Suxiao Jiuxin Pill Research Articles

The efficacy of Chinese herbal drugs for adults with angina pectoris: Bayesian network meta-analysis of 331 RCTs involving 36,467 individuals

Ethnopharmacological relevanceHundreds of randomized controlled trials (RCT) on Chinese herbal drugs (CHDs) including Shexiang baoxin pill (BXP), compound Danshen dripping pill (DSP), compound Danshen tablet (DST), Suxiao jiuxin pill (JXP), Naoxintong capsule (NXT), Tongxinluo capsule (TXL), and Di'ao xinxuekang capsule (XXK) and conventional chemical drugs, such as isosorbide dinitrate (ISDN), for angina pectoris are available but have not been evaluated by a PRISMA-compliant network meta-analysis (NMA). Aim of the studyThis study aimed to compare the efficacy of nine anti-anginal drugs through NMA on RCTs. MethodsRCTs of drug treatment for adult patients with angina pectoris for improvements in symptoms and electrocardiography were retrieved. Odds ratios and 95% credible intervals were computed to measure effect sizes. RCT quality was evaluated with the Cochrane risk of bias tool. Evidence synthesis was performed with Bayesian NMA. Essential analyses including subgroup analysis, sensitivity analysis, meta-regression analysis, publication bias analysis, and ranking analysis were conducted to assess the robustness of efficacies. Evidence strength was assessed with the GRADE approach. ResultsA total of 331 RCTs with 36,467 participants were eligible. The overall quality of all included RCTs was low. Overall efficacy estimates from different approaches of evidential synthesis found that BXP, TXL, and DSP were more efficacious than DST and ISDN. Essential analyses indicated consistent efficacy estimates, insignificant publication bias, and corroborative ranking results. The overall GRADE evidence strength was low. ConclusionThis comprehensive Bayesian NMA found BXP, TXL, and DSP to be the top three candidates among the seven tested CHDs for treating adults suffering from angina pectoris. However, the quality and the evidence strength of eligible RCTs were low. Further high-quality RCTs with more outcome measures and their NMAs are warranted. RegistrationPROSPERO CRD42014007035.

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

BackgroundMyocardial 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. PurposeThis 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. MethodsAn 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. ResultsSJP 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. ConclusionWe 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.

A pharmacokinetic study on twenty-one compounds in rat plasma by integrating UPLC-QQQ-MS/MS with GC-MS after oral administration of Suxiao Jiuxin pill

Suxiao Jiuxin pill (SX) was a commonly used traditional Chinese medical preparation for treating acute myocardial infarction. However, fewer studies have been reported for comprehensively investigating the pharmacokinetic of the non-volatile and volatile chemical constituents in SX. For current study, a comprehensive and sensitive quantitation of twenty-one compounds in rat plasma was developed and validated by integrating UPLC-QQQ-MS/MS with GC-MS after oral administration of SX. The pharmacokinetic study was performed under acceptable specificity, linearity, sensitivity, precision and accuracy, recovery, matrix effect and stability. Ultimately, borneol, isoborneol, senkyunolide A, senkyunolide Ⅰ, ligustilide, ferulic acid, neocnidilide, senkyunolide H and levistolide A were detected. Their ranges of Tmax, Cmax, T1/2z and AUC (0-∞) were 0.07 ∼ 0.33 h, 5.24 ∼ 683.35 μg/L, 0.29 ∼ 1.08 h and 6.55 ∼ 474.62 μg/L*h, respectively. This study would be meaningful in better revealing the therapeutic material basis of SX. Furthermore, it could provide reference on pharmacological effect of SX against acute myocardial infarction.

Evaluation of the efficacy and safety of Suxiao Jiuxin Pill in the treatment of stable angina: A randomized, double-blind, placebo-controlled, multi-center clinical trial

Ethnopharmacological relevanceThe Suxiao Jiuxin Pill (SJP) has been used for treating chronic stable angina (SA) for more than 40 years in China. SJP is composed of two Chinese herbs and has the effect of activating blood and promoting qi, according to traditional Chinese medicine (TCM) theory. Aim of the studyThe study aims to determine the effects of adjunct SJP on conventional therapy in patients with SA which provides a complementary choice and its evidence for clinical medication for treating SA. Materials and methodsParticipants with SA were recruited and randomized 1:1 to either the SJP group or the control group for 24 weeks. Both groups received conventional treatment according to local tertiary hospital protocols, and the participants received additional SJP (composed of Ligusticum wallichii and Borneol) or placebo in treatment and control groups respectively. The primary outcome was the curative efficacy rate at week 4. Secondary outcomes are the curative efficacy rate, the total score of angina pectoris symptoms, CCS Angina Classification improvement, Seattle Angina Questionnaire (SAQ) score, TCM syndrome scores (TCMSS), and the curative efficacy rate of TCMSS. Adverse events and adverse drug reactions were observed and recorded for safety analysis. ResultsA total of 324 participants with SA from 13 hospitals in China were enrolled in this trial. Compared with the control group, the curative efficacy rate of SA, the curative efficacy rate of TCMSS significantly increased, and the total score of angina pectoris symptoms and TCMSS significantly reduced in the SJP group at week 4, 12, and 24, accompanied by the statistically significant improvement in the curative efficacy rate based on CCS grade reduction (all P < 0.05). Furthermore, the SAQ score (physical limitation, angina stability, and treatment satisfaction) was evaluated as the quality of life significantly improved after treatment (P < 0.05). The medication compliance, concomitant medication, and rates of adverse events were similar between the two groups (P > 0.05). ConclusionThe present prospective, multicenter, randomized, double-blind, placebo-controlled, clinical trial confirms that adjunct SJP to conventional treatment increased the curative efficacy and life quality of SA patients with no significant adverse drug reactions during the clinical application. Clinical trial registration(ID, ChiCTR1900021876, URL = http://www.chictr.org.cn/showproj.aspx?proj=34955).

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 relevanceThe 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 studyThis study aimed to investigate the cardioprotective effect of SJP against isoproterenol-induced acute myocardial infarction (AMI) by integrating the gut microbiome and metabolome. MethodsA 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. ResultThe 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. ConclusionThe 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/GSK3β/mTOR pathway

BackgroundMany 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.MethodsIn 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 GSK3β 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.ResultsSJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3β/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3β/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 GSK3β/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3β/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).ConclusionSJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3β/mTOR pathway, thanks to its two major constituents TMP and BOR.

CaMKII, that binds with ligustilide, as a potential drug target of Suxiao jiuxin pill, a traditional Chinese medicine to dilate thoracic aorta

CaMKII, that binds with ligustilide, as a potential drug target of Suxiao jiuxin pill, a traditional Chinese medicine to dilate thoracic aorta

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

Ethnopharmacological relevanceShexiang 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. AimsThe 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 methodsThe 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. ResultsDIA-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. ConclusionsThe results of our study suggest that the cardioprotective effects of SBP and SJP are achieved through the modulation of focal adhesion, platelet activation pathways.

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

BackgroundCoronary 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/designThis 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.DiscussionIntegrated 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 registrationThis study protocol was registered on 14 March 2019. The registration number is ChiCTR1900021876 on the Chinese Clinical Trial Registry.

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

Ethnopharmacology relevanceSuxiao 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. ObjectiveThe aim of this study was to investigate the effect of SJP on the activity of drug-metabolizing enzyme CYP450 and transporters. Materials and methodsHuman 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. ResultsThe 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. ConclusionsBased 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.

Effectiveness and safety of Suxiao Jiuxin pill in treating acute coronary syndrome: a systematic review and Meta-analysis.

To evaluate the effectiveness and safety of Suxiao Jiuxin pill (SX) in acute coronary syndrome (ACS) treatment. An extensive search of four English databases (Medline/PubMed, Cochrane Library, Embase, and World Health Organization International Clinical Trials Registration Platform) and four Chinese databases (Chinese National Knowledge Infrastructure, Wanfang, China Science and Technology Journal, and Chinese Biomedical Literature Service System) was performed. Randomized, controlled trials (RCTs) involving SX combined with conventional therapy versus conventional therapy were included. The extracted data included populations, interventions, outcomes, and risk of bias. The cardiovascular events served as the primary outcome. Review Manager 5.3 software was used for data analysis. Relative risks (RRs) with 95% confidence intervals (CIs) were the effect measure. A total of eight RCTs with 979 patients were included. There were 559 patients with unstable angina (UA) in six RCTs and 420 patients with acute myocardial infarction (AMI) in two RCTs. Our review showed that SX plus conventional therapy might reduce the incidence of the total endpoint (RR: 0.34, 95% CI: 0.17, 0.68, P = 0.002), with no obvious adverse events (RR: 1.29, 95% CI: 0.60, 2.77, P = 0.52) compared with conventional therapy for patients with UA. Additionally, SX plus conventional therapy also reduced the incidence of the total endpoint (RR: 0.35, 95% CI: 0.18, 0.68, P = 0.002) compared with conventional therapy in patients with AMI. SX plus conventional therapy also reduced the incidence of ventricular fibrillation (RR: 0.23, 95% CI: 0.10, 0.57, P = 0.001) compared with conventional therapy in patients with AMI. Our results suggest that SX is beneficial for treating patients with UA or AMI. However, our findings should be treated with caution because of the poor methodological quality of the included trials. Therefore, more multicenter, large-sample, high-quality RCTs are required to provide high-quality evidence.

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

BackgroundSJP 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. AimsTo investigate the underlying protective mechanisms of SJP in an acute myocardial infarction (AMI) rat model using comprehensive metabolomics. Materials and methodsThe 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. ResultsThe 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. ConclusionThe 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.

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.

Ligustilide Targets SMAD3, Showing Immunomodulatory Effects in Improving Atherosclerosis

Backgrounds: Atherosclerosis is initiated by the local immune response to lipid deposition, and the most commonly administered antiatherogenic drugs are lipid regulators, particularly statins. However, statins appear to be weak in regulating immunity. Based on traditional ethnopharmacological evidence, in this paper, we aimed to find effective therapeutic agents other than statins and to elucidate their potential action mechanisms. Methods: A traditional Chinese Medicine (TCM), Suxiao Jiuxin Pill (SX) has been widely used in curing cardiovascular diseases for thirty years. With a combination of pharmacologic studies on atherosclerosis and RNA-Seq transcriptomics were employed to explore the pharmacodynamic differences of SX and atorvastatin in the ApoE-/- mouse. The active ingredients in SX, were screened by biomarker CD137, which is closely associated with anti-atherosclerosis. Then, chemical proteomics based on a ligustilide-derived photoaffinity probe were applied to identify potential targets and active mechanism for developing the therapeutic effects of SX. Findings: Differentially expressed genes (DEGs) that were modulated by SX to a greater degree than atorvastatin were primarily involved in immunomodulation, and secondarily in cholesterol export. Ligustlide was the active ingredient in SX and its potential target was SMAD3. Ligustilide suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the mutual interaction of SMAD3 and transforming growth factor-β (TGF-β) receptor І. Interpretation: Ligustilide suppresses local lipid accumulation and achieves immunomodulation by targeting SMAD3 and the TGF-β1/SMAD3 pathway, thereby improving atherosclerosis. Developing a novel SMAD3 inhibitor may be a therapeutic option for preventing atherosclerosis in combination therapy with statins Funding Statement: This work was financially supported by National Key Research and Development Program of China (No. 2018YFC1704805, 2018YFC1704505) and International Cooperation and Exchange of the National Natural Science Foundation of China (No. 81761168039). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All animal procedures conformed to the Council of Europe Convention Directive (2010/63/EU) for the protection of vertebrate animals used for scientific purposes. And all animal procedures were performed strictly according to the guidelines for the care and use of laboratory animals, which was approved by Tianjin University of Traditional Chinese Medicine (TCM-LAEC2016032).

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.

Suxiao Jiuxin pill promotes exosome secretion from mouse cardiac mesenchymal stem cells in vitro.

Cardiac mesenchymal stem cells (C-MSCs) are endogenous cardiac stromal cells that play a role in heart repair after injury. C-MSC-derived exosomes (Exo) have shown protective effects against apoptosis induced by acute myocardial ischemia/reperfusion. Suxiao Jiuxin pill (SJP) is a traditional Chinese medicine (TCM) formula used in China for the treatment of acute myocardial ischemia, which contains tetramethylpyrazine (TMP) and borneol (BOR) as major components. In this study, we investigated whether SJP treatment affected exosome release from C-MSCs in vitro. C-MSCs prepared from mice were treated with SJP (62.5 μg/mL), TMP (25 μg/mL) or BOR (15 μg/mL). Using an acetylcholinesterase activity assay, we found that both SJP and TMP treatment significantly increased exosome secretion compared to the control ethanol treatment. The neutral sphingomyelinase 2 (nSMase2) pathway was important in exosome formation and packaging. But neither the level of nSMase2 mRNA nor the level of protein changed following SJP, TMP or BOR treatment, suggesting that SJP stimulated exosome release via an nSMase2-independent pathway. The Rab27a and Rab27b GTPases controlled different steps of the exosome secretion pathway. We showed that SJP treatment significantly increased the protein levels of Rab27a, SYTL4 (Rab27a effector) and Rab27b compared with the control treatment. SJP treatment also significantly upregulated the mRNA level of Rab27b, rather than Rab27a. Moreover, SJP-induced increase of C-MSC-exosome release was inhibited by Rab27b knockdown, suggesting that SJP promotes exosome secretion from C-MSCs via a GTPase-dependent pathway. This study reveals a novel mechanism for SJP in modulating cardiac homeostasis.

Exosomes from Suxiao Jiuxin pill-treated cardiac mesenchymal stem cells decrease H3K27 demethylase UTX expression in mouse cardiomyocytes in vitro.

Suxiao Jiuxin pill (SJP) is a traditional Chinese medicine for the treatment of acute coronary syndrome in China, which contains two principal components, tetramethylpyrazine (TMP) and borneol (BOR). Thus far, however, the molecular mechanisms underlying the beneficial effects of SJP on the cardiac microenvironment are unknown. Cardiac mesenchymal stem cells (C-MSCs) communicate with cardiomyocytes (CMs) through the release of microvesicles (exosomes) to restore cardiac homeostasis and elicit repair, in part through epigenetic regulatory mechanisms. In this study, we examined whether SJP treatment altered C-MSC-derived exosomes (SJP-Exos) to cause epigenetic chromatic remodeling in recipient CMs. C-MSC isolated from mouse hearts were pretreated with SJP (SJP-Exos), TMP (TMP-Exos) or BOR (BOR-Exos). Then, HL-1 cells, a mouse cardiomyocyte line, were treated with exosomes from control C-MSCs (Ctrl-Exos), SJP-Exos, TMP-Exos or BOR-Exos. Treatment with SJP-Exos significantly increased the protein levels of histone 3 lysine 27 trimethylation (H3K27me3), a key epigenetic chromatin marker for cardiac transcriptional suppression, in the HL-1 cells. To further explore the mechanisms of SJP-Exo-mediated H3K27me3 upregulation, we assessed the mRNA expression levels of key histone methylases (EZH1, EZH2 and EED) and demethylases (JMJD3 and UTX) in the exosome-treated HL-1 cells. Treatment with SJP-Exo selectively suppressed UTX expression in the recipient HL-1 cells. Furthermore, PCNA, an endogenous marker of cell replication, was significantly higher in SJP-Exo-treated HL-1 cells than in Ctrl-Exo-treated HL-1 cells. These results show that SJP-Exos increase cardiomyocyte proliferation and demonstrate that SJP can modulate C-MSC-derived exosomes to cause epigenetic chromatin remodeling in recipient cardiomyocytes; consequently, SJP-Exos might be used to promote cardiomyocyte proliferation.

Efficacy of Suxiao Jiuxin Pill on Coronary Heart Disease: A Meta-Analysis of Randomized Controlled Trials.

Suxiao jiuxin pill is considered an effective ancillary drug in patients with coronary heart disease. Although numerous small, single-center clinical trials have been conducted, the benefits and harms of suxiao jiuxin pill remain controversial. We performed a meta-analysis to clarify the efficacy of suxiao jiuxin pill on patients with coronary heart disease. Randomized controlled trials were identified by using the Cochrane Library, PubMed, Web of Science, Embase, Wanfang, Weipu, and China Knowledge Resource Integrated databases (until June 2016). Pooled relative risks (RR), weighted mean differences (WMD), and 95% confidence intervals (95% CIs) were estimated using random-effects models. Forty-one trials involving 6276 patients were included in our analysis. Administration of suxiao jiuxin pill significantly improved electrocardiogram (ECG) results when compared with other therapies (RR 1.32, 95% CI 1.26 to 1.38, and P < 0.001). Subgroup analyses revealed that suxiao jiuxin pills improve ECG results more than salvia tablets (RR 1.54, 95% CI 1.41 to 1.67, and P < 0.001), isosorbide dinitrate (RR 1.14, 95% CI 1.21 to 1.44, and P = 0.001), nitroglycerin (RR 1.35, 95% CI 1.16 to 1.56, and P < 0.001), and other drugs (RR 1.32, 95% CI 1.21 to 1.44, and P < 0.001). Available evidence additionally suggests that suxiao jiuxin pills could significantly reduce total cholesterol (WMD −0.62 mmol/L, 95% CI −1.06 to –0.18 mmol/L, and P = 0.005) and low-density lipoprotein (LDL) levels (WMD −1.12 mmol/L, 95% CI −1.42 to −0.82 mmol/L, and P < 0.001) and increase high-density lipoprotein (HDL) levels (WMD 0.32 mmol/L, 95% CI 0.07 to 0.58 mmol/L, and P = 0.014). However, no significant differences were observed in total triglyceride levels, plasma viscosity, hematocrit, and fibrinogen. No incidences of adverse reactions were observed after administration of suxiao jiuxin pill. Improvements in ECG results and lipid profiles were also observed after suxiao jiuxin administration compared to other therapies. It also decreased low-cut and high-cut whole blood viscosity without significant adverse reactions.

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 β1, Akt1, ATPase Na+/K+ transporting subunit β2, 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 3β (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.