Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury

BACKGROUND AND PURPOSE

Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells.

EXPERIMENTAL APPROACH

Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors.

KEY RESULTS

NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor α-glycyrrhetinic acid (α-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of α-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by α-GA.

CONCLUSION AND IMPLICATIONS

Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.     

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

Aim:

β-adrenergic receptor (β-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 (Cx43), the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function.

Methods:

The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay.

Results:

Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective β₂-AR inhibitor ICI 118551, but not β₁-AR inhibitor {"type":"entrez-protein","attrs":{"text":"CGP20712","term_id":"874704353","term_text":"CGP20712"}}CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and G_i protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.

Conclusion:

Taken together, β₂-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A- and G_i-dependent manner.     

Connexin 43 stabilizes astrocytes in a stroke-like milieu to facilitate neuronal recovery

Aim:

Connexin 43 (Cx43) is a member of connexin family mainly expressed in astrocytes, which forms gap junctions and hemichannels and maintains the normal shape and function of astrocytes. In this study we investigated the role of Cx43 in astrocytes in facilitating neuronal recovery during ischemic stroke.

Methods:

Primary culture of astrocytes or a mixed culture of astrocytes and cortical neurons was subjected to oxygen glucose deprivation and reperfusion (OGD/R). The expression of Cx43 and Ephrin-A4 in astrocytes was detected using immunocytochemical staining and Western blot assays. Intercellular Ca²⁺ concentration was determined with Fluo-4 AM fluorescent staining. Middle cerebral artery occlusion (MCAO) model rats were used for in vivo studies.

Results:

OGD/R treatment of cultured astrocytes caused a decrement of Cx43 expression and translocation of Cx43 from cell membrane to cytoplasm, accompanied by cell retraction. Furthermore, OGD/R increased intracellular Ca²⁺ concentration, activated CaMKII/CREB pathways and upregulated expression of Ephrin-A4 in the astrocytes. All these changes in OGD/R-treated astrocytes were alleviated by overexpression of Cx43. In the cortical neurons cultured with astrocytes, OGD/R inhibited the neurite growth, whereas overexpression of Cx43 or knockdown of Ephrin-A4 in astrocytes restored the neurite growth. In MCAO model rats, neuronal recovery was found to be correlated with the recuperation of Cx43 and Ephrin-A4 in astrocytes.

Conclusion:

Cx43 can stabilize astrocytes and facilitate the resistance to the deleterious effects of a stroke-like milieu and promote neuronal recovery.     

Adenosine 5＇-triphosphate stimulates the increase of TGF-β1 in rat mesangial cells under high-glucose conditions via reactive oxygen species and ERK1/2

Aim:

To investigate the role of adenosine 5′-triphosphate (ATP)-induced generation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the production of transforming growth factor-β1 (TGF-β1) in cultured rat glomerular mesangial cells under high-glucose conditions.

Methods:

Subconfluent glomerular mesangial cells were serum-starved for 24 h and pretreated with suramin, diphenylenechloride iodonium (DPI) or PD98059 followed by stimulation with a high concentration of glucose (30 mmol/L D-glucose) or ATP (300 μmol/L). Extracellular and total ATP and ROS production were detected using commercially available kits. Phosphorylation of ERK1/2 was evaluated by Western blot. TGF-β1 mRNA expression was examined by real-time PCR.

Results:

Suramin had a dose-dependent inhibitory effect on the generation of ROS induced by high glucose. Extracellular ATP production by mesangial cells increased markedly after a 2-h incubation with high glucose. ROS production was upregulated in mesangial cells after 5 min incubation with 300 μmol/L ATP and was sustained for 120 min. ERK1/2 was significantly activated after 5 min incubation of mesangial cells with ATP, this activation was partially inhibited by DPI. The effects of high glucose on TGF-β1 mRNA were markedly inhibited by suramin, DPI or PD98059.

Conclusion:

Our results suggest that a high concentration of glucose increases the extracellular levels of ATP in mesangial cells within a short time-frame. ATP, in turn, activates ERK1/2, an effect which is at least partially dependent on ROS, which results in the upregulation of TGF-β1.     

Lipopolysaccharide-Induced Depressive-Like Behavior is Associated with α1-Adrenoceptor Dependent Downregulation of the Membrane GluR1 Subunit in the Mouse Medial Prefrontal Cortex and Ventral Tegmental Area

Background:

Chronic stress-induced depressive-like behavior is relevant to inflammatory immune activation. However, the neurobiological alterations in the brain following the central inflammatory immune activation remain elusive.

Methods:

Therefore, we investigated the neurobiological alterations during depressive-like behavior induced in mice by systemic administration of lipopolysaccharide (LPS; 1.2mg/kg administered twice at a 30-min interval via intraperitoneal injection).

Results:

At 24h after the second administration of LPS, an increased immobility time in the tail suspension test and the forced swimming test were observed, as well as reduced sucrose preference. Protein levels of the AMPA receptor GluR1 were significantly decreased at the plasma membrane in the medial prefrontal cortex (mPFC) and ventral tegmental area (VTA), while levels of the GluR2 were increased at the plasma membrane in the nucleus accumbens (NAc) at 24h after LPS. However, total GluR1 and GluR2 protein levels in the mPFC, VTA, and NAc were not affected by LPS. Moreover, LPS facilitated release of noradrenaline in the mPFC and VTA, but not in the NAc. Consistently, systemic administration of prazosin, an α₁-adrenoceptor antagonist, blocked the LPS-induced downregulation of the membrane GluR1 subunit in both the mPFC and VTA and also blocked the upregulation of the membrane GluR2 subunit in the NAc. Intracerebroventricular administration of prazosin 30min before LPS injection abrogated the LPS-induced depressive-like behaviors. In opposition, administration of propranolol, a β-adrenoceptor antagonist, did not affect the LPS-induced downregulation of GluR1, the upregulation of GluR2, or the depressive-like behavior.

Conclusions:

These results suggest that LPS-activated α₁-adrenoceptor-induced downregulation of membrane GluR1 in the mPFC and VTA is associated with inflammation-induced depressive-like behavior.     

Salvianolic acid B suppresses maturation of human monocyte-derived dendritic cells by activating PPARγ

BACKGROUND AND PURPOSE

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing maturation of human monocyte-derived dendritic cells (h-monDC).

EXPERIMENTAL APPROACH

h-monDC were derived by incubating purified human monocytes with GM-CSF and IL-4. h-monDC were pre-incubated with or without Sal B and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of PPARγ siRNA. Expression of h-monDC membrane molecules (CD40, CD86, CD1a, HLA-DR) were analysed by FACS, cytokines were measured by elisa and the TLR4-associated signalling pathway was determined by Western blotting.

KEY RESULTS

Ox-LDL promoted h-monDC maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production; and up-regulated TLR4 signalling. These effects were inhibited by Sal B. Sal B also triggered PPARγ activation and promoted PPARγ nuclear translocation, attenuated ox-LDL-induced up-regulation of TLR4 and myeloid differentiation primary-response protein 88 and inhibited the downstream p38-MAPK signalling cascade. Knocking down PPARγ with the corresponding siRNA blocked these effects of Sal B.

CONCLUSIONS AND IMPLICATIONS

Our data suggested that Sal B effectively suppressed maturation of h-monDC induced by ox-LDL through PPARγ activation.     

Emodin induces human T cell apoptosis in vitro by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction

Aim:

To elucidate the molecular mechanisms underlying the immunosuppressive effects of emodin isolated from Rheum palmatum L.

Methods:

Human T cells were isolated from the peripheral venous blood of 10 healthy adult donors. Cell viability was analyzed with MTT assay. AO/EB and Annexin V/PI staining and DNA damage assay were used to detect cell apoptosis. Fluorescence staining was used to detect the levels of ROS, the mitochondrial membrane potential and intracellular Ca²⁺. Colorimetry was used to detect the levels of MDA and total SOD and GSH/GSSG ratio. The expression and activity of caspase-3, -4, and -9 were detected with Western blotting and a fluorometric assay. Western blotting was also used to detect the expression of Bcl-2, Bax, cytochrome C, and endoplasmic reticulum (ER) markers.

Results:

Emodin (1, 10, and 100 μmol/L) inhibited the growth of human T cells and induced apoptosis in dose- and time dependent manners. Emodin triggered ER stress and significantly elevated intracellular free Ca²⁺ in human T cells. It also disrupted mitochondrial membrane potential, and increased cytosolic level of cytochrome C, and the levels of activated cleavage fragments of caspase-3, -4, and -9 in human T cells. Furthermore, emodin significantly increased the levels of ROS and MDA, inhibited both SOD level and GSH/GSSG ratio in human T cells, whereas co-incubation with the ROS scavenger N-acetylcysteine (NAC, 20 μmol/L) almost completely blocked emodin-induced ER stress and mitochondrial dysfunction in human T cells, and decreased the caspase cascade-mediated apoptosis.

Conclusion:

Emodin exerts immunosuppressive actions at least partly by inducing apoptosis of human T cells, which is triggered by ROS-mediated ER stress and mitochondrial dysfunction.     

Reviewer comments on Reflections on drug research by Sir James Black

Background and purpose:

Activation of muscarinic M₃ mucarinic acetylcholine receptors (M₃-mAChRs) has been previously shown to confer short-term cardioprotection against ischaemic injuries. However, it is not known whether activation of these receptors can provide delayed cardioprotection. Consequently, the present study was undertaken to investigate whether stimulation of M₃-mAChRs can induce delayed preconditioning in rats, and to characterize the potential mechanism.

Experimental approach:

Rats were pretreated (24 h), respectively, with M₃-mAChRs agonist choline, M₃-mAChRs antagonist 4-DAMP or M₂-mAChRs antagonist methoctramine followed by the administration of choline. This was followed by 30 min of ischaemia and then 3 h of reperfusion. Ischaemia-induced arrhythmias and ischaemia–reperfusion (I/R)-induced infarction were determined. The phosphorylation status of connexin43 (Cx43) after 30 min ischaemia, and the expression level of Hsp70, cyclooxygenase-2 (COX-2) and iNOS effected by administration of choline were also measured.

Key results:

Compared to the control group, pretreatment with choline significantly decreased ischaemia-induced arrhythmias, reduced the total number of ventricular premature beats, the duration of ventricular tachycardia episodes and markedly reduced I/R-induced infarct size. Furthermore, choline attenuated ischaemia-induced dephosphorylation of Cx43, and up-regulated the expression of Hsp70 and COX-2. Administration of 4-DAMP abolished these changes, while methoctramine had no effect.

Conclusions and implications:

Our results suggest that stimulation of M₃-mAChRs with choline elicits delayed preconditioning, which we propose is the result of up-regulation of the expression of COX-2 and inhibition of the ischaemia-induced dephosphorylation of Cx43. Therefore, M₃-mAChRs represent a promising target for rendering cardiomyocytes tolerant to ischaemic injury.     

Ca2+-calmodulin can activate and inactivate cardiac ryanodine receptors

Background and purpose:

Ca²⁺-calmodulin (Ca²⁺CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated.

Experimental approach:

We investigated the effects of low concentrations of Ca²⁺CaM (50–100 nmol·L⁻¹) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol·L⁻¹ CaM on sarcoplasmic reticulum (SR) Ca²⁺-release.

Key results:

Ca²⁺CaM activated or inhibited single RyR2, but activation was much more likely at low (50–100 nmol·L⁻¹) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol·L⁻¹ CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca²⁺-release events. Application of caffeine demonstrated that 50 nmol·L⁻¹ CaM reduced SR Ca²⁺ content.

Conclusions and implications:

We describe for the first time how Ca²⁺CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca²⁺-release and the use of caffeine suggests that this is a RyR2-mediated effect.     

Vascular hyperpolarization to β-adrenoceptor agonists evokes spreading dilatation in rat isolated mesenteric arteries

BACKGROUND AND PURPOSE

β-Adrenoceptor stimulation causes pronounced vasodilatation associated with smooth muscle hyperpolarization. Although the hyperpolarization is known to reflect K_ATP channel activation, it is not known to what extent it contributes to vasodilatation.

EXPERIMENTAL APPROACH

Smooth muscle membrane potential and tension were measured simultaneously in small mesenteric arteries in a wire myograph. The spread of vasodilatation over distance was assessed in pressurized arteries following localized intraluminal perfusion of either isoprenaline, adrenaline or noradrenaline.

KEY RESULTS

Isoprenaline stimulated rapid smooth muscle relaxation associated at higher concentrations with robust hyperpolarization. Noradrenaline or adrenaline evoked a similar hyperpolarization to isoprenaline if the α₁-adrenoceptor antagonist prazosin was present. With each agonist, glibenclamide blocked hyperpolarization without reducing relaxation. Focal, intraluminal application of isoprenaline, noradrenaline or adrenaline during block of α₁-adrenoceptors evoked a dilatation that spread along the entire length of the isolated artery. This response was endothelium-dependent and inhibited by glibenclamide.

CONCLUSIONS AND IMPLICATIONS

Hyperpolarization is not essential for β-adrenoceptor-mediated vasodilatation. However, following focal β-adrenoceptor stimulation, this hyperpolarization underlies the ability of vasodilatation to spread along the artery wall. The consequent spread of vasodilatation is dependent upon the endothelium and likely to be of physiological relevance in the coordination of tissue blood flow.

LINKED ARTICLES

This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3     

Stimulated release of a hyperpolarizing factor (ADHF) from mesenteric artery perivascular adipose tissue: involvement of myocyte BKCa channels and adiponectin

Background and Purpose

Perivascular adipose tissue (PVAT) releases adipocyte-derived hyperpolarizing factors (ADHFs) that may partly act by opening myocyte K⁺ channels. The present study in rat and mouse mesenteric arteries aimed to identify the myocyte K⁺ channel activated by PVAT and to determine whether adiponectin contributed to the hyperpolarizing effects of PVAT.

Experimental Approach

Myocyte membrane potential was recorded from de-endothelialized, non-contracted rat and mouse mesenteric arteries in the presence and absence of PVAT.

Key Results

The β₃-adrenoceptor agonist, CL-316,243 (10 μM), generated PVAT-dependent, iberiotoxin-sensitive myocyte hyperpolarizations resulting from BK_Ca channel opening and which were partially blocked by L-NMMA (100 μM). Adiponectin (5 μg·mL⁻¹) also produced iberiotoxin-sensitive hyperpolarizations in PVAT-denuded arterioles. Activation of myocyte AMP-activated protein kinase (AMPK) using 5 μM A-769662 also induced BK_Ca-mediated hyperpolarizations. Dorsomorphin abolished hyperpolarizations to CL-316,243, adiponectin and A-769662. In vessels from Adipo^−/− mice, hyperpolarizations to CL-316,243 were absent whereas those to A-769662 and adiponectin were normal. In rat vessels, adipocyte-dependent hyperpolarizations were blocked by glibenclamide and clotrimazole but those to NS1619 (33 μM) were unaltered.

Conclusions and Implications

Under basal, non-contracted conditions, β₃-adrenoceptor stimulation of PVAT releases an ADHF, which is probably adiponectin. This activates AMPK to open myocyte BK_Ca channels indirectly and additionally liberates NO, which also contributes to the observed PVAT-dependent myocyte hyperpolarizations. Clotrimazole and glibenclamide each reversed hyperpolarizations to adiponectin and A-769662, suggesting the involvement of myocyte TRPM4 channels in the ADHF-induced myocyte electrical changes mediated via the opening of BK_Ca channels.     

β, β-Dimethylacrylshikonin induces mitochondria-dependent apoptosis of human lung adenocarcinoma cells in vitro via p38 pathway activation

Aim:

β, β-Dimethylacrylshikonin (DMAS) is an anticancer compound extracted from the roots of Lithospermum erythrorhizon. In the present study, we investigated the effects of DMAS on human lung adenocarcinoma cells in vitro and explored the mechanisms of its anti-cancer action.

Methods:

Human lung adenocarcinoma A549 cells were tested. Cell viability was assessed using an MTT assay, and cell apoptosis was evaluated with flow cytometry and DAPI staining. The expression of the related proteins was detected using Western blotting. The mitochondrial membrane potential was measured using a JC-1 kit, and subcellular distribution of cytochrome c was analyzed using immunofluorescence staining.

Results:

Treatment of A549 cells with DMAS suppressed the cell viability in dose- and time-dependent manners (the IC₅₀ value was 14.22 and 10.61 μmol/L, respectively, at 24 and 48 h). DMAS (7.5, 10, and 15 μmol/L) dose-dependently induced apoptosis, down-regulated cIAP-2 and XIAP expression, and up-regulated Bax and Bak expression in the cells. Furthermore, DMAS resulted in loss of mitochondrial membrane potential and release of cytochrome c in the cells, and activated caspase-9, caspase-8, and caspase-3, and subsequently cleaved PARP, which was abolished by pretreatment with Z-VAD-FMK, a pan-caspase inhibitor. DMAS induced sustained p38 phosphorylation in the cells, while pretreatment with SB203580, a specific p38 inhibitor, blocked DMAS-induced p38 activation and apoptosis.

Conclusion:

DMAS inhibits the growth of human lung adenocarcinoma A549 cells in vitro via activation of p38 signaling pathway.     

Electrical conduction along endothelial cell tubes from mouse feed arteries: confounding actions of glycyrrhetinic acid derivatives

BACKGROUND AND PURPOSE

Electrical conduction along endothelium of resistance vessels has not been determined independently of the influence of smooth muscle, surrounding tissue or blood. Two interrelated hypotheses were tested: (i) Intercellular conduction of electrical signals is manifest in endothelial cell (EC) tubes; and (ii) Inhibitors of gap junction channels (GJCs) have confounding actions on EC electrical and Ca²⁺ signalling.

EXPERIMENTAL APPROACH

Intact EC tubes were isolated from abdominal muscle feed (superior epigastric) arteries of C57BL/6 mice. Hyperpolarization was initiated with indirect (ACh) and direct (NS309) stimulation of intermediate- and small-conductance Ca²⁺-activated K⁺ channels (IK_Ca/SK_Ca). Remote membrane potential (V_m) responses to intracellular current injection defined the length constant (λ) for electrical conduction. Dye coupling was evaluated following intracellular microinjection of propidium iodide. Intracellular Ca²⁺ dynamics were determined using Fura-2 photometry. Carbenoxolone (CBX) or β-glycyrrhetinic acid (βGA) was used to investigate the role of GJCs.

KEY RESULTS

Steady-state V_m of ECs was −25 mV. ACh and NS309 hyperpolarized ECs by −40 and −60 mV respectively. Electrical conduction decayed monoexponentially with distance (λ∼1.4 mm). Propidium iodide injected into one EC spread into surrounding ECs. CBX or βGA inhibited dye transfer, electrical conduction and EC hyperpolarization reversibly. Both agents elevated resting Ca²⁺ while βGA inhibited responses to ACh.

CONCLUSIONS AND IMPLICATIONS

Individual cells were effectively coupled to each other within EC tubes. Inhibiting GJCs with glycyrrhetinic acid derivatives blocked hyperpolarization mediated by IK_Ca/SK_Ca channels, regardless of Ca²⁺ signalling, obviating use of these agents in distinguishing key determinants of electrical conduction along the endothelium.     

Ibrolipim increases ABCA1/G1 expression by the LXRα signaling pathway in THP-1 macrophage-derived foam cells

Aim:

To determine the effects and potential mechanisms of ibrolipim on ATP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1) expression from human macrophage foam cells, which may play a critical role in atherogenesis.

Methods:

Human THP-1 cells pre-incubated with ox-LDL served as foam cell models. Specific mRNA was quantified using real-time RT-PCR and protein expression using Western blotting. Cellular cholesterol handling was studied using cholesterol efflux experiments and high performance liquid chromatography assays.

Results:

Ibrolipim 5 and 50 μmol/L significantly increased cholesterol efflux from THP-1 macrophage-derived foam cells to apoA-I or HDL. Moreover, it upregulated the expression of ABCA1 and ABCG1. In addition, LXRα was also upregulated by the ibrolipim treatment. In addition, LXRα small interfering RNA completely abolished the promotion effect that was induced by ibrolipim.

Conclusion:

Ibrolipim increased ABCA1 and ABCG1 expression and promoted cholesterol efflux, which was mediated by the LXRα signaling pathway.     

An integrated pathway interaction network for the combination of four effective compounds from ShengMai preparations in the treatment of cardio-cerebral ischemic diseases

Aim:

SMXZF (a combination of ginsenoside Rb1, ginsenoside Rg1, schizandrin and DT-13) derived from Chinese traditional medicine formula ShengMai preparations) is capable of alleviating cerebral ischemia-reperfusion injury in mice. In this study we used network pharmacology approach to explore the mechanisms of SMXZF in the treatment of cardio-cerebral ischemic diseases.

Methods:

Based upon the chemical predictors, such as chemical structure, pharmacological information and systems biology functional data analysis, a target-pathway interaction network was constructed to identify potential pathways and targets of SMXZF in the treatment of cardio-cerebral ischemia. Furthermore, the most related pathways were verified in TNF-α-treated human vascular endothelial EA.hy926 cells and H2O2-treated rat PC12 cells.

Results:

Three signaling pathways including the NF-κB pathway, oxidative stress pathway and cytokine network pathway were demonstrated to be the main signaling pathways. The results from the gene ontology analysis were in accordance with these signaling pathways. The target proteins were found to be associated with other diseases such as vision, renal and metabolic diseases, although they exerted therapeutic actions on cardio-cerebral ischemic diseases. Furthermore, SMXZF not only dose-dependently inhibited the phosphorylation of NF-κB, p50, p65 and IKKα/β in TNF-α-treated EA.hy926 cells, but also regulated the Nrf2/HO-1 pathway in H2O2-treated PC12 cells.

Conclusion:

NF-κB signaling pathway, oxidative stress pathway and cytokine network pathway are mainly responsible for the therapeutic actions of SMXZF against cardio-cerebral ischemic diseases.     

Differential effects of uridine adenosine tetraphosphate on purinoceptors in the rat isolated perfused kidney

BACKGROUND AND PURPOSE

Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine tetraphosphate (Up₄A) as a novel and highly potent endothelial-derived contracting factor. Up₄A induces strong vasoconstrictive effects in the renal vascular system mainly by P2X₁ receptor activation. However, other purinoceptors are also involved and were analysed here.

EXPERIMENTAL APPROACH

The rat isolated perfused kidney was used to characterize vasoactive actions of Up₄A.

KEY RESULTS

After desensitization of the P2X₁ receptor by α,β-methylene ATP (α,β-meATP), Up₄A showed dose-dependent P2Y₂-mediated vasoconstriction. Continuous perfusion with Up₄A evoked a biphasic vasoconstrictor effect: there was a strong and rapidly desensitizing vasoconstriction, inhibited by P2X₁ receptor desensitization. In addition, there is a long-lasting P2Y₂-mediated vasoconstriction. This vasoconstriction could be blocked by suramin, but not by PPADS or reactive blue 2. In preparations of the rat isolated perfused kidney model with an elevated vascular tone, bolus application of Up₄A showed a dose-dependent vasoconstriction that was followed by a dose-dependent vasodilation. The vasoconstriction was in part sensitive to P2X₁ receptor desensitization by α,β-meATP, and the remaining P2Y₂-mediated vasoconstriction was only inhibited by suramin. The Up₄A-induced vasodilation depended on activation of nitric oxide synthases, and was mediated by P2Y₁ and P2Y₂ receptor activation.

CONCLUSIONS AND IMPLICATIONS

Up₄A activated P2X₁ and P2Y₂ receptors to act as a vasoconstrictor, whereas endothelium-dependent vasodilation was induced by P2Y_1/2 receptor activation. Up₄A might be of relevance in the physiology and pathophysiology of vascular tone regulation.