Poncirus trifoliate fruit modulates pacemaker activity in interstitial cells of Cajal from the murine small intestine

Ethnopharmacological relevance

Poncirus fructus (PF) has been widely used as a traditional medicine in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility.

Aim of the study

Poncirus fructus (PF), also known as Poncirus trifoliata (L.) Raf. (Rutaceae), is widely used as a traditional medicine in Eastern Asia mainly to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. In a previous study, a methanol extract of PF was found to have particularly potent gastroprokinetic effects. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal tract, but the action mechanisms of PF extract in mouse small intestinal ICCs have not been investigated. Therefore, in the present study, we investigated the effects of a methanol extract of PF (MPF) in mouse small intestinal ICCs. In addition, we sought to identify the receptors involved.

Materials and Methods

Enzymatic digestions were used to dissociate ICCs from small intestines. The whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. In addition, we analyzed intracellular Ca²⁺ concentrations ([Ca²⁺]_i).

Results

MPF decreased the amplitudes of pacemaker potentials in ICCs, and depolarized resting membrane potentials in a concentration dependent manner. Y25130 (a 5-HT₃ receptor antagonist) and RS39604 (a 5-HT₄ receptor antagonist) blocked MPF-induced membrane depolarizations, whereas SB269970 (a 5-HT₇ receptor antagonist) did not. Pretreatment with Na⁺ or Ca²⁺-free solution or thapsigargin (a Ca²⁺-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed MPF-induced activity. [Ca²⁺]_i analysis showed that MPF increased [Ca²⁺]_i. Furthermore, treatments with PD 98059, SB203580, or JNK II inhibitor blocked MPF-induced membrane depolarizations in ICCs.

Conclusion

These results suggest that MPF modulates pacemaker potentials through 5-HT₃ and 5-HT₄ receptor-mediated pathways via external Na⁺ and Ca²⁺ influx, and via Ca²⁺ release from internal stores in a mitogen-activated protein kinase dependent manner. The study shows MPF is a good candidate for the development of a gastroprokinetic agent. In view of the effects of MPF on ICCs, further research is required, particularly to identify the active compound(s) involved and to determine their action mechanisms.     

Antiarrhythmic effects of dehydroevodiamine in isolated human myocardium and cardiomyocytes

Ethnopharmacological relevance

Dehydroevodiamine alkaloid (DeHE), a bioactive component of the Chinese herbal medicine Wu-Chu-Yu (Evodiae frutus), exerted antiarrhythmic effect in guinea-pig ventricular myocytes. We further characterize the electromechanical effects of DeHE in the human atrial and ventricular tissues obtained from hearts of patients undergoing corrective cardiac surgery or heart transplantation.

Materials and methods

The transmembrane potentials of human myocardia were recorded with a traditional microelectrode technique while sarcolemmal Na⁺ and Ca²⁺ currents in single human cardiomyocytes were measured by a whole-cell patch-clamp technique. The intracellular pH (pH_i) and Na⁺–H⁺ exchanger (NHE) activity were determined using BCECF-fluorescence in human atria.

Results

In human atria, DeHE (0.1–0.3 μM) depressed upstroke velocity, amplitude of action potential, and contractile force, both in slow and fast response action potential. Moreover, the similar depressant effects of DeHE were found in human ventricular myocardium. Both in isolated human atrial and ventricular myocytes, DeHE (0.1–1 μM) reversibly, concentration-dependently decreased the Na⁺ and Ca²⁺currents. Moreover, DeHE (0.1 and 0.3 μM) suppressed delayed afterdepolarizations and aftercontractions, induced by epinephrine and high [Ca²⁺]_o in atria. In human ventricular myocardium, the strophanthidin-induced triggered activities were attenuated by pretreating DeHE (0.3 μM). The resting pH_i and NHE activity were also significantly increased by DeHE (0.1–0.3 μM).

Conclusions

We concluded for the first time that, in the human hearts, DeHE could antagonize triggered arrhythmias induced by cardiotonic agents through a general reduction of the Na⁺ and Ca²⁺ inward currents, while increase of resting pH_i and NHE activity.     

Tanshinone IIA,a constituent of Danshen,inhibits the release of glutamate in rat cerebrocortical nerve terminals

Ethnopharmacological relevance

Danshen is a commonly used traditional Chinese medicine and has received considerable attention due to their beneficial effects on the health, including prevention of cardiovascular disease, and cancer. Tanshinone IIA, a major active constituent of Danshen, has been reported to have a neuroprotective profile.

Aim of the study

An excessive release of glutamate is considered to be related to neuropathology of several neurological diseases. In this study, we investigated whether tanshinone IIA could affect endogenous glutamate release and explored the possible mechanism.

Materials and methods

The experimental model was the isolated nerve terminals (synaptosomes) purified from the rat cerebral cortex. The release of glutamate was evoked by the K⁺ channel blocker 4-aminopyridine (4-AP) and measured by one-line enzyme-coupled fluorometric assay. We also used a membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and a Ca²⁺ indicator, Fura-2-acetoxymethyl ester, to monitor cytosolic Ca²⁺ concentrations ([Ca²⁺]_C).

Results

Tanshinone IIA inhibited the release of glutamate evoked by 4-AP in a concentration-dependent manner. Inhibition of glutamate release by tanshinone IIA was prevented by the chelating the extracellular Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate did not have any effect on the action of tanshinone IIA. Tanshinone IIA decreased the depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. Furthermore, the effect of tanshinone IIA on evoked glutamate release was prevented by the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene or the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP37157. Mitogen-activated protein kinase (MEK) inhibition also prevented the inhibitory effect of tanshinone IIA on evoked glutamate release.

Conclusion

These results show that tanshinone IIA inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca²⁺ entry and MEK signaling cascade.     

Relaxant effect of flavonoid naringenin on contractile activity of rat colonic smooth muscle

Ethnopharmacological relevance

Disturbed gastrointestinal (GI) motility can be associated with smooth muscle abnormalities and dysfunction. Exploring innovative approaches that can modulate the disturbed colonic motility are of great importance for clinical therapeutics. Naringenin, a flavonoid presented in many traditional Chinese herbal medicines, has been shown to have a relaxant effect on different smooth muscles. The aim of the present study was to investigate the effect of naringenin on regulation of GI motility.

Material and methods

Mechanical recording was used to investigate the effect of naringenin on isolated rat colonic smooth muscle spontaneous contractions. Whole cell patch clamp, intracellular [Ca²⁺] concentration ([Ca²⁺]_i) and membrane potential measurements were examined on primary cultures of colonic smooth muscle cells (SMCs). A neostigmine-stimulated rat model was utilized to investigate the effect of naringenin in vivo.

Results

Naringenin induced a concentration-dependent inhibition (1–1000 μM) on rat colonic spontaneous contraction, which was reversible after wash out. The external Ca²⁺ influx induced contraction and [Ca²⁺]_i increase were inhibited by naringenin (100 μM). In rat colonic SMCs, naringenin-induced membrane potential hyperpolarization was sensitive to TEA and selective large-conductance calcium-activated K⁺ (BK_Ca) channel inhibitor iberiotoxin. Under whole cell patch-clamp condition, naringenin stimulated an iberiotoxin-sensitive BK_Ca current, which was insensitive to changes in the [Ca²⁺]_i concentration. Furthermore, naringenin significantly suppressed neostigmine-enhanced rat colon transit in vivo.

Conclusion

Our results for the first time demonstrated the relaxant effect of flavonoid naringenin on colon smooth muscle both in vitro and in vivo. The relaxant effect of naringenin was attributed to direct activation of BK_Ca channels, which subsequently hyperpolarized the colonic SMCs and decreased Ca²⁺ influx through VDCC. Naringenin might be of therapeutic value in the treatment of GI motility disorders.     

Relaxant effect of 1-butanol fraction from Elaeagnus pungens leaf through inhibiting l-type Ca channel on guinea pig tracheal smooth muscle

Ethnopharmacological relevance

The leaf of Elaeagnus pungens thunb. (Family Elaeagnaceae) has been documented as an effective herb for the treatment of asthma and chronic bronchitis in traditional Chinese medicine. In the past years, only a few of preliminary studies reported the chemical constituents and pharmacology effects of the herb, but their action on the tracheal relaxation has not been investigated.

Aim of the study

To investigate the relaxing effect and mechanism of the extracts from Elaeagnus pungens leaves on guinea pig tracheal smooth muscle and bronchi smooth muscle cells.

Materials and methods

Four fractions of different polarities from Elaeagnus pungens leaves were tested to the tracheal strips on the resting tension or pre-contracted by histamine (20 μM) and acetylcholine (20 μM). Inhibitory effects of the 1-butanol fraction (400 mg/ml) on cumulative histamine and acetylcholine (0.2–20 μM) induced contraction were measured. In order to determine the mediators on the 1-butanol fraction effect, the relaxing effect of the 1-butanol fraction was evaluated in the absence and presence of β-adrenoceptor antagonists (1 μM propranolol), K⁺ channels-blockers (4-aminopyridine (2 mM), tetraethylammonium chloride (5 mM) or glibenclamide (10 μM)), the cyclooxygenase inhibitor (indomethacin, 10 μM), nitric oxide synthase inhibitor (Nω-nitro-l-arginine methyl ester, 100 μM) or l-type Ca²⁺ channel inhibitor (nifedipine, 1 μM). Moreover, [Ca²⁺]_i in bronchi smooth muscle cells was analyzed by measuring the fluorescence intensity with confocal system.

Results

1-Butanol fraction induced the highest relaxant effect among four fractions of different polarities from Elaeagnus pungens leaves, and significantly relaxed the tracheal strip in the concentration-dependent manner on the resting tension and pre-contracted by histamine phosphate and acetylcholine. It also produced an unparallel rightward shift of the cumulative concentration-response curve of histamine or acetylcholine. Furthermore, the relaxant effect of 1-butanol fraction was not affected by propranolol, glibenclamide, tetraethylammonium chloride, 4-aminopyridine, indomethacin and Nω-nitro-l-arginine methyl ester. However, 1-butanol fraction-induced relaxation decreased after adding nifedipine. It also concentration-dependently inhibited CaCl₂-induced contraction in the Ca²⁺-free, 60 mM K⁺-containing solution. Additionally, [Ca²⁺]_i in the BSMCs significantly reduced after administration of the 1-butanol fraction.

Conclusions

The 1-butanol fraction from Elaeagnus pungens leaves resulted in a relaxation in the non-precontracted and pre-contracted tracheal strips. The relaxant effect was not related to K⁺ channels, NO, cGMP or β-adrenoceptors, but related to the inhibition of Ca²⁺ influx through l-type Ca²⁺ channels.     

Korean red ginseng stimulates insulin release from isolated rat pancreatic islets

Ethnopharmacological relevance

Korean red ginseng (KRG), one of heat-processed Korean ginseng (Panax ginseng C.A. Meyer), has a long history as herbal remedy for antidiabetic effect.

Aim of the study

The effect and mechanism of KRG on stimulation of insulin release were investigated in isolated rat pancreatic islets.

Material and methods

Pancreatic islets isolated from rats were used to evaluate the insulinotropic action of KRG. The effect of Ca on the insulinotropic action of KRG was investigated.

Results

The aqueous ethanolic extract of KRG (AEE-KRG) (0.1–1.0 mg/ml) significantly evoked a stimulation of insulin release at 3.3 mM glucose compared to the control. Experiments at different glucose concentrations (8.4 and 16.7 mM) showed that AEE-KRG significantly stimulated on its own whereas it did not potentiate insulin secretion induced by glucose. The extracellular Ca²⁺-free condition, a L-type Ca²⁺ channel blocker and an ATP-sensitive K⁺ channel opener significantly inhibited insulin secretion evoked by AEE-KRG.

Conclusion

These findings suggest that KRG displays beneficial effects in the treatment of diabetes at least in part via the stimulation of insulin release in a glucose-independent manner.     

Vasodilatory effects of ethanol extract of Radix Paeoniae Rubra and its mechanism of action in the rat aorta

Ethnopharmacological relevance

Radix Paeoniae Rubra (RPR) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China. RPR is the radix of either Paeonia lactiflora Pall. or Paeonia veitchii Lynch. RPR has a wide variety of pharmacological actions such as anti-thrombus, anti-coagulation, and anti-atherosclerotic properties, protecting heart and liver. However, the mechanisms involved are to be defined.

Aim of the study

The aim of the present study was to define the effect of Paeonia lactiflora Pall. extracts on vascular tension and responsible mechanisms in rat thoracic aortic rings.

Materials and methods

Ethanol extract of Paeonia lactiflora Pall. (EPL) was examined for their vascular relaxant effects in isolated phenylephrine-precontracted rat thoracic aorta.

Results

EPL induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Vascular relaxation induced by EPL was significantly inhibited by removal of the endothelium or pretreatment of the rings with N^G-nitro-l-arginine methylester (l-NAME) or 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ). Extracellular Ca²⁺ depletion or diltiazem significantly attenuated EPL-induced vasorelaxation. Modulators of the store-operated Ca²⁺ entry (SOCE), thapsigargin, 2-aminoethyl diphenylborinate and Gd³⁺, and an inhibitor of Akt, wortmannin, markedly attenuated the EPL-induced vasorelaxation. Further, the EPL-induced vasorelaxation was significantly attenuated by pretreatment with tetraethylammonium, a non-selective K_Ca channels blocker, or glibenclamide, an ATP-sensitive K⁺ channels inhibitor, respectively. Inhibition of cyclooxygenases with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on the EPL-induced vasorelaxation.

Conclusions

The present study suggests that EPL relaxes vascular smooth muscle via endothelium-dependent and Akt- and SOCE-eNOS-cGMP-mediated pathways through activation of both K_Ca and K_ATP channels and inhibition of L-type Ca²⁺ channels.     

Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation

Ethnopharmacological relevance

Total saponins of Aralia elata (Miq) Seem (AS) from the Chinese traditional herb Longya Aralia chinensis L. can improve cardiac function, although the active mechanism remains poorly understood. The present study aimed to determine the direct effect of AS on cardiac function in dogs and the effects on Ca²⁺ transient and contractions in isolated rat cardiomyocytes.

Material and Methods

In anesthetized dogs, hemodynamic indexes and myocardial oxygen consumption were determined before and after AS was administered. In isolated adult rat cardiomyocytes, contractile and intracellular Ca²⁺ properties were determined simultaneously in real time by using an IonOptix MyoCam system.

Results

Our results showed that AS directly induced a positive inotropic effect and improved coronary blood flow and energy metabolism, indicating that AS induced a beneficial effect to treat myocardial ischemia/reperfusion injury. Moreover, AS increased sarcomere shortening, maximal velocity of shortening/relengthening (±dL/dt), amplitude of [Ca²⁺]_i transients and SERCA activity in a concentration-dependent manner. PKCε was also activated after the cells were treated with AS.

Conclusion

These findings revealed the positive inotropic effect of AS on canine myocardium and isolated rat cardiomyocytes. This effect was possibly associated with an increase in amplitude of the [Ca²⁺]_i transient and PKCε-dependent signaling pathway.     

Mechanisms of the dilator action of the Erigerontis Herba on rat aorta

Ethnopharmacological relevance

Erigerontis Herba is widely used as a traditional Chinese medicine and is commonly used for neuroprotection and vascular protection.

Aim of study

In this study, the vasodilator effects of Erigerontis Herba (DZXX) were investigated using rat isolated aorta rings.

Material and method

The involvement of endothelium in the vasorelaxation was studied by comparing response of endothelium-intact and endothelium-denuded aorta rings which precontracted with U46619. The involvement of K⁺ channels was studied by pretreatment of the aorta rings with various K⁺ channel inhibitors. The involvement of Ca²⁺ channel was studied by incubating aorta rings with Ca²⁺-free solution, primed with U46619 prior to elicit contraction by addition of Ca²⁺ solution.

Results

DZXX (0.2–2 mg/ml) induced a concentration-dependent relaxation on U44619-precontracted aorta rings with EC₅₀ of 0.354±0.036 mg/ml. Removal of endothelium or pretreatment with a BK_Ca inhibitor iberiotoxin, K_IR inhibitor barium chloride or K_v inhibitor 4-aminopyridine produced no effect on the DZXX-induced vasorelaxation. However, pretreatment with a K_ATP inhibitor glibenclamide or a non-selective K⁺ channel inhibitor tetraethylammonium produced significant inhibition on the DZXX-induced vasorelaxation by 29.9% and 21.3%, respectively. Pretreatment with DZXX (0.4, 1.2 and 2 mg/ml) produced a concentration-dependent inhibition on Ca²⁺-induced vasoconstriction.

Conclusions

These results suggest that the vasodilator effect of DZXX was endothelium-independent, mediated by decreasing the influx of Ca²⁺ by calcium channel inhibition and increasing the influx of K⁺ by opening of a K_ATP channel.     

Busuishengxue ranules mediate their effects upon non-severe aplastic anemia via mitogen-activated protein kinase/extracellular signal-regulated kinase pathway

Objective

To observe the clinical efficacy of Busuishengxue granules on non-severe aplastic anemia (NSAA) and investigate its effect on the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway.

Methods

Sixty NSAA patients were divided equally into two groups. Subjects in the experimental group were treated with Busuishengxue granules, and the control group with Zaizaoshengxue tablets. The treatment course was 6 months and curative efficacy was compared between the two groups as well as with 10 healthy individuals. Flow cytometry (FCM) was used to detect the intracellular concentration of Ca^{2 +} ([Ca^{2 +}]_i). Western blotting was employed to detect the expression of enzymes in the MAPK/ERK pathway.

Results

The efficacy of Busuishengxue granules was significantly better than that of Zaizaoshengxue tablets (P<0.05). Before treatment, expression of JNK, phospho-ERK 1/2 and p-JNK was higher, and [Ca^{2 +}]_i higher, than that of the control group (P< 0.05). After treatment with Busuishengxue granules, expression of all enzymes related to signal transduction pathways in the blood cells of NSSA patients were altered to different degrees.

Conclusion

Busuishengxue granules had a better effect with regard to improving symptom scores, increasing the number of blood leukocytes, and increasing hemoglobin levels than Zaizaoshengxue tablets, and they differed slightly in terms of increasing the number of platelets.     

The inhibitory activity of atractylenolide Ш, a sesquiterpenoid,on IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA)

Ethnopharmacological relevance

AT Ш, a sesquiterpenoid, is the major component of Atractylodes japonica Koidz that has been used as a traditional oriental medicine.

Aim of the study

We investigated the anti-allergic activity of AT Ш and its mechanism of action.

Materials and methods

The released amount of β-hexosaminidase in mast cells, a key parameter of degranulation, was measured. Anti-allergic potential of AT Ш was evaluated using passive cutaneous anaphylaxis in vivo. The anti-allergic mechanism of AT Ш was investigated by immunoblotting analysis, RT-PCR and measurement of [Ca²⁺]i in mast cells.

Results

AT Ш significantly inhibited IgE/Ag-mediated degranulation with an IC₅₀ value (36±4 μM) in RBL-2H3 cells without affecting cell viability. It also suppressed IgE/Ag-mediated passive cutaneous anaphylaxis (PCA) response with an ED₅₀ value (65±41 mg/kg) in vivo. AT Ш suppressed the production of interleukin (IL-4) and tumor necrosis factor (TNF)-alpha mRNAs more potent than the Src-family kinase inhibitor PP2 in RBL-2H3 cells at all concentrations. In order to elucidate the anti-allergic mechanisms of AT Ш in mast cells, we examined the activated levels of signaling molecules. AT Ш inhibited the phosphorylation of Lyn, Fyn, Syk, LAT, PLCγ, Gab2, Akt, p38, and JNK kinases expression. IgE/Ag-mediated [Ca²⁺]i elevation was significantly inhibited by AT Ш.

Conclusions

Our study suggests that AT Ш might be used as a therapeutic agent for allergic diseases.     

Vasorelaxant effects and mechanisms of action of Heracleum sphondylium L. (Apiaceae) in rat thoracic aorta

Ethnopharmacological relevance

Aerial parts of Heracleum sphondylium L. (HS) are used in traditional medicine to treat hypertension. To provide pharmacological basis for this use, we investigated the vasorelaxant effects of a dichloromethane extract of HS (HSDE) and the mechanisms involved.

Materials and methods

Activity of HSDE was evaluated on rat isolated thoracic aortic rings.

Results

HSDE induced vasorelaxation in phenylephrine (PE, 10⁻⁶ mol/L) and high KCl—(6×10⁻² mol/L) pre-contracted aortic rings that was independent on the presence of endothelium. HSDE markedly decreased extracellular Ca²⁺-induced contraction in high-KCl and PE pre-challenged rings. It also inhibited the intracellular Ca²⁺ release sensitive to PE (10⁻⁶ M). The relaxant effect of HSDE were blunted by 4-amino-pyridine (4-AP, 10⁻³ mol/L), an inhibitor of voltage-dependent K⁺ channels.

Conclusion

Our results provide the first evidence that a dichloromethane extract of Heracleum sphondylium L. exhibits vasorelaxant properties through endothelium-independent mechanisms involving the inhibition of Ca²⁺ mobilization and changes in Kv channel conductances. These data argue for its use as antihypertensive therapy in traditional medicine.     

Inhibitory effects of Bulnesia sarmienti aqueous extract on agonist-induced platelet activation and thrombus formation involves mitogen-activated protein kinases

Ethnopharmacological relevance

B. sarmienti has long been recognized in folk medicine as a medicinal plant with various medicinal uses. Traditionally, it has been appreciated for the skin-healing properties of its essence. The bark has also been employed to treat stomach and cardiovascular disorders and reported to have antitumor, antioxidant and anti-inflammatory activities. However, information on its antiplatelet activity is limited.

Aim of the study

To examined the effects of B. sarmienti aqueous extract (BSAE) in platelet physiology.

Materials and methods

The anti-platelet activity of BSAE was studied using rat platelets for in vitro determination of the extract effect on agonist-induced platelet aggregation, ATP secretion, [Ca²⁺]_i mobilization and MAP kinase phosphorylation. The extract in vivo effects was also examined in arterio-venous shunt thrombus formation in rats, and tail bleeding time in mice.

Result

HPLC chromatographic analysis revealed that B. sarmienti extract contained (+)-catechin (C), (−)-epigallocatechin (EGC), (−)-epicatechin (EC), and (−)-epicatechin gallate (ECG). BSAE, significantly and dose dependently, inhibited collagen, thrombin, or ADP-induced platelet aggregation. The 50 percent inhibitory concentrations (IC₅₀) of the extract for collagen, thrombin and ADP-induced platelet aggregation were 45.3 ± 2.6, 100 ± 5.6 and 110 ± 4.6 μg/ml, respectively. Collagen activated ATP release and thrombin-induced intracellular Ca²⁺ concentration were reduced in BSAE-treated platelets. In addition, the extract in vivo activity showed that BSAE at 100 mg/kg significantly attenuated thrombus formation in rat extracorporeal shunt model while mice tail bleeding time was not affected. Moreover, BSAE attenuated p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations.

Conclusion

BSAE inhibits platelet activation, granule secretion, aggregation, and thrombus formation without affecting bleeding time, and that this effect is mediated by inhibition of P38, JNK1 and ERK2 phosphorylations. The ability of BSAE to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the plant extract could be considered as a candidate to anti-platelet and antithrombotic agent.     

Synergistic interaction between the Ligusticum chuanxiong constituent butylidenephthalide and the nitric oxide donor sodium nitroprusside in relaxing rat isolated aorta

Ethnopharmacological relevance

Ligusticum chuanxiong Hort. (Umbelliferae), a traditional Chinese medicinal herb, is often prescribed together with nitric oxide donors for treating coronary heart diseases such as angina in China; however, studies concerning their pharmacological interaction are scarce.

Aim of the study

The objective of the present study was to examine the interaction between the Ligusticum chuanxiong major active constituent butylidenephthalide (BDPH) and the nitric oxide donor sodium nitroprusside (SNP) in vasorelaxation.

Materials and methods

Vasorelaxation was examined in rat isolated aorta using an organ bath system.

Results

BDPH and SNP interacted synergistically under 9,11-dideoxy-9α,11α-methanoepoxyprostaglandin H₂ (U-46619)-induced tone. This synergism became greater with increasing U-46619 concentrations where Ca²⁺ sensitization contributed more significantly, and less when U-46619 was replaced with phenylephrine where participation of Ca²⁺ sensitization was minimal. BDPH-SNP synergism remained intact in the absence of external Ca²⁺, indicating that regulation of Ca²⁺ influx was not a requirement for the manifestation of this interaction.

Conclusions

The present study demonstrated the synergistic relaxation between BDPH and SNP in rat isolated aorta. This interaction is related to an enhancement of the effectiveness of SNP in producing relaxation under tone induced mainly by Ca²⁺ sensitization.     

Atractylodes macrocephala Koidz promotes intestinal epithelial restitution via the polyamine—Voltage-gated K channel pathway

Ethnopharmacological relevance

Atractylodes macrocephala Koidz (AMK) has been used widely as a digestive and tonic in traditional Chinese medicine. AMK has shown noteworthy promoting effect on intestinal epithelial cell migration, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via IEC-6 cell migration model.

Materials and methods

A wounding model of IEC-6 cells was induced by a single-edge razor blade along the diameter of six-well polystyrene plates. The cells were grown in control cultures and in cultures containing spermidine (5 μmol/L, SPD, reference drug), alpha-difluoromethylornithine (2.5 mmol/L, DFMO, polyamine inhibitor), AMK (50, 100, and 200 μg/mL), DFMO plus SPD and DFMO plus AMK for 24 h. The membrane potential (MP) and cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) were detected by flow cytometry, and polyamines content was determined via high-performance liquid chromatography (HPLC). The expression of Kv1.1 mRNA and protein levels were assessed by RT-qPCR and Western blot analysis, respectively. Cell migration assay was carried out using the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK.

Results

(1) Treatment with AMK caused significant increases in cellular polyamines content, membrane hyperpolarization, an elevation of [Ca²⁺]_cyt and an acceleration of cell migration in IEC-6 cells, as compared to control group. (2) AMK not only reversed the inhibitory effects of DFMO on the polyamines content, MP, and [Ca²⁺]_cyt but also restored IEC-6 cell migration to control levels. (3) The Kv1.1 mRNA and protein expression were significantly increased by AMK treatment in control and polyamine-deficient IEC-6 cells.

Conclusions

The results of our current studies revealed that treatment with AMK significantly stimulates the migration of intestinal epithelial cells through polyamine-Kv1.1 channel signaling pathway, which could promote the healing of intestinal injury. These results suggest the potential usefulness of AMK to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.     

Confirmation of a proarrhythmic risk underlying the clinical use of common Chinese herbal intravenous injections

Ethnopharmacological relevance

The Chinese herbal intravenous injections (CHI) which are extracted from herb(s) are used clinically in China as putative therapies for a variety of diseases.

Aim of the study

The mechanism(s) which underline findings of severe adverse drug reactions (ADR) noted in more than a thousand published articles on CHIs, are still poorly understood. With 109 CHIs currently in clinical use, we investigated the proarrhythmic effects of three specific CHIs, Shuanghuanglian (SHL), Qingkailing (QKL) and Yinzhihuang (YZH), using in vivo and in vitro ion channel models.

Materials and methods

In vivo and in vitro guinea pig electrocardiogram, intracellular action potential and patch clamp recording techniques were carried out.

Results

Both SHL and QKL (both in one, five and ten times clinically relevant doses (CRD) for in vivo and clinically relevant concentrations (CRC) for in vitro) prolonged P–R intervals in a dose or concentration-dependent manner and SHL also prolonged QTc. YZH (ten and 20 times CRD and CRC) prolonged P–R intervals without changing QTc. Intracellular action potential recordings from guinea pig papillary muscle indicated SHL and QKL abolished the firing of action potentials at ten and 30 times CRC respectively. SHL significantly suppressed L-type Ca²⁺ current from left ventricular myocytes of guinea pig, hNav1.5 current and hERG current with 50% inhibiting concentrations (IC₅₀) of 6.0, 3.0 and 10.7 times CRC, respectively. Also, QKL significantly suppressed L-type Ca²⁺ and hNav1.5 currents with IC₅₀s of 10.7 and 13.8 times CRC. YZH significantly suppressed L-type Ca²⁺, hNav1.5 and hERG currents with IC₅₀s of 12.1, 32.9 and 141.7 times CRC, respectively.

Conclusions

The three CHIs studied caused bradyarrhythmia mainly by inhibiting Na⁺ current and L-type Ca²⁺ current.     

Salvia miltiorrhiza causes tonic contraction in rat ileum through Ca²⁺-calmodulin pathway

Ethnopharmacological relevance

Danshen, root of Salvia miltiorrhiza (SM), has been traditionally used in Chinese medicine for the treatment of heart, abdomen, gurgling in the intestines, and relieving fullness. However, the effects of SM on intestine have rarely been done to date.

Aim of the study

To investigate the contraction effect of SM on isolated rat ileum and its mechanisms involved.

Materials and methods

The isometric contractions of ileum segments were investigated in organ baths for spontaneous activity and response to ethanolic extracts of SM. To determine the contraction mechanism caused by SM extracts, atropine (a muscarinic receptor antagonist), tetrodotoxin (TTX, a sodium channel blocker), nifedipine (a calcium channel blocker), Ca²⁺ free Krebs solution with EGTA, or trifluoperazine (TFP, a calmodulin blocker) was administered and its response to cumulative dosages of SM extracts were examined. The effect of SM extracts on Ca²⁺ signaling in the intestinal epithelial cell-6 (IEC-6) was examined using fura-2 as a Ca²⁺ indicator.

Results

SM extracts caused dose-dependent tonic contraction on rat ileum in ex vivo organ bath studies. The contraction induced by SM extracts was not inhibited by atropine, TTX, nifedipine, or in Ca²⁺ free solution. However, the ileal contractions induced by SM extracts were significantly inhibited by TFP in a dose-dependent manner. In IEC-6 cells, the SM extracts induced extracellular Ca²⁺ entry and massive intracellular Ca²⁺ release in Ca²⁺-contained medium, and induced intracellular Ca²⁺ release in Ca²⁺-free medium.

Conclusion

These results demonstrate that SM extracts cause ileal contraction through the Ca²⁺-calmodulin pathway.     

双苯氟嗪对大鼠脑缺血再灌注损伤后胞浆[Ca2+]i的影响

 目的研究双苯氟嗪(Dip)对大鼠局灶性脑缺血再灌注损伤后脑神经细胞胞浆Ca²⁺浓度([Ca²⁺]_i)变化的影响。方法将400pmol的内皮素-1(ET-1)灌注到大鼠大脑中动脉附近制备大鼠局灶性脑缺血再灌注模型,以Fura-2/AM作为钙荧光指示剂,双波长荧光分光光度法检测灌注ET-1后不同时间大鼠大脑皮层和纹状体神经细胞胞浆[Ca²⁺]_i的变化及Dip对灌注ET-1后4h胞浆[Ca²⁺]_i变化的影响,并采用TYC染色法观察了Dip对灌注ET-1后24h脑梗死范围的影响。结果灌注400pmolET-1诱发大鼠局灶性脑缺血再灌注损伤后,大鼠大脑皮层和纹状体神经细胞胞浆[Ca²⁺]_i明显升高,并于灌注EF-1后4h达峰值,随着再灌注时间的延长,胞浆[Ca²⁺]_i逐渐降低;Dip20,40mg·kg^-1可以明显降低灌注ET-1后4h大脑皮层和纹状体神经细胞胞浆[Ca²⁺]_i的升高(P<0.01),Dip10mg·kg^-1组虽表现出一定的降低胞浆[Ca²⁺]_i的作用,但与溶剂组比较,差异无显著性(P>0.05);对脑梗死范围的测定结果显示,Dip可以缩小脑梗死范围,其作用呈现明显的剂量依赖关系(r=0.9797,P<0.01)。结论Dip对抗脑缺血再灌注损伤后胞浆[Ca²⁺]_i升高可能是其产生神经保护作用的重要机制。     

Modulation effect of Smilax glabra flavonoids on ryanodine receptor mediated intracellular Ca release in cardiomyoblast cells

Ethnopharmacological relevance

Smilax glabra rhizome, a plant material from Liliaceae family, is a widely used traditional Chinese medicine for anti-cardiac hypertrophy treatment. We have previously found that Smilax glabra flavonoids (SGF) exerted such anti-cardiac hypertrophy activity. However, the mechanism of this activity of SGF has not been clarified yet.

Materials and methods

This study was aimed to investigate the inhibitory role of SGF on intracellular Ca²⁺ release in rat cardiomyoblast cells (H9C2). Intracellular Ca²⁺ release was determined by Ca²⁺ indicator fluorescence (fluo 4-AM) in H9C2 cell line.

Results

SGF at concentrations of 0.25, 0.5, 1.0 mg/ml significantly inhibited the phenylephrine or angiotensin II induced intracellular Ca²⁺ release in a dose-dependent manner. Furthermore, SGF could also inhibit ryanodine receptor (RyR) agonist caffeine induced Ca²⁺ release and phenylephrine (PE)-induced Ca²⁺ release under the condition in which inositol trisphosphate (IP3) receptors were blocked with 2-Aminoethoxydiphenyl borate (2-APB). Nevertheless, SGF had no impact on PE-induced Ca²⁺ release under the condition in which RyRs were blocked with tetracaine.

Conclusions

Our results suggest that the protective effects of SGF are mediated via targeting inhibition of RyR mediated intracellular Ca²⁺ release.