The Study On The Role And Mechanism Of HMGB1 And TLR4-MyD88-NF-κB Signal Pathway In Vascular Injury During Ischemia And Reperfusion

Chapter 1 The role of HMGB1 in limb ischemia reperfusion injuryObjective:To investigate the effect of HMGB1 is involved in ALI patients with femoral artery incision ischemia reperfusion injury during thrombectomy,and whether the increased HMGB1 level can affect inflammation cytokine interleukin-6 and TNF-a levels,and involved in the IRI process.Methods:From January 2017 to February 2017,the 21 patients who ALI became ill for 6 hours in our hospital were treated with femoral artery incision and thrombolysis were the ALI operation group(21 cases of acute arterial embolism).In addition,10 patients with normal lower limbs were selected as the healthy control group.Ten minutes before operation,postoperative 24 and 48 hours to return after two groups of patients with venous blood 5 ml,after centrifugation at-80 ℃ refrigerator.In addition,liver and kidney function,heart color ultrasound and electrocardiogram examination were performed on the day of admission.Records of adverse clinical events occurred during hospitalization(heart failure,sudden cardiac death).After the specimen was collected,serum HMGB1,IL-6 and TNF-a were detected by ELISA,and compare the above factors at different time points.SPSS 20.0 software was used for statistical analysis.Results:1 The patients’ age(years),gender,smoking rates(%),hypertension prevalence rate(%),diabetes prevalence rate(%),blood lipid(mmol/L),creatinine((pmol/L),alanine aminotransferase(mmol/L)of two groups were no statistically significant difference level(P>0.05).2 The serum HMGB1 level in ALI operation group was higher than that of the healthy control group(P<0.01).The ALI surgical group and healthy control group of plasma HMGB1 levels in postoperative 24 hours was significantly higher compared to the moment before surgery(P<0.01).compared to postoperative 24 hours,the serum HMGB1 level at postoperative 48 hours was significantly decreased(P<0.01).There was no statistically significant difference between postoperative 48 hours and the moment before surgery(P>0.05).3 The serum IL-6 level in ALI operation group was higher than that of the healthy control group(P<0.01).The ALI surgical group and healthy control group of plasma IL-6 levels in postoperative 24 hours was significantly higher compared to the moment before surgery(P<0.01).There was no statistically significant difference between postoperative 48 and 24 hours(P>0.05).And compared to the moment before surgery,the serum HMGB1 level at postoperative 48 hours was significantly increased(P<0.01).4 The serum TNF-α level in ALI operation group was higher than that of the healthy control group(P<0.01).The ALI surgical group and healthy control group of plasma TNF-α levels in postoperative 24 hours was significantly higher compared to the moment before surgery(P<0.01).compared to postoperative 24 hours,the serum TNF-α level at postoperative 48 hours was significantly decreased(P<0.01).And compared to the moment before surgery,the serum HMGB1 level at postoperative 48 hours was significantly increased(P<0.01).5 The plasma HMGB1 and IL-6 levels corresponding time points were no statistical significance between ALI surgical group and health control group(P>0.05).Two groups of plasma HMGB1 and TNF-α levels corresponding time points were positively correlated(P<0.01).Conclusion:HMGB1 participated in the reperfusion injury process during the operation of the femoral artery incision in ALI patients.It may be involved in ischemia reperfusion injury by influencing the level of inflammatory cytokines IL-6 and TNF-α.Chapter 2 The role of HMGBl-TLR4-MyD88-NF-κB in the arterial injury of acute limb ischemia reperfusion.Objective:To establish a model of ischemia/reperfusion in rats and to study the role of HMGB1 in vascular injury in rats with ischemia and reperfusion.To clarify the mechanism of HMGB1-TLR4-MyD 8 8-NF-κB signaling pathway in rat limb ischemia/reperfusion.Methods:The Wistar male rats were randomly divided into sham operation group and ischemia reperfusion group.Sham operation group(sham):operation without ischemia/reperfusion operation.The acute ischemia/reperfusion model was developed by using the method of vascular clamping femoral artery combined with hemostasis.Ischemic reperfusion group was divided into:ischemia reperfusion group(IR);EP+ischemia reperfusion group(EP+IR);TAK-242+ischemia reperfusion group(TAK-242+IR).HE staining was used to observe the pathological changes of vascular tissue morphology and structure.Immunohistochemistry was used to detect the expression levels of HMGB1,TNF-α and IL-6.Serum HMGB1 protein level was detected by ELISA.The protein expression levels of HMGB1,TLR4,Myd88,and NF-κB in vascular tissues were detected by real-time fluorescence quantitative PCR and Western blot.Results:The model of acute ischemia reperfusion in rat hind limbs was successfully constructed.Compared with the normal group and the sham group,the expression of plasma HMGB1 in rat model group was significantly increased(P<0.01),and the expression of HMGB1,TNF-α and IL-6 were significantly increased in rat femoral artery wall.The HMGB1,TLR4,MyD88 and NF-κB expressions in ischemia reperfusion group were obviously higher than that in the control group(P<0.01).Compared the ischemia reperfusion group,the above indexes in the EP pretreatment group were significantly lower(P<0.01),but higher than control group(P<0.05).In TAK-242+IR group,the HMGB1 level was no obvious change than that of the ischemia reperfusion group,while the TLR4,MyD88 and NF-κB expressions were decreased in different degrees.(P<0.05),TLR4,MyD88 decreased significantly(P<0.01),and the NF-κB smaller decreased(P<0.05).Compared with EP+IS group,the expression of HMGB1 in TAK-242+IS group was higher(P<0.05),while TLR4 showed no significant difference(P>0.05),while the expression of MyD88 was slightly higher,but insignificant(P>0.05),and the expression of NF-kappa B was increased(P<0.05).Conclusion:1 The combination of blood vessel clamp and rubber band can successfully construct the rat model of acute limb ischemia and reperfusion,which can lead to inflammation of arterial vascular tissue and distant organs.2 The high expression of HMGB1 in the arterial vascular tissue and the distal organ of acute limb ischemia reperfusion was an important source of inflammatory cytokines.The effect may be achieved by up-regulating the expression of associated inflammatory factors.3 The role of HMGB1 in ischemia reperfusion artery injury was achieved through the HMGB1-TLR4-MyD88-NF-κB signaling pathway,but it is not entirely dependent on the HMGB1-TLR4-MyD88-NF-KB signaling pathway.4 HMGB1 inhibitors and TLR4 inhibitors could reduce artery injury of ischemia reperfusion process inflammation,reduced tissue injury,provided theoretical basis for clinical treatment.