Effect And Mechanism Of HMGB1 In Restenosis After Rat Carotid Artery Balloon Injury

Background and Purpose:Atherosclerosis(AS)is the most common clinical chronic inflammatory disease which is the main death cause of vascular disease.The incidence and mortality of cardiovascular and cerebrovascular diseases based on AS are increasing year by year.Arteriosclerosis obliterans(ASO)is partial manifestations of systemic atherosclerosis in the limb,resulting in chronic ischemia.The limbs show ischemic symptoms such as chill,numbness,pain intermittent claudication and ulcers or necrosis on toes or foot as well as other clinical manifestations.Transvaginal stentoplasty(PTA)is a rapid development treatment of ASO,but it is prone to restenosis(RS),which is the most important obstacle affecting long-term effects after PTA.The pathogenesis of RS is complex and still not clear.Establishment of a stable,economical,viable and ideal animal model similar to human arterial balloon dilatation angioplasty is theprecursor and basis for further study.High mobility group protein(HMG)is a kind of protein which can migrate rapidly in polyacrylamide gel.HMGB1 is a kind of DNA binding non-histone,which is widely distributed in eukaryotic nucleus.HMGB1 in the nucleus mainly non-specifically binds to DNA,which plays a role as skeletal protein,while inducing inflammation response is the most important effect of extracellular HMGB1.AS is the basis of all kinds of cardiovascular and cerebrovascular diseases.Multi-factor interactions are involved in the disease development.Progressive lipid accumulation leading to chronic vascular inflammation is the main pathological feature of RS,which exists in each period of RS,leading to inflammatory cells infiltration and inflammatory factors expressions.HMGB1 can promote the inflammatory response,while inflammatory response is the most important pathological feature of RS.DoesHMGB1 level have a relationship with vascular restenosis after balloon dilatation?We intend to assay HMGB1 level and investigate whether HMGB1 can increase inflammatory cells infiltration and inflammatory factors expressions,and then promote local vascular inflammatory response and RS progression.Epigallocatechin gallate(EGCG)is the most abundant type of polyphenols in tea.It has a strong anti-inflammatory effect and can inhibit HMGB1-induced inflammatory response.However,whether EGCG can inhibit HMGB1 expression to reduce balloon dilatation after vascular stenosis is unclear.In addition,extracellular HMGB1 can transmit signal to cell membrane by binding to cell surface specific membrane receptors,and then lead to biological effects.Both receptor for advanced glycation endproducts(RAGE)and toll like receptor(TLR)are the most important receptors for HMGB1.However,whether RAGE and TLR level on the surface of damaged blood vessels of RS is correlated with the severity of HMGB1 and RS needs further investigation.Method:Firstly,we intend to establish carotid artery restenosis model by using balloon injury method in rats.Healthy SD rats were randomly divided into 5 groups:control group(sham operation group),balloon dilatation group(Surgery group),EGCG(1 mg/kg)group,EGCG(2 mg/kg)group and EGCG(4 mg/kg)group.Rats in the EGCG group were injected intraperitoneally with EGCG after balloon dilatation.The rats in the control group was treated with sham operation only.Pathological analysis was performed to observe the morphology of blood vessels and neointima,endometrium,lumen,medial area and stenosis degree of carotid arteries.Quantitative PCR and western blot were used to detect the mRNA and protein expressions of HMGB1,RAGE and Toll-like receptor(TLR4).The expression levels of tumor necrosis factor-a(TNF-a),interleukin-1?(IL-1?),interleukin-6(IL-6),intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule(VCAM-1)in the peripheral blood were detected by ELISA.The commercialization kit was used to detect the expression of malonaldehyde(MDA)content and reactive oxygen species(ROS)production and EMSA was carried out to of nuclear factor NF-kb activity.Results:1.Three days after balloon dilatation,the endothelial cells of carotid artery began to exfoliate.Thrombosis was formed attached to vascular wall and a small amount of inflammatory cells were appeared.Over time,there were a large number of inflammatory cells infiltration and inflammatory factor expressions.The VSMCs proliferated,arranged in disorder and migrated to the intima.The arterial lumen showed severe stenosis 28 days after surgery.There were a great number of ECM deposition and a large number of foam cells.2.Lumen area:Two weeks after surgery,the lumen area was significantly reduced compared with the lumen area 14 days after operation(p<0.05).The neointima area was significantly higher than that in the control group(p<0.05)at 7 days after operation,and then the neointima area was gradually increased with the passage of time(p<0.05).Neointima area:The neointima area at 7 days after operation was significantly higher than that of the control group(p<0.05),and then the neointima area was increased with the passage of time(P<0.05).The neointima area of rats at 28 days was significantly different from that of rats at 14 days(p<0.05).Middle layer area:There was no significant difference in middle layer area between rats at 0 days after operation and 28 days after operation(p>0.05).Restenosis was mainly caused by thickening of the neoint layer,no significant thickening was observed in the middle layer.3.The expressions of HMGB1 gene and protein in carotid artery in the operation group were significantly higher than those in the sham operation group(p<0.01).After treated with different concentrations of EGCG,the level of HMGB1 in carotid arteries of rats was lower than that in the operation group(p<0.01).The expression of HMGB1 was gradually downregulated with increasing EGCG concentration,which showed a dose-dependent relationship.4.Western blot and EMSA showed that the expression and activity of NF-?B in carotid artery of balloon dilatation group were 4.19 folds higher than those in the sham operation group(p<0.01).After treated with different concentrations of EGCG,the expression and activity of NF-?B in carotid artery of operation group decreased gradually with the increase of EGCG concentration.5.The concentrations of IL-1??IL-6?TNF-a in tcarotid artery of balloon dilatation group were 254.12,178.68,813.17 pg/mgprot,which were significantly higher than those in the sham-operated group(40.51,35.12,78.68 pg/mgprot(p<0.01).The concentrations oflL-1??IL-6?TNF-?,IACM-1 and CAM-1 in rats treated with different concentrations of EGCG decreased gradually with the increase of EGCG concentration,showing a dose-dependent relationship.6.The content of ROS in the carotid artery of the balloon dilatation group was significantly higher than that in the sham operation group(p<0.001).After EGCG treatment,the ROS decreased and showed the trend that with the increase of EGCG concentration,ROS decreased gradually.The concentration of MDA in the carotid artery of the balloon dilatation group was 1.89 nmol/mgprot,which was significantly higher than that in the sham operation group(p<0.001).MDA concentration also showed a tendency to decrease gradually with the increase of EGCG dosage.7.The expressions of RAGE and TLR4 in carotid artery in the operation group were significantly higher than those in the sham operation group(p<0.01).After treated with different concentrations of EGCG,the level of RAGE and TLR4 in carotid arteries of rats was lower than that in the operation group(p<0.01).The expression of RAGE and TLR4 were gradually downregulated with increasing EGCG concentration,which showed a dose-dependent relationship.Conclusions:The carotid artery stenosis model was successfully constructed by balloon dilatation in 4 weeks.The expressions of HMGB1 and its receptor RAGE and TLR4 in the damaged tissue of carotid artery dilatation and stenosis were significantly higher than that in the control group,which promote infiltration of inflammatory cells and release of inflammatory factors to aggravate the inflammatory reaction and accelerate AS progression by the downstream signal pathway.EGCG can inhibit the excessive inflammation and oxidative stress induced by HMGB1,and has certain protective and reversal effect on balloon dilatation and restenosis.