Qihuang Zhuyu formula alleviates coronary microthrombosis by inhibiting PI3K/Akt/αIIbß3-mediated platelet activation.

BACKGROUND: Coronary microembolism (CME) is commonly seen in the peri-procedural period of Percutaneous Coronary Intervention (PCI), where local platelet activation and endothelial cell inflammation crosstalk may lead to micro thrombus erosion and rupture, with serious consequences. Qihuang Zhuyu Formula (QHZYF) is a Chinese herbal compound with high efficacy against coronary artery disease, but its antiplatelet mechanism is unclear. HYPOTHESIS/PURPOSE: This study aimed to elucidate the effects and mechanisms of QHZYF on sodium laurate-induced CME using network pharmacology and in vitro and in vivo experiments. METHODS: We employed high-performance liquid chromatography mass spectrometry to identify the main components of QHZYF. Network pharmacology analysis, molecular docking and surface plasmon resonance (SPR) were utilized to predict the primary active components, potential therapeutic targets, and intervention pathways mediating the effects of QHZYF on platelet activation. Next, we pretreated a sodium laurate-induced minimally invasive CME rat model with QHZYF. In vivo experiments were performed to examine cardiac function in rats, to locate coronary arteries on heart sections to observe internal microthrombi, to extract rat Platelet-rich plasma (PRP) for adhesion assays and CD62p and PAC-1 (ITGB3/ITGA2B) flow assays, and to measure platelet-associated protein expression in PRP. In vitro clot retraction and Co-culture of HUVECs with PRP were performed and the gene pathway was validated through flow cytometry and immunofluorescence. RESULTS: Combining UPLC-Q-TOF/MS technology and database mining, 78 compounds were finally screened as the putative and representative compounds of QHZYF, with 75 crossover genes associated with CME. QHZYF prevents CME mainly by regulating key pathways of the inflammation and platelets, including Lipid and atherosclerosis, Fluid shear stress, platelet activation, and PI3K-Akt signaling pathways. Five molecules including Calyson, Oroxin A, Protosappanin A,Kaempferol and Geniposide were screened and subjected to molecular docking and SPR validation in combination with Lipinski rules (Rule of 5, Ro5). In vivo experiments showed that QHZYF not only improved myocardial injury but also inhibited formation of coronary microthrombi. QHZYF inhibited platelet activation by downregulating expression of CD62p receptor and platelet membrane protein αIIbß3 and reduced the release of von Willebrand Factor (vWF), Ca2+ particles and inflammatory factor IL-6. Further analysis revealed that QHZYF inhibited the activation of integrin αIIbß3, via modulating the PI3K/Akt pathways. In in vitro experiments, QHZYF independently inhibited platelet clot retraction. Upon LPS induction, the activation of platelet membrane protein ITGB3 was inhibited via the PI3K/Akt pathway, revealing an important mechanism for attenuating coronary microthrombosis. We performed mechanistic validation using PI3K inhibitor LY294002 and Akt inhibitor MK-2206 to show that QHZYF inhibited platelet membrane protein activation and inflammation to improved coronary microvessel embolism by regulating PI3K/Akt/αIIbß3 pathways, mainly by inhibiting PI3K and Akt phosphorylation. CONCLUSION: QHZYF interferes with coronary microthrombosis through inhibition of platelet adhesion, activation and inflammatory crosstalk, thus has potential in clinical anti-platelet applications. Calyson, Oroxin A, Protosappanin A, Kaempferol and Geniposide may be the major active ingredient groups of QHZYF that alleviate coronary microthrombosis.

Qihuang Zhuyu Formula Attenuates Atherosclerosis via Targeting PPARγ to Regulate Cholesterol Efflux and Endothelial Cell Inflammation.

At present, due to the limitations of drug therapy targets for atherosclerosis, some patients fail to achieve satisfactory efficacy. Cholesterol efflux dysfunction and endothelial cell inflammation are considered to be important factors in the development of atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARγ), a promising therapeutic target for atherosclerosis, plays a dual role in regulating cholesterol efflux and endothelial cell inflammation. However, the use of PPARγ agonist in clinical practice is greatly limited as it could lead to water and sodium retention and hence result in congestive heart failure. Qihuang Zhuyu Formula (QHZYF) is a hospital preparation of Jiangsu Province Hospital of Chinese Medicine which has definite effect in the treatment of atherosclerosis, but its pharmacological mechanism has not been clear. In this study, we successfully predicted that QHZYF might regulate cholesterol efflux and endothelial inflammation via targeting PPARγ-mediated PPARγ/LXRα/ABCA1-ABCG1 and PPARγ/NF-κB p65 pathways by using UPLC-Q-TOF/MS, network pharmacology, bioinformatics analysis, and molecular docking technology. Subsequently, we confirmed in vivo that QHZYF could attenuate atherosclerosis in ApoE-/- mice and regulate the expression levels of related molecules in PPARγ/LXRα/ABCA1-ABCG1 and PPARγ/NF-κB p65 pathways of ApoE-/- mice and C57BL/6 wild-type mice. Finally, in in vitro experiments, we found that QHZYF could reduce lipid content and increase cholesterol efflux rate of RAW 264.7 cells, inhibit the inflammatory response of HUVECs, and regulate the expression levels of related molecules in the two pathways. In addition, the above effects of QHZYF were significantly weakened after PPARγ knockdown in the two kinds of cells. In conclusion, our study revealed that QHZYF attenuates atherosclerosis via targeting PPARγ-mediated PPARγ/LXRα/ABCA1-ABCG1 and PPARγ/NF-κB p65 pathways to regulate cholesterol efflux and endothelial cell inflammation. More importantly, our study offers a promising complementary and alternative therapy which is expected to make up for the limitation of current drug treatment methods and provide a valuable reference for new drugs development in the future.

Mitigation of heterocyclic amines by phenolic compounds in allspice and perilla frutescens seed extract: The correlation between antioxidant capacities and mitigating activities.

The effect of different levels of allspice and perilla frutescens seed extract (ASE and PSE) on the formation of heterocyclic amines (HCAs) in pan-fried chicken meat patties and the bioactive components found in ASE and PSE that contribute to the mitigation of HCAs were investigated in this study. DPPH radical scavenging activity was evaluated and the results indicated that APSE (ASE + PSE) showed the highest capacity to scavenge free radicals, and the most effective inhibition of HCAs formation. Furthermore, Single and mixed phenolic compounds exhibited a positive effect in scavenging free radicals and mitigating HCAs. The radical scavenging activity and HCAs inhibition effect of single phenolic compounds were highly correlated, whereas mixed phenolic compounds exhibited poor correlation. PCA analysis indicated that phenolic compounds had the maximum inhibitory effect on IQ, followed by Norharman and harman and the minimal effect on PhIP and 7,8-DiMeIQx.

Selenium-GPX4 axis protects follicular helper T cells from ferroptosis.

Follicular helper T (TFH) cells are a specialized subset of CD4+ T cells that essentially support germinal center responses where high-affinity and long-lived humoral immunity is generated. The regulation of TFH cell survival remains unclear. Here we report that TFH cells show intensified lipid peroxidation and altered mitochondrial morphology, resembling the features of ferroptosis, a form of programmed cell death that is driven by iron-dependent accumulation of lipid peroxidation. Glutathione peroxidase 4 (GPX4) is the major lipid peroxidation scavenger and is necessary for TFH cell survival. The deletion of GPX4 in T cells selectively abrogated TFH cells and germinal center responses in immunized mice. Selenium supplementation enhanced GPX4 expression in T cells, increased TFH cell numbers and promoted antibody responses in immunized mice and young adults after influenza vaccination. Our findings reveal the central role of the selenium-GPX4-ferroptosis axis in regulating TFH homeostasis, which can be targeted to enhance TFH cell function in infection and following vaccination.

Differences in Clinical Characteristics and Brain Activity between Patients with Low- and High-Frequency Tinnitus.

This study was aimed at delineating and comparing differences in clinical characteristics and brain activity between patients with low- and high-frequency tinnitus (LFT and HFT, respectively) using high-density electroencephalography (EEG). This study enrolled 3217 patients with subjective tinnitus who were divided into LFT (frequency < 4000 Hz) and HFT (≥4000 Hz) groups. Data regarding medical history, Tinnitus Handicap Inventory, tinnitus matching, and hearing threshold were collected from all patients. Twenty tinnitus patients and 20 volunteers were subjected to 256-channel EEG, and neurophysiological differences were evaluated using standardized low-resolution brain electromagnetic tomography (sLORETA) source-localized EEG recordings. Significant differences in sex (p < 0.001), age (p = 0.022), laterality (p < 0.001), intensity (p < 0.001), tinnitus type (p < 0.001), persistent tinnitus (p = 0.04), average threshold (p < 0.001), and hearing loss (p = 0.028) were observed between LFT and HFT groups. The tinnitus pitch only appeared to be correlated with the threshold of the worst hearing loss in the HFT group. Compared with the controls, the LFT group exhibited increased gamma power (p < 0.05), predominantly in the posterior cingulate cortex (PCC, BA31), whereas the HFT group had significantly decreased alpha1 power (p < 0.05) in the angular gyrus (BA39) and auditory association cortex (BA22). Higher gamma linear connectivity between right BA39 and right BA41 was observed in the HFT group relative to controls (t = 3.637, p = 0.027). Significant changes associated with increased gamma in the LFT group and decreased alpha1 in the HFT group indicate that tinnitus pitch is crucial for matching between the tinnitus and control groups. Differences of band frequency energy in brain activity levels may contribute to the clinical characteristics and internal tinnitus "spectrum" differences.

[Diagnostic value of lung ultrasound B-line score in acute heart failure].

OBJECTIVE: To investigate the value of bedside lung ultrasound B-line score in the diagnosis of acute heart failure (AHF). METHODS: A retrospectively analysis was conducted. The adult patients presenting with acute dyspnea in intensive care unit (ICU) of Affiliated Hospital of Nanjing University of Traditional Chinese Medicine from January 2016 to June 2017 were enrolled. An 8-zone lung ultrasound was performed and plasma B-type natriuretic peptide (BNP) level was tested in all patients. AHF was determined as the final diagnosis by two experienced ICU doctors according to the diagnostic criteria of AHF. Patients were divided into two groups: AHF group and non-AHF group. The levels of BNP and B-line score were compared between the two groups, and the diagnostic value of BNP and B-line score in AHF was evaluated. RESULTS: Fifty-six patients were included in this study, with 32 of men and 24 of women, and with an average age of 77.3±8.8. Thirty-six patients were diagnosed as AHF. The level of BNP and lung ultrasound B-line score in AHF group were higher than those in non-AHF group [BNP (ng/L): 1 640.4±1 078.4 vs. 236.9±124.9, B line score: 12.8±5.3 vs. 5.4±1.8, both P < 0.01]. There was a strong correlation between elevated BNP levels and an increased B-lines score (R2 = 0.712, P = 0.000). The receiver operating characteristic curve (ROC) showed that when the cut-off of lung ultrasound B-line score was 8.5, AHF could be discriminated from dyspnea caused by other diseases (sensitivity was 77.8%, specificity was 95%, positive likelihood ratio was 15.56, negative likelihood ratio was 0.23). The area under the ROC curve (AUC) of lung ultrasound B-line score was 0.917 [95% confidence interval (95%CI) = 0.847-0.987, P = 0.000], slightly lower than that of plasma BNP [0.979 (95%CI = 0.951-1.008)]. CONCLUSIONS: Lung ultrasound B-line score was highly specific, but moderately sensitive for identifying patients with AHF.

Bioassay-Guided Isolation and Identification of Xanthine Oxidase Inhibitory Constituents from the Leaves of Perilla frutescens.

Activity-directed fractionation and purification processes were employed to identify xanthine oxidase (XO) inhibitory compounds from the leaves of Perilla frutescens. The total extract was evaluated in vitro on XO inhibitory activity and in vivo in an experimental model with potassium oxonate-induced hyperuricemia in mice which was used to evaluate anti-hyperuricemic activity. The crude extract showed expressive urate-lowering activity results. Solvent partitioning of the total extract followed by macroporous resin column chromatography of the n-butanol extract yielded four extracts and eluted parts. Among them, only the 70% ethanol eluted part of the n-butanol extract showed strong activity and therefore was subjected to separation and purification using various chromatographic techniques. Five compounds showing potent activity were identified by comparing their spectral data with literature values to be caffeic acid, vinyl caffeate, rosmarinic acid, methyl rosmarinate, and apigenin. These results indicate that pending further study, these compounds could be used as novel natural product agents for the treatment of hyperuricemia.

Comparative study of the efficacy of the canalith repositioning procedure versus the vertigo treatment and rehabilitation chair.

CONCLUSION: Vertigo treatment and rehabilitation chair (TRV) may be suggested as the first choice for patients with posterior canal benign paroxysmal positional vertigo (p-BPPV). OBJECTIVE: To investigate the short- and long-term treatment efficacy of the canalith repositioning procedure (CRP) versus TRV for patients with p-BPPV. METHODS: A total of 165 patients with unilateral p-BPPV were assigned to either the CRP group or the TRV group. Patients were assessed at 1 week, 4 weeks, 3 months, and 6 months after their first treatment. The numbers of treatment sessions required for successful repositioning in both groups at 4 weeks, 3 months, and 6 months were recorded. RESULTS: Treatment efficacy of patients in the TRV group was significantly better than that of patients in the CRP group 1 week after the first treatment. The number of treatment sessions needed for successful repositioning was significantly lower in the TRV group than in the CRP group at 4 weeks and 3 months after the first treatment.

The genus Gnaphalium L. (Compositae): phytochemical and pharmacological characteristics.

The genus Gnaphalium, a herb distributed worldwide, comprises approximately 200 species of the Compositae (Asteraceae) family that belongs to the tribe Gnaphalieae. Some species are traditionally used as wild vegetables and in folk medicine. This review focuses on the phytochemical investigations and biological studies of plants from the genus Gnaphalium over the past few decades. More than 125 chemical constituents have been isolated from the genus Gnaphalium, including flavonoids, sesquiterpenes, diterpenes, triterpenes, phytosterols, anthraquinones, caffeoylquinic acid derivatives, and other compounds. The extracts of this genus, as well as compounds isolated from it, have been demonstrated to possess multiple pharmacological activities such as antioxidant, antibacterial and antifungal, anti-complement, antitussive and expectorant, insect antifeedant, cytotoxic, anti-inflammatory, antidiabetic and antihypouricemic properties. The present review compiles the information available on this genus because of its relevance to food and ethnopharmacology and the potential therapeutic uses of these species.

Effects of salvianolic acid and notoginseng triterpenes on angiogenesis in EA-hy926 cells in vitro.

AIM: To investigate the different effects of salvianolic acid and notoginseng triterpenes on proliferation, angiogenesis and expression of vascular endothelial growth factor in EA-hy926 cells in vitro. METHODS: EA-hy926 cells were cultured in vitro. Salvianolic acid and notoginseng triterpenes at concentrations of 0.4, 0.8 and 1.2 mg·L(-1) were used to culture EA-hy926 cells. EA-hy926 cells in a blank control group were grown in culture solution only. Viability of cells was assessed by CCK-8, and after treated for 12 h, capillary-like structures were examined. After 24 h culture, the expression of VEGF was detected by real-time PCR. RESULTS: Salvianolic acid at 0.4, 0.8 mg·L(-1), the same as notoginseng triterpenes, increased VEGF content in EA-hy926 cells. Expression of VEGF protein in the salvianolic acid at 1.2 mg·L(-1) group, was up-regulated as compared with notoginseng triterpenes group (P < 0.05). CONCLUSION: Salvianolic acid and notoginseng triterpenes can promote EA-hy926 cell proliferation, angiogenesis and expression of VEGF protein. This analysis also provided evidence that salvianolic acid had the better effects as compared with notoginseng triterpenes.

Protocatechualdehyde synergizes with aspirin at the platelet cyclooxygenase-1 level.

Polyphenol-aspirin interactions were recently identified; however, the interaction mode and underlying mechanisms remained elusive. Here, we quantitatively assessed the potential interactions among two important polyphenolic compounds, caffeic acid (CA) and protocatechualdehyde (Pro), and aspirin in the AA-induced platelet aggregation model by applying the isobologram and universal response surface approach (URSA) methods. A molecular docking approach and an originally developed platelet-associated aspirin clearance approach (PAACA) were then applied to explore the potential interaction mechanisms. Although Pro and CA themselves exhibited weak inhibitory effect on arachidonic acid (AA)-induced platelet aggregation and the production of thromboxane B2 (TXB2), both Pro and CA potentiated aspirin action in a synergistic manner. The most prominent synergism was found between Pro and aspirin. Pro formed a stable complex into the cyclooxygenase-1 (COX-1) channel by in silico docking and significantly promoted the platelet-associated aspirin clearance, suggesting that the Pro interaction with COX-1 was favorable to the binding of aspirin with COX-1. Taken together, our findings suggest that the capacity of Pro and potentially other structurally similar polyphenolic compounds on promoting the binding of aspirin on platelet COX-1 might be the main mechanism of their synergism with aspirin.

Enhancement of anti-allergic effects mediated by the Kampo medicine Shoseiryuto (Xiao-Qing-Long-Tang in Chinese) with lysed Enterococcus faecalis FK-23 in mice.

Kampo is a traditional Japanese medicine originating from ancient Chinese medicine which included the administration of herbal prescription, lifestyle advice and acupuncture. Orally administered Kampo prescriptions are believed to be influenced by diet and intestinal microbiota. However, reports on the Kampo administration effects are still limited. Shoseiryuto (TJ-19), which has anti-allergic and anti-inflammatory properties, is a Kampo prescription used clinically for the treatment of allergic bronchial asthma. We examined whether Shoseiryuto administration is affected by a probiotic product, lysed Enterococcus faecalis FK-23 (LFK). BALB/c mice were sensitized with cedar pollen allergen, and the peritoneal accumulation of eosinophils was induced. During a sensitization period of 21 days, varying amounts of Shoseiryuto (and saline as a control) were administered to the mice. The accumulation of eosinophils was significantly reduced by 30 mg/day doses of Shoseiryuto but not by 3 or 9 mg/day doses. Similarly, 3 mg/day Shoseiryuto, 30 mg/day LFK, 3 mg/day of Shoseiryuto co-administered with 30 mg/day of LFK, and saline control were compared. A significant reduction in the accumulation of eosinophils was observed at 3 mg/day Shoseiryuto co-administered with 30 mg/day of LFK. These results suggest that Shoseiryuto-mediated anti-allergic effects are enhanced by the probiotic (LFK). Although not significant statistically, serum allergen-specific and total IgE levels in the treatment group exposed to the mixed agent (ie. Shoseiryuto and LFK) were generally lower than those receiving either one alone. The results indicate a synergistic effect of a Kampo medicine (Shoseiryuto, Xiao-Qing-Long-Tang in Chinese) and lysed Enterococcus faecalis FK-23 on allergic responses in mice.

Effects of lysed Enterococcus faecalis FK-23 on allergen-induced peritoneal accumulation of eosinophils and serum total IgE concentration in inbred mice.

Recent clinical trials have shown the possibility of probiotics in prevention and treatment of allergic diseases. The purpose of this experimental study was to assess the influence of lysed Enterococcus faecalis FK-23 (LFK) on allergic responses in different mouse strains. We performed a comparative study on the effects of LFK for allergen-induced peritoneal accumulation of eosinophils and serum total IgE concentration by using BALB/c, C57BL/6, C3H/HeN and C3H/HeJ mice. There was no significant difference in total number of peritoneal accumulated cells induced by cedar pollen allergen between the control and LFK groups in any strain of mice (p > 0.05); however, the ratio of eosinophils to total accumulated cells was significantly decreased in LFK-treated mice of BALB/c (p = 0.016), C3H/HeN (p = 0.010) and C3H/HeJ (p = 0.004), but not C57BL/6 (p > 0.05). No significant difference in serum total IgE concentration was found between the control and LFK groups of different mouse strains (p > 0.05). These results reveal a different effect of LFK on suppressing allergen-induced local eosinophila in inbred strains of mice, suggesting the effectiveness of probiotics on limiting allergy might be under the influence of individual genetic background.

[Study on the theraputic effect of plants of Camellia genus on osteoporosis].

OBJECTIVE: To observe anti-osteoporotic effect of Plants of Camelia genus induced by retinoic acid in rats, in adqulis crude drug dosage, and to compare activities of them. METHODS: Extracts of Camellia japonica and Camellia oleifera were given to rats with osteoporosis induced by retinoic acid, some indexes of rats were measured and compared with those of modle group, control group and positive control group, including weight/length (G/L), bone density, earth and calcium content of bone, morphology change and serum calcium, tartrate-resistant acid phosphatase and alkaline phosphatase. We also compared effective intensity between different groups in adqulis crude drug dosage. RESULTS: Ethanol extracts of seed from Camellia japonica 0.51 g/kg could markedly enhance weight/length (G/L), bone density of femur, serum calcium and alkaline phosphatase level, with the decreasing of anti-tartaric acid tartrate-resistant acid phosphatase level. Meanwhile, they were accompanied by a significant increase of morphologic observed sclerotomal cell and by a significant decrease of osteoclast. Moreover, it was observed greatly that bone trabecula transformateed to normal morphous. The results of this study indicated that effects of ethanol extracts of seed from Camellia japonica on anti-osteoporosis with retinoic acid were the strongest. Ethanol extracts of seed from Camellia japonica , ethanol extracts of leaves from Camellia Oleifera, and aqueous extracts of leaves from Camellia Oleifera were stronger than positive control drug. The other extracts didnt show obvious anti-osteoporotic effects. Eventually the strength order of each group on anti-osteoporosis was as following: ethanol extracts of seed from Camellia japonica > ethanol extracts of leaves from Camellia Oleifera > aqueous extracts of leaves from Camellia Oleifera > aqueous extracts of seed from Camellia Oleifera > positive control drug > aqueous extracts of seed from Camellia Japonica. CONCLUSION: Plants of Camellia genus have different degree anti-osteoporosis effect, which can offer significant theory basis for progressive investigation and exploitation of them.