Establishment Of Coronary Artery Calcification Prediction Model And The Effects And Mechanism Of Danshen-Sanqi And Its Components On Arterial Calcification Based On TGF-β Pathway

Objective:1.Through retrospective research methods and logistic regression analysis,we can find the risk factors of coronary artery calcification and establish a predictive model for the occurrence of coronary artery calcification to provide a reference for clinical screening of high-risk patients with cardiovascular calcification.The TCM syndromes and clinical symptoms of patients with coronary artery calcification were counted,and the common pathological factors of coronary artery calcification were found.2.To study the effects of activating blood circulation and removing blood stasis drugs Danshen and Sanqi on the anti-arterial calcification and the effect on the TGFβ1/Smad2/3 signal pathway.And further study the effective component of Salvia miltiorrhiza protocatechuic aldehyde(PCA)to improve the effect of arterial calcification and related mechanisms,open up new ideas for the prevention and treatment of arterial calcification.Methods:1.Establishment of coronary artery calcification prediction model:A retrospective study was conducted to collect cases of coronary artery CT.According to the presence or absence of calcification plaque formation,they were divided into calcification plaque group(366 cases)and non-calcification plaque group(621 cases),respectively The baseline data,past history,family history,and hematological indicators of the calcification group and the non-calcification group were compared and analyzed.Logistic regression analysis was used to obtain the risk factors of coronary calcification plaque formation,and the coronary calcification prediction model was established and verified.Coronary artery calcification TCM syndrome analysis:The statistical description method is used to analyze the TCM syndrome differentiation and clinical symptom distribution of coronary artery calcification patients,and the common syndromes,pathogenic factors and common symptoms of coronary artery calcification are obtained.2.Anti-arterial calcification effect of Danshen-Sanqi drμg pair and active ingredients:use ApoE-/-mouse build animal calcification model,intervented with rosuvastatin,Danshen,Sanqi drug pair,protocatechuic aldehyde(PCA)and notoginseng Saponin R1(NgRl)monomer for 12weeks.Then observe the effect on arterial calcification by the pathological staining method,serum lipids and serum calcium levels were detected by the biochemical analyzer,and serum OPG,RANKL and TGFβ1 levels were detected by the ELISA method.3.The antagonistic effect of PCA and NgR1 on cell calcification and its influence on the TGFβ1/Smad2/3 signaling pathway:establish a calcification model of human aortic smooth muscle cells(HAVSMCs),treat cells with PCA+NgR1,PCA and NgR1.Then observe the effect of PCA+NgR1,PCA and NgR1 on calcification by pathological staining methods,detect the mRNA expression of osteogenic genes and TGFβ1/Smad2/3 signaling pathway related genes by PCR.4.Research on the mechanism of antagonistic arterial calcification of protocatechuic aldehyde(PCA):Establish a calcification model of human aortic smooth muscle cells(HAVSMCs),and treated with PCA.Then observe the effect of PCA on HAVSMCs calcification by pathological staining,as well as the effect on TGFβ1/Smad2/3 pathway and oxidative stress by Molecular biology methods5.Study on the mechanism of PCA inhibiting calcification through TGFβ1/Smad2/3 pathway and Nrf2:silence target genes to study the specific mechanism of PCA inhibiting calcification based on the TGFβ1/Smad2/3 pathway and Nrf2.Results:Prediction model establishment:(1)The predictive model of CAC is P=-1.034 lipid-lowering drugs+0.02 age+0.03 smoking+1.14 low-density cholesterol+0.01 alkaline phosphatase-2.418,and P≥5.128 is the occurrence of CAC,P<5.12 8 means there is no CAC.the common TCM syndromes of CAC:heart blood stasis syndrome,qi stagnation and blood stasis syndrome,phlegm blocking blood stasis syndrome,phlegm turbidity blocking collaterals syndrome,and phlegm-heat accumulation syndrome.The main pathogenic factor is Blood stasis;common clinical symptoms are chest tightness,dizziness,palpitation,chest and back pain,insomnia and dreaminess.2.In vivo experiments show that rosuvastatin,danshen-sanqi drug pairs,PCA+NgRl,PCA,NgRl can effectively reduce the aortic calcification of calcified mice,the serum levels of OPQ RANKL and TGF β1,blood lipids and serum calcium levels.3.In vitro experiments found that PCA+NgR1,PCA,and NgR1 could reduce the calcification of HAVSMCs induced by calcium and phosphorus,inhibite the mRNA expression of ALPL,COL1A1,BMPR2 and increase mRNA expression of MGP and SPP1.Further research found that PCA can inhibit the mRNA expression of TGFβ1,TGF βR1,TGFβR2,and Smad2/3.NgR1 has no effect on the TGFβ1/Smad2/3 signaling pathway.4.In vitro experiments found that PCA inhibits HAVSMCs calcification induced by calcium and phosphorus,reduces intracellular calcium content and inhibits the protein expression of Smad2/3.Further studies found that PCA increases the protein expression of SOD2,reduces the occurrence of ROS,promotes Nrf2 enter the nucleus,increases protein expression of P62,Nrf2,HO-1 and NQO1.After silencing Nrf2,PCA’s antioxidant effect and the inhibition of calcification disappears.Treat cells with LSKL and LY2109761 to interfere with the cell calcification model,the nuclear protein expression of Nrf2 without decrease significantly.5.In vitro experiments found that PCA can inhibit cell calcification induced by TGFβ1.After silencing Nrf2,PCA’s inhibitory effect on calcification disappears.It was further found that PCA can inhibit the protein expression of TGFβR1 and Smad2/3.In this calcification model,PCA can increase the protein and mRNA expression of SOD2,promote the protein expression of P62 and Nrf2,and increase the mRNA expression of HO-1 and NQO1.After P62 was silenced,PCA promoted the nucleus and protein expression of P62 and Nrf2,and the effect of increasing HO-1 and NQO1 protein expression disappeared.Conclusion:1.The common TCM syndrome of CAC is heart-blood stasis syndrome,and the main pathological factor is blood stasis.Coronary artery calcification-related risk factors:age,smoking,low-density cholesterol and alkaline phosphatase are positively correlated,lipid-lowering drugs are negatively correlated.2.Danshen Sanqi drug pair and PCA+NgR1,PC A,NgR1 can effectively inhibit the occurrence and development of arterial calcification.PCA can effectively inhibit the occurrence of oxidative stress and calcification.PCA inhibits the TGFfβ1/Smad2/3 signaling pathway,thereby promoting P62 expression,and promoting Nrf2 to enter the nucleus to exert antioxidant effects,thereby inhibiting the occurrence of calcification.