DADLE suppresses the proliferation of human liver cancer HepG2 cells by activation of PKC pathway and elevates the sensitivity to cis-diammine dichloridoplatium / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect of DADLE, a δ-opioid receptor agonist, on the proliferation of human liver cancer HepG2 cells and explore the mechanism involving PKC pathway.</p><p><b>METHODS</b>HepG2 cells were treated with DADLE at different doses (0.01, 0.1, 1.0 and 10 µmol/L). Cell viability was determined using methyl thiazolyl terazolium (MTT) assay. The expression of PKC mRNA and p-PKC protein were examined by RT-PCR and Western blot assay. After treated separately with DADLE plusing NAL or PMA, the cell cycle of HepG2 cells was analyzed by flow cytometer. MTT was used to detect their proliferation capacity and Western blot was used to examine the p-PKC expression. The growth inhibitory rate of HepG2 cells treated with DADLE and cis-diammine dichloridoplatinum (CDDP) was analyzed.</p><p><b>RESULTS</b>DADLE at different concentrations showed an inhibitory effect on the proliferation of HepG2 cells though inhibiting the expression of PKC mRNA and p-PKC protein. The results of flow cytometry showed that compared with the control group, the percentage of S + G(2)/M cells in DADLE-treated group was lowered by 3.94% (P < 0.01). Meanwhile, after treated with NAL and PMA, the percentage was elevated by 3.22% and 3.63%, respectively (P < 0.01). The MTT and Western blot assays showed that compared with the control group, the values of A570 and p-PKC protein levels in the HepG2 cells of DADLE-treated group were significantly decreased (P < 0.01). After treatment with NAL and PMA, the values of A570 and p-PKC protein levels were elevated significantly (P < 0.01). The growth inhibitory rate of DADLE + CDDP group was 79.9%, significantly lower than 25.2% and 43.2% of the DADLE and CDDP groups, respectively.</p><p><b>CONCLUSIONS</b>Activation of δ-opioid receptor by DADLE inhibits the apoptosis of human liver cancer HepG2 cells. The underlying mechanism may be correlated with PKC pathway. DADLE can enhance the chemosensitivity of HepG2 cells to CDDP.</p>

The Neuroprotective Effect of delta-opioid Receptor Stimulation with D-Ala2, D-Leu5 Enkephalin Against Ischemic Neuronal Injury

PURPOSE: Oxygen is indispensable for survival and aerobic metabolism in all mammalian cells. Inadequate oxygen triggers a multifaceted cellular response negatively impacting important physiological functions which are observed in clinical diseases such as stroke, drowning, cardiac arrest, hazardous gas poisoning, myocardial infarction and vascular dementia. In this study, we investigated the neuroprotective effect of a synthetic delta-opioid agonist, [D-Ala2, D-Leu5] enkephalin (DADLE), and its role in ischemic neuronal injury. METHODS: This experiment was conducted in vitro using a primary culture of rat cortical neurons. Ischemia induction was performed using a hypoxic chamber. To test the degree of neuronal viability, as protected by delta-opioid stimulation with DADLE under ischemia, we used three independent approaches including a lactate dehydrogenase assay, MTT assay, and an immunofluorescent staining assay for viable cells. In addition, the gene expressions of caspase-3 and heat shock protein 70 were analyzed using real-time PCR. RESULTS: Incubation of the cortical neurons with DADLE protected them from ischemia-induced cytotoxicity, as observed by all three independent viability assays. Also, we found that its neuroprotective effect might be related with suppression of the caspase-3 gene. CONCLUSION: The results of this study suggested that DADLE exhibits a neuroprotective effect against ischemia-induced neuronal cell death.

The Neuroprotective Effect of delta-opioid Receptor Stimulation with D-Ala2, D-Leu5 Enkephalin Against Ischemic Neuronal Injury

PURPOSE: Oxygen is indispensable for survival and aerobic metabolism in all mammalian cells. Inadequate oxygen triggers a multifaceted cellular response negatively impacting important physiological functions which are observed in clinical diseases such as stroke, drowning, cardiac arrest, hazardous gas poisoning, myocardial infarction and vascular dementia. In this study, we investigated the neuroprotective effect of a synthetic delta-opioid agonist, [D-Ala2, D-Leu5] enkephalin (DADLE), and its role in ischemic neuronal injury. METHODS: This experiment was conducted in vitro using a primary culture of rat cortical neurons. Ischemia induction was performed using a hypoxic chamber. To test the degree of neuronal viability, as protected by delta-opioid stimulation with DADLE under ischemia, we used three independent approaches including a lactate dehydrogenase assay, MTT assay, and an immunofluorescent staining assay for viable cells. In addition, the gene expressions of caspase-3 and heat shock protein 70 were analyzed using real-time PCR. RESULTS: Incubation of the cortical neurons with DADLE protected them from ischemia-induced cytotoxicity, as observed by all three independent viability assays. Also, we found that its neuroprotective effect might be related with suppression of the caspase-3 gene. CONCLUSION: The results of this study suggested that DADLE exhibits a neuroprotective effect against ischemia-induced neuronal cell death.

Effects of delta-opioid receptor stimulation on survival of cultured myocardial cells upon to serum deprivation / 中国应用生理学杂志

<p><b>AIM</b>To study upon to serum deprivation if delta-opioid receptor activation has direct effect on cultured impaired cardiomyocytes survival.</p><p><b>METHODS</b>Myocardial cells of neonatal rats were cultured in vitro. The cell viability was determined with crystal violet staining uptake. The percentage of S + G2 + M in cell cycle was determined by flow cytometry. Apoptosis rates were determined by flow cytometry (FCM). The expression of Caspase-3 were investigated by Western blotting.</p><p><b>RESULTS</b>Myocardial cells of neonatal rats were cultured of serum-free in vitro, apoptotic index was significantly increased, the expression of Caspase-3 was significantly increased, free-serum induced apoptosis in cardiac myocytes after 48 h. At concentrations of 10 nmol x L(-1) - 10 micromol x L(-1), a delta opoid receptor agonist [D-Ala2, D-Leu5]-enkephalin DADLE promoted the myocardial cells survival, in a concentration-dependent manner. The optimal response was achieved at 0.1 micromol x L(-1), which increase survival index of cardiac myocyte, percentage of S + G2 + M in cell cycle, decrease apoptotic index of cardiac myocyte, and the expression activate caspase-3. Delta-opioid receptor antagonist naltrindole at 10 micromol x L(-1) inhibited the promoting effects of DADLE, which decrease survival index of cardiac myocyte, and percentage of S + G2 + M in cell cycle, increase apoptotic index of cardiac myocyte and the expression of Caspase-3.</p><p><b>CONCLUSION</b>The protective of delta-opioid receptor activation can promote survival in cultured impaired myocardial cells.</p>

Effects of a delta-opioid Agonist on the Brainstem Vestibular Nuclear Neuronal Activity of Rats

This study was undertaken to investigate the effects of [D-Ala2, D-Leu5]-enkephalin (DADLE) on the spontaneous activity of medial vestibular nuclear neurons of the rat. Sprague-Dawley rats, aged 14 to 16 days, were anesthetized with ether and decapitated. After enzymatic digestion, the brain stem portion of medial vestibular nuclear neuron was obtained by micropunching. The dissociated neurons were transferred to a recording chamber mounted on an inverted microscope, and spontaneous action potentials were recorded by standard patch-clamp techniques. The spontaneous action potentials were increased by DADLE in 12 cells and decreased in 3 cells. The spike frequency and resting membrane potential of these cells were increased by DADLE. The depth of afterhyperpolarization was not affected by DADLE. The potassium currents were decreased in 20 cells and increased in 5 cells. These results suggest that DADLE increases the neuronal activity of the medial vestibular nuclear neurons by altering resting membrane potential.

Assessment of the possible capacity of the synthetic and-opioid receptor agonist DADLE in an experimental model of hypothermic ischemia and reperfusion in isolated rat heart - role of ATP-sensitive potassium channels

Efforts to prevent myocardial ischemia have focused on findings ways to block events associated with irreversible ischemic injury. The discovery of the endogenous cellular protective mechanism known as ischemic preconditioning [ipc] has risen hoping that natural pathways could be activated to help cells to overcome necrosis. Pharmacologic preconditioning has been tried to simulate ipc in cardio protection against ischemia. Aim: the aim of the present study is to assess the possible protective capacities of both [ipc] and the synthetic -opioid receptor agonist dadle in an experimental model of hypothermic ischemia and reperfusion [hir] in isolated rat heart, and to assess whether the effects of both protective measures are similarly mediated via k[atp] channels. Isolated rat hearts were divided into 7 groups: g1: is control normothermic [37°C] perfused hearts. G2 hearts were subjected to 45 min of hypothermic ischemia at 30°C, followed by 25 min of normothermic reperfusion [hir]. G3: ipc for 3 min followed by 5 min normothermic reperfusion then hir as g2. G4: dadle pretreatment [1 mg/kg] 30 min before isolated heart preparation, then the protocol of g2 was done. G5: DADLE pretreatment, then the protocol of G3. G6: glibenclamide [0.3 mg/kg] 60 min pretreatment followed by DADLE after 30 min, then the protocol of g2 was done g7: glibenclamide and dalde pretreatment, followed by protocol of g3. Both ipc and DADLE pretreatment and their combination in groups 3, 4, and 5, respectively, improved the post ischemic recovery in the form of significant increase in heart rate [hr], myocardial contractility, coronary flow [cf], and significant decrease in creative kinas [ck] in coronary effluent with a significant reduction of infarct size [is] to 6.6 +/- 1.5, 3.1 +/- 1.3, and 2.8 +/- 1.2%, respectively, when compared with g1 and g2 with is 17.2 +/- 2.2%. Glibenclamide pretreatment in g6 and g7 abolished this improvement in the post ischemic recovery when compared with groups 3, 4 and 5. The results of the present study indicate that ischemic prerconditioning [ipc] improves post ischemic recovery and reduces myocardial infract size in isolated rat heart. Pretreatment with -opioid receptor agnist DADLE mimicked the cardio protection induced by ipc, indicating an opiod receptor-mediated mechanism. Pharmacologic preconditioning by dasle and ipc has additional effects in improving the post ischemic recovery. Lastly, this cardioprotection induced by both can be abolished by the k[ATP] channel antagonist glibenclamide suggesting an involvement of the k[ATP] channel most probably mitochondrial k[ATP], as an important end-effecter of this potent cardio protective effect

Possible involvement of nitric oxide in estrogen-induced uterine edema in the immature rat

We examined whether nitric oxide mediates estrogen-induced uterine edema in the immature rat. Immature Wistar rats (19-21 days) received estradiol-17 beta (E2) in a single sc dose of 10 micrograms/animal and 6 h later the animals were sacrificed and the changes in uterine wet and dry weights were determined. E2 treatment caused a 93 per cent increase in uterine wet weight (control, N = 6, 39.88 +/- 3.2 mg; E2 treated, N = 6, 76.8 +/- 4.9 mg), but not in dry weight, suggesting that it induces uterine edema. Pretreatment with L-nitroarginine methyl ester (L-NAME), a competitive antagonist of nitric oxide synthetase, at doses of 10 and 20 mg/kg, ip, caused a dose-related reduction (59 and 86 per cent ) in the uterine wet weight increase induced by E2. Furthermore, L-arginine (300-600 mg/kg, sc), the nitric oxide precursor, was able to reverse L-NAME (20 mg/kg)-induced decreases in uterine weight by 47 and 62 per cent , respectively. The results suggest that nitric oxide is the principal mediator involved in estrogen-induced uterine edema in the immature rat