Dihydromyricetin Ameliorates Vascular Calcification In Rats With Chronic Kidney Disease And Underlying Mechanism

BackgroundVascular calcification,the ectopic deposition of hydroxyapatite crystals in the medial or intimal layers of arteries,is a prevalent cardiovascular complication of chronic kidney disease(CKD).It is a marker of increased risk of cardiovascular event and mortality.Initially,vascular calcification was regarded as a passive process of calcium and phosphate deposition.However,accumulating evidence has suggested that vascular calcification is an active cell-mediated regulated process sharing similarities with bone formation.Complex mechanisms are involved in vascular calcification,including hyperphosphatemia,hypercalcemia,an imbalance between calcification promoters and inhibitors,osteogenic and osteochondrogenic transformation of VSMCs,the release of matrix vesicles and the deposition of calcium-phosphate crystals in the extracellular matrix,apoptosis,inflammation,oxidative stress,endoplasmic reticulum(ER)stress,senescence,etc.A variety of signaling pathways have been identified being involved in regulation of vascular calcification,but the exact molecular mechanisms of vascular calcification still remain elusive.Consequently,no effective therapeutic strategies to prevent the progression of vascular calcification are yet available.Dihydromyricetin(DMY),a natural product extracted from Rattan tea,belongs to the flavonoids.It has been shown to possess various effects such as anti-inflammatory,antioxidant,anti-tumor,anti-microbial,regulating glucose and lipid metabolism,anti-alcoholism and hepatoprotective.However,whether DMY has a role in vascular calcification in CKD remains unclear.In this study,we aimed to explore the effect of DMY on vascular calcification using in vitro,ex vivo,in vivo models,and investigate the underlying mechanisms.ObjectiveThis study aims to explore the effect of DMY on vascular calcification in CKD and the underlying mechanism.MethodsCalcifying medium(CM)containing high phosphorus and calcium was used to induce calcification of VSMCs and arterial rings.Rat and human VSMCs were treated with different concentrations of DMY(20 μM,40 μM,80 μM).Rat aortic rings were treated with DMY at the concentration of 80 μM.MTS was used to test the effect of different concentrations of DMY on the viability of VSMCs.Alizarin red staining was used to assess mineral deposition.The calcium content of cells and aortic rings was determined by Calcium Content Detection Kit according to the manufacturer’s protocol.Quantitative real-time fluorescence PCR(RT-qPCR)was used to measure the expression of bone-related molecules RUNX2 and BMP2.Western blot analysis was used to determine the protein expression of bone-related molecules such as RUNX2,BMP2,COLI and OCN.The level of p-AKT expression in rat VSMCs was measured by immunofluorescence assay.In addition,AKT activator and inhibitor or small interfering RNA were used to overexpress or knock down AKT to investigate the role of AKT signal in vascular calcification.CKD rat model was established in in vivo experiment.Blood samples were collected to evaluate serum creatinine(CRE)by using Creatinine Assay Kit.Micro-CT and alizarin red staining were used to assess calcification of rat aortic arteries.Results1.Alizarin red staining and calcium content analysis showed that DMY dose-dependently inhibited rat VSMCs calcification.Additionally,RT-qPCR analysis revealed that DMY decreased the mRNA expression of osteogenic differentiation related genes including RUNX2 and BMP2.Concomitantly,Western blot analysis exhibited decreased RUNX2,BMP2,COLI and OCN expression by DMY treatment.Taken together,these results indicated that DMY effectively attenuated osteogenic trans-differentiation and calcification of rat VSMCs.Moreover,ex vivo study revealed that DMY inhibited calcification of rat aortic rings,and significantly down-regulated COL I,RUNX2 and BMP2 expression.In addition,alizarin red staining of whole aortas and sections showed that DMY ameliorated aortic calcification in CKD rats.Similarly,micro-CT and calcium content analysis confirmed that DMY treatment significantly decreased the mineral density in CKD rats,indicating that DMY ameliorated aortic calcification.2.Western blot analysis revealed that the protein level of p-AKT was significantly elevated in CM-treated VSMCs and calcified arteries,while DMY dramatically down-regulated p-AKT protein expression.Similarly,immunofluorescence analysis showed increased p-AKT expression in VSMCs cultured in CM,which was drastically inhibited by DMY treatment.Alizarin red staining showed that AKT activator enhanced VSMCs calcification compared with CM-treated cells.However,this effect was abrogated by DMY treatment.Furthermore,inhibition of AKT signaling by AKT inhibitor or small interfering RNA efficiently attenuated calcification,which was similar to that after treatment with DMY alone,and DMY had a better inhibitory effect on calcification as compared to AKT inhibitor.Conclusions1.DMY inhibits osteogenic differentiation and calcification of rat VSMCs,and the calcification of rat aortic rings.2.DMY inhibits aortic artery calcification in rats with CKD.3.DMY inhibits vascular calcification in CKD by targeting AKT signaling.