Mechanism Of Hydroxysafflower Yellow A In The Regulation Of Vascular Smooth Muscle Cell Calcification

Objective:Chronic kidney disease(CKD)is a major disease that endangers global human health.Vascular calcification caused by abnormal mineral bone metabolism is a unique and common complication and an important cause of cardiovascular events in patients with CKD,leading to a significant increase in all-cause and cardiovascular mortality.HSYA is the main effective ingredient of traditional Chinese medicine safflower.Safflower has anti-inflammatory,anti-apoptotic and anti-oxidative effects.The purpose of this study was to explore whether HSYA could improve the calcification of rat vascular smooth muscle cells induced by HP and its possible mechanism.Methods:The effect of hydroxysafflor yellow A on the survival and vitality of rat vascular smooth muscle cells was detected by CCK8 method.Westernblot was used to detect the effect of safflor yellow A on calcification of vascular smooth muscle cells in rats.The experiment was divided into three groups: control group(NC),high-phosphorus-induced calcification group(HP),and HSYA intervention group(HSYA).Alizarin red staining and calcium determination kit(o-cresolphthalein ketone colorimetric method)were used to detect the calcium deposition in each group of cells;Western blot was used to detect the cell calcification indicator alkaline phosphatase(ALP),Runt-related transcription factor 2(RUNX2),nuclear factor κB receptor activating factor ligand(RANKL),alpha smooth muscle actin(α-SMA),and TLR4 /NF-κB pathway and inflammatory response-related indicators Toll-like receptor 4(TLR4),white blood cells Changes of interleukin 8(IL-8),tumor necrosis factor alpha(TNF-α).Nuclear and cytoplasmic proteins were extracted,and Western blot was used to detect nuclear NF-κB p65 and cytoplasmic p65,as well as changes in p65 and phosphorylated p65 in total cell proteins.Immunofluorescence was used to observe the expression of NF-κB in each group of cells.The expression of IL-1β and IL-6were detected by PCR.ROS kit,superoxide dismutase(SOD),and malondialdehyde(MDA)kits were used to detect changes in cell antioxidant enzymes and oxidation end products.Results:Alizarin red staining and calcium determination kit(o-cresolphthalein ketone colorimetric method)detected the calcium deposition in each group of cells showed that β-glycerol phosphate induced calcification model was successful,and hydroxysafflor yellow A reduces calcified nodules induced by β-glycerol phosphate calcification model.Westernblot results showed that the expression of calcification indicators ALP,RUNX2,and RANKL in the cells of HSYA group was significantly lower than that of HP group;the expression of α-SMA was higher than that of HP group(all P <0.01).The expression levels of TLR4,TNF-α,IL-8 and p-p65 / p65 in the HSYA group were lower than those in the HP group,p65 nucleation decreased,and p65 expression increased in the cytoplasm(all P <0.05).Immunofluorescenceresults showed that β-glycerol phosphate induction increased P65 nucleation.After HSYA treatment,P65 nucleation decreased.The contents of ROS and SOD in the cells of HSYA group were significantly higher than those of HP group,and the contents of MDA were significantly lower than those of HP group(both P <0.01).Conclusions:1.HSYA can delay high-phosphorus-induced calcification of vascular smooth muscle cells.2.HSYA inhibited the TLR4 / NF-KB signaling pathway and delayed the entry of NF-kB P65 into the nucleus during the delay of high-phosphate-induced vascular smooth muscle calcification.HSYA delays oxidative stress response during high phosphorus-induced vascular smooth muscle calcification.