducted to investigate the etiology, pathophysiology, treatment of TAO, However, The specific etiology and pathological mechanisms that are responsible for the development of vascular lesions in TAO remain not elucidated.High mobility group box1(HMGB1) is an important mediator of inflammation and inflammatory cytokines. During cell activation and cell death, HMGB1can translocate to the cytoplasm, as well as the extracellular space to take pro-inflammatory effects via interaction with two types of cell-surface receptors: toll-like receptor (TLR) and receptor for advanced glycation end-products (RAGE). HMGB1interaction with these receptors transduces intracellular signals and mediates cellular responses including chemotactic cell movement and release of pro-inflammatory cytokines and adhesion molecules. HMGB1is a kind of signal transduction and amplification factor in a variety of inflammation, which can enhance the intensity of inflammatory response and prolong the inflammatory pathological process. Elevated levels of HMGB1as a pro-inflammatory cytokine in serum and tissues occur during sterile tissue injury such as arthritis, ischemia-reperfusion injury and during infection such as lethal endotoxemia, sepsis. Previous research showed that HMGB1is involved in many cardiovascular diseases such as atherosclerosis, myocardial infarction and induces endothelial cell damage. The incubation of HMGB1and endothelial cells can increase the expression of ICAM-1, VCAM-1and RAGE in time-dependent manner, prompt the secretion of TNF alpha, monocyte chemotactic protein(MCP-1), interleukin-6(IL-6), and can induce endothelial release plasminogen activator inhibitor-1(PAI1)causing platelet deformation. But the role of HMGB1in buerger’s disease has not been reported.HMGB1has2DNA-binding motifs:A and B box. Structure-function analyses demonstrated that the active cytokine domain of HMGB1is localized to the DNA-binding B box, whereas the A box competes with HMGB1for binding sites on the surface of activated macrophages and attenuates the biologic function of the full length HMGB1. Recently, it was shown that the recombinant A box (rA box) as a specific antagonist of HMGB1is protective in established preclinical inflammatory disease models.At present, there is no report about HMGB1in the development of thromboangiitis obliterans. Based on the backgrounds mentioned above, in the present study, we explored the role of HMGB1in rat model of TAO induced by the intravascular injection of sodium laurate, we also investigate whether the recombinant A box play its unique role in prevention and treatment of TAO in order to provide a theoretical basis for TAO pathogenesis and find effective therapeutic measures.STUDY CONTENTS1. To establish the model of thromboangiitis obliterans in rat.2. To investigate whether HMGB1and RAGE was involved in the development of thromboangiitis obliterans.3. Blockade of HMGB1inhibited the progression of thromboangiitis obliterans.4. Analysis of the corresponding mechanisms of the process mentioned above.METHODS: 1. Animal treatment:Rats were received sodium laurate solution injection in the left femoral artery while the sham operated group was treated with normal saline as vehicle, then recombinant A box was intraperitoneal injected to observe the effect in different doses.2. Main pathological signs:The gross appearance in rat hind legs was checked daily after operation. The degree of the disease on the15th day was graded according to the system developed by Murakami et al.3. Tissue collection:The animals were euthanized after treatment. The femoral artery and blood samples were collected for further analysis.4. Histology:To observe the patency of the vessel and calculate the area of the thrombus through H&E staining.5. Assessment of hematology:Blood cell counts and Blood coagulation were analyzed in clinical laboratory.6. Plasma protein levels:Plasma HMGB1, TXB2, and6-keto-prostaglandin Fl alpha (6-K-PGF1α) were measured with ELISA Kits according to the manufacture’s instructions.7. Analysis of expression of proteins in the femoral artery:1) The protein levels and distributions of HMGB1and RAGE were examinated by Immunocytochemistry and Immunofluorescence.2) The protein levels of HMGB1and RAGE were examinated by Western blot.8. Analysis of expression of Inflammatory cytokines and adhesion factor in the femoral artery:1) The protein levels and distributions of Interleukin-6(IL-6), intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule1(VCAM-1) were examinated by immunocytochemistry.2) The protein levels of above factors were examinated by Western blot.9. Analysis of expression of mRNA in the femoral artery:The mRNA levels of HMGB1, RAGE, IL-6, ICAM-1and VCAM-1were determined enzymatically using quantitative real-time RT-PCR. RESULTS:1. Rat TAO model induced by sodium laurate injection1.1On the15th day, TAO model showed typical signs and symptoms of TAO, while in sham operated and normal rats, no ischemic signs occurred.1.2Stained sections of TAO model rats showed many inflammatory cell infiltration and thrombi that resulted in narrowing or complete occlusion of the vessel lumen.2. HMGB1and RAGE was involved in the development of thromboangiitis obliterans.2.1Compared with sham-treated animals, plasma HMGB1was increased significantly in the TAO model (p<0.01). There was no difference between normal and sham operated group.2.2We performed immunocytochemistry and double immunofluorescence staining to analyze the expression of HMGB1and RAGE in the femoral arterys in normal, sham-operated and TAO group. Our results showed that HMGB1was expressed in intima and media in TAO rats and appeared frequently within the cytoplasm and diffusely distributed within lesions, which probably reflected secreted HMGB1. As compared with sham operated rats, TAO rats showed increased expression of the HMGB1receptor RAGE mainly in intima.2.3We also performed Western blot and PCR analysis on femoral arteries tissue lysates from all groups. Compared with sham operated group, the protein and mRNA expression of HMGB1and its receptor RAGE in the TAO model group was significantly increased (p<0.01).3. Blockade of HMGB1inhibited the progression of thromboangiitis obliterans3.1The lesion values in TAO model group were significantly higher than those in sham operated group, while the lesion grades were significantly attenuated in low or high dose rA box injection group with dose-dependent manner compared with the TAO model group.3.2H&E staining revealed that the low dose of rA box mildly and high dose rA box significantly reduced the number and levels of thrombus (p<0.05). 3.3ELISA showed plasma HMGB1level decreased with high-dose rA box injection as compared with TAO rats (p<0.05).3.4Immunohistochemical and immunofluorescence staining showed a marked reduction of HMGB1and RAGE expression in the vascular wall with high-dose rA box injection as compared with TAO rats. Low-dose injection decreased HMGB1and RAGE expression slightly in the vascular wall. These results were consistent with the protein and mRNA expression of HMGB1and RAGE analyzed by Western bolt and qRT-PCR, Thus, rA box could attenuate the gross appearance of TAO by reducing the expression of HMGB1and RAGE dose-dependengtly.4. The mechanisms of HMGB1in thromboangiitis obliterans and protective effect of rA box4.1We found protein staining of IL-6, ICAM-1and VCAM-1in femoral arteries higher in TAO rats than sham operated rats (p<0.05).4.2The result of Western blot and PCR analysis on femoral arteries tissue lysates from all groups showed that compared with sham operated group, the protein and mRNA expression of IL-6, ICAM-1and VCAM-1in the TAO model group was significantly increased (p<0.05).4.3Treatment with high-dose but not low-dose rA box reduced the protein levels of IL-6, ICAM-1and VCAM-1significantly (p<0.05). mRNA levels were similar to those for protein levels. Therefore, rA box inhibited HMGB1to decrease the expression of inflammatory mediators in TAO rats.4.4We investigated blood cell counts and blood coagulation in each group. Blood platelet count was markedly higher in TAO rats than sham operated rats(p<0.05); Prothrombin, thrombin and activated partial thromboplastin times were all significantly shortened and fibrinogen level was increased in TAO rats as compared with sham operated rats(p<0.05). However, counts of red blood cells, leucocyte and neutrophils did not differ among the groups.4.5Blood platelet count was slightly decreased with low-dose rA box (p>0.05) and greatly decreased (p<0.05) with high-dose treatment as compared with TAO rats. The values of PT, TT, APTT, FIB were reversed to almost normal with high-dose rA box treatment (p<0.05), with the effect of low-dose rA box relatively weak.4.6TXB2and6-K-PGF1α are the stable metabolite of TXA2and PGI2, which, respectively induces and inhibits platelet aggregation. We found the plasma6-K-PGF1α level was lower and that of TXB2and TXB2/6-K-PGF1α ratio were markedly higher in TAO rats than sham operated or normal rats, However, rA box treatment increased6-K-PGF1α level and decreased TXB2level and TXB2/6-K-PGF1α ratio with low-and high-dose rA box treatment as compared with TAO rats(p<0.05)CONCLUSION:1. HMGB1and its receptor RAGE involve in sodium laurate induced TAO rats.2. rA box as the antagonist of HMGB1improve the pathological condition.3. rA box block the expression of HMGB1and RAGE to inhibit the release and injury of inflammatory mediators as well as improve the hypercoagulable state of blood. |
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