Pei Luo

Ginseng protects rodent hearts from acute myocardial ischemia-reperfusion injury through GR/ER-activated RISK pathway in an endothelial NOS-dependent mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE Ginseng (Panax ginseng C.A. Meyer) is widely used in Asian communities for treating cardiovascular diseases. However, the mechanism by which it protects the myocardium in ischemia-reperfusion (I/R) injury remains unclear. In this study, we aim to investigate whether a standardized ginseng extract (RSE) protects rodent hearts against I/R injury and if glucocorticoid and/or estrogen receptor-mediated activation of Akt and Erk1/2 (the reperfusion injury salvage kinase pathway, RISK) and subsequent nitric oxide (NO) synthesis signaling are involved in this effect. MATERIALS AND METHODS Rats or gene-deleted mice were subjected to 30 min ischemia by occluding the left anterior descending coronary artery and 90 min reperfusion. Infarct size, serum level of creatine kinase (CK), lactate dehydrogenase (LDH), and NO, expression and phosphorylation of glucocorticoid receptor (GR), estrogen receptor (ER), phosphatidylinositol-3 kinase (PI3K), Akt, NO synthase (NOS), extracellular signal-regulated kinase (Erk) 1/2, p38, and c-Jun NH2 terminal kinases (JNK) were examined in rat or mice treated with or without RSE in the absence or presence of pharmacological inhibitors. RESULTS RSE significantly reduced infarct size in a dose-dependent manner and reduced the incidence of arrhythmia, increased serum NO production, reduced serum activities of creatine kinase and lactate dehydrogenase. The infarct size reduction effect of RSE was abolished by RU468 (an inhibitor of GR), tamoxifen (an inhibitor of ER), LY294002 (an inhibitor of PI3K), Akt inhibitor IV (an inhibitor of Akt protein kinase), U0126 (an inhibitor of Erk1/2) and NG-nitro-l-arginine methyl ester hydrochloride (an inhibitor of NOS), but not actinomycin D (an inhibitor of transcription process). RSE also significantly increased the activation of GR/ER, PI3K-Akt-eNOS cascades and Erk1/2 signaling in rat heart. However, RSE did not markedly reduce infarct size in endothelium NOS(-/-) mice. This differs from its effect in inducible NOS(-/-) and wild type mice, suggesting that endothelium NOS is required for the beneficial effect of RSE on the heart. CONCLUSION Our findings showed for the first time that RSE protects hearts subjected to acute I/R injury and the infarct size reduction effect of RSE is associated with GR and/or ER-mediated Akt and Erk1/2 activation in an endothelium NOS-dependent manner.

Total ginsenosides increase coronary perfusion flow in isolated rat hearts through activation of PI3K/Akt-eNOS signaling

BACKGROUND Ginseng is the most popular herb used for treatment of ischemic heart diseases in Chinese community; ginsenosides are considered to be the major active ingredients. However, whether ginsenosides can enhance the coronary artery flow of ischemic heart and, if so, by what mechanisms they do this, remains unclear. METHODS Isolated rat hearts with ischemia/reperfusion injury in Langendorff system were employed for examining the effect of total ginsenosides (TGS) on coronary perfusion flow (CPF). In addition, human aortic endothelial cells (HAECs) were used for mechanistic study. Levels of various vasodilative molecules, intracellular calcium concentration ([Ca²+](i)), and expressions and activation of proteins involving regulation of nitric oxide (NO) signaling pathways in heart tissues and HAECs were determined. RESULTS TGS dose-dependently and significantly increased CPF and improved systolic and diastolic function of the ischemia/reperfused rat heart, while inhibitors of NO synthase (NOS), soluble guanylate cyclase (sGC), heme oxygenase (HO), cyclooxygenase (COX), and potassium channel abolished the vasodilation effect of TGS. Positive control verapamil was effective only in increasing CPF. TGS elevated levels of NO and 6-keto-prostaglandin F₁α, a stable hydrolytic product of prostacyclin I₂ (PGI₂), in both coronary effluents and supernatants of HAECs culturing medium, and augmented [Ca²+](i) in HAECs. TGS significantly up-regulated expression of phosphoinositide 3-kinase (PI3K) and phosphorylations of Akt and endothelial NOS (eNOS) as well. CONCLUSIONS TGS significantly increased CPF of ischemia/reperfused rat hearts through elevation of NO production via activation of PI3K/Akt-eNOS signaling. In addition, PGI₂, EDHF and CO pathways also partially participated in vasodilation induced by TGS.

Rg1 prevents myocardial hypoxia/reoxygenation injury by regulating mitochondrial dynamics imbalance via modulation of glutamate dehydrogenase and mitofusin 2

PURPOSE Mitochondrial dysfunction is a prominent feature of ischemia heart disease but the underlying mechanism of dynamics (fusion/fission) is still unclear. Here we investigated a novel function and underlying mechanism of Rg1 on an in vitro cardiomyocyte model of hypoxia/reoxygenation (H/R). METHODS Cellular cytotoxicity was evaluated by MTT, mitochondrial viable staining, and cardiac marker detection. Mitochondrial function was evaluated by ATP content measurement, MMP determination, ROS, OCR and ECAR assay. Mitochondrial dynamics was investigated by Live-cell imaging with time-lapse fluorescence microscopy and morphological features were evaluated by the high-content image analysis. Mitochondrial fusion and fission-related proteins, GDH were determined by Western blot, RT-PCR and immunofluorescence. RESULTS Rg1 moderated GDH dysregulation and then protected against H/R-induced cellular damage and mitochondrial dysfunction in a dose-dependent manner. Rg1 significantly increased mitochondrial length, reduced the number of cells with fragmented mitochondria and up-regulated the MFN2 expression finally leading to preventing the imbalance of mitochondrial dynamics following H/R. Knock-down of MFN2 by specific siRNA completely abolished the ability of Rg1 to cell survival by H/R. CONCLUSION Rg1 through modulation of GDH and MFN2 maintained mitochondrial dynamics that resulted in protection against H/R-induced cardiomyocyte injury. All these results put forward a new protective mechanism of Rg1 on the therapeutic potential in cardiac I/R disorders.

Characterization and quantification of the chemical compositions of Scutellariae Barbatae herba and differentiation from its substitute by combining UHPLC–PDA–QTOF–MS/MS with UHPLC–MS/MS

The aim of this study was to investigate the possibility of using the non-official species Scutellaria indica L. as the substitute for the official species Scutellaria barbata D. Don. A sensitive, precise and accurate method was developed to identify their chemical compositions from the crude extracts using UHPLC-PDA-QTOF-MS/MS. 36 peaks were detected and 28 peaks have been tentatively and structurally characterized by comparing their retention times, UV spectra and HR-MS data with those of the reference substances and/or the data of the literatures. Additionally, 5 flavonoids have been quantified by multiple reaction monitoring (MRM) in the negative ionization mode. The results demonstrated that there were 23 common peaks between these two species. However, the other 13 peaks from S. barbata could not be detected in S. indica. Furthermore, the content of 5 flavonoids is significantly different. Scutellarein cannot be detected in S. indica, which was considered as the characteristic component for distinguishing the two species. Our data, therefore, clearly demonstrate that S. indica may not be used as the substitute for S. barbata and further investigation is needed to determine through investigation of their therapeutic efficacy.

Stauntoside B inhibits macrophage activation by inhibiting NF-κB and ERK MAPK signalling

Inflammation is a defensive reaction of body to resist foreign invasion. However, it has been demonstrated that excessive and continuous inflammatory responses contribute to various inflammatory diseases, including rheumatoid arthritis. Nuclear factor-κB (NF-κB) regulates the expression of an array of inflammatory mediators, cytokines and chemokine genes in activated macrophages. Therefore, NF-κB has become an attractive drug target for controlling inflammation. In this study, stauntoside B, a C21 steroidal glycosides compound isolated from a Chinese medicine Cynanchi Stauntonii, was for the first time found to suppress macrophage activation induced by lipopolysaccharide (LPS) in RAW264.7 cells and rat primary peritoneal macrophages and could be a potent NF-κB inhibitor. The results showed that stauntoside B significantly reduced the release of inflammatory mediators in activated RAW264.7 cells and rat peritoneal macrophages, including nitric oxide (NO) and prostaglandin E2 (PGE2). The mRNA expressions of pro-inflammatory mediators and cytokines, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), microsomal prostaglandin synthetase-1 (mPGES-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) as well as the production of TNF-α and IL-6 were also inhibited by stauntoside B. Mechanistic investigation implies that the anti-inflammatory activity of stauntoside B could result from the suppression of LPS-induced IKKα/β activation, IκBα phosphorylation, p65 (ser536) NF-κB phosphorylation, and ERK MAPK activation by stauntoside B treatment in activated macrophages. Meanwhile, stauntoside B could induce apoptosis in LPS-activated macrophages. The current study suggests stauntoside B being a valuable candidate drug for the treatment of inflammatory diseases, especially for NF-κB activation associated inflammatory diseases.

Hepatoprotective activity of Gentiana veitchiorum Hemsl. against carbon tetrachloride-induced hepatotoxicity in mice.

AIM To study the hepatoprotective effect of methanol extract of Gentiana veitchiorum (MGV) against CCl4-induced oxidative stress and liver injury in mice. METHOD The acute hepatic model was developed by injection of 20% CCl4 in mice. ICR mice were divided into six groups, including control, CCl4, CCl4(+) silymarin, and CCl4(+) MGV (100, 200, and 400 mg·kg(-1)) groups. Hepatic enzymes including AST, ALT and ALP levels in serum, and antioxidant enzymes, including SOD, CAT and GPX activity in liver tissue, were determined. Histopathological examination and Western blot analysis were performed. RESULTS Oral administration of MGV at 200 and 400 mg·kg(-1) for 15 days dose-dependently inhibited the serum elevations of AST, ALT, and ALP, and recovered the reduction of SOD, CAT, and GPX in liver tissue. Hematoxylin and eosin staining examination performed in liver tissues suggested that MGV treatment ameliorated histopathological changes in CCl4-induced mice. Western blotting analysis implied that MGV increased HO-1 expression and recovered TNF-α alternation. CONCLUSION G. veitchiorum can protect the liver against CCl4-induced damage in mice, and this hepatoprotective effect was due at least in part to its ability through scavenging CCl4-associated free radical activities. The study provided in vivo evidence that G. veitchiorum can be used as a safe, cheap, and effective agent to reduce acute liver damage, supporting its folk medicine use.

Determination of Contents of Catechins in Oolong Teas by Quantitative Analysis of Multi-components Via a Single Marker (QAMS) Method

The catechins in oolong tea are related to its flavor and health benefits. In this paper, a new strategy for a quantitative analysis of multi-components via a single marker (QAMS) was developed and validated to analyze six catechins (epigallocatechin, catechin, epicatechin, epigallocatechin gallate, gallocatechin gallate, and epicatechin gallate) in oolong tea by HPLC. Catechin was chosen as the internal reference substance. The relative correction factors (RCFs) between catechin and the other five catechins were investigated for the QAMS calculation using five different types of chromatographic columns. Meanwhile, the content of each of the six analytes was also determined by a conventional external standard method. Finally, the angle cosine value revealed that there was no significant difference between the QAMS method and the external standard method. The QAMS method established in this study solved the problem of the availability and high cost of some standard substances and would be useful for providing an efficient and feasible quality assessment method for oolong tea and other botanic samples.

Resveratrol Ameliorates Mitochondrial Elongation via Drp1/Parkin/PINK1 Signaling in Senescent-Like Cardiomyocytes

Resveratrol is widely known for its antiaging properties and exerts cardiovascular protective effects in different experimental models. The role of resveratrol in regulating mitochondrial functions and dynamics during the cardiac aging process remains poorly understood. In this study, the effects of resveratrol on mitochondrial morphology and mitochondrial depolarization and on expressions of Drp1, parkin, PINK1, and LC3 were investigated in H9c2 cells after D-galactose treatment that induced senescent-like cardiomyocytes. The results show that downregulation of Drp1 markedly increased mitochondrial elongation. Senescent-like cardiomyocytes were more resistant to CCCP-induced mitochondrial depolarization, which was accompanied by suppressed expression of parkin, PINK1, and LC3-II. Resveratrol treatment significantly increased Drp1 expression, ameliorated mitochondrial elongation, and increased the mitochondrial translocations of parkin and PINK1. In addition, resveratrol significantly enhanced LC3-II expression and decreased TOM20-labeled mitochondrial content. Resveratrol also suppressed the phosphorylation of parkin and PINK1, which may relate to its abilities to degrade the impaired mitochondria in senescent-like cardiomyocytes. These findings show that suppressing mitochondrial elongation in a Drp1-dependent manner is involved in the effect of resveratrol on attenuating the development of aging cardiomyocytes. Activation of parkin and PINK1 may be a potential mechanism of resveratrol for treating cardiovascular complications related to aging.

Purity assessment of ginsenoside Rg1 using quantitative 1H nuclear magnetic resonance

HIGHLIGHTSA rapid, sensitive, precise, accurate, and reliable qNMR method is developed for the purity assessment of ginsenoside Rg1.Three different approaches were compared in eliminating the interference signal caused by the acidic proton exchange.The purity obtained by the qNMR method was cross‐validated by using the DSC, HPLC‐UV, HPLC‐ELSD, and UHPLC‐TOF/MS methods. ABSTRACT Ginseng herbs comprise a group of the most popular herbs, including Panax ginseng, P. notoginseng and P. quinquefolius (Family Araliaceae), which are used as traditional Chinese medicine (TCM) and are some of the best‐selling natural products in the world. The accurate quantification of ginsenoside Rg1 is one of the major aspects of its quality control. However, the purity of the commercial Rg1 chemical reference substance (CRS) is often measured with high‐performance chromatography coupled with an ultraviolet detector (HPLC‐UV), which is a selective detector with unequal responses to different compounds; thus, this detector introduces probable error to purity assessments. In the present study, quantitative nuclear magnetic resonance (qNMR), due to its absolute quantification ability, was applied to accurately assess the purity of Rg1 CRS. Phenylmethyl phthalate was used as the internal standard (IS) to calibrate the purity of Rg1 CRS. The proton signal of Rg1 CRS in methanol‐d4 at 4.37 ppm was selected to avoid interfering signals, enabling accurate quantitative analysis. The relaxation delay, number of scans, and NMR windowing were optimized for data acquisition. For post‐processing, the Lorentz/Gauss deconvolution method was employed to increase the signal accuracy by separating the impurities and noise in the integrated region of the quantitative proton. The method validation showed that the developed method has acceptable sensitivity, linearity, precision, and accuracy. The purity of the commercial Rg1 CRS examined with the method developed in this research was 90.34 ± 0.21%, which was obviously lower than that reported by the manufacturer (>98.0%, HPLC‐UV). The cross‐method validation shows that the commonly used HPLC‐UV, HPLC‐ELSD (evaporative light scattering detector) and even LC‐MS (mass spectrometry) methods provide significantly higher purity values of Rg1 CRS compared with the qNMR method, and the accuracy of these LC‐based methods largely depend on the amount of the sample that was loaded and the properties of the impurities.

Suppressing mPGES-1 expression by sinomenine ameliorates inflammation and arthritis

ABSTRACT Recently, microsomal prostaglandin E synthase 1 (mPGES‐1) has attracted much attention from pharmacologists as a promising strategy and an attractive target for treating various types of diseases including rheumatoid arthritis (RA), which could preserve the anti‐inflammatory effect while reducing the adverse effects often occur during administration of non‐steroidal anti‐inflammatory drugs (NSAIDs). Here, we report that sinomenine (SIN) decreased prostaglandin (PG)E2 levels without affecting prostacyclin (PG)I2 and thromboxane (TX)A2 synthesis via selective inhibiting mPGES‐1 expression, a possible reason of low risk of cardiovascular event compared with NSAIDs. In addition, mPGES‐1 protein expression was down‐regulated by SIN treatment in the inflamed paw tissues both in carrageenan‐induced edema model in rats and the collagen‐II induced arthritis (CIA) model in DBA mice. More interestingly, SIN suppressed the last step of mPGES‐1 gene expression by decreasing the DNA binding ability of NF‐&kgr;B, paving a new way for drug discovery.