Celastrol Alleviates Vascular Calcification In Rats With Chronic Kidney Disease

Background:Cardiovascular disease is the most common complication in patients with chronic kidney disease(CKD).Vascular calcification is highly associated with cardiovascular mortality in CKD.Previous studies have demonstrated that the mechanisms of vascular calcification share many similarities with bone formation.Under the stimulation of high calcium and phosphate levels,oxidative stress and inflammatory cytokines,vascular smooth muscle cells(VSMCs)undergo a process of osteogenic differentiation.Oxidative stress has been shown to play a critical role in the progression of vascular calcification in CKD.However,to date there is no effective therapy for vascular calcification in patients with CKD.Celastrol(Cel)is a natural product extracted from the plant tripterygium wilfordii,and has important anti-oxidant and anti-inflammatory functions.Heme oxygenase-1(HMOX-1),an antioxidant defense enzyme,has anti-inflammatory and antioxidant effects,and plays a protective role in the pathogenesis of atherosclerosis.However,whether Cel inhibits oxidative stress and attenuates vascular calcification in CKD through modulation of HMOX-1 remains unclear.In this study,we used VSMC,arterial ring and CKD rat models to investigate the role of Cel in vascular calcification in CKD,and elucidate the underlying mechanism.Objective:This study aims to investigate the effect of celastrol on vascular calcification in CKD and the underlying mechanism.Methods:In in vitro experiment,high phosphorus and calcium was used to induce calcification of VSMCs and arterial rings.Rat VSMCs,and rat and human arterial rings were treated with a variety of concentrations of Cel(1,10,20,and 50 nM).Alizarin red staining and formic acid were used to quantitatively assess calcium deposition.Calcium levels were determined by measuring optical density using a colorimetric assay.Western blot analysis was used to measure the expression levels of bone-related marker proteins.After small interfering RNA was used to knock down HMOX-1 and HMOX-1 inhibitor was used to treat cells,the extent of VSMC calcification was examined by alizarin red staining.In in vivo study,rats were randomly divided into control group,model group and Cel treatment group(1 mg/kg/day).The levels of serum creatinine in rats were measured using creatinine kit.Micro-CT and alizarin red staining were used to detect calcification of rat aortas.The levels of reactive oxygen species(ROS)in rat aortic smooth muscle cells were measured by immunofluorescence.Results:Alizarin red staining and gene expression analysis showed that Cel dose-dependently inhibited rat VSMC calcification and osteogenic differentiation.Similarly,ex vivo study revealed that Cel inhibited calcification of rat and human arterial rings.In addition,micro-computed tomography,alizarin red staining and calcium content analysis confirmed that Cel inhibited aortic calcification in CKD rats.Interestingly,Cel treatment increased the mRNA and protein levels of HMOX-1,and reduced the levels of ROS in VSMCs.Furthermore,both pharmacological inhibition of HMOX-1 and knockdown of HMOX-1 by siRNA independently counteracted the inhibitory effect of Cel on vascular calcification.Moreover,knockdown of HMOX-1 prevented Cel treatment-mediated reduction in ROS levels.Conclusions:1.Cel inhibits osteogenic differentiation and calcification of rat vascular smooth muscle cells.2.Cel inhibits calcification of rat and human arterial rings.3.Cel inhibits aortic calcification in rats with chronic kidney disease.4.Cel inhibits vascular calcification by activating HMOX-1 signaling pathway.