Experimental Study Of LiCl Inhibiting Abdominal Aortic Aneurysm In Rats By Regulating GSK3?/SIRT1/NF-?B Signaling

Objective:In this experiment,the model of abdominal aortic aneurysm?Abdominal aortic aneurysm,AAA?was prepared by the classic method of external application of abdominal aorta with CaCl₂.Explore the therapeutic effect of glycogen synthase kinase?GSK3??inhibitor LiCl on AAA and its potential mechanism.Methods:1.In vivo experiment:divided into two parts:LiCl prevention experiment of rat abdominal aortic aneurysm and Li Cl treatment experiment of rat abdominal aortic aneurysm.The LiCl prevention part is divided into:N?normal control?group,Li Cl control group?normal rat injection of LiCl solution?,AAA group?build rat AAA model?,Li Cl prevention group?AAA model building day injection of LiCl solution for 4 weeks?.Li Cl treatment part is divided into:N group,LiCl control group,AAA group,LiCl treatment group?inject LiCl solution for 4 weeks after AAA model construction for 4weeks?.Blood samples were taken from the treated rats in each group to prepare for the detection of relevant indicators.2.Stereomicroscope and HE staining were used to detect the diameter of the abdominal aorta.Orcein staining was used to observe the morphological changes of the elastic plate.DHE staining was used to detect the activity of oxidized free radicals in the abdominal aorta of each group.The GSK3?,SIRT1,acrtyl-p53,MMP-2,MMP-9,elastin,NF-?B were quantitatively analyzed.3.Rat vascular smooth muscle cells?VSMCs?were cultured under in vitro conditions,and were divided into:N group?normal control group?,TNF-?group,TNF-?+LiCl group,TNF-?+SB216763 group,TNF-?+Wortmannin group,TNF-?+SRT 1720 group,TNF-?+EX 527 group.Group N served as a normal control group without any treatment;first,VSMCs were treated with TNF-??20 ng/ml 24 h?alone to simulate the inflammatory environment when AAA occurred in vivo.After that,in addition to the TNF-?group,cells in other groups were treated with LiCl?15mmol/L 24h?,SB216763?25?mol/L24h?,SRT 1720?1?mol/L 24h?,EX 527?10?mol/L 24h?,and wortmannin?1?m/L24h?for processing.Proteins were extracted from the cultured cells and subjected to Western Blot detection.Results:1.In vivo LiCl prevention part:First,through the observation of the stereomicroscope,the diameter of the abdominal aortic blood vessel in the AAA group was significantly expanded compared with the N group,and it was significantly calcified.After LiCl treatment,the calcification was significantly reduced and the degree of diameter expansion was reduced.HE staining was used to observe the shape of the abdominal aortic wall.From the results,it can be seen that the diameter of the artery in the AAA group was larger than that in the N group,with thinning and rupture of the wall.Lichen red staining showed that the elastic plate of AAA group was obviously broken,and LiCl treatment can significantly inhibit the fracture of the elastic plate of the arterial wall.DHE detected active oxygen content and found that the AAA group contains a large amount of active oxygen compared with the N group,which is reduced in the LiCl prevention group.The active oxygen content in the LiCl control group is about the same as the N group.Western Blot detection revealed that LiCl as an inhibitor of GSK3?increased the expression of p-GSK3?in the LiCl prevention group compared with the AAA group.At the same time,the Western Blot detection found that the expression of SIRT1 protein in the AAA group was lower than that of the N group,while the expression level of SIRT1 in the LiCl prevention group was significantly higher than that of the AAA group.It indicates that the expression of MMP-2,MMP-9 and NF-kB in AAA is significantly higher in AAA than in the normal group,and it is significantly reduced after LiCl prevention;while the expression of elastin in AAA is significantly lower than that in AAA group.Increased.2.In the LiCl treatment part of the in vivo experiment:after the detection of HE and lichen red,the diameter of the LiCl treatment group was significantly reduced compared with the AAA group,and the elastin rupture situation was also improved.Western Blot detection found that the expression of MMP-2and MMP-9 in arterial tissues in LiCl treatment group was also significantly lower than that in AAA group.3.In vitro experiments,the expression of GSK3?was regulated by GSK3?inhibitors?LiCl,SB216763?and agonists?Wortmannin?.It can be seen that the expression of SIRT1 protein in the detection of Western Blot will be affected by the expression of GSK3?protein.?LiCl,SB216763?will be significantly up-regulated,and the expression level of SIRT1 will be significantly down-regulated after treatment with GSK3?agonist?Wortmannin?,and acetyl-p53/p53,which represents SIRT1 activity,will also change accordingly.In further Western Blot detection experiments,it can be seen that cells treated with GSK3?inhibitors?LiCl,SB216763?can reverse the down-regulation of NF-kB protein expression and the up-regulation of elastin caused by the inflammatory environment after TNF-?treatment;This phenomenon is exacerbated after treatment with GSK3?agonist?Wortmannin?.In summary,the regulation effect of GSK3?on SIRT1 was verified through in vitro experiments.4.In vitro experiments,by regulating the expression of SIRT1 in VSMCs,it was found in Western Blot detection that VSMCs treated with SIRT1 agonist?SRT 1720?can significantly reverse the expression of NF-?B protein caused by TNF-?treatment Rise and the downregulation of elastin's expression.Cells treated with SIRT1 inhibitors?EX 527?exacerbated this phenomenon.Therefore,we infer that GSK3?achieves the treatment of AAA by regulating SIRT1.Conclusions:LiCl can alleviate the symptoms of AAA induced by CaCl2.As an inhibitor of GSK3?,LiCl can reduce the symptoms of AAA through the GSK3?/SIRT1/NF-?B signaling pathway.