Effect And Mechanism Of Astaxanthin On Vascular Calcification In Chronic Kidney Disease

Objective: Chronic Kidney Disease(CKD)has a high incidence,and the population base is large in our country,chronic kidney disease patients are a relatively large group.There is a high incidence of Vascular calcification(VC)in patients with chronic kidney disease,which is one of the main causes of cardiovascular events.The mechanism of vascular calcification is complex,and the treatment options are limited.Currently reported drugs can only delay vascular calcification,but cannot reverse it.In this study,bioinformatics analysis screened Astaxanthin(Asx)to explore the effect and mechanism of astaxanthin on vascular calcification in chronic kidney disease,providing new methods and ideas for the treatment of vascular calcification in patients with CKD.Methods: 1.Pre-clinical drugs related to the clinical treatment of vascular calcification were collected through preliminary investigation,and their targets were obtained through bioinformatics analysis.Then the intersection of drugs and targets related to vascular calcification diseases was selected to screen out the drugs most likely to have effects on vascular calcification.2.To construct a rat model of CKD vascular calcification induced by 5/6 nephrectomy combined with 1.5% high phosphorus diet.On the third day after surgery,10mg/kg and 20mg/kg astaxanthin were given every other day by gavage,respectively.Ultrasonic tests were performed on the rats at 8 and12 weeks after surgery.At 12 weeks after operation,blood and abdominal aorta samples were collected after rats were sacrificed under anesthesia.The serum was isolated and the levels of serum creatinine,urea nitrogen and serum phosphorus were detected.HE,alizarin red,Von-kossa and other pathological staining were performed on rat abdominal aorta.3.The GSE146638 data set in GEO database was analyzed by combining mesh pharmacological methods to predict the potential mechanism pathway of astaxanthin intervention in vascular calcification.4.The targets related to vascular calcification,astaxanthin and ferrostopsis were further analyzed using reticular pharmacological databases such as TTD,Drug Bank and Dis Gent,and their intersection combinations were combined for KEGG enrichment analysis,and the pathways related to oxidative stress and ferrostopsis were enriched.5.Mitochondrial phenotypes related to ferrostopsis in vascular smooth muscle cells of CKD patients and rats were observed by transmission electron microscopy.6.AKP,LPO,SOD,MDA and other lipid peroxides related to ferrostopsis were detected in rat serum.7.Expressions of RUNX2,RANKL,Sm22α,GPX4 and XCT in rat vascular tissues were detected by immunohistochemical staining(IHC)and Western Blot(WB).Results: 1.Through bioinformatics analysis,astaxanthin was screened as a possible therapeutic effect on vascular calcification in CKD.2.The rat model of CKD vascular calcification induced by 5/6 nephrectomy combined with 1.5% high phosphorus diet was successfully established.At the 12 th week after the operation,the vascular wall of the abdominal aorta was significantly thickened and the vascular elasticity was significantly reduced.Alizarin red and Von-kossa staining showed calcium salt deposition in the abdominal aortic media of CKD rats.After astaxanthin treatment,no obvious vascular calcification was observed in CKD rats in the treatment group: compared with the calcification group,no thickening was observed in the abdominal aortic vascular wall of rats in the astaxanthin treatment group,and the vascular elasticity was significantly better.Alizin red and Von-kossa staining results respectively indicated that calcium salt deposition in the abdominal aortic media of rats was significantly reduced or no calcium salt deposition in the rats after astaxanthin treatment.3.Combined with mesh pharmacological analysis of GSE146638 data set in GEO database,astaxanthin may have an effect on ferrostopsis.4.Bioinformatics analysis of network pharmacologic databases,such as TTD,Drug Bank,Dis Gent,etc.,enriched the pathways related to oxidative stress and ferrostopsis.5.Ferrostopsis was observed in the blood vessels of both CKD patients and rats by transmission electron microscopy: mitochondrial volume decreased,crists decreased or disappeared,and mitochondrial double-layer membrane density increased or burst.6.Compared with sham operation group,serum levels of lipid peroxides such as AKP,LPO and MDA were significantly increased in calcification group,while serum levels of lipid peroxides related to ferrostopsis were significantly decreased in astaxanthin intervention group.The antioxidant index SOD decreased significantly in calcification group and increased significantly in astaxanthin intervention group.7.The results of immunohistochemical staining(IHC)and Western Blot(WB)showed that compared with the sham operation group,the expressions of GPX4,XCT and other proteins related to ferrostopsis in the blood vessels of rats in the calcification group were significantly reduced,while the expressions of GPX4,XCT and other proteins related to ferrostopsis indexes were significantly increased in the blood vessels of rats in the astaxanthin intervention group.The expressions of RUNX2,RANKL and SM22α were significantly increased in the calcification group,while the expressions of RUNX2,RANKL and SM22α were not significantly increased in the Sham group and astaxanthin intervention group.Conclusions: 1.Abdominal aortic calcification can be successfully induced by 5/6nephrectomy plus high phosphorus diet in rats.2.Astaxanthin can slow the progression of vascular calcification in CKD rats;3.Astaxanthin can inhibit oxidative stress and ferroptosis during vascular calcification in CKD rats.