Yangxin granules alleviate doxorubicin-induced cardiotoxicity by suppressing oxidative stress and apoptosis mediated by AKT/GSK3ß/ß-catenin signaling.

BACKGROUND: Yangxin granules (YXC), a Chinese herbal medicine, have been confirmed to have clinical benefits in the treatment of heart failure. This study examined the effects and molecular mechanisms of YXC in the treatment of doxorubicin-induced cardiotoxicity in vitro. METHODS: H9c2 cardiomyocytes were pretreated with YXC (5, 10, or 20 mg/mL) or the AKT inhibitor MK-2206 (50 nM) before doxorubicin treatment (1 µM). Cell apoptosis, viability, inflammatory factor expression (TNF-α, IL-1ß, and IL-6), and oxidative stress mediator levels including superoxide dismutase, reactive oxygen species, and malondialdehyde were detected. RESULTS: YXC increased the viability of H9c2 cells. In addition, doxorubicin inhibited AKT/GSK3ß/ß-catenin signaling, whereas YXC increased the expression of phosphorylated AKT and GSK3ß, and ß-catenin in doxorubicin-treated H9c2 cells. Moreover, T-cell factor/lymphoid enhancer factor signaling downstream of ß-catenin was also activated by YXC. YXC pretreatment also inhibited doxorubicin-induced inflammation, oxidative stress, and apoptosis. However, MK-2206 reversed the effects of YXC in doxorubicin-treated H9c2 cells. CONCLUSIONS: YXC alleviates doxorubicin-induced inflammation, oxidative stress, and apoptosis in H9c2 cells. These effects might be mediated by the AKT/GSK3ß/ß-catenin signaling pathway. YXC might have preventive effects against doxorubicin-induced heart failure.

Determination of four nitrofuran metabolites in gelatin Chinese medicine using dispersive solid phase extraction and pass-through solid phase extraction coupled to ultra high performance liquid chromatography-tandem mass spectrometry.

This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including semicarbazide (SEM), 1-aminohydantoin (AHD), 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ) in gelatin Chinese medicine. A C18 column with the mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ were found to be 0.2 µg/kg, 0.3 µg/kg, 0.2 µg/kg and 0.2 µg/kg, whereas their limit of quantification were 0.6 µg/kg, 0.8 µg/kg, 0.6 µg/kg and 0.5 µg/kg. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was purified through dispersive solid phase extraction using 1000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent, and then further purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the clean-up procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in water: acetonitrile (95:5, v/v). The recoveries of these nitrofuran metabolites under the 1 µg/kg, 2 µg/kg and 10 µg/kg spiking levels were between 77.4% and 95.6%. These metabolites after the extraction were stable at 4 °C for 24 h. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg).

Hypoxia-induced apoptosis of cardiomyocytes is restricted by ginkgolide B-downregulated microRNA-29.

Ginkgolide B exerts a cardioprotective function against ischemia-caused apoptosis in myocardial infarction. Here we sought out to address a functional mechanism associated with microRNA-29 (miR-29). Rat cardiomyocytes (H9c2 cells) were cultured in ginkgolide B-conditioned medium prior to hypoxic induction. To construct miR-29-overexpressed cells, miR-29 mimic was transfected into H9c2 cells. The cells were harvested for assaying survivability and apoptosis by CCK-8 and FITC-Annexin V staining methods. Western blot was applied to identify apoptotic hallmarks and signaling transducers. RT-PCR was carried out for investigating miR-29 expression. Cardiomyocytes were sensitive to hypoxic apoptosis, while ginkgolide B intensified the abilities of cardiomyocytes to resist hypoxia by increasing survivability and repressing apoptosis. Specifically, ginkgolide B repressed Bax and cleaved caspase 3 while enhanced Bcl-2. Ginkgolide B buffered the expression of miR-29 induced by hypoxia. However, ginkgolide B showed a slight role in survivability and apoptosis in the cells overexpressing miR-29. Meanwhile, ginkgolide B triggered the phosphorylation of PI3 K and AKT, as well as induced Sp1, while this beneficial role was abrogated in the cells treated by miR-29 mimic. Our results confirmed that ginkgolide B might have therapeutic significance by repressing hypoxic apoptosis. Ginkgolide B-elicited miR-29 inhibition might be the basis of this beneficial role.

Astragaloside III activates TACE/ADAM17-dependent anti-inflammatory and growth factor signaling in endothelial cells in a p38-dependent fashion.

Astragaloside III (AS-III) is a triterpenoid saponin contained in Astragali Radix and has potent anti-inflammatory effects on vascular endothelial cells; however, underlying mechanisms are unclear. In this study, we provided the first piece of evidence that AS-III induced phosphorylation of TNF-α converting enzyme (TACE) at Thr735 and enhanced its sheddase activity. As a result, AS-III reduced surface TNFR1 level and increased content of sTNFR1 in the culture media, leading to the inhibition of NF-κB signaling pathway and attenuation of downstream cytokine gene expression. Furthermore, AS-III induced TACE-dependent epidermal growth factor receptor (EGFR) transactivation and activation of downstream ERK1/2 and AKT pathways. Finally, AS-III induced activation of p38. Both TACE activation and EGFR transactivation induced by AS-III were significantly inhibited by p38 inhibitor SB203580. Taken together, we concluded that AS-III activates TACE-dependent anti-inflammatory and growth factor signaling in vascular endothelial cells in a p38-dependent fashion, which may contribute to its cardiovascular protective effect.

[Inhibition of Paeoniflorin on TNF-α-induced TNF-α Receptor Type I /Nuclear Factor-κB Signal Transduction in Endothelial Cells].

OBJECTIVE: To study the inhibitory effect of paeoniflorin (PAE) on TNF-α-induced TNF receptor type I (TNFR1)-mediated signaling pathway in mouse renal arterial endothelial cells (AECs) and to explore its underlying molecular mechanisms. METHODS: Mouse AECs were cultured in vitro and then they were treated by different concentrations PAE or TNF-α for various time periods. Expression levels of intercellular cell adhesion molecule-1 (ICAM-1) were detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 6-h TNF-α 30 ng/mL), the low dose PAE group (cultured by 2-h PAE 0.8 µmo/L plus 6-h TNF-α 30 ng/mL), the middle dose PAE group (cultured by 2-h PAE 8 µmol/L plus 6-h TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 6-h TNF-α 30 ng/mL) with Western blot analysis. Nuclear translocation of transcription factor NF-κB (NE-κB) was detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 45-mm TNF-α 30 ng/mL), and the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 45-min TNF-α 30 ng/mL) by immunofluorescent staining. Expression levels of the phosphorylation of extracellular signal-regulated (protein) kinase (ph-ERK) and p38 (ph- p38) were detected in the normal group (cultured by serum-free culture media) and the high dose PAE group (2-h PAE 80 µmol/L culture) by Western blot. NF-κB inhibitor-α (IκBα) protein expressions were detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 30-min TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 µmol/L plus 30-min TNF-α 30 ng/mL), the p38 inhibitor group (SB group, pretreatment with SB238025 25 µmol/L for 30 min, then treated by PAE 80 µmol/L for 2 h, and finally treated by TNF-α 30 ng/mL for 30 min), the ERK inhibitor group (PD group, treated by PD98059 50 µmol/L for 30 min, then treated by PAE 80 µmol/L for 2 h, and finally treated by TNF-α 30 ng/mL for 30 min) by Western blot. RESULTS: Compared with the normal group, ICAM-1 protein expression levels obviously increased (P < 0.01). Compared with the TNFα group, ICAM-1 protein expression levels were obviously inhibited in the high dose PAE group (P < 0.05). Protein expression levels of ph-p38 and ph-ERK were obviously higher in the hIgh dose PAE group (P < 0.05). Compared with the normal group, IκBα protein expression levels obviously decreased in the TNF-α group (P < 0.01). Compared with the TNFα group, TNF-α-induced IκBα degradation could be significantly inhibited in the high dose PAE group (P < 0.01); the inhibition of PAE on IκBα degradation could be significantly inhibited in the SB group (P < 0.05). NF-κB/p65 signal was mainly located in cytoplasm in the normal group. NF-κB/p65 was translocated from cytoplasm to nucleus after stimulated by 45 min TNF-α in the TNF-α group, while it could be significantly inhibited in the high dose PAE group. CONCLUSIONS: PAE inhibited TNF-α-induced expression of lCAM-1. Its action might be associated with inhibiting TNFR1/NF-κB signaling pathway. p38 participated and mediated these actions.

Rolipram stimulates angiogenesis and attenuates neuronal apoptosis through the cAMP/cAMP-responsive element binding protein pathway following ischemic stroke in rats.

Rolipram, a phosphodiesterase-4 inhibitor, can activate the cyclic adenosine monophosphate (cAMP)/cAMP-responsive element binding protein (CREB) pathway to facilitate functional recovery following ischemic stroke. However, to date, the effects of rolipram on angiogenesis and cerebral ischemia-induced neuronal apoptosis are yet to be fully elucidated. In this study, the aim was to reveal the effect of rolipram on the angiogenesis and neuronal apoptosis following brain cerebral ischemia. Rat models of ischemic stroke were established following transient middle cerebral artery occlusion and rolipram was administered for three, seven and 14 days. The results were examined using behavioral tests, triphenyl tetrazolium chloride staining, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to evaluate the effects of rolipram therapy on functional outcome, angiogenesis and apoptosis. Western blot analysis was used to show the phosphorylated- (p-)CREB protein level in the ischemic hemisphere. The rolipram treatment group exhibited a marked reduction in infarct size and modified neurological severity score compared with the vehicle group, and rolipram treatment significantly promoted the microvessel density in the ischemic boundary region and increased p-CREB protein levels in the ischemic hemisphere. Furthermore, a significant reduction in the number of TUNEL-positive cells was observed in the rolipram group compared with the vehicle group. These findings suggest that rolipram has the ability to attenuate cerebral ischemic injury, stimulate angiogenesis and reduce neuronal apoptosis though the cAMP/CREB pathway.

Hydroxy-Safflower Yellow A inhibits the TNFR1-Mediated Classical NF-κB Pathway by Inducing Shedding of TNFR1.

Hydroxy-safflower yellow A (HSYA) is the major active component of safflower, a traditional Asia herbal medicine well known for its cardiovascular protective activities. The purpose of this study was to investigate the effect of HSYA on TNF-α-induced inflammatory responses in arterial endothelial cells (AECs) and to explore the mechanisms involved. The results showed that HSYA suppressed the up-regulation of ICAM-1 expression in TNF-α-stimulated AECs in a dose-dependent manner. High concentration (120 µM) HSYA significantly inhibited the TNF-α-induced adhesion of RAW264.7 cells to AECs. HSYA blocked the TNFR1-mediated phosphorylation and degradation of IκBα and also prevented the nuclear translocation of NF-κB p65. Moreover, HSYA reduced the cell surface level of TNFR1 and increased the content of sTNFR1 in the culture media. TNF-α processing inhibitor-0 (TAPI-0) prevented the HSYA inhibition of TNFR1-induced IκBα degradation, implying the occurrence of TNFR1 shedding. Furthermore, HSYA induced phosphorylation of TNF-α converting enzyme (TACE) at threonine 735, which is thought to be required for its activation. Conclusively, HSYA suppressed TNF-α-induced inflammatory responses in AECs, at least in part by inhibiting the TNFR1-mediated classical NF-κB pathway. TACE-mediated TNFR1 shedding can be involved in this effect. Our study provides new evidence for the antiinflammatory and anti-atherosclerotic effects of HSYA. Copyright © 2016 John Wiley & Sons, Ltd.