The Therapeutic Effect Of Ag490 On Homocysteine-Induced Thromboangiitis Obliterans By Inhibiting The JAK2/STAT3 Pathway

Objectives:1.In order to confirm that hyperhomocysteinemia(HHcy)may be one of the causes of thromboangiitis obliterans(TAO),the difference of plasma homocysteine(Hcy)level between patients with TAO and healthy population was studied by evidence-based medicine Meta-analysis.2.By using Hcy to establish the injury model of human umbilical vein endothelial cell lines(HUVECs),the mechanism of Hcy causing injury to endothelial cells and the inhibitory effect of AG490 on this process were studied,which laid an experimental basis for subsequent studies.3.By establishing a rat model of TAO,the mechanism of Hcy promoting the formation of TAO in rats and the drugs that effectively inhibit it were studied,so as to provide theoretical basis for clarifying the etiology and pathogenesis of human TAO and seeking effective therapeutic measures in clinical practice.Material and Methods:1.The Pub Med,Web of Science,and Science Direct databases(up to February2021)were used to search for studies evaluating the relationship between plasma Hcy levels and the risk of TAO.2.HUVECs were used for cell experiments:CCK-8 was used to detect the cell proliferation activity and toxicity,and the toxic effect of Hcy solution at different concentrations and incubation time on endothelial cells was detected,as well as the inhibitory effect of AG490 at different concentrations on the cytotoxic effect of Hcy on endothelial cells.Endothelial cells were divided into three groups for culture:(1)Hcy group(medication administration team,Hcy concentration was 2m M,culture time was 24 h);(2)AG490+Hcy group(treatment group,cells were incubated with20μm AG490 for 4 h,and then 2m M homocysteine solution was added for further culture for 24 h);(3)control group.Cell proteins and total RNA were extracted from the cultured cells,and protein and gene levels of JAK2,STAT3,p-STAT3,HMGB1,ICAM1 and VCAM1 were detected by Western Blot and RT-PCR.3.Mature male SD rats were used in animal experiment.The rats were divided into 5 groups:(1)control group;(2)Normal saline(NS)+NS+operation group(model group,normal saline injected through caudal vein for 14 days+intraperitoneal injection of normal saline 2 hours before operation+operation+normal saline injected through caudal vein after operation for 14 days);(3)Hcy+NS+operation group(administration group,Hcy injected through caudal vein for 14 days+intraperitoneal injection of NS 2 hours before operation+operation+Hcy injected through caudal vein after operation for 14 days);(4)Hcy+AG490+operation group(treatment group,Hcy injection through caudal vein for 14 days+AG490 intraperitoneal injection 2 hours before operation+operation+Hcy injection through caudal vein after surgery for 14days);(5)Sham operation group.In each group,the rat model of TAO was constructed by injecting sodium laurate solution into the left posterior femoral artery.At the end of the experiment period,the degree and range of the affected limb skin temperature,color,arterial pulse,swelling,gangrene and mummification were observed and graded.Hematoxylin-eosin staining was used to examine the femoral artery of the affected limbs in each group.Protein and total RNA were extracted from the femoral artery of the affected limbs of rats in each group.The protein levels and gene levels of JAK2,STAT3,p-STAT3,HMGB1,ICAM1 and VCAM1 were detected by Western Blot and RT-PCR.Results:1.Based on inclusion and exclusion criteria,five eligible studies were finally identified,including 180 patients with TAO patients and 564 age-and sex-matched healthy participants.In the forest plot of the Meta-analysis,plasma Hcy levels were higher in patients with TAO than in the control group(MD:3.19,95%CI:0.26-6.12,P<0.00001,I~2:90%).Through the analysis of differences between subgroups,it was found that the excessive heterogeneity might be caused by the different races included in the study(P=0.001).2.The results of cell experiments showed that:(1)With the increase of Hcy concentration and the prolonging of action time,the cell viability of HUVECs decreased gradually.Suitable concentration of AG490 can increase the viability of Hcy-injured endothelial cells,but too high concentration of AG490 can make the viability of Hcy-injured endothelial cells even lower.(2)The protein and m RNA expressions of JAK2,STAT3,p-STAT3,HMGB1,ICAM1 and VCAM1 in group(1)were significantly higher than those in group(3),while the protein and m RNA expressions of group(2)were significantly lower than those in group(1).3.Animal experiment results show that:(1)through the femoral artery injection of sodium laurate solution method,can be successfully constructed TAO disease animal model in rats,and to observe the rat limb symptoms found that tail vein injection of Hcy can increase performance of rat limb vasculitis,and preoperative intraperitoneal injection of Ag490 can effectively reduce Hcy induced(2)Hematoxylin-eosin staining showed that group(1)and group(5)had normal femoral artery.Group(3)compared with group(1)and group(5),there were a lot of thrombosis in the femoral artery,the intima of the femoral artery was obviously thickened,and a lot of inflammatory cells were infiltrated around the femoral artery.Group(2)and group(4)compared with group(3),thrombosis in femoral artery was smaller,intima thickening was less,and inflammatory cell infiltration was less.(3)The molecular extract of femoral artery in the affected limb of rats was detected.The protein and m RNA expressions of JAK2,STAT3,p-STAT3,HMGB1,ICAM1 and VCAM1 in group(2)were significantly higher than those in group(1),and the protein and m RNA expressions of group(3)were significantly higher than those in group(2),while the protein and m RNA expressions of group(4)were significantly lower than those in group(3).Conclusions:1.HHcy may be one of the causes of TAO,but large randomized controlled trials and prospective studies are needed to supplement the accuracy of the results.2.The toxicity of Hcy to HUVECs increased with the increase of drug concentration and the prolongation of action time.3.Elevated plasma Hcy concentration in rats can aggravate the symptoms of affected limb TAO,and the injection of AG490 can effectively reduce the symptoms of Hcy-induced angiitis4.The toxic effect of Hcy on endothelial cells and aggravation of rat TAO may be due to the abnormal activation of JAK2/STAT3 pathway,which leads to the overexpression of inflammatory factors in endothelial cells and rat femoral artery of the affected limb,thus causing inflammatory response.5.Ag490 at appropriate concentration has therapeutic effect on Hcy-induced HUVECs injury and rat TAO by inhibiting the JAK2/STAT3 pathway.Meanwhile,as AG490 is a specific inhibitor of JAK2,it was confirmed that the damage of Hcy to endothelial cells and the aggravation of TAO induced by Hcy may be caused by the inflammatory response induced by the activation of JAK2/STAT3 pathway.