Antidigoxin antiserum prevents endogenous digitalis-like compound-mediated reperfusion injury via modulating sodium pump isoform gene expression.

Endogenous digitalis-like compound (EDLC) is an endogenous ligand of the digitalis receptor and can remarkably inhibit Na+/K+-ATPase activity. Antidigoxin antiserum (ADA), a selective EDLC antagonist, may lessen myocardial reperfusion injury; however, the molecular mechanisms underlying the effect remain unclear. Therefore, this study investigated whether ADA may prevent myocardial reperfusion injury and modulate gene expression of sodium pump alpha isoforms. Cardiac function was examined in isolated rat hearts subjected to ischemia and reperfusion (I/R). The infarct size, EDLC level, Na+/K+-ATPase activity, and the levels of mRNA for sodium pump alpha isoforms were measured in vivo I/R rat hearts in the presence or absence of ADA. It was found that ADA significantly improved the recovery of cardiac function, decreased infarct size, decreased EDLC level, and recovered Na+/K+-ATPase activity in I/R hearts. Further studies showed that sodium pump alpha1, alpha2, and alpha3 isoform mRNA levels were significantly reduced in I/R hearts, and pretreatment with ADA induced a large increase in the mRNA levels. These results indicate that EDLC may participate in depressing Na+/K+-ATPase activity and sodium pump alpha isoform gene expression in I/R heart. It is suggested that treatment with ADA may prevent EDLC-mediated reperfusion injury via modulating sodium pump isoform gene expression.

[Protective effects of adiponectin on myocardial ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate the protective effects of adiponectin on myocardial ischemia-reperfusion injury and the potential mechanisms in rats. METHODS: Thirty-two male rats aged 8 weeks were randomly assigned to sham operation (sham), myocardial ischemia-reperfusion (MIR), diltiazem treatment (diltiazem) or adiponectin administration (APN) groups (n = 8 each). MIR rats underwent left anterior descending artery (LAD) occlusion for 30 min followed by 60 min reperfusion. Diltiazem (7 microg/g) and APN (120 ng/g) were given by caudal intravenous injection at the end of 30 min ischemia and the beginning of reperfusion for rats in diltiazem or APN groups. Animals were sacrificed after 60 min reperfusion for determining the myocardial nitric oxide (NO), Caspase 3, activity of AMP-activated protein kinase(AMPK) and concentration of peroxisome proliferators-activated receptor gamma (PPARgamma). Apoptotic cells were stained by Caspase 3 Activity Assay Kit and mitochondria in myocardial cells were observed by transmission electron microscope (TEM). RESULTS: The myocardial Caspase 3 level was significantly increased [(168.50 +/- 30.08) micromol/L vs. (53.25 +/- 11.41) micromol/L, P < 0.01], AMPK activity, PPARgamma and NO concentrations were significantly reduced in MIR group compared with those in sham group (all P < 0.05) [(0.74 +/- 0.59) IU/ml vs. (25.63 +/- 4.61) IU/ml, P < 0.01; 0.1894 vs. 0.7949, P < 0.01; (6.359 +/- 1.355) micromol/L vs. (10.396 +/- 1.901) micromol/L, P < 0.01], these effects could be significantly reversed by APN. In comparison with MIR group, the levels of Caspase 3 in cardiac muscles were significantly lowered in Adiponectin group [(88.75 +/- 6.92) micromol/L vs. (168.50 +/- 30.08) micromol/L, P < 0.01], whereas the level of AMPK and PPARgamma, NO concentration in the cardiac muscle was remarkably increased [(27.22 +/- 4.76) IU/ml vs. (0.74 +/- 0.59) IU/ml, P < 0.01; 0.8613 vs. 0.1894, P < 0.01; (15.755 +/- 1.045) micromol/L vs. (6.359 +/- 1.355) micromol/L, P < 0.01]. APN also preserved the function and structure of mitochondria in rats post ischemia/reperfusion injury. The protective pharmacologic actions of APN were superior to that of diltiazem. CONCLUSION: Adiponectin could protect myocardial tissues from myocardial ischemia-reperfusion injury in rats, possibly by upregulating myocardial AMPK and PPARgamma expressions and preventing myocardial cells from apoptosis.

[Platelet-derived growth factor-BB inhibited p21(WAF1) expression partially through transforming growth factor-beta signalling system in vascular smooth muscle cell].

OBJECTIVE: To assess if the modulating effect of platelet-derived growth factor (PDGF)-BB on p21(WAF1) was mediated by upregulating transforming growth factor (TGF)-beta(1) expression in vascular smooth muscle cells (VSMC). METHODS: TGF-beta(1) mRNA and protein expressions were measured by reverse transcription-PCR and ELISA, the protein expressions of p21(WAF1) and the downstream TGF-beta signalling including TGF-beta type I receptor (ALK-5 in VSMC), Smurf2, pSmad2/3, Smad4, Smad7 were detected by Western blot. RESULTS: PDGF-BB significantly upregulated the expressions of TGF-beta(1) at mRNA (0.79-fold) and protein (1.98-fold) levels in VSMC, significantly inhibited the expression of p21(WAF1) (-67 +/- 12)%, and enhanced the expressions of ALK-5, pSmad2/3, Smad4, Smurf2 protein by 1.21-fold, 0.95-fold, 0.69-fold and 2.55-fold respectively, inhibited Smad7 expression (-65 +/- 9)%, these alterations were partially restored by anti-TGF-beta(1) neutralizing antibody. CONCLUSIONS: These findings suggested that PDGF-BB inhibited p21(WAF1) expression in VSMC partially through upregulating TGF-beta(1) expression via PDGF-BB and TGF-beta signalling pathways.

[Effect of inhibiting endoxin by antidigoxin antiserum on myocardial ischemia/reperfusion injury in rats].

AIM: To study the effect of antidigoxin antiserum on oxygen stress induced by myocardial ischemia/reperfusion (MI/R) injury in rats. METHODS: Sprauge Dawley rats were submitted to ligate left anterior descending coronary artery 30 min followed by 45 min reperfusion. Experiment animals were randomly divided into seven groups including sham group, MI/R group, normal salina group, verapamil group and three antidigoxin antiserum groups from low to high dose. The left ventricular myocardial tissue sample of ischemia were processed and measured the level of endoxin and malondialdehyde (MDA), the activities of Na+, K(+) -ATPase and superoxin dismutase (SOD). The myocardia morphology was observed. RESULTS: The levels of endoxin and MDA increased and the activities of Na+, K(+) -ATPase and MDA were inhibited significantly in MI/R and saline groups. Including verapamil group in comparison to MI/R and saline groups, MDA level decreased and SOD activities partly reserved, meanwhile, only in three antidigoxin antiserum groups, the myocardial endoxin level was remarkably decreased, Na+, K(+) -ATPase activities were drastically increased. The myocardial histological morphology was significantly improved. CONCLUSION: Antidigoxin antiserum, an endoxin mutual clone antibody, had the effect of attenuating the damage of oxygen free radicals induced by MI/R via to antagonizing the inhibition effect of endoxin on myocardial membrane Na+, K(+) -ATPase activities.

Effects of antidigoxin antiserum on endoxin levels, apoptosis and the expression of Bax and Bcl-2 protein in ischaemia-reperfusion myocardium.

The aim of the present study was to investigate the effects of antidigoxin antiserum (ADA), an endoxin special antagonist, on endoxin levels, apoptosis and the expression of the apoptosis-related protein bcl-2 and bax in myocardial ischaemia-reperfusion (MIR). The left anterior descending coronary artery was subjected to 30 min ischaemia followed by 45 min reperfusion in open-chest anaesthetized rats. The rats were divided randomly into seven groups: a sham-operated group, an MIR group, a vehicle control (normal saline) group, and groups receiving verapamil (5 mg/kg) or ADA (9, 18 and 36 mg/kg). The drugs were injected into rats via the femoral vein before reperfusion was commenced. Myocardial endoxin levels were measured by radioimmunoassay. Apoptotic cells was detected using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling method. The expression of the apoptosis-related proteins bcl-2 and bax was detected by immunohistochemistry and their semiquantification scores were recorded by a computer image analysis system. Myocardial endoxin levels, the number of apoptotic cells and bax protein expression were increased in the MIR group compared with the sham group. Although bcl-2 protein expression was elevated in the MIR group, there was no significant difference between the MIR and sham groups. However, the ratio of bcl-2/bax was significantly decreased in the MIR group. In the group receiving 36 mg/kg ADA, myocardial endoxin levels, the number of apoptotic cells and bax protein expression were significantly decreased; bcl-2 protein expression was enhanced. The bcl-2/bax ratio was increased. The results suggest that ADA inhibited myocardial apoptosis induced by MIR in rats. The mechanisms involved require further investigation, but the present study may suggest that ADA prevents bax upregulation and enhances bcl-2 upregulation by antagonizing the effects of endoxin.

Endoxin antagonist lessens myocardial ischemia reperfusion injury.

OBJECTIVE: To elucidate whether endoxin is one of important factors involved in myocardial ischemia reperfusion (MIR) injury, the change of myocardial endoxin level was determined in rats with MIR injury model and the effects of anti-digoxin antiserum (ADA), an endoxin specific antagonist, on MIR injury were studied. METHODS: MIR injury model was obtained by ligating left anterior descending coronary artery 30 min followed by 45 min reperfusion. Sprague-Dawley rats were randomly divided into six groups of 10 rats, each. Sham group, MIR group, normal saline group, ADA 9, 18 and 36 mg.kg(-1). ECG was continuously recorded. After reperfusion left ventricular myocardium samples of ischemic area were processed immediately. Myocardial endoxin level, Na(+)-K(+)-ATPase, Ca(2+)-ATPase, Mg(2+)-ATPase activities, and intramitochondrial Ca(2+) content were measured. RESULTS: Myocardial endoxin level was significantly increased; Na(+)-K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase activities were remarkably decreased; intramitochondrial Ca(2+) content was remarkably raised; ST segments of ECG were significantly elevated and occurrence and scores of ventricular arrhythmias were significantly increased in early stage of reperfusion in rats with MIR. In all groups with ADA, myocardial endoxin level was remarkably decreased; Na(+)-K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities were drastically increased; intramitochondrial Ca(2+) content was declined; ST segments and ventricular arrhythmias were improved. CONCLUSION: Myocardial endoxin level was increased in MIR, which implies that the elevated endoxin may be one of major factors inducing MIR injury. This postulate is supported by the observation that ADA has protective and therapeutic effects against MIR injury probably by antagonizing the action of endoxin. The underlying mechanism may be ascribed to restoration of energy metabolism, and attenuation of intracellular Ca(2+) overload.

Endoxin-mediated myocardial ischemia reperfusion injury in rats in vitro.

Myocardial ischemia reperfusion results in an increase in intracellular sodium concentration, which secondarily increases intracellular calcium via Na(+)-Ca2+ exchange, resulting in cellular injury. Endoxin is an endogenous medium of digitalis receptor and can remarkably inhibit Na+/K(+)-ATPase activity. Although the level of plasma endoxin is significantly higher during myocardial ischemia, its practical significance is unclear. This research is to investigate whether endoxin is one of important factors involved in myocardial ischemia reperfusion injury. Ischemia reperfusion injury was induced by 30 min of global ischemia and 30 min of reperfusion in isolated rat hearts. Heart rate (HR), left ventricular developed pressure (LVDP), and its first derivative (+/-dp/dtmax) were recorded. The endoxin contents, intramitochondrial Ca2+ contents, and the Na+/K(+)-ATPase activity in myocardial tissues were measured. Myocardial damages were evaluated by electron microscopy. The endoxin and intramitochondrial Ca2+ contents in myocardial tissues were remarkably higher, myocardial membrane ATPase activity was remarkably lower, the cardiac function was significantly deteriorated, and myocardial morphological damages were severe in myocardial ischemia reperfusion group vs. control. Anti-digoxin antiserum (10, 30 mg/kg) caused a significant improvement in cardiac function (LVDP and +/-dp/dtmax), Na+/K(+)-ATPase activity, and myocardial morphology, and caused a reduction of endoxin and intramitochondrial Ca2+ contents in myocardial tissues. In the present study, the endoxin antagonist, anti-digoxin antiserum, protected the myocardium against the damages induced by ischemia reperfusion in isolated rat hearts. The results suggest that endoxin might be one of main factors mediating myocardial ischemia reperfusion injury.

Effects of valsartan with or without benazepril on blood pressure, angiotensin II, and endoxin in patients with essential hypertension.

AIM: To evaluate the effects of valsartan (Val) with or without benazepril (Ben) on blood pressure and plasma levels of angiotensin (Ang II) and digoxin-immunoreactive factors (endoxin) in patients with essential hypertension. METHODS: Ninety patients with essential hypertension were randomly divided into 3 groups (n=30 per group): Ben group (Ben 10 mg/d, po); Val group (Val 80 mg/d, po); combination drug therapy group (Val 80 mg/d+Ben 10 mg/d, po); all patients were treated for 12 weeks. Age and sex-matched 20 normal subjects were served as control group. RESULTS: The levels of plasma endoxin and Ang II in patients with essential hypertension were remarkably higher than those in normal subjects. The levels of plasma Ang II and endoxin were all obvious positive correlation with systolic blood pressure (SBP) and diastolic blood pressure (DBP) (Ang II: r=0.5151, 0.7978; endoxin: r=0.4706, 0.7274, respectively). Within 6 weeks of drug intervene, SBP and DBP were remarkably decreased in 3 groups. After 6 weeks, SBP and DBP were continuously decreased in Ben group and Val+Ben group, but not in Val group. Level of plasma Ang II was remarkably decreased as SBP and DBP decreased in Ben group and Val+Ben group; level of plasma Ang II was remarkably increased in Val group. CONCLUSION: Val with or without Ben remarkably decreased SBP and DBP in patients with essential hypertension within 6 weeks. Antihypertensive efficacy was weakened after long-term use of Val alone. The antihypertensive effect of Val+Ben group was the most remarkable among 3 groups and could avoid the side effects of high plasma Ang II.