Inhibition of miR-128-3p by Tongxinluo Protects Human Cardiomyocytes from Ischemia/reperfusion Injury via Upregulation of p70s6k1/p-p70s6k1.

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism. Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 µg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection of HCMs with a hsa-miR-128-3p mimic eliminated the protective effects of TXL. Conclusions: The miR-128-3p/p70s6k1 signaling pathway is involved in protection by TXL against HCM apoptosis during H/R. Overexpression of p70s6k1 is, therefore, a potential new strategy for alleviating myocardial reperfusion injury.

Exosomes: promising sacks for treating ischemic heart disease?

Ischemic heart disease(IHD) is the leading cause of death worldwide. Despite the development of continuously improving therapeutic strategies, morbidity and mortality of patients with IHD remain relatively high. Exosomes are a subpopulation of vesicles that are universally recognized as major mediators in intercellular communication. Numerous preclinical studies have shown that these tiny vesicles were protective in IHD, through such actions as alleviating myocardial ischemia-reperfusion injury, promoting angiogenesis, inhibiting fibrosis, and facilitating cardiac regeneration. Our review focused on these beneficial exosome-mediated processes. In addition, we discuss in detail how to fully exploit the therapeutic potentials of exosomes in the field of IHD. Topics include identifying robust sources of exosomes, loading protective agents into exosomes, developing heart-specific exosomes, optimizing isolation methods, and translating the cardioprotective effects of exosomes into clinical practice. Finally, both the advantages and disadvantages of utilizing exosomes in clinical settings are addressed.

Quantitative Proteomics Analysis of Ischemia/Reperfusion Injury-Modulated Proteins in Cardiac Microvascular Endothelial Cells and the Protective Role of Tongxinluo.

BACKGROUND: The protection of endothelial cells (ECs) against reperfusion injury has received little attention. In this study, we used Tandem Mass Tag (TMT) labeling proteomics to investigate the modulated proteins in an in vitro model of cardiac microvascular endothelial cells (CMECs) subjected to ischemia/reperfusion (I/R) injury and their alteration by traditional Chinese medicine Tongxinluo (TXL). METHODS: Human CMECs were subjected to 2 h of hypoxia followed by 2 h of reoxygenation with different concentrations of TXL Protein expression profiles of CMECs were determined using tandem mass spectrometry. We evaluated several proteins with altered expression in I/R injury and summarized some reported proteins related to I/R injury. RESULTS: TXL dose-dependently decreased CMEC apoptosis, and the optimal concentration was 800 µg/mL. I/R significantly altered proteins in CMECs, and 30 different proteins were detected between a normal group and a hypoxia and serum deprivation group. In I/R injury, TXL treatment up-regulated 6 types of proteins including acyl-coenzyme A synthetase ACSM2B mitochondrial (ACSM2B), cyclin-dependent kinase inhibitor 1B (CDKN1B), heme oxygenase 1 (HMOX1), transcription factor SOX-17 (SOX17), sequestosome-1 isoform 1 (SQSTM1), and TBC1 domain family member 10B (TBC1D10B). Also, TXL down-regulated 5 proteins including angiopoietin-2 isoform c precursor (ANGPT2), cytochrome c oxidase assembly factor 5 (COA5), connective tissue growth factor precursor (CTGF), cathepsin L1 isoform 2 (CTSL), and eukaryotic elongation factor 2 kinase (LOC101930123). These types of proteins mainly had vital functions, including cell proliferation, stress response, and regulation of metabolic process. CONCLUSIONS: The study presented differential proteins upon I/R injury through a proteomic analysis. TXL modulated the expression of proteins in CMECs and has a protective role in response to I/R.

Tongxinluo exerts protective effects via anti-apoptotic and pro-autophagic mechanisms by activating AMPK pathway in infarcted rat hearts.

NEW FINDINGS: What is the central question of this study? In a rat model of acute myocardial infarction (AMI), we investigated the effect of Tongxinluo (TXL) treatment. Does TXL activate autophagy and attenuate apoptosis of cardiomyocytes through the AMPK pathway to facilitate survival of cardiomyocytes and improve cardiac function? What is the main finding and its importance? Major findings are as follows: (i) TXL treatment preserved cardiac function and reduced ventricular remodelling, infarct size and inflammation in rat hearts after AMI; (ii) TXL treatment dramatically increased autophagy and inhibited apoptosis in myocardium; and (iii) the AMPK signalling pathway played a crucial role in mediating the beneficial effects of TXL. Tongxinluo (TXL) has been demonstrated to have a protective role during ischaemia-reperfusion after acute myocardial infarction, but the long-term effects and underlying mechanisms are still unknown. The aim of this study was to investigate whether TXL could have an effect on apoptosis or autophagy of cardiomyocytes through the AMP-activated protein kinase (AMPK) pathway. Male Sprague-Dawley rats (n = 75) were randomly divided to sham, control, TXL (4 mg kg-1  day-1 orally), compound C (i.p. injection of 10 mg kg-1  day-1 ) and TXL + compound C groups. The extent of fibrosis, infarct size and angiogenesis were determined by pathological and histological studies. Four weeks after acute myocardial infarction, TXL treatment significantly increased ejection fraction, promoted angiogenesis in the peri-infarct region and substantially decreased fibrosis and the size of the infarcted area (P < 0.05). Treatment with TXL also increased AMPK/mTOR phosphorylation, upregulated expression of the autophagic protein LC3 and downregulated expression of the apoptotic protein Bax in the infarcted myocardium (P < 0.05). Addition of the AMPK inhibitor, compound C, counteracted these beneficial effects significantly (P < 0.05). The cardioprotective benefits of TXL against myocardial infarction are related to the inhibition of apoptosis and promotion of autophagy in rat hearts after acute myocardial infarction. This effect may occur through the AMPK signalling pathway.

Green tea consumption and risk of cardiovascular and ischemic related diseases: A meta-analysis.

BACKGROUND: The effects of green tea intake on risk of cardiovascular disease (CVD) have not been well-defined. The aim of this meta-analysis was to evaluate the association between green tea consumption, CVD, and ischemic related diseases. METHODS: All observational studies and randomized trials that were published through October 2014 and that examined the association between green tea consumption and risk of cardiovascular and ischemic related diseases as the primary outcome were included in this meta-analysis. The quality of the included studies was evaluated according to the Cochrane Handbook 5.0.2 quality evaluation criteria. RESULTS: A total of 9 studies including 259,267 individuals were included in the meta-analysis. The results showed that those who didn't consume green tea had higher risks of CVD (OR=1.19, 95% CI: 1.09-1.29), intracerebral hemorrhage (OR=1.24, 95% CI: 1.03-1.49), and cerebral infarction (OR=1.15, 95% CI: 1.01-1.30) compared to <1 cup green tea per day. Those who drank 1-3 cups of green tea per day had a reduced risk of myocardial infarction (OR=0.81, 95% CI: 0.67-0.98) and stroke (OR=0.64, 95% CI: 0.47-0.86) compared to those who drank <1 cup/day. Similarly, those who drank ≥4 cups/day had a reduced risk of myocardial infarction compared to those who drank <1 cup/day (OR=0.68, 95% CI: 0.56-0.84). Those who consumed ≥10 cups/day of green tea were also shown to have lower LDL compared to the <3 cups/day group (MD=-0.90, 95% CI: -0.95 to -0.85). CONCLUSIONS: Our meta-analysis provides evidence that consumption of green tea is associated with favorable outcomes with respect to risk of cardiovascular and ischemic related diseases.

Tongxinluo decreases apoptosis of mesenchymal stem cells concentration-dependently under hypoxia and serum deprivation conditions through the AMPK/eNOS pathway.

Tongxinluo (TXL), a traditional Chinese medicine, is widely used to treat cardiovascular diseases in China. Our previous study has demonstrated the pro-survival role of TXL on mesenchymal stem cells (MSCs) in vivo. But whether TXL could decrease apoptosis of MSCs in vitro, and the underlying mechanism are still unknown. Moreover, AMPK/eNOS pathway is crucial in regulating cell apoptosis. Therefore, we designed the study to investigate whether TXL could decrease MSCs apoptosis under hypoxia and serum deprivation (H/SD) conditions and to determine the role of AMPK/eNOS pathway. To test the hypothesis, MSCs were treated with TXL (50-400 µg/mL) under H/SD for 6 hours. For inhibitor studies, the cells were preincubated with AMPK inhibitor compound C. Results indicated that TXL decreased MSCs apoptosis concentration-dependently evidenced by reduced Annexin V+/PI- cells and increased red/green ratio of JC-1. Further, TXL enhanced the phosphorylation of AMPK and eNOS. Whereas, treatment with compound C decreased the phosphorylation of AMPK and eNOS and was accompanied by attenuated anti-apoptotic effect of TXL. In conclusion, TXL protected MSCs against H/SD-induced injury at least in part through the AMPK/eNOS pathway, which provides a novel explanation for the multi-effect of TXL on cardiovascular system.

Protein kinase A-mediated cardioprotection of Tongxinluo relates to the inhibition of myocardial inflammation, apoptosis, and edema in reperfused swine hearts.

BACKGROUND: Our previous studies have demonstrated that Tongxinluo (TXL), a traditional Chinese medicine, can protect hearts against no-reflow and reperfusion injury in a protein kinase A (PKA)-dependent manner. The present study was to investigate whether the PKA-mediated cardioprotection of TXL against no-reflow and reperfusion injury relates to the inhibition of myocardial inflammation, edema, and apoptosis. METHODS: In a 90-minute ischemia and 3-hour reperfusion model, minipigs were randomly assigned to sham, control, TXL (0.05 g/kg, gavaged one hour prior to ischemia), and TXL + H-89 (a PKA inhibitor, intravenously and continuously infused at 1.0 µg/kg per minute) groups. Myocardial no-reflow, necrosis, edema, and apoptosis were determined by pathological and histological studies. Myocardial activity of PKA and myeloperoxidase was measured by colorimetric method. The expression of PKA, phosphorylated cAMP response element-binding protein (p-CREB) (Ser(133)), tumor necrosis factor α (TNF-α), P-selectin, apoptotic proteins, and aquaporins was detected by Western blotting analysis. RESULTS: TXL decreased the no-reflow area by 37.4% and reduced the infarct size by 27.0% (P < 0.05). TXL pretreatment increased the PKA activity and the expression of Ser(133) p-CREB in the reflow and no-reflow myocardium (P < 0.05). TXL inhibited the ischemia-reperfusion-induced elevation of myeloperoxidase activities and the expression of TNF-α and P-selectin, reduced myocardial edema in the left ventricle and the reflow and no-reflow areas and the expression of aquaporin-4, -8, and -9, and decreased myocytes apoptosis by regulation of apoptotic protein expression in the reflow and no-reflow myocardium. However, addition of the PKA inhibitor H-89 counteracted these beneficial effects of TXL. CONCLUSION: PKA-mediated cardioprotection of TXL against no-reflow and reperfusion injury relates to the inhibition of myocardial inflammation, edema, and apoptosis in the reflow and no-reflow myocardium.

Resveratrol in cardiovascular disease: what is known from current research?

Resveratrol is a well-known antioxidant that exists in grape skin/seed, red wine, and the root of Polygonum cuspidatum, a traditional Chinese and Japanese medicinal material. Studies have found that resveratrol has many interesting properties, including anti-carcinogenic properties, anti-microbial and antiviral effects, the ability to reverse dyslipidemia and obesity, the ability to attenuate hyperglycemia and hyperinsulinemia, and the ability to protect endothelial function. Heart failure is the final consequence of the majority of cardiovascular diseases, and resveratrol has been shown to directly attenuate heart contraction. The cardiovascular protective capacities of resveratrol are associated with multiple molecular targets and may lead to the development of novel therapeutic strategies for atherosclerosis, ischemia/reperfusion, metabolic syndrome, and heart failure. This article will mainly review recently published basic researches about the protective cardiovascular effects of resveratrol because these results may lead to the development of new clinical therapeutics in patients.

No-reflow protection and long-term efficacy for acute myocardial infarction with Tongxinluo: a randomized double-blind placebo-controlled multicenter clinical trial (ENLEAT Trial).

BACKGROUND: No-reflow after emergency percutaneous coronary intervention (PCI) for acute ST segment elevation myocardial infarction (STEMI) is related to the severe prognosis. The aim of this study was to evaluate the efficacy of Tongxinluo, a traditional Chinese medicine, on no-reflow and the infarction area after emergency PCI for STEMI. METHODS: A total of 219 patients (female 31, 14%) undergoing emergency PCI for STEMI from nine clinical centers were consecutively enrolled in this randomized, double-blind, placebo-controlled, multicenter clinical trial from January 2007 to May 2009. All patients were randomly divided into Tongxinluo group (n = 108) and control group (n = 111), given Tongxinluo or placebo in loading dose 2.08 g respectively before emergency PCI with aspirin 300 mg and clopidogrel 300 mg together, then 1.04 g three times daily for six months after PCI. The ST segment elevation was recorded by electrocardiogram at hospitalization and 1, 2, 6, 12, 24 hours after coronary balloon dilation to evaluate the myocardial no-flow; myocardial perfusion scores of 17 segments were evaluated on day 7 and day 180 after STEMI with static single-photon emission computed tomography (SPECT) to determine the infarct area. RESULTS: There was no statistical significance in sex, age, past history, chest pain, onset-to-reperfusion time, Killip classification, TIMI flow grade just before and after PCI, either in the medication treatment during the follow up such as statin, ß-blocker, angiotensin converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) between two groups. There was significant ST segment restoration in Tongxinluo group compared to the control group at 6 hours ((-0.22 ± 0.18) mV vs. (-0.18 ± 0.16) mV, P = 0.0394), 12 hours ((-0.24 ± 0.18) mV vs. (-0.18 ± 0.15) mV, P = 0.0158) and 24 hours ((-0.27 ± 0.16) mV vs. (-0.20 ± 0.16) mV, P = 0.0021) reperfusion; and the incidence of myocardial no-reflow was also reduced significantly at 24-hour reperfusion (34.3% vs. 54.1%, P = 0.0031). The myocardial perfusion scores of 17 segments evaluated by static SPECT was improved significantly on day 7 and day 180 after STEMI in Tongxinluo group compared to the control group (0.61 ± 0.40 vs. 0.76 ± 0.42, P = 0.0109 and 0.51 ± 0.42 vs. 0.66 ± 0.43, P = 0.0115, respectively). There was no significant difference in severe adverse events between two groups. CONCLUSION: Tongxinluo as a kind of traditional Chinese medicine could reduce myocardial no-reflow and infarction area significantly after emergency PCI for STEMI with conventional medicine therapy.

Tongxinluo reduces myocardial no-reflow and ischemia-reperfusion injury by stimulating the phosphorylation of eNOS via the PKA pathway.

The objective of the present study was to investigate whether pretreatment with single low loading dose of tongxinluo (TXL), a traditional Chinese medicine, 1 h before myocardial ischemia could attenuate no-reflow and ischemia-reperfusion injury by regulating endothelial nitric oxide synthase (eNOS) via the PKA pathway. In a 90-min ischemia and 3-h reperfusion model, minipigs were randomly assigned to the following groups: sham, control, TXL (0.05 g/kg, gavaged 1 h before ischemia), TXL + H-89 (a PKA inhibitor, intravenously infused at a dose of 1.0 µg·kg(-1)·min(-1) 30 min before ischemia), and TXL + N(ω)-nitro-L-arginine (L-NNA; an eNOS inhibitor, intravenously administered at a dose of 10 mg/kg 30 min before ischemia). TXL decreased creatine kinase (CK) activity (P < 0.05) and reduced the no-reflow area from 48.6% to 9.5% and infarct size from 78.5% to 59.2% (P < 0.05), whereas these effects of TXL were partially abolished by H-89 and completely reversed by L-NNA. TXL elevated PKA activity and the expression of PKA, Thr(198) phosphorylated PKA, Ser(1179) phosphorylated eNOS, and Ser(635) phosphorylated eNOS in the ischemic myocardium. H-89 repressed the TXL-induced enhancement of PKA activity and phosphorylation of eNOS at Ser(635), and L-NNA counteracted the phosphorylation of eNOS at Ser(1179) and Ser(635) without an apparent influence on PKA activity. In conclusion, pretreatment with a single low loading dose of TXL 1 h before ischemia reduces myocardial no-reflow and ischemia-reperfusion injury by upregulating the phosphorylation of eNOS at Ser(1179) and Ser(635), and this effect is partially mediated by the PKA pathway.

[Effect of tongxinluo on mini-swine cytokines and myocardial no-reflow in early reperfusion of acute myocardial infarction].

OBJECTIVE: To assess the effect of Tongxinluo on cytokines and myocardial no-reflow in early reperfusion of acute myocardial infarction (AMI). METHODS: Forty mini-swine were divided into five groups randomly, sham group, control group, low dose (0.1 g/kg), medium dose (0.2 g/kg) and high dose (0. 4 g/kg) group of Tongxinluo (which were administered 2 h before reperfusion), eight swine in each group. Animals except those in the sham group were subjected to 1.5 h of coronary occlusion followed by 3 h of reperfusion. Serum contents of P-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), interleukin 6 (IL-6) and interleukin 10 (IL-10), as well as myocardial contrast echocardiography (MCE) were evaluated at baseline, and after 1.5 h of AMI and 3 h of reperfusion. RESULTS: (1) Compared with that of the control group, high dose of Tongxinluo could reduce serum contents of P-selectin and ICAM-1 at 1.5 h of AMI (all P<0.05), and P-selectin, ICAM-1, VCAM-1, and IL-6 at 3 h of reperfusion significantly (all P< 0.05), accompanied by significantly elevated IL-10 (P<0.05). (2) Compared with that of control group, high dose of Tongxinluo could reduce no-reflow area at 3 h of reperfusion significantly [(6.59 +/- 1.73) cm2 vs (4.68 +/- 1.53) cm2, P<0.05]. CONCLUSION: High dose of Tongxinluo could effectively reduce serum contents of adhesion and pro-inflammatory cytokines, regulate anti-inflammatory factor levels, and attenuate no-reflow area in the early reperfusion of AMI. It thus provided experimental basis for its clinical application.

Pretreatment with Tongxinluo protects porcine myocardium from ischaemia/reperfusion injury through a nitric oxide related mechanism.

BACKGROUND: The traditional Chinese medicine Tongxinluo can protect myocardium against ischaemia/reperfusion injury, but the mechanism of its action is not well documented. We examined the involvement of nitric oxide in the protective role of Tongxinluo. METHODS: Miniswine were randomized to four groups of seven: sham, control, Tongxinluo and Tongxinluo coadministration with a nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA, 10 mg/kg i.v.). Three hours after administration of Tongxinluo, the animals were anaesthetised and the left anterior descending coronary artery ligated and maintained in situ for 90 minutes followed by 3 hours of reperfusion before death. Area of no reflow and necrosis and risk region were determined pathologically by planimetry. The degree of neutrophil accumulation in myocardium was obtained by measuring myeloperoxidase activity and histological analysis. Myocardial endothelial nitric oxide synthase activity and vascular endothelial cadherin content were measured by colorimetric method and immunoblotting analysis respectively. RESULTS: Tongxinluo significantly increased the local blood flow and limited the infarct and size of no reflow. Tongxinluo also attenuated myeloperoxidase activity and neutrophil accumulation in histological sections and maintained the level of vascular endothelial cadherin and endothelial nitric oxide synthase activity in the reflow region when compared with control group. The protection of Tongxinluo was counteracted by coadministration with L-NNA. CONCLUSIONS: Tongxinluo may limit myocardial ischaemia and protect the heart against reperfusion injury. Tongxinluo regulates synthesis of nitric oxide by altering activity of endothelial nitric oxide synthase.

[Effects of tongxinluo on mini-swine vascular endothelial integrity and myocardial no-reflow in early reperfusion of acute myocardial infarction].

OBJECTIVE: To assess the effects of tongxinluo on vascular endothelial integrity and myocardial no-reflow in early reperfusion of acute myocardial infarction. METHODS: Forty mini-swines were divided into five groups randomly, sham group, control group, low dose (0.1 g/kg), medium dose (0.2 g/kg) and high dose (0.4 g/kg) groups of Tongxinluo. It was administered at 2 hours pre-reperfusion. Animals except in sham group were subjected to 1.5 hour of coronary occlusion followed by 3 hours of reperfusion. Content of VE-cadherin, beta-catenin, matrix metalloproteinase (MMP)-2 and 9 in myocardium were evaluated; no-reflow area was examined with myocardial contrast echocardiography (MCE) at 1.5 hour of AMI and 3 hours of reperfusion. RESULTS: (1) Compared with that of normal myocardium, content of VE-cadherin and beta-catenin decreased in reperfusion and no-reflow myocardium while MMP-2 and 9 increased significantly (all P < 0.05); (2) Compared with that of control group, a high dose of Tongxinluo could increase significantly the content of VE-cadherin in both reperfusion and no-reflow myocardium, (22.2 +/- 3.2)% vs (32.0 +/- 3.9)% and (14.5 +/- 2.8)% vs (28.3 +/- 2.2)% respectively, beta-catenin, (20.5 +/- 3.5)% vs (27.3 +/- 2.9)% and (13.3 +/- 2.1)% vs (20.6 +/- 2.4)%, while reduce MMP-2, (48.3 +/- 4.1)% vs (29.4 +/- 3.5)% and (57.3 +/- 4.3)% vs (38.2 +/- 4.0)% respectively, MMP-9, (55.6 +/- 4.0)% vs (34.3 +/- 3.5)% and (62.4 +/- 4.8)% vs (44.4 +/- 4.1)%, all P < 0.05; (3) Compared with that of control group, a high dose of Tongxinluo could reduce significantly both no-reflow area, (6.6 +/- 1.7) cm2 vs (4.7 +/- 1.5) cm2, P < 0.05, and percentage (90.8 +/- 3.8)% vs (71.4 +/- 4.1)%, P < 0.05, at 3 hours of reperfusion. CONCLUSION: A high dose of tongxinluo could effectively maintain the integrity of vascular endothelium and attenuate no-reflow area in early reperfusion of acute myocardial infarction.

Effects of Tongxinluo-facilitated cellular cardiomyoplasty with autologous bone marrow-mesenchymal stem cells on postinfarct swine hearts.

BACKGROUND: Treatment of ischemic heart disease remains an important challenge, though there have been enormous progresses in cardiovascular therapeutics. This study was conducted to evaluate whether Tongxinluo (TXL) treatment around the transplantation of mesenchymal stem cells (MSCs) can improve survival and subsequent activities of implanted cells in swine hearts with acute myocardial infarction (AMI) and reperfusion. METHODS: Twenty-eight Chinese mini-pigs were divided into four groups including a control group (n = 7); group 2, administration of low-dose TXL alone from the 3rd day prior to AMI to the 4th day post transplantation (n = 7); group 3, MSCs alone (n = 7) and group 4, TXL + MSCs (n = 7). AMI models were made by occlusion of the left anterior descending coronary artery for 90 minutes. Autologous bone marrow-MSCs (3 x 10(7) cells/animal) were then injected into the post-infarct myocardium immediately after AMI and reperfusion. The survival and differentiation of implanted cells in vivo were detected by immunofluorescent analysis. The data of cardiac function were obtained at baseline (1 week after transplantation) and endpoint (6 weeks after transplantation) by single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). Apoptosis was detected by TUNEL assay and the oxidative stress level was investigated in the post-infarct myocardium at endpoint. RESULTS: At endpoint, there was less fibrosis and inflammatory cell infiltration with more surviving myocardium in group 4 than in the control group. In group 4 the survival and differentiation of implanted MSCs were significantly improved more than that seen in group 3 alone (P < 0.0001); the capillary density was also significantly greater than in the control group, group 2 or 3 both in the infarcted zone (P < 0.0001) and the peri-infarct zone (P < 0.0001). MRI showed that parameters at baseline were not significantly different between the 4 groups. At endpoint, regional wall thickening and the left ventricular ejection fraction were increased while the left ventricular mass index, dyskinetic segments and infarcted size were decreased only in group 4 compared with control group (P < 0.0001). SPECT showed that the area of perfusion defect was significantly decreased at endpoint only in group 4 compared with control group (P < 0.0001). TUNEL assay indicated that TXL administration significantly decreased cell apoptosis in peri-infarct myocardium in groups 2 and 4. Furthermore, superoxide dismutase (SOD) significantly increased and malondialdehyde (MDA) decreased in groups 2 and 4 by the administration of TXL. CONCLUSIONS: Our study demonstrates the following: (1) immediate intramyocardial injection of MSCs after AMI and reperfusion resulted in limited survival and differentiation potential of implanted cells in vivo, thus being incapable of beneficially affecting post-hearts; (2) TXL-facilitation resulted in a significant survival and differentiation potential of implanted cells in vivo via inhibition of apoptosis and oxidative stress, accompanied by significant benefits in cardiac function.

[Effect of tongxinluo ultramicro-pulverization on myocardial post-reperfusion no-reflow in mini-swine model of acute myocardial infarction].

OBJECTIVE: To assess the effect of Tongxinluo (TXL) ultramicro-pulverization in preventing and treating post-reperfusion no-reflow in mini-swine model of acute myocardial infarction. METHODS: Forty mini-swines were randomly divided into 5 groups, the control group, the three (low-, middle- and high-dose) TXL groups and the sham-operation group, with 8 in each group. After pigs in the three TXL groups were administered with TXL in a dose of 0.05g/kg, 0.2g/kg and 0.5g/kg once a day for 3 days respectively, they were made into acute myocardial ischemia/reperfusion model by ligating left anterior descending coronary artery for 3h followed with 1h of untying. Hemodynamic examination and myocardial contrast echocardiography (MCE) were conducted before and after ligation, and after reperfusion, finally, pathological analysis was done. RESULTS: Post-reperfusion ventricular function injury was significantly improved in the three TXL groups, as compared with the control group, the no-reflow area determined by hemodynamic and MCE decreased from 78.5 +/- 4.4% and 82.3 +/- 1.9% in control to 43.4 +/- 3.2% and 44.6 +/- 3.3% (low-dose), 25.2 +/- 2.4% and 25.7 +/- 4.0% (middle-dose), 24.0 +/- 1.9% and 24.9 +/- 4.2% (high-dose), respectively (P < 0.05 or P < 0.01), and the myocardial infracted area was reduced from 98.5 +/- 1.4% to 89.8 +/- 4.6%, 80.2 +/- 3.1% and 79.9 +/- 3.1%, respectively (P < 0.05, P < 0.01). CONCLUSION: TXL ultramicro-pulverization can effectively prevent and treat no-reflow after myocardial acute infarction following reperfusion, and reduce the infracted area.

[Effects of Tongxinluo and Simvastatin on the stabilization of vulnerable atherosclerotic plaques of aorta in aortic atherosclerosis and molecular mechanism thereof: a comparative study with rabbits].

OBJECTIVE: To evaluate the plaque stabilization effects of Tongxinluo and Simvastatin in aortic atherosclerosis, and to explore molecular mechanism. METHODS: Twenty-three New Zealand white rabbits underwent aortic balloon injury and fed with high-cholesterol diet for 16 weeks and then randomly divided into 3 groups: Tongxinluo group (n = 6, undergoing gastric perfusion of Tongxinluo 1 gxkg(-1)xd(-1)), simvastatin group (n = 9, undergoing gastric perfusion of simvastatin 2 mgxkg(-1)xd(-1)), and model group (n = 8, without drug administration). Another 6 rabbits were used as normal controls. Peripheral blood samples were collected 10 weeks before the administration, and 3 and 16 weeks after the administration to detect the levels of total cholesterol (TG), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), and by the end of experiment peripheral blood samples were collected to detect the levels of serum endothelin (ET) and nitric oxide (NO). Then the rabbits were killed and their aortas were taken out to undergo pathological examination. Western blotting was used to detect the protein expression of Metalloproteinase (MMP)-1 and cyclooxygenase (COX)-2 and RT-PCR was used to detect the mRNA expression of FasL and bcl-2. RESULTS: By the end of the experiment the levels of blood lipids were raised by 1 approximately 2 times in the normal group (P < 0.05), and raised more significantly in the model and intervention groups (P < 0.05 approximately 0.01), especially the TC level of the model group was raised by 40 tomes. The levels of blood lipids of the simvastatin group were significantly lower than those of the model group (P < 0.05 approximately 0.01), however, between the Tongxinluo and model groups there were no significant differences in the levels of blood lipids. In the model group the blood level of ET was raised significantly and the level of NO was significantly decreased (both P < 0.01). The ET levels of the 2 intervention groups were both significantly lower than that of the model group (both P < 0.01), and the NO levels of the 2 intervention groups were both significantly higher than those of the normal group (P < 0.01 approximately 0.05), however, there were no significant differences in the ET and NO levels between these 2 intervention groups (all P > 0.05). Sclerotic plaques were distributed intensely at high degrees in the model group. The sclerotic changes of the 2 intervention groups were significantly milder. The contents of collagen of the 2 intervention groups were 0.11 +/- 0.08 and 0.22 +/- 0.11 respectively, both significantly higher then that of the model group (0.01 +/- 0.01, both P < 0.05). The intima/media ratios of aorta of the 2 intervention groups were 0.25 +/- 0.13 and 0.28 +/- 0.12 respectively, both significantly lower than that of the model group (0.60 +/- 0.37). The macrophage amount in the plaque (RAM-11 positively stained area) of the 2 intervention groups were 0.11 +/- 0.10 and 0.06 +/- 0.43 respectively, both significantly lower than that of the model group (0.24 +/- 0.14). The levels of protein expression of MMP-1 and COX-2 in the atherosclerotic lesions of the model group were both significantly higher than those of the normal group and the MMP-1 and COX-2 protein expression levels of the 2 intervention groups were all significantly lower than those of the model group (P < 0.05 approximately 0.01), however, there were no significant differences between these 2 groups (both P > 0.05). Significant linear correlation existed in the MMP-1 and COX-2 positively stained areas (r = 0.533, P = 0.007) and protein expression level (r = 0.833, P < 0.01). Compared with the normal group, the mRNA expression level of FasL in the aorta tissue of the model group was significantly higher (2.44 +/- 0.44) and the mRNA expression level of bcl was significantly lower (0.17 +/- 0.11). Compared with the model group, the mRNA expression levels of FasL of the 2 intervention groups were 0.47 +/- 0.36 and 1.32 +/- 0.61 respectively, both significantly lower and the mRNA expression levels of bcl were 0.64 +/- 0.16 and 1.66 +/- 0.94 respectively, both significantly higher (all P < 0.05 approximately 0.01), with the FasL mRNA expression of the Tongxinluo group significantly higher than that of the simvastatin group (P < 0.05). CONCLUSION: Tongxinluo and simvastatin have the same effects of stabilizing the vulnerable plaques, and the mechanism may be related with inhibition of expression of COX-2 and MMP and reduction of the apoptosis in the atherosclerotic plaque.

[Effect of tongxinluo on endothelin-1 in the mini-swine model of acute myocardial infarction and reperfusion].

OBJECTIVE: To evaluate the change of endothelin-1 (ET-1) in the mini-swine model of acute myocardial infarction (AMI) and reperfusion and the effect of Tongxinluo (TXL) on it, and to explore the possible mechanism of no-reflow. METHODS: Forty mini-swines were randomized into 5 groups: the model group, the small,middle and large dose of TXL groups and the sham-operated group, 8 in each group. The AMI reperfusion model was established by coronary ligation for 3 hrs followed with relaxation for 1 hr. Plasma ET-1 content before and after AMI, and after reperfusion was determined respectively by radioimmunoassay. The ET-1 mRNA expression in myocardial tissue of normal, ischemic and no-reflow area were respectively quantified by reverse transcription-polymerase chain reaction. RESULTS: (1) Compared with before AMI, levels of plasma ET-1 at the time points of 5 min and 3 hrs after AMI, 5 min and 1 hrs after reperfusion in the model group were significantly raised, showing an increasing tendency (all P < 0.01). But the increment in the middle and large dose of TXL groups were all lower than that in the model group (P < 0.05). (2) In the model and the TXL groups, levels of ET-1 in myocardial tissue of ischemic and no-reflow area were significantly higher than those in the normal area, and the increment in no-reflow area was higher than that in ischemic area (all P < 0.01). Compared with the model group, significant lowering of ET-1 in ischemic area was only shown in the middle and large dose of TXL groups (P < 0.01). (3) In the model and the TXL groups, ET-1 mRNA expression in ischemic area was significantly higher (all P < 0.01), while it in no-reflow area was significantly lower than that in the normal area respectively (all P < 0.01). The raised ET-1 mRNA expression in the middle and large dose TXL groups was significantly lowered when compared with that in the model group (P < 0.01). CONCLUSION: The endothelium injury might be one of the important mechanisms for no-reflow phenomenon. TXL might reduce the no-reflow by protecting endothelium cells. was significantly higher (all P < 0.01), while it in no-reflow area was significantly lower than that in the normal area respectively (all P < 0.01). The raised ET-1 mRNA expression in the middle and large dose TXL groups was significantly lowered when compared with that in the model group (P < 0.01). Conclusion The endothelium injury might be one of the important mechanisms for no-reflow phenomenon. TXL might reduce the no-reflow by protecting endothelium cells.

[Clinical study on spontaneous improvement after blood flow reconstruction interfered by tongxinluo capsule in patients with early stage acute myocardial infarction].

OBJECTIVE: To observe and evaluate the effect of tongxinluo capsule (TXL) on recovery of ventricular wall with segmental dyskinesia in patients with early stage acute myocardial infarction (AMI). METHODS: One hundred and twelve AMI patients after percutaneous coronary intervention (PCI) or fibrinolytic therapy, were randomly divided into 2 groups, the control group (CG) treated with conventional medicine and the interfered group (IG) treated with conventional medicine plus TXL. The changes of ventricular wall motion, left ventricular end diastolic volume (LVEDV) and left ventricular ejection fraction (LVEF) were observed at different time points (1-w, 2-w, 1-m, 3-m and 6-m) after PCI by using two dimensional echocardiography (2DE). RESULTS: The ventricular dyskinetic segment recovery rate at 1-w, 2-w, 1-m and 6-m in IG was 11.9%, 18.1%, 18.8% and 70.02% respectively, which was significantly higher than the respective rates in CG (4.1%, 8.3%, 11.1% and 51.68%, P < 0.01), but the 3-m recovery rate in the two groups was insignificantly different. LVEDV increase rate in the two groups at 1-w was insignificantly different, but it significantly increased at 2-w and 1-m, and showed a higher rate in CG (P < 0.05). However, at 3-m and 6-m, it significantly decreased in IG but was insignificantly changed in CG. Improvement of LVEF was insignificant at 1-w, 2-w and 1-m in both groups, but at 3-m and 6-m, LVEF was significantly improved in the interfered group (P < 0.01), but still showed no obvious change in the control group. CONCLUSION: Conventional western medicine combined with TXL can significantly decrease the infarction area, improve left ventricular diastolic function in patients with AMI.

[Beneficial effects of Tong-xin-luo (herb) on myocardial no-reflow after acute myocardial infarction and reperfusion: experiment of mini-swine model].

OBJECTIVE: To evaluate the beneficial effects of Tong-xin-luo on myocardial no-reflow after acute myocardial infarction (AMI) and reperfusion. METHODS: Forty mini-swine were randomized into 5 equal groups: control group, low-dose group (pretreated with Tong-xin-luo 0.05 g.kg(-1).d(-1) for 3 days), medium-dose group (pretreated with Tong-xin-luo 0.2 g .kg(-1).d(-1) for 3 days), high-dose group (pretreated with Tong-xin-luo 0.5 g.kg(-1).d(-1) for 3 days), and sham-operation group. The swine in the former four groups were subjected to 3 hours of coronary occlusion followed by 60 minutes of reperfusion. Left ventricle systolic pressure (LVSP), left ventricle end diastolic pressure (LVEDP), rate of maximum pressure change in left ventricle (+/- dp/dt(max)), cardiac output (CO), and heart rate (HR) were measured 5 min before AMI in all groups and 180 min after AMI and 60 min after reperfusion in the groups 1-4. Coronary blood volume (CBV) was recorded 5 min before AMI in all groups and immediately and 60 min after reperfusion in the group 1-4. Myocardial contrast echography (MCE) was used before AMI, 3 h after AMI, and 60 min after reperfusion in the group 1-4 so as to calculate the left ventricle wall area (LVWA), ligation area (LS), and %LA. Sixty minutes after reperfusion thioflavin-S was injected into the left ventricle to dye the reperfusion area, then the descending anterior branch was re-ligated at the original site and Evan's blue was injected into the left ventricle to dye the area outside the reperfusion area blue. The heart was taken out immediately to undergo pathological examination and calculation of LVWA, LS, area of no-reflow (SNR), LA, ANR. necrosis area (NS), and NA. RESULTS: (1) In the control group, systolic and diastolic blood pressures (SBP and DBP), LVSP, +/- dp/dt(max), and CO significantly decreased (P < 0.05 or P < 0.01), while LVEDP significantly increased (P < 0.01) 3 hour after AMI, and then LVSP was significantly recovered while +/- dp/dt(max) further significantly decreased (both P < 0.05) 60 minutes after reperfusion. In the 3 Tongxinluo groups, the changes of LVSP, +/- dp/dt(max), CO and LVEDP were the same as those in the control group 3 hours after AMI, and 60 minutes after reperfusion, +/- dp/dt(max), CO and LVEDP were recovered significantly in the high-dose group to degrees better than those in the control group (all P < 0.05). (2) In the control group, the LS values measured by MCE in vivo and by pathological evaluation were similar (P > 0.05), and the SNR was 78.5% by MCE in vivo and was 82.3% by pathological evaluation with the final NS reaching 98.5% of LS. There was no significant difference in LS by both MCE and pathological evaluation between the Tongxinluo groups and control group, though the values of SNR by both methods in the medium and high-dose groups were 41.1% and 42.4% and 24.1% and 25.0% respectively, all significantly lower than those in the control group and low-dose group (P < 0.05 or P < 0.01) with the values in the high-dose group being significantly lower than those in the median-dose group (P < 0.05 and P < 0.01). The final NS of pathological evaluation was also significantly decreased to 90.2%and 81.2% of LS (P < 0.05). In the control group, CBV was significantly decreased to 45.8% and 50.6% of the baseline value immediately at the beginning of reperfusion and 60 minutes after reperfusion (both P < 0.01). In the high-dose group, CBV was also significantly decreased to 76% and 73.5% of the baseline value immediately at the beginning of reperfusion and 60 minutes after reperfusion (both P < 0.05), however, both significantly higher than those in the control group (both P < 0.01). CONCLUSION: Tongxinluo is effective in preventing myocardial no-reflow, improving left ventricular function and reducing infarct area during AMI and reperfusion.