Treg Immunomodulation Contributes to the Anti-atherosclerotic Effects of Huxin Formula in ApoE-/- Mice.

OBJECTIVE: To explore the effects of Huxin formula (HXF) in curtailing atherosclerosis and its underlying mechanism. METHODS: According to random number table method, 24 specific pathogen free male ApoE-/- mice were randomly divided into model group, HXF low-dose (HXF-L) group (8.4 g/kg daily), HXF high-dose (HXF-H) group (16.8 g/kg daily), and pravastatin (8 mg/kg daily) group in Experiment I (n=6 per group). C57BL/6J mice served as the control group (n=6). ApoE-/- mice in HXF-L, HXF-H, pravastatin groups were fed a Western diet and administered continuously by gavage for 12 weeks, while C57BL/6J mice in the control group were fed conventional lab mouse chow for 12 weeks. Further, Tregs were depleted by weekly intraperitoneal injection of purified anti-mouse CD25 antibody (PC61, 250 µg per mouse) for 4 weeks in Experiment II (n=6 per group). Oil Red O and Masson staining were used to evaluate the plaque area and aortic root fibrosis. The CD4+CD25+Foxp3+Treg counts in the lymph nodes and spleen cells were detected using flow cytometric analysis. The transforming growth factor-ß1 (TGF-ß1), interleukin (IL)-10, and IL-6 serum levels were examined by MILLIPLEX® MAP technology. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot were utilized to assess the expression of TGF-ß mRNA and protein in the aorta. The expression of CD4+T lymphocytes, macrophages and smooth muscle cells in the aortic root were detected by immunofluorescence staining. RESULTS: HXF reduced plaque area in ApoE-/- mice (P<0.01). HXF increased the Treg counts in the lymph nodes and spleen cells (P<0.05 or P<0.01). Moreover, HXF alleviated inflammatory response via elevating IL-10 and TGF-ß 1 serum levels (P<0.05), while decreasing the IL-6 serum levels in ApoE-/- mice (P>0.05). Also, HXF upregulated the expression of TGF-ß mRNA and protein in the aorta (P<0.05). Additionally, HXF attenuated CD4+T lymphocytes, macrophages and smooth muscle cells in aortic root plaque (P<0.01). Furthermore, the depletion of Tregs with CD25 antibody (PC61) curtailed the reduction in plaque area and aortic root fibrosis by HXF (P<0.01). CONCLUSION: HXF relieved atherosclerosis, probably by restraining inflammatory response, reducing inflammatory cell infiltration and attenuating aortic root fibrosis by increasing Treg counts.

XinLi formula, a traditional Chinese decoction, alleviates chronic heart failure via regulating the interaction of AGTR1 and AQP1.

BACKGROUND: XinLi formula (XLF) is a traditional Chinese medicine used in clinical practice to treat chronic heart failure (CHF) in humans, with remarkable curative effect. However, the mechanism remains unknown. PURPOSE: The goal of the current investigation was to determine how XLF affected CHF in a rat model of the condition brought on by ligation of the left anterior descending coronary artery, and to investigate the underlying mechanism. STUDY DESIGN AND METHODS: Cardiac function was detected by echocardiography. The contents of myocardial enzymes, Ang II, ALD, TGF-ß1, and inflammatory factors were measured by ELISA. Myocardial injury and myocardial fibrosis were evaluated by HE and Masson staining. Myocardial edema was assessed by cardiac mass index and transmission electron microscopy. Using Western blot and immunohistochemistry to examining the protein expression of inflammasome, TGF-ß1, AGTR1, and AQP1 in the left ventricle. Furthermore, the interaction of AGTR1 and AQP1 was evaluated by co-immunoprecipitation. RESULTS: XLF attenuated myocardial enzymes and myocardial injury, and improved cardiac function in rats with CHF after myocardial infarction. It also reduced Ang II and ALD levels in CHF rats, and suppressed the expression of AGTR1 and TGF-ß1, finally alleviated myocardial fibrosis. By mechanism, XLF inhibited the expression of NLRP3 inflammasome proteins, reduced the plasma contents of IL-1ß, IL-18, IL-6 and TNF-α. Additionally, XLF inhibited the expression of AQP1 and the interaction of AGTR1 and AQP1, alleviating myocardial edema. The common structure of the main chemical constituents of XLF were glycoside compounds with glycosyl. CONCLUSION: XLF ameliorated CHF, which was evidenced by the alleviation of myocardial fibrosis by inhibiting AGTR1/NLRP3 signal, as well as the attenuation of myocardial edema by suppressing the interaction of AGTR1 and AQP1.

LOX-1 Regulation in Anti-atherosclerosis of Active Compounds of Herbal Medicine: Current Knowledge and the New Insight.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) have recently been identified to be closely related to the occurrence and development of atherosclerosis (AS). A growing body of evidence has suggested Chinese medicine takes unique advantages in preventing and treating AS. In this review, the related research progress of AS and LOX-1 has been summarized. And the anti-AS effects of 10 active components of herbal medicine through LOX-1 regulation have been further reviewed. As a potential biomarker and target for intervention in AS, LOX-1 targeted therapy might provide a promising and novel approach to atherosclerotic prevention and treatment.

Regulatory T Cells: A New Target of Chinese Medicine in Treatment of Atherosclerosis.

Inflammation and immune disorders are integral to the occurrence and progression of atherosclerosis (AS). With the role of regulatory T cells (Tregs) in immune regulation attracting attention, it has been widely accepted that Treg decrease and dysfunction are involved in AS pathogenesis. Chinese medicine (CM) has the advantages of being dual-directional, multi-targeted, and having minimal side effects in immune regulation. The anti-atherosclerosis effects of CM via Treg modulation have been revealed in clinical and animal studies. Therefore, this article reviews existing research on Tregs, the relationship between Tregs and AS, and the progress of CM for treating and prevention of atherosclerotic cardio-cerebrovascular diseases by regulating Tregs. Although the underlying mechanisms remain to be elucidated, CM treatment targeting Treg cells might provide a promising and novel future approach for prevention and treatment of AS.

Systems Pharmacology Dissection of Traditional Chinese Medicine Wen-Dan Decoction for Treatment of Cardiovascular Diseases.

Cardiovascular diseases (CVDs) have been recognized as first killer of human health. The underlying mechanisms of CVDs are extremely complicated and not fully revealed, leading to a challenge for CVDs treatment in modern medicine. Traditional Chinese medicine (TCM) characterized by multiple compounds and targets has shown its marked effects on CVDs therapy. However, system-level understanding of the molecular mechanisms is still ambiguous. In this study, a system pharmacology approach was developed to reveal the underlying molecular mechanisms of a clinically effective herb formula (Wen-Dan Decoction) in treating CVDs. 127 potential active compounds and their corresponding 283 direct targets were identified in Wen-Dan Decoction. The networks among active compounds, targets, and diseases were built to reveal the pharmacological mechanisms of Wen-Dan Decoction. A "CVDs pathway" consisted of several regulatory modules participating in therapeutic effects of Wen-Dan Decoction in CVDs. All the data demonstrates that Wen-Dan Decoction has multiscale beneficial activity in CVDs treatment, which provides a new way for uncovering the molecular mechanisms and new evidence for clinical application of Wen-Dan Decoction in cardiovascular disease.

3,7-Bis(2-hydroxyethyl)icaritin, a potent inhibitor of phosphodiesterase-5, prevents monocrotaline-induced pulmonary arterial hypertension via NO/cGMP activation in rats.

Pulmonary arterial hypertension (PAH) is a chronic progressive disease which leads to elevated pulmonary arterial pressure and right heart failure. 3,7-Bis(2-hydroxyethyl)icaritin (ICT), an icariin derivatives, was reported to have potent inhibitory activity on phosphodiesterase type 5 (PDE5) which plays a crucial role in the pathogenesis of PAH. The present study was designed to investigate the effects of ICT on monocrotaline (MCT)-induced PAH rat model and reveal the underlying mechanism. MCT-induced PAH rat models were established with intragastric administration of ICT (10, 20, 40 mg/kg/d), Icariin (ICA) (40 mg/kg/d) and Sildenafil (25 mg/kg/d). The mean pulmonary arterial pressure (mPAP) and right ventricle hypertrophy index (RVHI) were measured. Pulmonary artery remodeling was assessed by H&E staining. Blood and lung tissue were collected to evaluate the level of endothelin 1 (ET-1), nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The expressions endothelial nitric oxide synthase (eNOS) and PDE5A in lung tissues were determined by Western blot analysis. The results showed that ICT reduced RVHI and mPAP, and reversed lung vascular remodeling in rats with MCT-induced PAH. ICT also reversed MCT-induced ET-1 elevation, NO and cGMP reduction in serum or lung tissue. Moreover, ICT administration significantly induced eNOS activation and PDE5A inhibition. ICT with lower dose had better effects than ICA. In summary, ICT is more effective in preventing MCT-induced PAH in rats via NO/cGMP activation compared with ICA. These findings demonstrate a novel mechanism of the action of ICT that may have value in prevention of PAH.

Ginsenoside Rb1 prevents homocysteine-induced EPC dysfunction via VEGF/p38MAPK and SDF-1/CXCR4 activation.

Hyperhomocystinemia (HHcy) is known as an independent risk factor for cardiovascular disease. Our previous study showed that ginsenoside Rb1, the major active constituent of ginseng, prevents homocysteine (Hcy)-induced endothelial damage. However, the role of ginsenoside Rb1 in Hcy-induced dysfunction in endothelial progenitor cells (EPCs) remains unknown. In the study, we found that ginsenoside Rb1 reversed the Hcy-induced impairment of adhesive and migratory ability in EPCs which were significantly abolished by CXCR4 antagonist AMD3100 and VEGFR2 inhibitor SU5416. Ginsenoside Rb1 significantly reversed Hcy-induced SDF-1 reduction in the supernatant and in the serum. Ginsenoside Rb1 reversed downregulation of SDF-1 and VEGFR2 protein expression, inhibition of p38MAPK phosphorylation induced by Hcy. Re-endothelialization in balloon-injured carotid arteries significantly increased with EPCs transplant, and was even better with Rb1 treatment. This effect was significantly abolished by AMD3100. AMD3100 also decreased the number of CM-DiI labeled EPCs in injured arteries. Here we show for the first time that Rb1 prevents Hcy-induced EPC dysfunction via VEGF/p38MAPK and SDF-1/CXCR4 activation. These findings demonstrate a novel mechanism of the action of Rb1 that may have value in prevention of HHcy associated cardiovascular disease.

Effect of Shenzhu Guanxin Recipe () on patients with angina pectoris after percutaneous coronary intervention: A prospective, randomized controlled trial.

OBJECTIVE: To evaluate the efficacy and safety of a combination therapy using Chinese medicine (CM) Shenzhu Guanxin Recipe (, SGR) and standard Western medicine treatment (SWMT) in patients with angina pectoris after percutaneous coronary intervention (PCI). METHODS: Double-blind randomized controlled trial was used in this experimental procedure. One hundred and eighty-seven patients with coronary heart disease receiving SWMT after PCI were randomly assigned to the treatment (SGR) and control (placebo) groups. Outcome measures including angina pectoris score (APS), CM symptom score, and Seattle Angina Questionnaire (SAQ) score were evaluated in 1, 2, 3 and 12 months, and the death rate, restenosis and other emergency treatments were observed. The mixed-effects models were employed for the data analysis. RESULTS: In the treatment group, a larger within-treatment effect size (d=1.74) was found, with a 76.7% reduction in APS from pretreatment to 12-month follow-up assessment compared with the control group (d=0.83, 53.8% symptom reduction); betweentreatment (BT) effect size was d=0.66. CM symptom scores included an 18.3% reduction in the treatment group (d=0.46), and a 16.1% decrease in the control group (d=0.31); d=0.62 for BT effect size. In regard to scores of SAQ, the BT effect size of cognition level of disease was larger in the treatment group (d=0.63), followed by the level of body limitation of activity (d=0.62), condition of angina pectoris attacks (d=0.55), satisfaction level of treatments (d=0.31), and steady state of angina pectoris (d=0.30). Two cardiovascular related deaths and one incidental death were recorded in the control and treatment groups, respectively. No significant difference in any cardiovascular event (including death toll, frequency of cardiovascular hospitalization or emergency room visits) was found between the two groups. CONCLUSION: The combination therapy of SGR and SWMT is effective and safe in patients with angina pectoris after PCI when compared with SWMT alone.

Efficacy and safety of Huxin formula in patients after CABG: a multicenter, double-blind, randomized clinical trial.

BACKGROUND: Coronary artery bypass grafting (CABG) is widely used in the treatment of coronary artery disease. A multicenter, double-blind, randomized, controlled clinical trial was designed to evaluate the efficacy and safety of Huxin Formula post CABG. PATIENTS AND METHODS: 270 inpatients with coronary heart disease participated in this study. CABG patients in the control group were treated with placebo, while patients in the experimental group were treated with Huxin Formula 1 week after the surgery. All patients were treated for 6 months and followed up for another 6 months. The main outcomes (death, nonfatal myocardial infarction, repeat revascularization, and readmission) were assessed 360 days after treatment, and secondary outcomes (frequency and scores of angina pectoris, etc.) were assessed 0, 90, 180, 270, and 360 days after treatment. RESULTS: Our results showed no significant difference between the 2 groups for the primary endpoints. In patients with cardiac function class II (New York Heart Association), the score of angina pectoris was significantly lower (3.88 ± 3.86 vs. 5.45 ± 3.59) and the frequency of angina pectoris attacks was less (0.96 ± 1.01 vs. 1.36 ± 0.94) after 90 days of treatment with Huxin Formula compared to placebo (p < 0.05). In patients with 3 coronary vessel lesions, the cardiac function class (1.14 ± 0.35 vs. 1.05 ± 0.21) after 360 days was significantly higher in the control group compared to the treatment group (p < 0.05). There were no obvious adverse reactions. CONCLUSION: Huxin Formula may improve cardiac function of patients with 3 coronary vessel lesions and relieve symptoms of patients with cardiac function class II but failed to show superiority in primary outcomes.

Vascular fibrosis in atherosclerosis.

Vascular fibrosis, characterized by reduced lumen diameter and arterial wall thickening attributable to excessive deposition of extracellular matrix (ECM), links with many clinical diseases and pathological progresses including atherosclerosis. It involves proliferation of vascular smooth muscle cell (VSMC), accumulation of ECM and inhibition of matrix degradation. The risk factors associated with cardiovascular disease, including hypertension, hyperglycemia, dyslipidemia and hyperhomocysteinemia (HHcy), are also suggested as initiation and progression factors of vascular fibrosis. Vascular fibrosis has been found to relate to renin-angiotensin-aldosterone system (RAAS), oxidative stress, inflammatory factors, growth factors and imbalance of endothelium-derived cytokine secretion. Angiotensin II (Ang II) and aldosterone, the circulating effector hormones of RAAS, are recognized as responsible for the pathophysiology of vascular fibrosis. Transforming growth factor-beta (TGF-beta) plays a critical role in ECM accumulation and vascular remodeling via up-regulating the production of several agents including connective tissue growth factor (CTGF) and fibroblast growth factor. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) results in collagen accumulation and adverse matrix remodeling. Aberrant expression or function of peroxisome proliferator-activated receptor gamma (PPAR gamma) is also associated with, and very likely contributes to, the progression of pathological fibrosis and vascular remodeling. In this review, we discuss the pathogenesis of vascular fibrosis in atherosclerosis with focus on the networking among main responsible mediators. The main pathophysiologic factors leading to vascular fibrosis will also be discussed.

Ginsenoside Rb1 prevents homocysteine-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition.

Hyperhomocysteinemia (HHcy), a risk factor for cardiovascular disease, is associated with endothelial dysfunction. Ginsenoside Rb1, the major active constituent of ginseng, potently attenuates homocysteine (Hcy)-induced endothelial damage. However, the underlying mechanism remains unknown. In this study, we have investigated the effect of Ginsenoside Rb1 on Hcy-induced endothelial dysfunction and its underlying signal pathway in vivo and in vitro. Ginsenosides prevented Hcy-induced impairment of endothelium-dependent relaxation and Rb1 reversed Hcy-induced reduction of NO production in a dose-dependent manner as detected by nitrate reductase method. Rb1 activated serine-1177 phosphorylation of endothelial nitric oxide synthase (eNOS) and serine-473 phosphorylation of Akt, while inhibited threonine-495 phosphorylation of eNOS as detected by western blotting. Rb1-induced phosphorylation of serine-1177 was significantly inhibited by wortmannin, PI3K inhibitor or SH-5, an Akt inhibitor, and partially reversed by Phorbol 12-myristate 13-acetate (PMA), a PKC activator. PMA also stimulated phosphorylation of threonine-495 which was inhibited by Rb1. Here we show for the first time that Rb1 prevents Hcy-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition. These findings demonstrate a novel mechanism of the action of Rb1 that may have value in prevention of HHcy associated cardiovascular disease.