| Background:Thromboangiitis obilterans(TAO),also known as Buerger's disease,occurs mostly in young people with a history of smoking.The pathological characteristic was chronic segmental inflammatory occlusion of medium and small sized arteries and veins,often accompanied by thrombosis,mostly seen in the lower extremities.It is mainly manifested as ischemic lesions or even gangrene at the distal end of limbs,such as the toe end.However,with the progression of lesions,the necrotic range may extend to the proximal end,involving the foot or even the ankle.Some patients may develop characteristic wandering superficial phlebitis.In many cases,the symptoms of gangrene become more severe,and the range of gangrene gradually becomes larger,leading to amputation of limbs and imposing a heavy burden on families and society.Despite extensive research by many scholars,the mechanism of TAO has not been fully elucidated.Current studies have found that many inflammatory factors in the blood of TAO patients are significantly increased and play a pro-inflammatory role.For example,C-reactive protein(CRP)can promote endothelial cell damage and increase peripheral vascular resistance.Tumor necrosis factor-a(TNF-a)can promote endothelial dysfunction,lymphocyte and neutrophil activation.ThromboxaneA2(TXA2)has the effect of causing vasospasm,promoting platelet aggregation and thrombosis.Il-6 produced by activated T cells enhances the inflammatory response by activating neutrophils.Vascular endothelial cells activate and express Intercellular adhesion molecule 1(ICAM-1)and Vascular cell adhesion molecule 1(VCAM-1).They can not only mediate the adhesion between inflammatory cells and endothelial cells,but also mediate the adhesion between immune complexes and endothelial cells,thus promoting the progression of inflammation and immune disorders,leading to vascular injury and thrombosis.Endothelial cells also secrete E-selection,which recruits inflammatory cells.Endothelial cells can also synthesize and release vWF which can mediate platelet adhesion in endothelial tissue.Under the action of inflammatory factors,endothelial cells and endodermal fibrous layer are thickened and there are a large number of inflammatory cells infiltrating,resulting in thickening of arterial wall characterized by intima hyperplasia.These studies indicate that inflammatory factors play an important role in the pathogenesis of TAO,but the specific mechanism of action remains to be further studied.Current studies show that the receptor for advanced glycation end products(RAGE)is a multiligand receptor on the cell membrane,which is widely distributed in adults.Major ligands,such as advanced glycation end products AGEs,HMGB1,S100/cadherin and amyloid? peptide(A? peptide),could activate various intracellular signaling pathways,resulting in a series of diseases.Among them,High mobility group box 1(HMGB 1)is a kind of non-histone DNA binding protein widely distributed in the nucleus of eukaryotes.Recent studies have confirmed that extracellular HMGB 1 can be used as an important inflammatory mediators to initiate and maintain the inflammatory organ response.Domestic studies have confirmed for the first time that HMGB 1 expression level is significantly increased in the TAO model rat and plays an important pro-inflammatory role through its receptor RAGE.However,studies on the intracellular signal transduction pathway by which RAGE is activated by HMGB 1 have not been reported at home and abroad.Data showed that nuclear factor-kappa B(NF-kappa B),as a transcription factor,can bind to promoters or enhancers of various genes in the nucleus and promote their transcription and expression.Studies have confirmed that RAGE,combined with a variety of ligands,can activate NF-?B and activate and regulate the transcription of a variety of genes involved in inflammatory responses,leading to inflammatory and immune responses.Therefore,we hypothesized that HMGB 1,when combined with receptor RAGE,promotes inflammation and vascular damage by activating the intracellular NF-?B signal pathway in the rat model of TAO,Currently,RAGE has attracted increasing attention as a substrate for Proteolytic enzyme.A disintegrin and metalloprotease 10(ADAM10),as a multi-region type I transmembrane Zn-dependent metalloproprotease,can not only hydrolyze related proteins,but also participate in cell signal transmission.Current evidence shows that full-length RAGE can be transformed into soluble RAGE by metalloproteinase ADAM10.It has been proved by experiments that recombinant soluble RAGE can inhibit the activation of RAGE on cell surface by trapping RAGE ligands.Therefore,RAGE shedding may be of therapeutic value for inflammatory diseases.Therefore,we hypothesized whether ADAM 10 had a similar mechanism in vascular diseases.The research on ADAM10 and vascular lesions is at the preliminary stage.Studies have confirmed that statins can not only reduce blood lipid,but also increase sRAGE to reduce the risk of coronary heart disease.The mechanism of action is achieved by activating ADAM10.Therefore,we hypothesized that ADAM10 could inhibit RAGE binding to HMGB1 in a rat model of TAO,and play a therapeutic role by blocking RAGE/NF-?B signal pathway and increasing the competitive antagonistic sRAGE.Objective:Given for the researches of' the pathogenesis and pathology of TAO and the role of RAGE/NF-?B in mediating inflammation,we studied the the relationship between RAGE/NF-?B and TAO,explored the the influence and realated molecular mechanism of ADAM 10 for RAGE,clarified the pathogenesis of TAO and provided the theory basis for looking for effective treatment through the establishment of rat model of TAO in this thesis.Research contents:Part I:The research on the correlation between HMGB1,RAGE and NF-?B and thromboangiitis obliteratus.Part II:The research on the reactivity of TAO rat model to ADAM10.Part III:The research on the mechanism of action of ADAM10 for the model of TAO rat.Methods:Part?:1.Sodium laurate was injected into the femoral artery of the hind limbs of rats to construct a rat model of thromboangiitis obliterans.The rats were divided into normal group,sham operation group and TAO model group.2.After 14 days,the three groups of rats were scored by gross pathology of the hind limbs.The hemodynamics of femoral artery in rats were detected by color doppler ultrasound.Blood cell count,hemorheology and blood coagulation were measured by blood cell analyzer,automatic rheometer and blood coagulation analyzer.Morphological changes of femoral artery with HE staining were observed.Ultrastructural histological changes of vascular injury(smooth muscle cells and vascular endothelial cells)were observed by electron microscopy.3.Protein levels of HMGB1,TXB2 and sRAGE in plasma were detected by ELISA.Double staining fluorescence was used to locate the protein expression in frozen tissue sections.Western Blot and RT-PCR were used to detect the contents of HMGB1,RAGE,NF-B protein and mRNA.Part II:1.Sodium laurate was injected into the femoral artery of rat hind limbs to construct a rat model of thromboangiitis obliteratus,and the rat model was treated with intraperitoneal injection for 14 days with PBS,3 ug/kg weight and 6 ug/kg weight for ADAM10.The rats were divided into sham operation group,TAO model group,ADAM10 low dose inj ection group and ADAM10 high dose injection group.2.After 14 days of treatment,gross pathological scores of the hind limbs of the four groups of rats were scored;The hemodynamics of femoral artery in rats were detected by color doppler ultrasound.Blood cell count,hemorheology and blood coagulation were measured by blood cell analyzer,automatic rheometer and blood coagulation analyzer.Morphological changes of femoral artery with HE staining were observed.Ultrastructural histological changes of vascular injury(smooth muscle cells and vascular endothelial cells)were observed by electron microscopy.Part ?:1.A rat model of thromboangiitis obliteratus was constructed,and the rat model was treated with intraperitoneal injection for 14 days with PBS,3 ug/kg ADAM 10 and 6 ug/kg ADAM10.The rats were divided into sham operation group(sham),TAO model group(TAO),ADAM10 low dose injection group(ADAM10-LD)and ADAM10 high dose inj ection group(ADAM10-HD).2.After 14 days of treatment,the protein levels of HMGB1,TXB2 and sRAGE in the plasma of the four groups of rats were detected by ELISA.Western Blot and RT-PCR were used to detect the contents of HMGB1,RAGE,NF-?B protein and mRNA in rat femoral artery.Results:Part I:1.The rat of TAO model was successfully constructed:Compared with the normal group,TAO model showed:(1)Apparent identification:rats injected with sodium laurate 14 days later showed varying degrees of symptoms of thromboangiitis obliterans in their hind limbs,and scoring of their hind limbs showed that the scores of rats injected with sodium laurate were all above grade III.(2)Color doppler ultrasonography:color doppler ultrasonography of the femoral artery showed that after the injection of sodium laurate,the velocity of femoral artery decreased and the resistance index increased.(3)Blood test:The platelets,blood viscosity and blood coagulation function in the blood increased;(4)Tissue detection:HE staining results of the artery showed that,compared with the control group,there was thrombosis in the femoral artery lumen of rats in the TAO group,the lumen became narrow and occluded,and inflammatory cell infiltration in the vascular wall and thrombus could be observed.Electron microscopy showed that compared with the control group,a large number of mitochondrial ridges in smooth muscle cells were severely broken in the TAO group,and vacuolar degeneration was observed in the cytoplasm.Meanwhile,mitochondrial ridge rupture and vacuolar degeneration were observed in the vascular endothelial cells of the TAO group.The normal group and the sham operation group did not show the above performance.2.Plasma HMGB1,sRAGE and TXB2 detection:the plasma HMGB1 and TXB2 levels in the model group were significantly higher than those in the normal group and the sham operation group.sRAGE levels were lower than the normal group and the sham operation group.3.Double-staining immunofluorescence examination was performed on femoral artery tissues of rats in each group:The expression of HMGB1 in femoral artery tissues of the model group was higher than that of the normal group and the sham operation group,and the expression of HMGB1 mainly distributed in the endothelial and media smooth muscle cells and partially dispersed to the extracellular layer.RAGE expression was consistent with the variation trend of HMGB1,and was mainly expressed in endothelial cells.The expression of NF-?B was higher than normal group and sham operation group,mainly located in endothelial cells.There was no significant difference between the normal group and the control group.4.Western Blot was performed on the femoral artery tissues of rats in each group:The levels of HMGB1,receptor protein RAGE,and intracellular signaling molecule NF-?B in the femoral artery tissues of the TAO group were significantly higher than those of the normal group and the sham group.5.RT-PCR was performed on the homogenates of rat femoral artery tissues in each group:The mRNA levels of HMGB1,RAGE,and NF-?B in the femoral artery tissues of the TAO group were significantly higher than those of the normal group and the sham operation group,which was consistent with the trend of protein expression.Part II:1.Gross pathological scores of the hind limbs of the four groups of rats were scoredThe pathological grade of TAO model group was significantly higher than that of the sham operation group,and both low dose and high dose ADAM 10 treatment could reduce the grade of TAO rats,and the condition of the high dose group was significantly milder than that of the low dose group.2.Color doppler ultrasound was used to detect the hemodynamics of the hind limbs of ratsThe blood:flow velocity and resistance index of the TAO model group were significantly lower than those of the sham operation group and the resistance index is on the opposite.ADAM10 application could significantly improve the blood flow velocity of the femoral artery of TAO rats and reduce the resistance index,and the effect was more obvious in the high-dose group.3.Detection of blood cells,blood rheological indexes and coagulation indexes in ratsCompared with the model group,the platelet content,whole blood viscosity(high,medium and low),plasma viscosity and erythrocyte aggregation index of the ADAM10 treatment groups decreased significantly,while the fibrinogen content decreased significantly.And there was a significant correlation with dose.4.The intracellular structure of the femoral artery was observed by electron microscopyCompared with the model group,ADAM10 treatment group showed less mitochondrial ridge rupture in smooth muscle cells and less vacuolar degeneration in cytoplasm in a dose-dependent manner.At the same time,the ADAM10 treatment group showed reduced mitochondrial ridge rupture and vacuolar degeneration in the vascular endothelial cells of rats,which was also dose-dependent.5.HE staining results of rat femoral artery were analyzedCompared with the model group,the area of thrombus in the femoral artery lumen of the ADAM 10 treatment group decreased,the lumen area increased,and the intima thickness decreased to different degrees.In addition,it was observed that the vascular wall and inflammatory cell infiltration in the thrombus were significantly reduced,showing a dose-dependent effect.Part ?:1.Plasma protein level detectionThe ADAM 10 treatment for the rat TAO model showed that the HMGB1 content in the rat plasma decreased,and the HMGB1 content decreased significantly with the increase of ADAM 10 dose.Similarly,the content of TXB2 in rat plasma decreased,and with the increase of ADAM 10 dose,the content of TXB2 also decreased significantly.At the same time,it was found that the content of sRAGE in rat plasma was significantly increased,and with the increase of ADAM10 dose,the content of sRAGE was significantly increased.2.The expressions of HMGB1,RAGE,NF-?B were detected by double immunofluorescence stainingBy sodium laurate treatment of TAO group rats for 14 days by intraperitoneal injection of different doses of ADAM10 treatment,it can be seen that,compared with TAO group,both low dose injection ADAM10 group and high dose injection ADAM 10 group showed lower expression of HMGB1,RAGE,and NF-?B significantly,The expression of HMGB1,RAGE and NF-?B in ADAM 10 high dose group was lower than that in ADAM 10 low dose group.3.The protein levels of HMGB1,RAGE and NF-?B were quantitatively detected by Western-BlotCompared with the TAO group,the expression levels of HMGB1,RAGE and NF-?B protein in the low dose ADAM 10 group and the high dose ADAM10 group were significantly decreased,and the expression levels of HMGB1,RAGE and NF-?B in the high dose ADAM10 group were significantly lower than those in the low dose ADAM 10 group.4.HMGB1,RAGE,NF-?B mRNA expression in rat femoral artery tissue homogenate were detected by RT-PCR.Compared with the TAO group,the mRNA levels of HMGB1,RAGE and NF-?B in the low dose ADAM10 group and the high dose ADAM10 group were significantly decreased in a dose-dependent manner,which was consistent with the protein detection results.Conclusions:1.In rat models of TAO,HMGB 1,combined with receptor RAGE,promotes inflammation and vascular damage by activating the intracellular NF-kB signal pathway.2.ADAM 10 treatment can reduce the inflammatory response in TAO rat model and reduce the risk of thrombosis in a dose-dependent manner.3.In rat models of TAO,ADAM 10 can inhibit the binding of RAGE to HMGB1 by cleaving RAGE,thereby blocking RAGE/NF-?B signal pathway and playing a therapeutic role.Meanwhile,sRAGE,the product of enzymatic hydrolysis,plays a synergistic therapeutic role by combining the RAGE ligand HMGB1 competitively. |
PDF Full Text Request