Metformin and tanshinone llA as ethnodrugs protect heart against injury by inhibiting microRNA-1 / 中国药理学与毒理学杂志

OBJECTIVE To explore the protection mechanism of metformin and tanshinone IIA on myocardial injury. METHODS The cultured neonatal rat ventricular cells (NRVCs) were exposed to 100 μmol·L-1H2O2to simulate the in vitro model of ischemia-reperfusion injury.MTT,TUNEL and Viability/Cytotoxicity Assay were used to evaluate the effect of metformin on the viability of cardiomyocytes after treated with H2O2. The target of miR-1 was verified by Dual luciferase reporter assay. ChIP analyses was adopted to reveal the relationship between C/EBP β and miR-1.Tanshinone IIA was administrated daily for 7 d before ligation of the left anterior descending artery (LAD) and lasted for 3 months after LAD.Whole-cell patch-clamp techniques were used to measure the inward rectifying K+current(IK1)in rat isolated ventricular myocytes.GRP94,p-AMPKα,C/EBP β,CHOP,Caspase-3,Kir2.1,p38 MAPK, Cx43, MEF2 and SRF levels were analyzed by Western blot and miR-1 level was quantified by Real-time PCR.RESULTS The expression of miR-1 was significantly increased in NRVCs exposed to H2O2 in vitro. miR-1 was shown to target the 3′-untranslated region (UTR) of GRP94, which results in the accumulation of un/misfolded proteins,leading to the endoplasmic reticulum(ER)stress.C/EBP β directly induces the upregulation of miR-1 by binding to its promoter.Furthermore,metformin,a direct allosteric AMPK activator, significantly reduces C/EBP β and miR-1 levels comparing with control group. Similarly, tanshinone IIA decreased the incidence of arrhythmias and relieved ischemia-induced injury.Moreover, tanshinone IIA depressed the elevated miR-1 level and inhibited the activation of p38 MAPK and heart special transcription factors SRF and MEF2 in ischemic cardiomyocytes. CONCLUSION Metformin protects cardiomyocytes against H2O2damage through AMPK/C/EBPβ/miR-1/GRP94 pathway.Tanshi-none IIA play a role in protection cardiomyocytes from ischemic injury based on inhibiting miR-1 expres-sion through p38 MAPK signal pathway.

Effects of emodin on the intracellular calcium concentration (Ca2+i) and L-type calcium current of the single ventricular mytocytes from guinea pig / 药学学报

<p><b>AIM</b>To study the effects of emodin on intracellular calcium concentration ([Ca2+]i) and L-type calcium current of the single ventricular myocytes from guinea pig.</p><p><b>METHODS</b>Enzymatic dissociation was used to isolate single ventricular myocytes from adult guinea pig. They were loaded with Ca2(+)-sensitive fluorecent indicator Fluo-3/AM. [Ca2+]i represented by fluorescent intensity (FI) was measured by laser scanning confocal microscope. Whole cell patch clamp technique was used to record ICa-L.</p><p><b>RESULTS</b>At resting status, [Ca2+]i was not affected by emodin (1-100 mumol.L-1). Emodin at the concentration of 1 mumol.L-1 was shown to increase the [Ca2+]i induced by 60 mmol.L-1 KCl. The peak value of fluorescent intensity was increased from 1,877 +/- 551 to 2,905 +/- 739 (n = 8, P < 0.05). Emodin at the concentration of 10 mumol.L-1 had no effect on the increase of [Ca2+]i induced by 60 mmol.L-1 KCl. However, the increase of [Ca2+]i induced by KCl was reduced to 1,214 +/- 335 (n = 8, P < 0.05) by 100 mumol.L-1 emodin. The density of ICa-L was increased from (-6.2 +/- 1.3) pA/pF to (-8.3 +/- 0.3) pA/pF (n = 6, P < 0.05) by 1 mumol.L-1 emodin at the test pulse of 0 mV. The current was not altered by 10 mumol.L-1 emodin. But it was inhibited from (-6.6 +/- 1.0) pA/pF to (-3.80 +/- 0.16) pA/pF (n = 6, P < 0.05) by 100 mumol.L-1 emodin at the test pulse of +10 mV.</p><p><b>CONCLUSION</b>Emodin has two-way regulation on [Ca2+]i and ICa-L of cardiomyocytes in guinea pig.</p>

The ion targets of arrhythmias induced by ouabain and aconitine in guinea pig and rat ventricular myocytes / 药学学报

<p><b>AIM</b>To observe the effects of ouabain and aconitine on APD and ion channels in isolated guinea pig and rat ventricular myocytes; to elucidate the action mechanisms of these two drugs and set up new arrhythmic models on cellular level.</p><p><b>METHODS</b>In isolated ventricular myocytes of guinea pig and rat, the effects of ouabain and aconitine on APD, ICa-L, Ik, Ito and Ik1 were observed using the whole cell patch clamp technique.</p><p><b>RESULTS</b>Ouabain (5 micromol x L(-1)) obviously prolonged the APD90, increased ICa-L, decreased Ik and Ik1 in guinea pig ventricular myocytes. Aconitine (1 micromol x L(-1)) lengthened the APD90, increased ICa-L, decreased Ito and increased Ik1 in rat ventricular myocytes.</p><p><b>CONCLUSION</b>The targets on ouabain- and aconitine-induced arrhythmias included APD, ICa-L, Ik, Ito, and Ik1. APD, ICaL, Ik and Ito must be the powerful ones, both in arrhythmic and antiarrhythmic courses. The ouabain- and aconitine- induced arrhythmic models on cellular level were built to study the antiarrhythmic mechanisms of chemicals and evaluate new drugs. These two new-type models in vitro were stable, liable, repeatable and economic, which were superior to those typical models in vivo.</p>

Effects of cyclovirobuxine D on intracellular Ca2+ and L-type Ca2+ current in rat ventricular cardiomyocytes / 药学学报

<p><b>AIM</b>To determine the effects of cyclovirobuxine D (CD) on intracellular Ca2+ mobilization and L-type Ca2+ current (I(Ca-L)) in isolated rat cardiomyocytes.</p><p><b>METHODS</b>The effects of CD on the amplitude of I(Ca-L) and intracellular Ca2+ mobilization induced by KCl and caffeine were studied with the method of patch-clamp technique and laser scanning confocal microscopy in rat ventricular myocytes.</p><p><b>RESULTS</b>CD decreased the amplitude of I(Ca-L) in a concentration-dependent manner. At 10 mV, 1 and 10 micromol x L(-1) CD decreased I(Ca-L) density from (- 9.9 +/- 1.8) pA/pF to (-6.4 +/- 1.4) and (-4.2 +/- 0.6) pA/pF, respectively. Confocal experiments showed that intracellular fluorescent intensity (FI) value of [Ca2+] in control resting level was not changed by 1 and 10 micromol x L(-1) CD. [Ca2+] increase in response to KCl could not be reduced by CD. The rise of [Ca2+]i in response to caffeine was further enhanced by pretreatment with CD.</p><p><b>CONCLUSION</b>CD decreased I(Ca,L) in a concentration-dependent manner and increased [Ca2+]i release induced by caffeine in rat ventricular cardiomyocytes.</p>

Dihydromyricetin induce apoptosis in AGZY-83-a tumor cells / 中国药理学通报

Aim To investigate the effects of dihydromyricetin on the apoptosis in AGZY-83-a tumor cells and its relevant mechanisms.Methods The survival rate of AGZY-83-a cells was assayed by MTT dye reduction.The cellular apoptosis was analyzed by TUNEL method and caspase-3 activity.The intracellular free calcium i was detected to explore the apoptotic mechanism of dihydromyricetin.Results In the MTT assay,dihydromyricetin 50?mol?L-1 significantly inhibited survival ratios of AGZY-83-a cells at dose-and time-dependent manner.It was demonstrated in TUNEL assay that dihydromyricetin could induce the apoptosis of AGZY-83-a cells in a concentration-dependent manner.The examination of Caspase-3 activity indicated that the dihydromyricetin could induce the apoptosis of AGZY-83-a cells,which was dose-dependent activation of Caspase-3 in AGZY-83-a cells.The detection of the intracellular i showed that the average FI of the i could be markedly increased to 20-fold as the basic condition.Conclusions Dihydromyricetin can induce the apoptosis in AGZY-83-a cells,which is associated with the overload of the intracellular i.