Inhibitory Effect Of Puerarin On Vascular Calcification In Rats With Chronic Kidney Disease And Underlying Mechanism

Background:Chronic kidney disease(CKD),one of the most serious diseases,dramatically increases the risk of vascular calcification.Vascular calcification results in left ventricular hypertrophy,diastolic dysfunction and heart failure,and dramatically increases the mortality of CKD patients.The mechanism of vascular calcification is complex.Vascular inflammation,which can be mediated by NLRP3/Caspase1/IL-1β,NF-κB and reactive oxygen species(ROS)pathways,plays an important role in the development and progression of vascular calcification.Previous studies showed that puerarin has powerful anti-inflammation,anti-oxidation and anti-apoptosis effect and inhibits calcification of mouse vascular smooth muscle cells in vitro.However,whether puerarin can inhibit vascular calcification in vivo and the underlying mechanisms remain unclear.Objective:To research whether puerarin can inhibit vascular calcification in rats with chronic kidney disease by anti-inflammation and the related pathwaysMethods:Rat vascular smooth muscle cells(RVSMCs)of the control group were cultured by normal medium.Calcification of RVSMCs was induced by calcifying medium(the CM group)and puerarin(1,10,and 100 μM)was used to treat RVSMCs(the puerarin group).Rats with chronic kidney disease were established(5/6 renal nephrectomy,treated with vitamin D3 by gavage and fed high Ca and P diet)and randomly divided into two groups(n = 10 in each group):CKD model group(the Model group)and puerarin treatment group(the Puerarin group).Rats from the Puerarin group were also treated with puerarin(400 mg/kg·BW)by gavage once a day for 4 weeks.Normal rats were served as control(the Sham group,n = 10)and fed normal diet.Calcium content was detected by alizarin red staining and Calcium Detection Kit.Moreover,the expression of bone-related molecules Runx2 and BMP2 was measured by real-time fluorescence quantitative PCR(qPCR)and western blot.In addition,the levels of inflammation were determined through detecting expression of pro-inflammation cytokines including IL-1β,IL-6,IL-18 and TNFa in RVSMCs.Futhermore,the expression of NLRP3,Caspase1,IL-1β and NF-κB p-p65 and the generation of ROS were also measured by qPCR,western blot or immunofluorescence.Results:Calcifying medium effectively induced deposition of calcium in RVSMCs,which was significantly inhibited by puerarin,showed by alizarin red staining and Calcium Detection Kit.Similarly,vascular calcification of rats in the Model group was the most serious,the Puerarin group was the less serious and the Sham group was the least serious.In addition,puerarin obviously inhibited expression of bone-related molecules Runx2 and BMP2 in vitro and in vivo.Moreover,mRNA levels of pro-inflammation cytokines including IL-1β,IL-6,IL-18 and TNFa in RVSMCs treated with calcifying medium were distinctly increased,which could be markedly downregulated by puerarin treatment.Futhermore,the expression of NLRP3,Caspasel,IL-1β NF-κB p-p65 and the generation of ROS were also inhibited by puerarin treatment.Conclusions:This is the first report to show that puerarin inhibits vascular calcification in vivo.Furthermore,puerarin inhibits vascular calcification by attenuating vascular inflammation possibly through targeting NLRP3/Caspase 1/IL-1β and NF-κB pathways and inhibiting the generation of ROS.Puerarin has great potential to treat vascular calcification in CKD patients clinically.