Bazi Bushen ameliorates age-related energy metabolism dysregulation by targeting the IL-17/TNF inflammatory pathway associated with SASP.

BACKGROUND: Chronic inflammation and metabolic dysfunction are key features of systemic aging, closely associated with the development and progression of age-related metabolic diseases. Bazi Bushen (BZBS), a traditional Chinese medicine used to alleviate frailty, delays biological aging by modulating DNA methylation levels. However, the precise mechanism of its anti-aging effect remains unclear. In this study, we developed the Energy Expenditure Aging Index (EEAI) to estimate biological age. By integrating the EEAI with transcriptome analysis, we aimed to explore the impact of BZBS on age-related metabolic dysregulation and inflammation in naturally aging mice. METHODS: We conducted indirect calorimetry analysis on five groups of mice with different ages and utilized the data to construct EEAI. 12 -month-old C57BL/6 J mice were treated with BZBS or ß-Nicotinamide Mononucleotide (NMN) for 8 months. Micro-CT, Oil Red O staining, indirect calorimetry, RNA sequencing, bioinformatics analysis, and qRT-PCR were performed to investigate the regulatory effects of BZBS on energy metabolism, glycolipid metabolism, and inflammaging. RESULTS: The results revealed that BZBS treatment effectively reversed the age-related decline in energy expenditure and enhanced overall metabolism, as indicated by the aging index of energy expenditure derived from energy metabolism parameters across various ages. Subsequent investigations showed that BZBS reduced age-induced visceral fat accumulation and hepatic lipid droplet aggregation. Transcriptomic analysis of perirenal fat and liver indicated that BZBS effectively enhanced lipid metabolism pathways, such as the PPAR signaling pathway, fatty acid oxidation, and cholesterol metabolism, and improved glycolysis and mitochondrial respiration. Additionally, there was a significant improvement in inhibiting the inflammation-related arachidonic acid-linoleic acid metabolism pathway and restraining the IL-17 and TNF inflammatory pathways activated via senescence associated secretory phenotype (SASP). CONCLUSIONS: BZBS has the potential to alleviate inflammation in metabolic organs of naturally aged mice and maintain metabolic homeostasis. This study presents novel clinical therapeutic approaches for the prevention and treatment of age-related metabolic diseases.

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats.

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley (SD) rats were randomly divided into six groups: a sham-operated group, a model group (OVX), a progynova group, and BZBS groups (1, 2, and 4 d/kg/d). An ovariectomy was performed on all rats except those in the sham-operated group. Micro-computed tomography (micro-CT) scanning, hematoxylin and eosin (H&E) staining, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) detection were performed after 4 months of BZBS treatment. As a result, compared with the OVX group, rats treated with BZBS showed an increased number and area of trabecular bone and bone marrow cells, and a decreased number of adipose cells. The bone volume, trabecular number, and trabecular thickness of the right tibia in the medication groups increased and the trabecular space decreased. The 17ß-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, ß-CTX, and TRACP-5b were decreased. In the medication groups, the RANKL and sclerostin levels were decreased, while the osteoprotegerin (OPG) level was increased. In conclusion, this protocol systematically evaluated the therapeutic effects and potential molecular mechanisms of Chinese herbal compounds in ovariectomized rats with a variety of techniques.

Shensong Yangxin Capsule Reduces the Susceptibility of Arrhythmia in db/db Mice via Inhibiting the Inflammatory Response Induced by Endothelium Dysfunction.

Purpose: The aim of our study was to investigate the mechanism by which the Chinese compound Shensong Yangxin Capsule (SSYX) reduces susceptibility to arrhythmia in db/db mice. Methods: The db/db mice without drug treatment served as the model group. Other-treated db/db mice were administered SSYX for 8 weeks. Electrocardiogram (ECG), electrical mapping, pathological changes, immunofluorescence staining, real-time quantitative PCR, and Western blot analyses were then conducted. Results: SSYX decreased arrhythmia susceptibility and shortened the abnormal ECG parameters of db/db mice. Meanwhile, SSYX restored irregular conduction direction and shortened the conduction time of the isolated heart. HE and Masson staining showed that SSYX alleviated inflammatory infiltration and collagen fiber deposition. Western blot showed that SSYX decreased the protein expression of ICAM-1, VCAM-1, and MCP-1 and increased the protein expression of occludin, ZO-1, eNOS, and Cx43. SSYX also increased the content of NO, decreased ET-1, TNF-α, IL-1ß, IL-6, MCP-1, and CCR-2 mRNA expression, and increased Kv 4.2, Kv 4.3, Cav 1.2, and Nav 1.5 mRNA expression. Furthermore, SSYX decreased the fluorescence intensity of F4/80 and iNOS, increased the fluorescence intensity of CD31 and eNOS, and improved the Cx43 and α-actinin connection structure in cardiac tissues. The above therapeutic effects of SSYX were inhibited by L-NAME. Conclusion: SSYX reduced the susceptibility of db/db mice to arrhythmia by inhibiting the inflammatory response and macrophage polarization, and this effect of SSYX occurred through protection of endothelial cell function.

Bazi Bushen mitigates epigenetic aging and extends healthspan in naturally aging mice.

Bazi Bushen (BZBS), a traditional Chinese medicine, has been proven effective in the treatment of age-related disease in mouse models. However, whether its therapeutic effects are due to antiaging mechanism has not yet been explored. In the present study, we investigated the antiaging effects of BZBS in naturally aging mice by using behavioral tests, liver DNA methylome sequencing, methylation age estimation, and frailty index assessment. The methylome analysis revealed a decrease of mCpG levels in the aged mouse liver. BZBS treatment tended to restore age-associated methylation decline and prune the methylation pattern toward that of young mice. More importantly, BZBS significantly rejuvenated methylation age of the aged mice, which was computed by an upgraded DNA methylation clock. These results were consistent with enhanced memory and muscular endurance, as well as decreased frailty score and liver pathological changes. KEGG analysis together with aging-related database screening identified methylation-targeted pathways upon BZBS treatment, including oxidative stress, DNA repair, MAPK signaling, and inflammation. Upregulation of key effectors and their downstream effects on elevating Sod2 expression and diminishing DNA damage were further investigated. Finally, in vitro experiments with senescent HUVECs proved a direct effect of BZBS extracts on the regulation of methylation enzymes during cellular aging. In summary, our work has revealed for the first time the antiaging effects of BZBS by slowing the methylation aging. These results suggest that BZBS might have great potential to extend healthspan and also explored the mechanism of BZBS action in the treatment of age-related diseases.

Bazi Bushen capsule attenuates cognitive deficits by inhibiting microglia activation and cellular senescence.

CONTEXT: Bazi Bushen capsule (BZBS) has anti-ageing properties and is effective in enhancing memory. OBJECTIVE: To find evidence supporting the mechanisms and biomarkers by which BZBS functions. MATERIALS AND METHODS: Male C57BL/6J mice were randomly divided into five groups: normal, ageing, ß-nicotinamide mononucleotide capsule (NMN), BZBS low-dose (LD-BZ) and BZBS high-dose (HD-BZ). The last four groups were subcutaneously injected with d-galactose (d-gal, 100 mg/kg/d) to induce the ageing process. At the same time, the LD-BZ, HD-BZ and NMN groups were intragastrically injected with BZBS (1 and 2 g/kg/d) and NMN (100 mg/kg/d) for treatment, respectively. After 60 days, the changes in overall ageing status, brain neuron morphology, expression of p16INK4a, proliferating cell nuclear antigen (PCNA), ionized calcium-binding adapter molecule 1 (Iba1), postsynaptic density protein 95 (PSD95), CD11b, Arg1, CD206, Trem2, Ym1 and Fizz1, and the senescence-associated secretory phenotype (SASP) factors were observed. RESULTS: Compared with the mice in the ageing group, the HD-BZ mice exhibited obvious improvements in strength, endurance, motor coordination, cognitive function and neuron injury. The results showed a decrease in p16INK4a, Iba1 and the upregulation of PCNA, PSD95 among brain proteins. The brain mRNA exhibited downregulation of Iba1 (p < 0.001), CD11b (p < 0.001), and upregulation of Arg1 (p < 0.01), CD206 (p < 0.05), Trem2 (p < 0.001), Ym1 (p < 0.01), Fizz1 (p < 0.05) and PSD95 (p < 0.01), as well as improvement of SASP factors. CONCLUSIONS: BZBS improves cognitive deficits via inhibition of cellular senescence and microglia activation. This study provides experimental evidence for the wide application of BZBS in clinical practice for cognitive deficits.

Jinlida Granules Reduce Obesity in db/db Mice by Activating Beige Adipocytes.

Recent studies indicate existence of beige adipocytes in adults. Upon activation, beige adipocytes burn energy for thermogenesis and contribute to regulation of energy balance. In this study, we have analyzed whether Jinlida granules (JLD) could activate beige adipocytes. JLD suspended in 0.5% carboxymethyl cellulose (CMC) was gavage fed to db/db mice at a daily dose of 3.8 g/kg. After 10 weeks, body weight, biochemical, and histological analyses were performed. In situ hybridization, immunofluorescence, and western blotting were conducted to test beige adipocyte activation in mice. X9 cells were induced with induction medium and maintenance medium containing 400 µg/mL of JLD. After completion of induction, cells were analyzed by Nile red staining, time polymerase chain reaction (PCR), western blotting, and immunofluorescence to understand the effect of JLD on the activation of beige adipocytes. A molecular docking method was used to preliminarily identify compounds in JLD, which hold the potential activation effect on uncoupling protein 1 (UCP1). JLD treatment significantly improved obesity in db/db mice. Biochemical results showed that JLD reduced blood glucose (GLU), triglyceride (TG), and low-density lipoprotein cholesterol (LDL) levels as well as liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in mice. Hematoxylin and eosin staining (H&E) showed that JLD reduced hepatocyte ballooning changes in the liver. Immunofluorescence showed that JLD increased the expression of the thermogenic protein, UCP1, in the beige adipose tissue of mice. JLD also increased the expression of UCP1 and inhibited the expression of miR-27a in X9 cells. Molecular docking results showed that epmedin B, epmedin C, icariin, puerarin, and salvianolic acid B had potential activation effects on UCP1. The results suggest that JLD may activate beige adipocytes by inhibiting miR-27a expression, thereby promoting thermogenesis in beige adipocytes. This study provides a new pharmacological basis for the clinical use of JLD.

Integrating Evidence of the Traditional Chinese Medicine Collateral Disease Theory in Prevention and Treatment of Cardiovascular Continuum.

Cardiovascular disease has become a major public health problem. The concept of "cardiovascular continuum" refers to the continuous process from the risk factors that lead to arteriosclerosis, vulnerable plaque rupture, myocardial infarction, arrhythmia, heart failure, and death. These characteristics of etiology and progressive development coincide with the idea of "preventing disease" in traditional Chinese medicine (TCM), which corresponds to the process of systemic intervention. With the update of the understanding via translational medicine, this article reviews the current evidence of the TCM collateral disease theory set prescriptions in both mechanical and clinical aspects, which could lead to the development of new therapeutic strategies for prevention and treatment.

Chinese medicine Bazi Bushen capsule improves lipid metabolism in ovariectomized female ApoE-/- mice.

BACKGROUND: Dyslipidemia is one of the mechanisms of atherosclerosis (AS). Depletion of estrogen plays a key role in the pathogenesis of postmenopausal AS in women, and the blood lipid levels of women are closely related to endogenous estrogen levels. Phytoestrogens (PEs) exert estrogenic effects, including protection against AS, without the adverse effects of estrogen administration. Bazi Bushen capsule (BZBS) is a traditional Chinese medicine herbal compound prescription that has been shown to contain 11 unique PEs. In the present study, we assessed the effects of BZBS against lipid metabolism disorders. METHODS: All ApoE-/- mice underwent ovary ligation and bilateral ovariectomy (Ovx) to induce surgical menopause (Ovx/ApoE-/- mice), whereas the C57BL/6J mice underwent sham surgery (needle threading). Ovx/ApoE-/- mice were given a high-fat diet without estrogen and C57BL/6J mice were given a normal diet for 12 weeks. Ovx/ApoE-/- mice treated with G1, a highly selective G-protein-coupled estrogen receptor1 (GPER1) agonist with proven activity against AS, were used as positive controls. Estrogen levels were measured and uterine atrophy index was calculated to determine the success of the model. Serum levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured in each group. The orthogonal projections to latent structures discriminant analysis (OPLS-DA) model was used to separate the groups, MetaboAnalyst was then used to analyze the metabolic pathway, and the most representative metabolites were finally identified. RESULTS: Removal of bilateral ovaries resulted in successful surgical menopause models, where BZBS increased estrogen levels but did not increase the risk of uterine proliferation. BZBS attenuated dyslipidemia, including decreased TG, TC, and LDL-C levels, but increased HDL-C levels. The OPLS-DA model successfully distinguished the groups with good predictive ability and revealed their tendency to separate from each other. MetaboAnalyst suggested that both the G1 group and high-dose BZBS (HD-BZ) could improve the effect of lipid metabolism: the glycerophospholipid metabolism pathway was mainly improved by the G1 group, while the inositol phosphate metabolism pathway was mainly improved by the HD-BZ group. For the four compounds with the highest content, the concentrations of docosahexaenoic acid (DHA), 3-hydroxybutyric acid, and 5(Z), 8(Z), 11(Z)-eicosatrienoic acid were dramatically lower in the model group compared to the control group. Lysophosphatidylethanolamine (18:0) was higher in the model group than in the control group. BZBS corrected these effects. CONCLUSIONS: BZBS treatment reduced serum lipid levels and improved fatty acid metabolism in high-fat diet-fed, surgically induced menopausal ApoE-/- mice.

Hospitalization in patients with type 2 diabetes mellitus in Taiwan: A nationwide population-based observational study.

BACKGROUND/PURPOSE: We investigated hospitalization rates of patients with type 2 diabetes mellitus (T2DM) and individuals without diabetes mellitus (non-DM) in a disease-specific manner from 2005 to 2014 in Taiwan. METHODS: This population-based study was conducted using data from the National Health Insurance Research Database. We analyzed the hospitalization rates of patients with and without T2DM. We collected up to five diagnostic codes given at discharge for each hospitalization, and the first one was considered the main diagnosis. Odds ratios were determined to assess the risk of hospitalization according to disease-specific classifications in patients with T2DM compared with those without T2DM. RESULTS: The hospitalization rates of non-DM patients was stable from 2005 to 2014. By contrast, the rate of hospitalization among patients with T2DM decreased from 395.4 (per 1000 person-years) in 2005 to 336.9 (per 1000 person-years) in 2014. An increase in hospitalization rates for malignancies and sepsis/infection (other than pneumonia) was observed from 2005 to 2014 in both patients with and without T2DM. Although patients with T2DM had a higher hospitalization risk for all the disease-specific classifications than non-DM patients, this difference in risk decreased from 2005 to 2014 for all diseases except pneumonia. CONCLUSION: Hospitalization rates for malignancies and sepsis/infection (other than pneumonia) continually increased from 2005 to 2014 in Taiwan. Although patients with T2DM had a greater risk of disease-specific hospitalization than those without, this difference in risk decreased from 2005 to 2014 for all diseases except for pneumonia.

Qiliqiangxin Attenuates Oxidative Stress-Induced Mitochondrion-Dependent Apoptosis in Cardiomyocytes via PI3K/AKT/GSK3ß Signaling Pathway.

Qiliqiangxin capsule (QLQX) is a well-known traditional Chinese medicine that exhibits cardioprotective effects in heart failure patients. However, it remains unclear whether and by which mechanism QLQX attenuates oxidative stress-induced mitochondria-dependent myocardial apoptosis. In vivo, Sprague Dawley (SD) rats received left anterior descending coronary artery ligation for 4 weeks to establish a model of heart failure after acute myocardial infarction, and then were treated with QLQX for another 4 weeks. We evaluated cardiac function, oxidative stress injury, as well as the expressions of mitochondria-dependent apoptosis and its signaling factors. The results indicated that QLQX protected cardiac function and attenuated oxidative stress-induced myocardial apoptosis. Meanwhile, QLQX elevated the Bcl-2 expression, declined the expressions of Bax, cytochrome c, apoptotic protease activating factor-1 (Apaf-1), cleaved-caspase 9 and cleaved-caspase 3, and up-regulated the ratios of phospho-AKT/AKT and phospho-glycogen synthase kinase-3ß (GSK3ß)/GSK3ß. In vitro, H9c2 cardiomyocytes were pretreated with QLQX, then exposed to H2O2 for 24 h. QLQX promoted the proliferation of H9c2 cardiomyocytes induced by H2O2 and reversed oxidative stress damage. Moreover, QLQX inhibited the apoptosis rate and the pro-apoptosis protein expressions, but improved the Bcl-2 expression as well as the ratios of phospho-AKT/AKT and phospho-GSK3ß/GSK3ß. Meanwhile, it further ameliorated mitochondrion-related apoptosis by inhibiting the mitochondrial fission, mitochondrial permeability transition pore (MPTP) opening, and mitochondrial membrane potential (MMP) decline in H9c2 cardiomyocytes induced by H2O2. In addition, all the effects of QLQX on H2O2-induced mitochondria-dependent apoptosis could be blocked by the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. We conclude that QLQX may ameliorate oxidative stress-induced mitochondria-dependent apoptosis in cardiomyocytes through PI3K/AKT/GSK3ß signaling pathway.

Chinese medicine Jinlida granules improve high-fat-diet induced metabolic disorders via activation of brown adipose tissue in mice.

AIMS: Activation of brown adipose tissue (BAT) thermogenesis could contribute to energy expenditure, which is critical for the treatment of obesity and type 2 diabetes mellitus (T2DM). In the present study, we aimed to systematically investigate whether traditional Chinese medication Jinlida (JLD) granules could improve metabolic disorders and activate BAT thermogenesis in C57BL/6 J mice fed with a high-fat diet (HFD). METHODS: In the present study, JLD (3.8 g/kg) in 0.5% of carboxymethyl cellulose (CMC) solution was administrated daily by oral gavage to HFD-induced mice for 15 weeks. The body weight, biochemical analysis, histology analysis, intraperitoneal glucose and insulin tolerance (OGTT and ITT) tests were measured to explore metabolic disorders. Cold tolerance test, real-time PCR (qRT-PCR), immunohistochemistry, and western blot were performed to evaluate BAT function. RESULTS: As results, JLD treatment significantly ameliorated HFD-induced obesity and fat mass gain, maintained glucose and lipid homeostasis, and improved hepatic steatosis and inflammation. More importantly, we observed that JLD markedly activated BAT thermogenesis in HFD-induced obese mice. Moreover, our data confirmed that JLD promoted mitochondrial biogenesis and fatty acid oxidation metabolism in BAT. CONCLUSIONS: These data suggested that JLD could improve metabolic disorders in associated with activation of BAT thermogenesis via enhancement of mitochondrial biogenesis and fatty acid oxidation metabolism, thus providing a new pharmacological evidence for the clinical usage of JLD in T2DM treatment.

New therapy with XLQ® to suppress chronic prostatitis through its anti-inflammatory and antioxidative activities.

Chronic prostatitis is a common urological disease. The etiology of this disease and effective therapy for its treatment are yet to be elucidated. We investigated the functions of XLQ® in chronic nonbacterial prostatitis using a complete Freund's adjuvant-induced rat model. Prostates and blood samples were collected for further evaluation after oral gavage with XLQ ® or a vehicle for 4 weeks. The results showed that XLQ ® significantly decreased the prostate index, ameliorated the histopathologic changes, and reduced CD3+ and CD45+ cell infiltration in the prostate stroma. Further study showed that XLQ ® suppressed the expression of proinflammatory cytokines, such as interleukin (IL)-1ß, IL-2, IL-6, IL-17A, monocyte chemoattractant protein-1, and tumor necrosis factor-α. XLQ ® showed a strong antioxidant capacity by enhancing the activities of antioxidative enzymes (e.g., total superoxide dismutase, catalase, and glutathione peroxidase) and decreasing the level of lipid peroxidation products (malondialdehyde). Moreover, XLQ ® can suppress the activation of nuclear factor-κB and P38-mitogen-activated protein kinase signaling pathways. In summary, XLQ ® has affirmative effects on chronic prostatitis, which could be attributed to its anti-inflammatory and antioxidative capacities. On the basis of these results, XLQ ® can be developed as an effective and safe therapy for chronic prostatitis.

A metabolomics study of Qiliqiangxin in a rat model of heart failure: a reverse pharmacology approach.

The Chinese medicine Qiliqiangxin (QL) has been shown to have a protective role in heart failure. Here, we explore the underlying working mechanism of the key therapeutic component in QL using a rat model of heart failure. Heart failure after myocardial infarction was induced surgically and confirmed using echocardiography; a separate group of rats underwent sham surgery. The rats with heart failure were randomly assigned to receive QL, the angiotensin-converting enzyme inhibitor benazepril, or placebo groups. Blood samples were collected from the rats at four time points for up to 8 weeks and used for biochemical analysis and mass spectrometry‒based metabolomics profiling. In total, we measured nine well-known biochemical parameters of heart failure and 147 metabolites. In the rats with heart failure, QL significantly improved these biochemical parameters and metabolomics profiles, significantly increasing the cardioprotective parameter angiopoietin-like 4 and significantly lowering inflammation-related oxylipins and lysophosphatidic acids compared to benazepril. Mechanistically, QL may improve outcome in heart failure by controlling inflammatory process and cardiac hypertrophy. Clinical studies should be designed in order to investigate these putative mechanisms in patients.

Tongxinluo Improves Apolipoprotein E-Deficient Mouse Heart Function.

BACKGROUND: Our previous studies have shown that Tongxinluo (TXL), a compound Chinese medicine, can decrease myocardial ischemia-reperfusion injury, protect capillary endothelium function, and lessen cardiac ventricle reconstitution in animal models. The aim of this study was to illuminate whether TXL can improve hypercholesterolemia-impaired heart function by protecting artery endothelial function and increasing microvascular density (MVD) in heart. Furthermore, we will explore the underlying molecular mechanism of TXL cardiovascular protection. METHODS: After intragastric administration of TXL (0.1 ml/10 g body weight) to C57BL/6J wild-type mice (n = 8) and ApoE-/- mice (n = 8), total cholesterol, high-density lipoprotein-cholesterol, very-low-density lipoprotein (VLDL)-cholesterol, triglyceride, and blood glucose levels in serum were measured. The parameters of heart rate (HR), left ventricular diastolic end diameter, and left ventricular systolic end diameter were harvested by ultrasonic cardiogram. The left ventricular ejection fraction, stroke volume, cardiac output, and left ventricular fractional shortening were calculated. Meanwhile, aorta peak systolic flow velocity (PSV), end diastolic flow velocity, and mean flow velocity (MFV) were measured. The pulsatility index (PI) and resistant index were calculated in order to evaluate the vascular elasticity and resistance. The endothelium-dependent vasodilatation was evaluated by relaxation of aortic rings in response to acetylcholine. Western blotting and real-time quantitative reverse transcription polymerase chain reaction were performed for protein and gene analyses of vascular endothelial growth factor (VEGF). Immunohistochemical detection was performed for myocardial CD34 expression. Data in this study were compared by one-way analysis of variance between groups. A value of P < 0.05 was considered statistically significant. RESULTS: Although there was no significant decrease of cholesterol level (F = 2.300, P = 0.240), TXL inhibited the level of triglyceride and VLDL (F = 9.209, P = 0.024 and F = 9.786, P = 0.020, respectively) in ApoE-/- mice. TXL improved heart function of ApoE-/- mice owing to the elevations of LVEF, SV, CO, and LVFS (all P < 0.05). TXL enhanced aortic PSV and MFV (F = 10.774, P = 0.024 and F = 11.354, P = 0.020, respectively) and reduced PI of ApoE-/- mice (1.41 ± 0.17 vs. 1.60 ± 0.17; P = 0.037). After incubation with 10 µmol/L acetylcholine, the ApoE-/- mice treated with TXL aortic segment relaxed by 44% ± 3%, significantly higher than control group mice (F = 9.280, P = 0.040). TXL also restrain the angiogenesis of ApoE-/- mice aorta (F = 21.223, P = 0.010). Compared with C57BL/6J mice, the MVD was decreased in heart tissue of untreated ApoE-/- mice (54.0 ± 3.0/mm2 vs. 75.0 ± 2.0/mm2; F = 16.054, P = 0.010). However, TXL could significantly enhance MVD (65.0 ± 5.0/mm2 vs. 54.0 ± 3.0/mm2; F = 11.929, P = 0.020) in treated ApoE-/- mice. In addition, TXL obviously increased the expression of VEGF protein determined by Western blot (F = 20.247, P = 0.004). CONCLUSIONS: TXL obviously improves the ApoE-/- mouse heart function from different pathways, including reduces blood fat to lessen atherosclerosis; enhances aortic impulsivity, blood supply capacity, and vessel elasticity; improves endothelium-dependent vasodilatation; restraines angiogenesis of aorta-contained plaque; and enhances MVD of heart. The molecular mechanism of MVD enhancement maybe relate with increased VEGF expression.

Folic Acid in Stroke Prevention in Countries without Mandatory Folic Acid Food Fortification: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND AND PURPOSE: Additional folic acid (FA) treatment appears to have a neutral effect on reducing vascular risk in countries that mandate FA fortification of food (e.g., USA and Canada). However, it is uncertain whether FA therapy reduces stroke risk in countries without FA food fortification. The purpose of this study was to comprehensively evaluate the efficacy of FA therapy on stroke prevention in countries without FA food fortification. METHODS: PubMed, EMBASE, and clinicaltrials.gov from January 1966 to August 2016 were searched to identify relevant studies. Relative risk (RR) with 95% confidence interval (CI) was used as a measure of the association between FA supplementation and risk of stroke, after pooling data across trials in a random-effects model. RESULTS: The search identified 13 randomized controlled trials (RCTs) involving treatment with FA that had enrolled 65,812 participants, all of which stroke was reported as an outcome measure. After all 13 RCTs were pooled, FA therapy versus control was associated with a lower risk of any future stroke (RR, 0.85; 95% CI, 0.77 to 0.95). FA alone or combination of FA and minimal cyanocobalamin (≤0.05 mg/day) was associated with a lower risk of future stroke (RR, 0.75; 95% CI, 0.66 to 0.86) whereas combination of FA and cyanocobalamin (≥0.4 mg/day) was not associated with a lower risk of future stroke (RR, 0.95; 95% CI, 0.86 to 1.05). CONCLUSIONS: FA supplement reduced stroke in countries without mandatory FA food fortification. The benefit was found mostly in patients receiving FA alone or combination of FA and minimal cyanocobalamin.

Chinese medicine Tongxinluo capsule alleviates cerebral microcirculatory disturbances in ischemic stroke by modulating vascular endothelial function and inhibiting leukocyte-endothelial cell interactions in mice: A two-photon laser scanning microscopy study.

OBJECTIVE: The aim of this study was to examine the effect of TXL, a Chinese medicine prescription, on cerebral microcirculatory disturbances after pMCAO in mice using TPLSM and further explore the underlying mechanisms. METHODS: Adlut male C57BL/6J mice were subjected to pMCAO and orally administered with TXL (3.0, 1.5 and 0.75 g/kg/d) at 1, 3, and 21 hours after pMCAO. The following parameters were examined at 6 and 24 hours after pMCAO: neurological deficits, infarct volume, BBB permeability, cerebral microvessel structure, brain microcirculation (TPLSM imaging), vasoactive factors, and adhesion molecules. RESULTS: TXL improved neurological deficits, reduced infarct volume, attenuated BBB disruption, protected cerebral microvessel structure, increased cerebral capillary flow velocity and volume flux, and inhibited leukocyte-endothelial cell interactions at 6 or 24 hours after pMCAO. The therapeutic efficacy was exerted in a dose-dependent manner. Further study revealed that TXL (high dose) regulated the expression of PGI2, TXA2, and ET-1, and suppressed ICAM-1 and P-selectin. CONCLUSIONS: TXL alleviates cerebral microcirculatory disturbances against ischemic injury by modulating endothelial function and inhibiting leukocyte-endothelial cell interactions. These effects are associated with regulating the expression of PGI2, TXA2, and ET-1, and suppressing ICAM-1 and P-selectin expression.

Observation of changes in the number of myocardial capillaries in rabbits after treatment of acute myocardial infarction by Tongxinluo superfine powder.

OBJECTIVE: To investigate the effects of Tongxinluo superfine powder on cardiac function, infarct size and the number of myocardial capillaries in a rabbit model of acute myocardial infarction. METHODS: A total of 32 New Zealand white rabbits were randomly divided into four groups: sham operation group, model group, treatment group, and pre-treatment, the experiment of pre-treatment group was performed 6 weeks early than the treat) group,The four groups use a unified modeling technique. An acute myocardial infarction model was established through external application of 70% ferric chloride on the coronary artery. After 7 d, electrocardiogram, ultrasonography of cardiac function, micro-computed tomography, pathology and other data were collected. RESULTS: In the treatment and pre-treatment groups, ejection fraction, left ventricular short axis shortening rate, left ventricular end-systolic diameter and cardiac output significantly improved, the number of capillaries significantly increased, and infarct size significantly decreased. In addition, the results suggest that the value of intra-ventricular pressure and the situation of electrocardiogram also changed to different degrees with the increasing of treatment of cycle. CONCLUSION: Tongxinluo superfine powder can protect the myocardium, improve the blood supply of the myocardium and reduce the degree of myocardial injury, during acute stage of myocardial infarction.

Tongxinluo Regulates Expression of Tight Junction Proteins and Alleviates Endothelial Cell Monolayer Hyperpermeability via ERK-1/2 Signaling Pathway in Oxidized Low-Density Lipoprotein-Induced Human Umbilical Vein Endothelial Cells.

Vascular hyperpermeability resulting from distortion of endothelial junctions is associated with a number of cardiovascular diseases. Endothelial tight junction regulates the paracellular permeability of macromolecules, a function of Human Umbilical Vein Endothelial Cells (HUVEC) monolayers that can be regulated by oxidized Low-density Lipoprotein (ox-LDL). However, the understanding of drug regulation of vascular hyperpermeability is so far limited. This study thus aimed to investigate the role of Tongxinluo (TXL) in the maintenance of the vascular endothelial paracellular permeability. Here, changes in permeability were determined by measuring the paracellular flux of FITC-dextran 40000 (FD40), while protein expression and intercellular distribution were examined by western blot and immunofluorescence assay, respectively. We found that TXL alleviated the ox-LDL-induced increase in flux of FD40 and then reduced the hyperpermeability. Moreover, ox-LDL-induced disruptions of ZO-1, occludin, and claudin1 were also restored. This is via the activation of ERK1/2 in the vascular endothelial cells. Our results provide insights into the molecular mechanism by which TXL alleviates ox-LDL-induced hyperpermeability and provide the basis for further investigations of TXL as regulators of vascular barrier function.

Heat shock protein 27 is a potential indicator for response to YangZheng XiaoJi and chemotherapy agents in cancer cells.

Heat shock protein 27 (HSP27) is a member of the heat shock protein family which has been linked to tumour progression and, most interestingly, to chemotherapy resistance in cancer patients. The present study examined the potential interplay between HSP27 and YangZheng XiaoJi, a traditional Chinese medicine used in cancer treatment. A range of cell lines from different tumour types including pancreatic, lung, gastric, colorectal, breast, prostate and ovarian cancer (both wild-type and resistant) were used. Levels and activation of HSP27 and its potential associated signalling pathways were evaluated by protein array and western blotting. Knockdown of HSP27 in cancer cells was achieved using siRNA. Localisation and co-localisation of HSP27 and other proteins were carried out by immunofluorescence. Cell growth and migration were evaluated in their response to a range of chemotherapeutic agents. The present study first identified, by way of protein array, that YangZheng XiaoJi was able to inhibit the phosphorylation of HSP27 protein in cancer cells. We further demonstrated that HSP27, which is co-localised with caspase-9, can be blocked from localising in focal adhesions and co-localising with caspase-9 by YangZheng XiaoJi. The study also demonstrated that YangZheng XiaoJi was able to sensitise cancer cells including those cells that were resistant to chemotherapy, to chemotherapeutic agents. Finally, knocking down HSP27 markedly reduced the migration of cancer cells and increased the sensitivity of cancer cells to the inhibitory effect on cellular migration by YangZheng XiaoJi. YangZheng XiaoJi can act as an agent in first sensitising cancer cells to chemotherapy and secondly to overcome, to some degree, chemoresistance when used in an appropriate fashion in patients who have active HSP27.

[Effect of Tongxinluo on Apoptosis of Rat Cardiac Microvascular Endothelial Cells].

OBJECTIVE: To observe the protective effects of Tongxinluo (TXL) on apoptosis of rat cardiac microvascular endothelial cells (RCMECs) resulting from homocysteine (Hcy) induced endoplasmic reticulum stress (ERS), and to determine the signaling pathway behind its protection. METHODS: Primary cultured RCMECs were isolated from neonatal rats using tissue explant method. The morphology of RCMECs was observed using inverted microscope, identified and differentiated by CD31 immunofluorescence method. Selected were well growing 2nd-4th generations of RCMECs. The optimal action time was determined by detecting the expression of glucose regulated protein 78 (GRP78) using immunofluorescence method. In the next experiment RCMECs were divided into 5 groups, i.e., the blank control group, the Hcy induced group (Hcy 10 mmol/L, 10 h), the Hcy + TXL group (Hcy 10 mmol/L + TXL 400 µg/mL), the Hcy +LY294002 group (Hcy 10 mmol/L + LY294002 5 µmol/L, LY294002 as the inhibitor of PI3K), the Hcy + LY294002 + TXL group (Hcy 10 mmol/L + LY294002 5 µmol/L + TXL 400 µg/mL). The apoptosis rate of RCMECs was detected by flow cytometry. mRNA and protein expressions of GRP78, C/ EBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase12) were detected by real-time reverse transcription PCR (RT-PCR) and Western blot respectively. Expression levels of phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T- P13K) , phosphorylation of kinase B (P-Akt) , and total kinase B (T-Akt) were detected by Western blot. RESULTS: Ten hours Hcy action time was determined. Compared with the blank control group, the apoptosis rate was increased (22.77%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, protein expressions of P-PI3K and P-Akt,ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy induced group (P < 0.05, P < 0.01). Compared with the Hcy induced group, the apoptosis rate was decreased (10.17%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were decreased, and expression levels of P-PI3K, P-Akt, P-PI3K/T-PI3K, and P-Akt/T-Akt were increased in the Hcy + TXL group (P < 0.05, P < 0.01). Compared with the Hcy + TXL group, the apoptosis rate was increased (17.9%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, expression levels of P-PI3K and P-Akt, ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy + TXL + LY294002 group (P < 0.05, P < 0.01). CONCLUSION: TXL could inhibit the apoptosis of RCMECs resulting from Hcy-induced ERS and its mechanism might be associated with activating PI3K/Akt signaling pathway.