Mechanisms Underlying The Effects Of Salvianic Acid A On Cardiovascular Protection: Possible Involvement Of Calcium Concentration Regulation And Inhibition Of Oxidative Stress And Inflammation

Ischemic cardiomyopathy（ICM）is one of the most widespread and dangerous cardiovascular diseases,the incidence and mortality of ICM are high with increasing age.ICM pathogenesis is complex,and myocardial infarction（MI）,atherosclerosis（AS）,and endothelial injury are the most common causes.Continuous ischemia in MI patients leads to myocardial ischemia,decreased oxygen supply,reduced ATP production,insufficient energy supply,and failure of calcium pumps.The increased calcium(Ca²⁺)flow accompanied by significantly reduced Ca²⁺-reuptake by the sarcoplasmatic reticulum causes calcium overload and eventually becomes the main inducing factor of cardiomyocyte structure destruction,leading to myocardium mitochondrial injury,myocardial necrosis,arrhythmia,and additional cardiac function deterioration,eventually resulting in ICM.In addition,the increase in cardiocyte Ca²⁺concentrations can cause a significant increase in myocardial contractility and ultimately lead to an increase in myocardial oxygen consumption and ICM deterioration.Therefore,the drug that regulates intracellular Ca²⁺homeostasis has the potential to protect against the harmful effects of ICM.Oxidative stress,vascular inflammation,and the risk of cardiovascular events are frequently discussed in the literature.Oxidative stress and inflammatory reaction are closely related.The interactions between oxidative stress and inflammatory reaction causes endothelial dysfunction.Endothelial dysfunction in turn promotes a pro-inflammatory environment as evidenced by increased endothelial expression of adhesion molecules and imbalance of arachidonic acid metabolites and chemoattractant molecules.Endothelial dysfunction has been proven to be a key factor in the early AS process.Therefore,drugs with anti-inflammatory,anti-oxidant,and anti-lesion effects are promising candidates for ICM treatment.In traditional Chinese medicine,Radix Salviae Milthiorrhizae is widely applied in the treatment of cardiovascular diseases such as myocardial ischemia,MI,and AS.Salvianic acid A（SAA）is a water-soluble compound that is considered the main active pharmacological active constituent in the dried roots or rhizomes of Salvia miltiorrhiza Bge.Recent studies have shown that Radix Salviae Milthiorrhizae aqueous extract（containing 2.15 mg/mL SAA）can play a cardioprotective role by inhibiting L-type calcium channels（LTCCs）and myocardial contractility,and its effect is similar to calcium channel blockers.Experimental studies have shown that SAA has powerful effects on reducing inflammatory response and scavenging reactive oxygen species（ROS）,but research on the mechanisms of cardiovascular protection of SAA is more scattered and limited.We speculate that SAA exerts its ICM-protective effects by inhibiting oxidative stress,vascular inflammation,and regulating intracellular Ca²⁺concentration.This paper mainly discusses the protective effects and mechanisms of SAA on ICM from the aspects of animal and cell experiments:（1）Protective effects and underlying mechanisms of SAA against acute MI in rats;（2）Protective effects and underlying mechanisms of SAA on AS in rats;and（3）Protective effects and underlying mechanisms of SAA on endothelial cell injury.Part 1 Protective effects and underlying mechanisms of SAA against acute MI in ratsObjective:The aim of this study was to investigate the protective effects of SAA against isoproterenol（ISO）-induced MI in rats and to elucidate its potential myocardial protective mechanism based on SAA regulation on cardiocyte Ca²⁺homeostasis.Methods:All Sprague-Dawley rats were randomly divided into groups（n=8）:control group（Con）,isoproterenol treatment group（ISO）,low-dose SAA treatment group（3 mg/kg/d,L-SAA）,high-dose SAA treatment group（10 mg/kg/d,H-SAA）and verapamil treatment group（2 mg/kg/d,Ver）.The same volume of normal saline was given to the Con and ISO groups.All of the treatments were administered by intraperitoneal injection（i.p.）.After seven consecutive days of preconditioning,the rats were subcutaneously injected with ISO（85 mg/kg/d,except for Con group）for the next two days.IonOptix Myocam detection system and whole cell patch clamp techniques were used to detect the regulatory effects of SAA on cardiocyte calcium homeostasis in cardiocytes.Results:1.SAA significantly reduced ST segment elevation and heart rate in rats with MI induced by ISO.2.SAA significantly improved pathological symptoms such as myocardial cell swelling and degeneration and loss of transverse striations in the ISO-induced MI model.3.SAA significantly reduced serum levels of creatinine kinase and lactic dehydrogenase in the ISO-induced MI model.4.IonOptix recording showed that SAA(3×10^-7 M)could significantly inhibit the peak calcium transient value in rat ventricular myocytes co-incubated with fura-2-AM with an inhibition rate of 50.64±1.33%.5.SAA at 3×10^-7 M could significantly increase the time to 10%of the peak（Tp）and significantly decrease the time to 10%of the baseline（Tr）in rat ventricular myocytes.6.IonOptix recording showed that SAA(3×10^-7 M)could significantly inhibit the contraction amplitude（with inhibition rates of 33.48±0.75%）of ventricular myocytes in rats.7.The whole cell patch clamp recording showed that SAA‘s inhibition rates(3×10^-6,10^-5,3×10^-5,3×10^-4,and 10^-4 M)of I_Ca-L were 9.5%±0.9%,17.6%±1.1%,29.6%±1.8%,34.6%±1.9%,and 39.5%±1.9%,respectively,with an IC₅₀ of 1.47±10^-5 M.8.SAA at 3×10^-6 and 3×10^-4 M up-regulated the I–V relationship curve without changing the activation and peak potentials.Part 2 Protective effects and underlying mechanisms of SAA on AS in ratsObjective:The primary aim of this study was to investigate the effects of SAA on high-fat diet-induced AS and molecular mechanisms involved in oxidative stress and inflammation.Methods:A high-fat diet that consisted of normal laboratory chow containing 40%fat（saturated fatty oil）and 5%cholesterol for a period of 6weeks along with a single intraperitoneal injection of vitamin D3 in a dose of600,000 IU/kg body mass were administered to establish the AS rat model.All SD rats were randomly divided into groups（n=10）:Control group（Con）,atherosclerotic group（AS）,low-dose SAA group（3 mg/kg/d,L-SAA）,high-dose SAA group（10 mg/kg/d,H-SAA）and simvastatin group（5 mg/kg/d,Sim）.AS was induced in all groups except Con,L-SAA,H-SAA,and Sim groups were given corresponding drugs for six weeks.The Con and AS groups were given the same volume of distilled water.All the treatments were administered by intragastric administration.Results:1.The testing results of four lipid indices in serum demonstrated that SAA could significantly alleviate high fat-induced dyslipidemia in rats.2.Hematoxylin and eosin staining and Oil Red O staining showed that SAA inhibited the development of aortic intimal lesion in AS rats.3.SAA treatment improved antioxidant status in rats fed with high-fat diet by reducing the aortic malondialdehyde（MDA）content and improving aortic superoxide dismutase（SOD）,catalase（CAT）,and glutathione peroxidase（GSH-Px）activities.4.The ELISA results showed that serum interferon（IL）-1βand-6 and tumor necrosis factor（TNF）-αlevels in the AS rats were significantly up-regulated,whereas SAA treatment suppressed IL-1βand-6 and TNF-αoverexpression in a dose-dependent manner.5.The WB results showed that the expression levels of toll-like receptor（TLR）4,myeloid differentiation factor（MyD）-88,nuclear factor（NF）-κB（p65）,p47phox,and p22phox in the AS rats were significantly elevated,whereas their over-expression was down-regulated by SAA.6.The WB results showed that IκBα,nuclear factor erythroid 2–related factor（Nrf）2,and heme oxygenase（HO）-1 expression levels in the AS rats were significantly decreased,whereas their expressions were up-regulated by SAA.Part 3 Protective effects and underlying mechanisms of SAA on endothelial cell injuryObjective:The main purpose of this study was to explore the protective effect of SAA on ox-low density lipoprotein（LDL）-induced human umbilical vein endothelial cell（HUVEC）injury and to elucidate the potential protective mechanisms based on SAA regulation of related proteins.Methods:The endothelium injury model was induced by 80μg/mL ox-LDL treatment for 24 h in the HUVEC‘s logarithmic growth phase.HUVECs were randomly divided into groups:1)Control:HUVECs were incubated for 27 h with standard ECM culture solution;2)Endothelial injury model:HUVECs were incubated for 3 h with standard ECM culture solution+24 h incubation with 80μg/m L ox-LDL;3)Low-dose SAA treatment:HUVECs were incubated for 3 h with standard ECM culture solution(containing 10^-5 M SAA)+24 h incubation with 80μg/mL ox-LDL;4)High-dose SAA treatment:HUVECs were incubated for 3 h with standard ECM culture solution(containing 3×10^-5 M SAA)+24 h incubation with 80μg/mL ox-LDL;5)Sim:HUVECs were incubated for 3 h with standard ECM culture solution(containing 10^-7 M Sim)+24 h incubation with 80μg/mL ox-LDL.Results:1.The potential cytotoxic results on HUEVCs showed that SAA treatment had little effect on cell viability at the concentrations of 10^-5 and3×10^-5 M used in this study.2.The WB results showed that the expression levels of lectin-like ox LDL receptor-1（LOX1）,nicotinamide adenine dinucleotide phosphate oxidase 4（NOX4）,and NF-κB in ox-LDL-induced HUVECs were significantly elevated,whereas their expressions were down-regulated by SAA.3.The WB results showed that the expression levels of vascular cell adhesion protein（VCAM）-1 and intercellular adhesion molecule（ICAM）-1 in ox-LDL-induced HUVECs were significantly elevated,whereas their expressions were down-regulated by SAA.4.ox-LDL exposure significantly increased ROS generation in HUVECs,while pretreatment with SAA(10^-5,3×10^-5 M)significantly decreased ROS generation in HUVECs.Conclusions:1.SAA can significantly alleviate the ISO-induced MI,inhibit calcium transient,reduce myocardial contractility and decrease Ca²⁺concentrations of cardiocyte by inhibiting LTCCs,therefore protect cardiocyte from MI injury.2.SAA can significantly alleviate dyslipidemia and improve high-fat-induced antioxidant and anti-inflammatory capacities in AS rats.The mechanism underlying ICM protection may be connected to inhibition of TLR4/NF-κB and upregulation of Nrf2/HO-1 signal pathways.3.SAA can alleviate ox-LDL-induced cytotoxicity in HUVECs,thereby preventing endothelial cell injury and ICM.The potential mechanisms involve modulation of LOX-1,NOX4,NF-κB,VCAM-1,and ICAM-1 activities and inhibition of ROS generation.