Suxiao Jiuxin Pill alleviates myocardial ischemia/reperfusion-induced autophagy via miR-193a-3p/ALKBH5 pathway.

BACKGROUND: Myocardial ischemia/reperfusion injury (MIRI) poses a formidable challenge to cardiac reperfusion therapy due to the absence of effective clinical interventions. Methylation of N6-methyladenosine (m6A), which is the most common post-transcriptional modifications occurring within mammalian mRNA, is believed to be involved in MIRI by modulating autophagy. MicroRNAs (miRNAs) play a crucial role in regulating gene expression at the post-transcriptional level and have been implicated in the regulation of m6A methylation. Suxiao Jiuxin Pill (SJP) is extensively used in China for the clinical treatment of angina pectoris and confers benefits to patients with acute coronary syndrome who have received percutaneous coronary intervention. However, the precise mechanisms underlying SJP intervention in MIRI remain unclear. PURPOSE: This study aimed to demonstrate, both in vivo and in vitro, that SJP could alleviate autophagy in MIRI by regulating miR-193a-3p to target and upregulate the demethylase ALKBH5. METHODS: An in vitro hypoxia/reoxygenation model was established using H9c2 cells, while an in vivo MIRI model was established using Wistar rats. A lentivirus harboring the precursor sequence of miR-193a-3p was employed for its overexpression. Adeno-associated viruses were used to silence both miR-193a-3p and ALKBH5 expressions. Cardiac function, infarct size, and tissue structure in rats were assessed using echocardiography, triphenyl tetrazolium chloride (TTC) staining, and HE staining, respectively. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was employed to detect the levels of apoptosis in rat cardiac tissue. m6A methylation levels were assessed using colorimetry. GFP-RFP-LC3B was used to monitor autophagic flux and transmission electron microscopy was used to evaluate the development of autophagosomes. Western Blot and qRT-PCR were respectively employed to assess the levels of autophagy-related proteins and miR-193a-3p. RESULTS: SJP alleviated autophagy, preserved cardiac function, and minimized myocardial damage in the hearts of MIRI rats. SJP attenuated autophagy in H/R H9C2 cells. Elevated levels of miR-193a-3p were observed in the cardiac tissues of MIRI rats and H/R H9C2 cells, whereas SJP downregulated miR-193a-3p levels in these models. ALKBH5, a target gene of miR-193, is negatively regulated by miR-193a-3p. Upon overexpression of miR-193a-3p or silencing of ALKBH5, m6A methylation decreased, and the autophagy-attenuating effects of SJP and its components, senkyunolide A and l-borneol, were lost in H/R H9C2 cells, whereas in MIRI rats, these effects were not abolished but merely weakened. Further investigation indicated that the METTL3 inhibitor STM2475, combined with the silencing of miR-193a-3p, similarly attenuated autophagy in the hearts of MIRI rats. This suggests that a reduction in m6A methylation is involved in autophagy alleviation. CONCLUSION: We demonstrated that SJP mitigates autophagy in MIRI by downregulating miR-193a-3p, enhancing ALKBH5 expression, and reducing m6A methylation, a mechanism potentially attributed to its constituents, senkyunolide A and l-borneol.

Suxiao Jiuxin Pill attenuates acute myocardial ischemia via regulation of coronary artery tone.

Suxiao Jiuxin Pill (SJP) is a well-known traditional Chinese medicine drug used to manage heart diseases. This study aimed at determining the pharmacological effects of SJP in acute myocardial infarction (AMI), and the molecular pathways its active compounds target to induce coronary artery vasorelaxation. Using the AMI rat model, SJP improved cardiac function and elevated ST segment. LC-MS and GC-MS detected twenty-eight non-volatile compounds and eleven volatile compounds in sera from SJP-treated rats. Network pharmacology analysis revealed eNOS and PTGS2 as the key drug targets. Indeed, SJP induced coronary artery relaxation via activation of the eNOS-NO pathway. Several of SJP's main compounds, like senkyunolide A, scopoletin, and borneol, caused concentration-dependent coronary artery relaxation. Senkyunolide A and scopoletin increased eNOS and Akt phosphorylation in human umbilical vein endothelial cells (HUVECs). Molecular docking and surface plasmon resonance (SPR) revealed an interaction between senkynolide A/scopoletin and Akt. Vasodilation caused by senkyunolide A and scopoletin was inhibited by uprosertib (Akt inhibitor) and eNOS/sGC/PKG axis inhibitors. This suggests that senkyunolide A and scopoletin relax coronary arteries through the Akt-eNOS-NO pathway. In addition, borneol induced endothelium-independent vasorelaxation of the coronary artery. The Kv channel inhibitor 4-AP, KCa2+ inhibitor TEA, and Kir inhibitor BaCl2 significantly inhibited the vasorelaxant effect of borneol in the coronary artery. In conclusion, the results show that Suxiao Jiuxin Pill protects the heart against acute myocardial infarction.

Microbiome-metabolome reveals that the Suxiao Jiuxin pill attenuates acute myocardial infarction associated with fatty acid metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: The Suxiao Jiuxin pill (SJP) is a Chinese medical patent drug on the national essential drug list of China, with well-established cardiovascular protective effects in the clinic. However, the mechanisms underlying the protective effects of SJP on cardiovascular disease have not yet been elucidated clearly, especially its relationship with the gut microbiota. AIM OF THE STUDY: This study aimed to investigate the cardioprotective effect of SJP against isoproterenol-induced acute myocardial infarction (AMI) by integrating the gut microbiome and metabolome. METHODS: A rat model of AMI was generated using isoproterenol. Firstly, the effect of antibiotic (ABX) treatment on the blood absorption and excretion of the main components of SJP were studied. Secondly, 16S rRNA sequencing and untargeted metabolomics were used to discover the improvement of SJP treatment on gut microbiota and host metabolism in AMI rats. Finally, targeted metabolomics was used to verify the effects of SJP treatment on host metabolism in AMI rats. RESULT: The results showed that ABX treatment could affect the blood absorption and fecal excretion of the main active components of SJP. At the same time, SJP can restore the richness and diversity of gut microbiota, and multiple gut microbiota (including Jeotgalicoccus, Lachnospiraceae, and Blautia) are significantly associated with fatty acids. Untargeted metabolomics also found that SJP could restore the levels of various fatty acid metabolites in serum and cecal contents (p < 0.01, FC > 1.5 and VIP >1). Targeted metabolomics further confirmed that 41, 21, and 39 fatty acids were significantly altered in serum, cecal contents, and heart samples, respectively. Interestingly, these fatty acids belong to the class of eicosanoids, and SJP can significantly downregulate these eicosanoids in AMI rats. CONCLUSION: The results of this study suggest that SJP may exert its cardioprotective effects by remodeling the gut microbiota and host fatty acid metabolism.

Suxiao Jiuxin Pill alleviates myocardial ischemia-reperfusion injury through the ALKBH5/GSK3ß/mTOR pathway.

BACKGROUND: Many studies have shown effective protection from myocardial ischemia-reperfusion injury (MIRI) in animal models, but few, if any, treatments have yielded a substantial reduction in clinical. Several studies showed significant therapeutic effects for the Chinese patent medicine Suxiao Jiuxin Pill (SJP) in MIRI, although the specific molecular mechanisms remain undefined. Recently, increasing evidence indicates an important role for m6A modification in autophagy regulation in MIRI, and SJP has not been investigated in this regard. METHODS: In vivo experiments were performed in a Wistar rat MIRI model. In vitro assays were conducted in hypoxia/reoxygenation (H/R)-treated H9c2 cells. H9c2 cells with ALKBH5 and GSK3ß silencing were constructed by lentivirus transfection. TUNEL and Annexin V/PI assays were carried out for apoptosis detection. Then, m6A modification was detected with the EpiQuik m6A RNA methylation quantification kit, and GFP-RFP-LC3B was used to observe dynamic changes in autophagy. The autophagosome structure was assessed by Transmission electron microscopy. qPCR and immunoblot were performed for mRNA and protein analyses, receptively. RESULTS: SJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3ß/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3ß/mTOR pathway. Additionally, SJP enhanced ALKBH5 expression in H/R cardiomyocytes, which is important in impaired m6A modification. Interestingly, ALKBH5 knockdown enhanced autophagy and apoptosis in H/R-induced cells, whereas SJP reversed these effects. Further experiments showed that autophagic activity and apoptosis enhanced by ALKBH5 deficiency are GSK3ß/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3ß/mTOR pathway in H/R-induced cardiomyocytes. These effects of SJP were common to its two main constituents, including tetra-methylpyrazine (TMP) and borneol (BOR). CONCLUSION: SJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3ß/mTOR pathway, thanks to its two major constituents TMP and BOR.

Serum proteomic analysis reveals the cardioprotective effects of Shexiang Baoxin Pill and Suxiao Jiuxin Pill in a rat model of acute myocardial infarction.

ETHNOPHARMACOLOGICAL RELEVANCE: Shexiang Baoxin Pill (SBP) and Suxiao Jiuxin Pill (SJP) are traditional Chinese medicines used to treat cardiovascular disease (CVD) in China. However, the mechanism of their therapeutic effect on CVD has not been clearly elucidated yet. AIMS: The aim of this study is to investigate the cardioprotective effect of SBP and SJP in the treatment of acute myocardial infarction (AMI) model rats by applying serum proteomic approach. MATERIALS AND METHODS: The rat model of AMI was generated by ligating the left anterior descending coronary artery. 42 rats were randomly divided into four groups: sham-operating (Sham, n = 10) group, model (Mod, n = 8) group, Shexiang Baoxin pills pretreatment (SBP, n = 12) group and Suxiao Jiuxin pills pretreatment (SJP, n = 12) group. Data Independent Acquisition (DIA) proteomic approach was utilized to investigate the serum proteome from the rat individuals. The differentially expressed proteins were subsequently obtained with bioinformatic analysis. RESULTS: DIA-MS identified 415 proteins within 42 samples, and 84 differentially expressed proteins may contribute to the therapeutic effects of SBP and SJP. GOBP and KEGG pathway analysis of 84 differentially expressed proteins revealed that the proteins were mainly involved in platelet activation and adhesion processes. All 84 differentially expressed proteins presented the same changing tendency in the SBP and SJP groups when compared with the Mod group. Among these 84 proteins, 25 proteins were found to be related to CVD. Among these 25 proteins, ACTB, ACTG1, FGA, FGB, FGG, PF4 and VWF were found to be involved in platelet aggregation and activation. FN1, HSPA5 and YWHAZ were associated with adhesion. CONCLUSIONS: The results of our study suggest that the cardioprotective effects of SBP and SJP are achieved through the modulation of focal adhesion, platelet activation pathways.

Evaluation of potential herb-drug interactions based on the effect of Suxiao Jiuxin Pill on CYP450 enzymes and transporters.

ETHNOPHARMACOLOGY RELEVANCE: Suxiao jiuxin pill (SJP) is a Chinese medical drug with anti-inflammatory, anti-apoptotic, and vasodilatory function. It is widely used in combination with other drugs for the treatment of coronary heart disease (CHD) and angina. Nevertheless, the effect of SJP on Cytochrome P450 (CYP450) enzymes and transporters' activity related to drug metabolism is rarely studied. OBJECTIVE: The aim of this study was to investigate the effect of SJP on the activity of drug-metabolizing enzyme CYP450 and transporters. MATERIALS AND METHODS: Human primary hepatocytes were used in present study. Probe substrates of CYP450 enzymes were incubated in human liver microsomes (HLMs) with and without SJP while IC50 values were calculated. The inhibitory effect of SJP on the activity of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 was evaluated. The inducing effect of SJP on the activity of CYP1A2, 2B6 and 3A4 was accessed. The inhibition of SJP on human OATP1B1 was investigated through cell-based assay. The inhibition of SJP on human MDR1 and BCRP was also estimated by means of the vesicles assay. RESULTS: The results showed that the SJP under the concentration of 1000 µg/mL could inhibit the activity of CYP1A2, 2B6, 2C19, and 3A4, with IC50 values of 189.7, 308.2, 331.2 and 805.7 µg/mL, respectively. There was no inhibitory effect found in the other 3 liver drug enzyme subtypes. In addition, SJP showed no induction effect on CYP1A2, 2B6 and 3A4, however it had a significant inhibitory effect on human-derived OATP1B1 at the concentration of 100 and 1000 µg/mL, with the IC50 value of 21.9 µg/mL. Simultaneously, the SJP inhibited BCRP at high concentration of 1000 µg/mL but did not affect human MDR1. CONCLUSIONS: Based on these research results above, it is suggested that the SJP can affect some of the CYP450 enzymes and transporters' activity. When used in combination with related conventional drugs, potential herb-drug interactions should be considered.

Efficacy and safety of Suxiao Jiuxin Pill in the treatment of stable angina (Qi stagnation and blood stasis syndrome): study protocol of a randomized, double-blind, placebo-controlled, multi-center clinical trial.

BACKGROUND: Coronary heart disease (CHD) has become one of the biggest health problems in the world. Stable angina is a common clinical type of CHD with poor prognosis and high mortality. Although there are various interventions for stable angina, none of them can significantly reduce mortality. Both basic and clinical research have shown that Suxiao Jiuxin Pill (SJP) can relieve the symptoms of angina pectoris and improve the clinical efficacy, but there is a lack of high-quality clinical research to provide research-based evidence. We design a randomized, double-blind, placebo-controlled trial to evaluate the efficacy of SJP for stable angina. METHODS/DESIGN: This is a prospective, randomized, double-blind, placebo-controlled, and multicenter trial. The trial will enroll 324 participants with chronic stable angina (Qi Stagnation and Blood Stasis syndrome). All participants will have received the conventional therapy of chronic stable angina. Participants will be randomized into two groups, conventional therapy plus SJP group and conventional therapy plus placebo group. Eligible participants will receive either SJP or placebo (five pills administered orally, three times daily) in addition to conventional treatment for 24 weeks. The primary outcomes are the symptom improvement rate of angina from baseline to 4 weeks after inclusion and major adverse cardiovascular events (MACE). The secondary outcomes are angina classification (CCS), improvement of traditional Chinese medicine (TCM) syndromes, Seattle Angina Scale score, the dosage of emergency drugs and the stopping rate, and electrocardiogram (EKG) efficacy. Adverse events will be monitored throughout the trial. DISCUSSION: Integrated traditional Chinese and Western Medicine is commonly used for angina in China. This study will evaluate the clinical effectiveness and safety of SJP for angina. The results of the trial will provide high-level clinical research-based evidence for the application of SJP instable angina. TRIAL REGISTRATION: This study protocol was registered on 14 March 2019. The registration number is ChiCTR1900021876 on the Chinese Clinical Trial Registry.

Effects of the Suxiao Jiuxin pill on acute myocardial infarction assessed by comprehensive metabolomics.

BACKGROUND: SJP is the commercial Chinese medicine included in the Chinese Pharmacopoeia, with well-established cardiovascular protective effects in the clinic. However, the mechanisms underlying the protective effects of SJP on cardiovascular disease have not yet been clearly elucidated. AIMS: To investigate the underlying protective mechanisms of SJP in an acute myocardial infarction (AMI) rat model using comprehensive metabolomics. MATERIALS AND METHODS: The rat model of AMI was generated by ligating the left anterior descending coronary artery. After 2 weeks treatment with SJP, the entire metabolic changes in the serum, heart, urine and feces of the rat were profiled by HPLC-QTOF-MS/MS. RESULTS: The metabolic profiles in different biological samples (heart, serum, urine and feces) were significantly different among groups, in which a total of 112 metabolites were identified. AMI caused comprehensive metabolic changes in amino acid metabolism, galactose metabolism and fatty acid metabolism, while SJP reversed more than half of the differential metabolic changes, mainly affecting amino acid metabolism and fatty acid metabolism. Correlation analysis found that SJP could significantly alter the metabolic activity of 12 key metabolites, regarded as potential biomarkers of SJP treatment. According to the results of network analysis, 6 biomarkers were considered to be hub metabolites, which means that these metabolites may have a major relationship with the SJP therapeutic effects on AMI. CONCLUSION: The combined comprehensive metabolomics and network analysis, indicated that the protective effect of SJP on cardiovascular disease was associated with systemic metabolic modulation, in particular regulation of amino acid and fatty acid metabolism.

Effects on Suxiao Jiuxin Pills in the Treatment of Patients with Acute Coronary Syndrome Undergoing Early Percutaneous Coronary Intervention: A Multicenter Randomized Double-Blind Placebo-Controlled Trial.

Objectives: Acute coronary syndrome (ACS) is an acute disease with high mortality. Although early percutaneous coronary intervention (PCI) is proved to be the practical approach in treating ACS, the incidence of cardiovascular events is still far from satisfactory. The combination of Suxiao Jiuxin Pill (SJP) and Western medicine is one conventional approach in the treatment of ACS. Many elementary and clinical trials have proved the efficacy and safety in the improvement of cardiocerebral vascular conditions. The aim of this project is to evaluate the safety and efficacy of SJP on ACS with early PCI patients. Trial Design: This is a multicenter randomized, double-blind placebo-controlled trial. Trial registration: ChiCTR-TRC-13003053. Settings: Hospitals. Subjects: A total of 200 ACS with early PCI patients were randomly divided into SJP group (n = 100) and placebo group (n = 100). Interventions: The SJP group was treated with routine treatment and SJP (taking eight SJP pills orally each time, three times per day). The placebo group was treated with routine treatment along with equal amounts of SJP placebo. The course of treatment was 6 months and a follow-up visit at 12 months. Outcome Measures: Assessments of major adverse cardiovascular events (MACE), safety assessments, adverse events, left ventricular systolic function (LVEF), Seattle angina questionnaire (SAQ), troponin C (cTNI), C-reactive protein (CRP), fibrinogen (Fib), and cystatin C (cysC). Results: The SJP group had a relatively low incidence of MACE than the placebo (p < 0.05, odds ratio: 1.916, 95% confidence interval [0.999-3.674]). LVEF was significantly higher in the SJP group than the placebo group on the 360-day follow-up (p < 0.01). SJP had a significant increase score in the SAQ subscale of physical limitation, angina frequency, and treatment satisfaction (p < 0.05). There is no significant difference in the cTNI and CRP level between the two groups. The serum concentration of Fib and cysC in the SJP was significantly decreased compared with the placebo (p < 0.05). The numbers of adverse events between the two groups were not statistically different (p > 0.05). Conclusions: SJP is associated with a reduction in MACE, and an improvement of heart function and quality of life in ACS patients with early PCI, and is probably safe to use.

Phthalides, senkyunolide A and ligustilide, show immunomodulatory effect in improving atherosclerosis, through inhibiting AP-1 and NF-κB expression.

Atherosclerosis is initiated by the local inflammation response to lipid deposition, and the most commonly administered antiatherogenic drugs are statins. Based on traditional Chinese medicine (TCM) evidence, we aimed to find effective therapeutic agents other than statins. A TCM, Suxiao Jiuxin Pill (SX), has been widely used in curing cardiovascular diseases for thirty years. In this paper, a combination of pharmacologic studies and RNA-Seq transcriptomics were employed to explore the pharmacodynamic advantages of SX over atorvastatin in the ApoE-/- mouse. 113 differentially expressed genes that were modulated by SX to a greater degree than atorvastatin were primarily involved in immunomodulation. The expression of BTK, AKT1, c-jun and CD137 was effectively regulated by SX with better effect than atorvastatin. Then a dual-luciferase reporter assay for NF-κB inhibition was applied to identify active components in SX. As a result, Senkyunolide A (Sen A) and Ligustilide (Lig), the key immunomodulatory ingredients in SX, were found to inhibit the expression of CD137 which is a diagnostic biomarker in atherosclerosis. It was further confirmed that Lig effectively suppressed the expression of AP-1 and NF-κB and the phosphorylation of AKT. Therefore, Lig achieved its CD137 inhibition through suppressing the expression of AP-1 and AKT/NF-κB signaling pathway, which partly explains the immunomodulation of SX in atherosclerosis. Above all, phthalides may be the primary components of SX improving immune and inflammation response in atherosclerosis.

Searching for synergistic calcium antagonists and novel therapeutic regimens for coronary heart disease therapy from a Traditional Chinese Medicine, Suxiao Jiuxin Pill.

Coronary heart disease is a vital cause of morbidity and mortality worldwide, and calcium channel blockers (CCBs) are important drugs that can be used to treat cardiovascular diseases. Suxiao Jiuxin Pill (SX), a traditional Chinese medicine, is widely used as an emergency drug for coronary heart disease therapy. However, understanding its potential mechanism in intracellular calcium concentration ([Ca2+]i) modulation remains a challenge. To identify the active pharmacological ingredients (APIs) and reveal a novel combination therapy for ameliorating cardiovascular diseases, the ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) combined with a dual-luciferase reporter [Ca2+]i assay system was applied. Ligustrazine, ferulic acid, senkyunolide I, senkyunolide A and ligustilide were identified as potential calcium antagonists in SX, and the combination of ligustrazine and senkyunolide A showed synergetic calcium antagonistic activity. Additionally, the synergetic mechanism was further investigated by live-imaging analysis with the Ca2+ indicator fluo-4/AM by monitoring fluorescence changes. Our results indicated that ligustrazine can block voltage-operated Ca2+ channels (VDCCs) effectively and senkyunolide A can exert an inhibition effect mostly on ryanodine receptors (RYRs) and partly on VDCCs. Finally, an arterial ring assay showed that the combination of ligustrazine and senkyunolide A exerted a better vasodilatation function than using any components alone. In this study, we first revealed that a pair of natural APIs in combination acting on VDCCs and RYRs was more effective on vasodilatation by regulating [Ca2+]i.

Anti-apoptotic effect of Suxiao Jiuxin Pills against hypoxia-induced injury through PI3K/Akt/GSK3ß pathway in HL-1 cardiomyocytes.

BACKGROUND: Suxiao Jiuxin Pill (SX), Chinese traditional medicine primarily consisting of tetramethylpyrazine and borneol, has been shown to protect against ischemic heart diseases. Nevertheless, the involved mechanism still remains unclear. The following study aimed to investigate the potential protective effect and molecular mechanisms of SX on apoptosis in HL-1 cardiomyocytes. METHODS: Simulated hypoxia was established by culturing HL-1 cardiomyocytes in DMEM with no glucose or serum in a hypoxic chamber with 95% N2 and 5% CO2 for 24 h. HL-1 cardiomyocytes were divided into 5 groups: control, hypoxic injury, hypoxic injury + insulin (PI3K agonist, 10 µM), hypoxic injury + SX (100 µg/mL), and hypoxic injury + SX + LY294002 (PI3K inhibitor, 10 µM) (n = 3 wells/group). The anti-apoptotic effect of SX was evaluated by Annexin V/PI analysis. Mitochondrial membrane potential (ΔΨm) was detected by JC-1 assay. The protein expression of PI3K, phosphorylated PI3K (p-PI3K), Akt, phosphorylated Akt (p-Akt), GSK3ß and phosphorylated GSK3ß (p-GSK3ß) were detected by western blot. RESULTS: SX exhibited anti-apoptotic effect in HL-1 cardiomyocytes; nonetheless, the effect was blocked by PI3K inhibitor LY294002. Also, the anti-apoptotic effect of SX was mediated by increased mitochondrial membrane potential (ΔΨm). Furthermore, p-PI3K, p-Akt, and p-GSK3ß expressions were significantly increased after SX treatment, while they were all reduced after administration of LY294002. CONCLUSION: SX protects HL-1 cardiomyocytes from apoptosis induced by hypoxia, partly through enhancing the phosphorylation of PI3K/Akt/GSK3ß signaling pathway.

Suxiao Jiuxin Pill protects cardiomyocytes against mitochondrial injury and alters gene expression during ischemic injury.

Suxiao Jiuxin Pill (SX), a traditional Chinese medicine compound consisting primarily of tetramethylpyrazine and borneol, has been reported to protect against ischemic heart disease. However, the effects of SX on mitochondrial injury and gene expression in various signaling pathways are unclear. The aim of the present study was to investigate the effects of SX on mitochondrial injury and to screen the expression of genes potentially altered by SX using a cell culture model of ischemic injury. Simulated ischemia was established by culturing HL-1 cardiomyocytes in Dulbecco's modified Eagle medium without glucose or serum in a hypoxic chamber containing 95% N2 and 5% CO2 for 24 h. HL-1 cardiomyocytes were divided into 3 groups: Control, ischemic injury and ischemic injury + SX (100 µg/ml; n=3 wells/group). Mitochondrial membrane potential was detected by staining with JC-1 dye. The mRNA expression levels of adenylyl cyclase (Adcy) 1-9, adrenoceptor ß1, Akt1, ATPase Na+/K+ transporting subunit ß2, calcium voltage-gated channel auxiliary subunit α2δ (Cacna2d)2, Cacna2d3, calcium channel voltage-dependent γ subunit 8, cytochrome C oxidase subunit 6A2 (Cox6a2), fibroblast growth factor receptor (Fgfr) 4, Fgf8, Fgf12, Gnas complex locus, glycogen synthase kinase 3ß (Gsk3b), mitogen-activated protein kinase (Mapk)11-14, Mapk kinase kinase kinase 1 (Map4k1), Mas1, nitric oxide synthase 3 (Nos3), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (Pik3ca), phospholipase A2 group 4A, rap guanine nucleotide exchange factor 4 and ryanodine receptor 2 were detected using reverse transcription-quantitative polymerase chain reaction. The protein expression levels of phosphoinositide 3-kinase (PI3K), MAS-1 and phosphorylated-endothelial NOS were also examined by immunofluorescence staining. The decrease in mitochondrial membrane potential in the cell culture model of ischemic injury (P<0.001) was significantly attenuated by SX treatment (P<0.001). Furthermore, increases in the mRNA expression levels of Adcy2 (P<0.05), 3 (P<0.01) and 8 (P<0.05) in the ischemic injury model were significantly attenuated by SX treatment (P<0.01), and SX treatment significantly decreased the mRNA expression levels of Adcy1 (P<0.01) and 6 (P<0.05) in ischemic cells. Decreases in the mRNA expression levels of Cox6a2 (P<0.001), Gsk3b (P<0.01) and Pik3ca (P<0.001) in the ischemic injury model were also significantly attenuated by SX treatment (P<0.05, P<0.01 and P<0.001, respectively). In addition, the decrease in the protein expression of PI3K (P<0.001) was significantly attenuated by SX treatment (P<0.001). The present findings indicate that SX may protect cardiomyocytes against mitochondrial injury and attenuate alterations in the gene expression of Adcy2, 3 and 8, Cox6a2, Gsk3b and Pik3ca during ischemic injury.

Study on anti-atherosclerotic effect of Suxiao Jiuxin Pill and its mechanism.

BACKGROUND: Suxiao Jiuxin Pill is composed of Ligusticum wallichii, Borneolum Syntheticum and other drugs; it has qi promoting and blood circulation activating, meridian dredging and pain relieving efficacies. The objective of this paper is to study the effect of Suxiao Jiuxin Pill (quick-acting heart reliever), in atherosclerosis (AS) rat model and explore the mechanism for its prevention and treatment of AS. MATERIALS AND METHODS: AS rat model was established by high cholesterol diet and single intra-peritoneal injection of increased dose of vitamin D3. RESULTS: Compared with the model group, Suxiao Jiuxin Pill medium-and high-dose groups and atorvastatin group can effectively regulate lipid metabolism. CONCLUSION: We conclude that Suxiao Jiuxin Pill has a good hypo-lipidemic effect, and can inhibit the occurrence and development of AS.