miRNA-185 regulates retained fetal membranes of cattle by targeting STIM1.

Retained fetal membranes (RFM) of cows is an important reproductive disturbance, and is related to miRNA-185. Stromal interaction molecule 1 (STIM1), a potential target gene of miRNA-185, could influence placenta release via regulating Ca2+ concentration intracellular. The aim of this study was to explore the mechanism of RFM by investigating the regulatory relationship between miRNA-185 and STIM1 in primary uterine caruncel epithelial (UCE) cells. Serum samples of healthy Holstein dairy cows (n = 20) and RFM cows (n = 12), with a similar age, parity, weight, and milk yield, were collected to detect Ca2+ concentration at prepartum 1-5 d and postpartum 6, 12 and 24 h. Caruncle tissues were collected from healthy (n = 6) and RFM cows (n = 6) at 12 h after calving. Quantitative polymerase chain reaction (Q-PCR) and western blotting (WB) were performed to detect the mRNA and protein levels of STIM1, respectively. UCE cells were cultured by the explant culture method, further purified, and subsequently treated with PmirGLO-STIM1-Mut + miRNA-185 mimics and mirGLO-STIM1-Mut + miRNA-185 NC. Q-PCR and WB were performed to detect mRNA and protein levels of STIM1 with treated miRNA-185 mimics. Serum levels of Ca2+ from RFM cows were abnormally decreased at prepartum 1 d and postpartum 6, 12 and 24 h. Expression level of STIM1 was down-regulated in the caruncle tissue of RFM cows. The luciferase activity was decresed about 30.9% by miRNA-185 mimics (p < 0.01), and the mRNA and protein levels of STIM1 were downregulated miRNA-185-mimics. It was suggesting that miRNA-185 might play an important role in RFM through regulating the expression of STIM1.

miRNA-185 regulates the VEGFA signaling pathway in dairy cows with retained fetal membranes.

Retention of fetal membranes (RFM) of cows is an important reproductive disturbance, and is related to miRNAs. Vascular endothelial growth factor (VEGF)A, regulated by miRNA-185, can activate arachidonic acid (ARA) release via the VEGFA signaling pathway, which influences RFM. The aim of this study was to explore the pathogenic mechanism of RFM by investigating the regulatory relationship between miRNA-185 and the VEGFA signaling pathway. Serum samples of healthy Holstein dairy cows (n = 20) and RFM cows (n = 12), with a similar age, parity, weight, and milk yield, were collected to detect VEGFA and ARA concentrations at 6, 12, and 24 h after calving. Caruncle tissues were collected from healthy (n = 6) and RFM cows (n = 6) at 12 h after calving. Quantitative polymerase chain reaction (qPCR) and western blotting (WB) were performed to detect the mRNA and proteins levels, respectively, of genes involved in the VEGFA signaling pathway. Uterine caruncle epithelial (UCE) cells were cultured by the explant culture method, further purified, and subsequently treated with miRNA-185 mimics, miRNA-185 mimics + MEK inhibitor, or left untreated as a control for detection of the mRNA and protein levels of genes involved in the VEGFA signaling pathway. The cellular supernatant was collected for measurement of ARA levels at 12, 24 and 48 h after treatment. Serum levels of VEGFA and ARA from RFM cows were abnormally increased at 12 h after calving, as compared to those in healthy dairy cows. Expression levels of most of the investigated genes (VEGFA, PLC, PRK, RAF, MEK, MAPK, and PLA) were down-regulated in the caruncle tissue of RFM cows. However, P-p44/42 MAPK was up-regulated in the caruncle tissues of cows with RFM (p < .01). In UCE cells treated with the miRNA-185 mimics, expression of VEGFA, PLC, RAF, MEK, MAPK and PLA was significantly down-regulated, while that of P-p44/42 MAPK was significantly up-regulated. Expression of genes involved in the VEGFA signaling pathway was similar to that in the in vivo assay. In UCE cells treated with the miRNA-185 mimics + MEK inhibitors, expression of VEGFA, PLC, RAF, MEK, MAPK and P-p44/42 MAPK was significantly down-regulated, while that of PLA was significantly up-regulated. Meanwhile, the release of ARA was increased (p < .01). These results demonstrate that miRNA-185 can regulate the VEGFA signaling pathway, especially via abnormal expression of P-p44/42 MAPK, which influences the release of the fetal placenta after calving.

Alterations of fatty acid ß-oxidation capability in the liver of ketotic cows.

Dairy cows are highly susceptible to ketosis after parturition. In the present study, we evaluated the expression of fatty acid ß-oxidation-related enzymes in the liver of ketotic (n=6) and nonketotic (n=6) cows. Serum levels of nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), and glucose were determined by using standard biochemical techniques. The mRNA abundance and protein content of acyl-CoA synthetase long-chain (ACSL), carnitine palmitoyltransferase I (CPT I), carnitine palmitoyltransferase II (CPT II), acyl-CoA dehydrogenase long chain (ACADL), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), and acetyl-CoA carboxylase (ACC) were evaluated by real-time PCR and ELISA. We found that serum glucose levels were lower in ketotic cows than in nonketotic cows, but serum BHBA and NEFA concentrations were higher. Messenger RNA and protein levels of ACSL were significantly higher in livers of ketotic cows than those in nonketotic cows. In contrast, mRNA levels of CPT I and mRNA and protein levels of CPT II, ACADL, HMGCS, and ACC were decreased in the liver of ketotic cows. Serum NEFA concentration positively correlated with ACSL protein levels and negatively correlated with protein levels of CPT II, HMGCS, ACADL, and ACC. In addition, serum BHBA concentration negatively correlated with protein levels of CPT II, HMGCS, and ACADL. Overall, fatty acid ß-oxidation capability was altered in the liver of ketotic compared with nonketotic cows. Furthermore, high serum NEFA and BHBA concentrations play key roles in affecting pathways of fatty acid metabolism in the liver.

Characteristics of different phenotypes of polycystic ovary syndrome based on the Rotterdam criteria in a large-scale Chinese population.

OBJECTIVE: To analyse the phenotypic spectrum of polycystic ovary syndrome (PCOS) and determine the association between metabolic, hormonal and new ultrasonographic criteria. DESIGN: Clinical cross-sectional study. SETTING: University teaching hospital. POPULATION: A total of 804 Chinese women, among whom 719 cases were diagnosed as PCOS based on the 2003 Rotterdam criteria. Eighty-five women with regular menstrual cycles and without hyperandrogenism were recruited as controls. METHODS: PCOS patients were divided into four subgroups: (i) oligo- and/or anovulation (O), hyperandrogenism (H), and polycystic ovary morphology (P); (ii) O + H; (iii) H + P; and (iv) O + P. MAIN OUTCOME MEASUREMENTS: Clinical history, ultrasonographic (ovarian follicle number and volume), hormonal and metabolic parameters. RESULTS: The composition of the two new phenotypes created by the European Society for Human Reproduction and Embryology/The American Society for Reproductive Medicine (ESHRE/ASRM) 2003 was 65.6% (O + P and H + P). BMI and F-G scores were highest in the O + H + P group and lowest in O + P and controls. Serum testosterone concentrations and insulin resistance were highest in cases with O + H + P and O + H, intermediate in cases with H + P, and lowest in cases with O + P and controls. The prevalence of metabolic syndrome in the five groups was 28.5% (O + H + P), 25.5% (O + H), 8.3% (H + P), 7.2% (O + P) and 3.5% (controls), respectively. CONCLUSIONS: Nonclassic phenotypes for PCOS (O + P, H + P and O + H + P) were more frequent than the classic phenotype (O + H). The nonhyperandrogenic PCOS phenotype (O + P), one of the new phenotypes created by the Rotterdam criteria, may represent a form of PCOS associated with milder metabolic profile compared with the other phenotypes.

[Clinical analysis of 318 cases of new-mode cesarean section].

OBJECTIVES: To study types of new mode cesarean section (CS) and its clinical value. METHODS: Four hundred and sixty-eight pregnant women with operative indications were randomly divided into three groups: the new mode CS group whose peritoneum was not sewed, the new mode CS group whose peritoneum was sewed, and the lower segmental CS group. The duration of operation, bleeding volume, morbidity after operation, and B type ultrasound for examining the incision were observed. RESULTS: There were significant difference in the duration of operation, bleeding volume, and morbidity after operation between the lower segmental CS and the two types of the new mode CS (P < 0.05). There was no significant differences in the bleeding volume and morbidity after operation between the two types of the new mode CS (P > 0.05), but there were significant difference in the duration of operation and the incision examined by B type ultrasound (P < 0.05). CONCLUSION: The new mode CS has shorter operative period, less bleeding, weak pain, faster recovery of peristalsis, and lower incidence of post-operative morbidity.

Urethane-induced hyperglycemia.

AIM: To study the effects of urethane, at anesthetic dose, on the blood glucose levels in normal rats and hyperglycemic rats, and its effects on the hypoglycemic action of exogenous insulin in alloxan-treated rats. METHODS: Blood glucose concentration was measured with the glucose oxidase method. RESULTS: Urethane at anesthetic dose 1.5 g.kg-1 increased the blood glucose levels in fasting (to 2.6 +/- 0.3 g.L-1, P < 0.01) or glucose-loaded (to 3.9 +/- 0.4 g.L-1, P < 0.01) rats. It did not modify the hyperglycemia induced by epinephrine (normal islet beta-cells) or alloxan (impaired islet beta-cells). In the rats treated with alloxan, blood glucose level decreased to 1.8 +/- 0.7 g.L-1 at 200 min after administration of insulin from control level of 7.0 +/- 2.3 g.L-1, but the hypoglycemic action of exogenous insulin was abolished by urethane. CONCLUSION: Hyperglycemic action of urethane was due to its inhibiting effect on the hypoglycemic effect of insulin, except for its known mechanism of increased sympathetic release.

Selective effects of alfuzosin and doxazosin with intraduodenal administration on urethral pressure of cats.

AIM: To observe the selective effects of alfuzosin (Alf) and doxazosin (Dox) on the urethral pressure by different administration routes. METHODS: The urethral pressure of the anesthetized cat was increased by electric stimulation of the hypogastric nerve. The different effects of Alf or Dox on the arterial blood pressure and urethral pressure between intraduodenal administration (i.d.) and intravenous infusion (i.v.) were compared. RESULTS: When the hypogastric nerve was stimulated by electric stimulation (10 Hz, 25 V), the ratios of ED20(BP)/ED50(UP) i.d. to ED20(BP)/ED50(UP) i.v. were 10.9:4.3 for Alf, and 3.1:2.1 for Dox. The reduction in urethral pressure induced by i.d. Alf was greater than that by i.v. Alf. Dox did not show any difference in its effects by 2 administration routes. CONCLUSION: Intraduodenal administration of Alf, but not Dox, selectively decreased the urethral pressure elevated by electric stimulation. The uroselectivity of i.d. Alf was not due to the species difference in its bioavailability and biotransformation.

Effects of 3-morpholinosydnonimine-N-ethylcarbamide on hypoxia-induced mechanical and electric responses of isolated pig coronary artery.

AIM: To study effects of 3-morpholinosydnonimine-N -ethylcarbamide (SIN-1) on hypoxia-induced mechanical and electric activities of the isolated pig coronary artery. METHODS: Mechanical tension and membrane potential were measured simultaneously. RESULTS: Hypoxia initially caused a transient vascular smooth muscle cell membrane hyperpolarization followed by a membrane depolarization in isolated pig coronary artery. Subsequent addition of SIN-1 100 mumol . L-1 or verapamil (Ver) 10 mumol . L-1 led to membrane repolarization and relaxation of the vascular smooth muscle. Nitro-L-arginine (NLA) 0.2 mmol . L-1 and KCI 40 mmol . L-1 also induced membrane depolarization and vasoconstriction, which were similarly suppressed by SIN-1 or Ver. CONCLUSION: Hypoxic contractile response in isolated pig coronary artery is mediated by an increased Ca2+ influx via suppression of nitric oxide release.

[Effects of m-nisoldipine on arterial baroreflex sensitivity in anesthetized rats].

Baroreflex sensitivity (BRS) in anesthetized rats was measured as the slope of the regression line of the concomitant maximal change in heart rate (delta R-R) and blood pressure (delta BP) induced by bolus i.v. phenylephrine or sodium nitroprusside, m-nisoldipine (m-Nis) i.v. 5, 10, 20 micrograms.kg-1 depressed the BRS (2.6 +/- 0.2, 2.4 +/- 0.3, 1.6 +/- 0.2, or 1.7 +/- 0.1, 1.6 +/- 0.2, and 1.4 +/- 0.2 ms.kPa-1, respectively) compared with before medication (P < 0.01). m-Nis may act directly on carotid sinus to depress the BRS, but not through cardiac beta or M receptor nor through central nervous system.

Effects of inhibitor of endothelium-derived relaxing factor on hypoxic contraction of isolated pig coronary artery.

Exposure of isolated pig coronary artery with endothelium intact to hypoxia Krebs-Henseleit solution aerated with 95% N2 + 5% CO2 caused a transient contractile response, and the coronary artery without endothelium exhibited a gradual decrease in basal tension. The endothelium-dependent contractile response to hypoxia was almost completely blocked by nitro-L-arginine (0.2 mmol.L-1), and inhibited by methylthioninium chloride (10 mumol.L-1). The inhibitory effect of the NLA was partially reversed by L-arginine (2 mmol.L-1). Sodium nitroprusside (10 mumol.L-1) was also completely antagonized and nicorandil (0.3 mol.L-1) remarkably reduced the hypoxic contractile response. Tetraethylammonium (10 mmol.L-1) and glibenclamide (1 mumol.L-1) had little effect on hypoxia-induced vascular contraction, whereas cromakalim (1 mumol.L-1) produced obvious relaxing effect on hypoxic response. These results suggest that suppression of basally released nitric oxide (NO) is an important mechanism of coronary vasoconstriction induced by hypoxia.

Effects of m-nisoldipine on contraction and 45Ca influx in isolated rat aorta.

m-Nisoldipine caused a concentration-dependent depression of the contractile response and 45Ca influx evoked by KCl in isolated rat thoracic aorta. The IC50 value for contraction and 45Ca influx were 0.69 (95% confidence limits 0.32-1.5) nmol.L-1 and 0.35 (95% confidence limits 0.06-2.0) nmol.L-1, respectively. There was a positive correlation between the inhibition of KCl-evoked contraction and 45Ca influx (r = 0.996). m-Nisoldipine (0.1-10 mumol.L-1) did not influence the 45Ca influx into resting cells and failed to inhibit noradrenaline (1.0 mumol.L-1)-evoked contraction and 45Ca influx. The results suggest that the relaxant effect of m-nisoldipine on rat aorta may be closely related to the blockade of Ca2+ entry through a potential-dependent calcium channel.

Effects of hydrochlorothiazide on contraction and 86Rb efflux in rat aorta.

Hydrochlorothiazide (HCT) (0.1, 0.3 mmol.L-1) inhibited the contraction of rat aortic strips induced by low (< 40 mmol.L-1), not higher concentrations of KCl. HCT (0.3 mmol.L-1) did not inhibit the CaCl2-induced contraction of the aortic strips depolarized with high K+ (KCl 80 mmol.L-1). The inhibitory effect of HCT (0.1 mmol.L-1) on KCl (20 mmol.L-1)-induced contraction was markedly antagonized by BaCl2 (0.1 mmol.L-1) and tetraethylammonium (TEA) (0.3 mmol.L-1), but not by glibenclamide (Gli, 0.01 mmol.L-1). With norepinephrine (NE) or 5-HT as agonists, HCT (0.3 mmol.L-1) also inhibited the contractions of rat aortic strips. In the 2 components of NE-induced contraction, HCT inhibited only the tonic component depending on Ca2+ influx, but not the phasic component elicited by the release of intracellular Ca2+. The inhibitory action of HCT was endothelium-independent. That the HCT (3 mmol.L-1) increased the 86Rb efflux rate coefficient was antagonized by BaCl2 (0.1 mmol.L-1), but not by Gli (0.01 mmol.L-1). The results indicated that the inhibitory effect of HCT on the contraction of rat aorta was attributable to the opening of membrane potassium channels.

Antihypertensive effects of m-nisoldipine and nisoldipine on conscious renal hypertensive rats and dogs.

The antihypertensive effects of m-nisoldipine (m-Nis) and nisoldipine (Nis) by ig 0.3, 1.0, 3.0 and 1.0, 3.0, 9.0 mg.kg-1 respectively on renal hypertensive rats (RHR) and 0.1, 0.3, 1.0 mg.kg-1 orally (for both drugs) on renal hypertensive dogs (RHD) were studied. Both m-Nis and Nis depressed blood pressure (BP) dose-dependently in RHR and RHD. The reduction of blood pressure correlated well with the m-Nis concentration in plasma of RHD. On the basis of ED20 (HR)/ED20 (BP), the hypotensive effect of m-Nis on systolic blood pressure (SBP) was only 1.6 times as great as that of Nis on RHR (P less than 0.05), but in RHD, both drugs showed the same potency (P greater than 0.05). In both models, m-Nis showed much more potent effect on diastolic blood pressure (DBP) than Nis (P less than 0.01), and possessed stronger hypotensive effects on DBP than on SBP (P less than 0.05 and P less than 0.01); but for Nis, its effects on SBP and DBP appeared to be in the same order (P greater than 0.05). The fall in BP was accompanied by a transient increase of heart rates (HR) with m-Nis and Nis in RHR and RHD. The chronic antihypertensive effects of m-Nis and Nis were also remarkable with 1.0 mg.kg-1 daily at 9 AM for 21 d. During this period, the BP and HR lowered to nearly normal level. After withdrawal of m-Nis and Nis, the hypotensive effects lasted nearly 1 wk.

[Effects of m-nisoldipine and nisoldipine on isolated uterine muscle].

m-Nisoldipine (m-Nis) and nisoldipine (Nis) inhibited spontaneous contraction of isolated non-pregnant human myometrium, IC50 being 3.7 +/- 0.7 mumol.L-1 for m-Nis and 6.0 +/- 0.9 mumol.L-1 for Nis. Both drugs also suppressed spontaneous and oxytocin-induced contraction of isolated pregnant rat uterus. In isolated uterus from virgin rat, both drugs inhibited the contractions induced by KCl, acetylcholine (ACh), and oxytocin (Oxy), the values of pD'2 against KCl, ACh, Oxy being 9.0 +/- 0.2, 6.1 +/- 0.1, and 5.9 +/- 0.1 for m-Nis and 9.1 +/- 0.3, 5.7 +/- 0.2, and 5.6 +/- 0.1 for Nis respectively. The results indicate that both m-Nis and Nis may be clinically useful for the treatment of dysmenorrhea and premature labour.

Effects of glibenclamide and tolbutamide on ischemia- and ouabain-induced arrhythmias and membrane potentials of ventricular myocardium from rat and guinea pig.

Glibenclamide (Gli) 0.3, 1, 3 mg.kg-1 and tolbutamide (Tol) 3, 10, 30 mg.kg-1 iv 10 min before ischemia or ouabain infusion prevented ventricular fibrillation induced by ischemia in rat and arrhythmias induced by ouabain in guinea pig. Gli 10 mumol.L-1 and Tol 1 mmol.L-1 increased APD and ERP in rat ventricular muscle. Gli 0.1, 1, 10 mumol.L-1 and Tol 0.01, 0.1, 1 mmol.L-1 prevented and reversed the shortening of APD and ERP induced by hypoxia in guinea pig ventricular muscle. These effects of Gli and Tol were dose-dependent. The results confirmed that Gli and Tol were effective on arrhythmias induced by ischemia and ouabain by blocking ATP-sensitive potassium channel.

Effect of ubiquinone on ischemic arrhythmia in conscious rats.

Ubiquinone 6.2, 12.5 or 2.5 mg.kg-1 respectively twice iv 24 h and 30 min before coronary artery ligation, ameliorated the ischemic arrhythmia in conscious rats, and there was a close positive correlation between the ubiquinone concentration in myocardium and plasma and its anti-arrhythmic effect. Ubiquinone iv 3.1, 6.2, and 12.5 mg.kg-1 increased, while 25 mg.kg-1 decreased 6-keto-PGF1 alpha, and 12.5 and 25 mg.kg-1 decreased TXB2, which was in accordance with inhibitory effects on the synthesis of 6-keto-PGF1 alpha and TXB2 in vitro. But the ratio of metabolites of PGI2/TXA2 in vivo was increased in all ubiquinone groups. These results indicated that ubiquinone possesses protective effects on ischemic arrhythmia of conscious rats and the beneficial effects on myocardial ubiquinone content and PGI2/TXA2 seem to contribute to its myocardial protective action.

Pharmacokinetics of m-nisoldipine in rabbits and rats.

A reverse phase HPLC method was devised for determination of m-Nis in plasma. A mobile phase of methanol-KH2PO4 with a flow rate of 1 ml/min was used. Diazepam was used as the internal standard. A two-compartment model featured the pharmacokinetic process of m-Nis after its iv injection to rats (30 micrograms/kg) and rabbits (50 micrograms/kg). The pharmacokinetic parameters were: T 1/2 alpha = 4.3 min, T 1/2 beta = 63.6 min, Vd = 0.805 L/kg, Cl = 9 ml/(min.kg) in rats; T 1/2 alpha = 5.0 min, T 1/2 beta = 78.3 min, Vd = 1.191 L/kg, Cl = 11 ml/(min.kg) in rabbits. The pharmacokinetics for m-Nis after ig 200 micrograms/kg to rats described one-compartment model with parameters: T 1/2 = 84.8 min, Tmax = 31.2 min, Cmax = 49.97 micrograms/L, Vd = 0.792 L/kg and Cl = 25 ml/(min.kg).

Effects of m-nisoldipine on anoxia-potentiated histamine and acetylcholine-induced contractions of the porcine isolated coronary artery.

Anoxia (95% N2 + 5% CO2) potentiated the contractile response to KCl 20 mmol/L, histamine (His) 5 mumol/L and acetylcholine (ACh) 0.5 mumol/L in isolated porcine coronary arterial rings. Calcium antagonists m-nisoldipine (m-Nis) and nisoldipine (Nis) 0.4-250 nmol/L produced a concentration-dependent decrease in both KCl, His and anoxia-potentiated KCl2 His or ACh-induced contractions. Chlorpheniramine 10 mumol/L but not cimetidine 10 mumol/L and atropine 10 mumol/L abolished contractions induced by His and ACh respectively. All 3 agents did not affect KCl response and the anoxia facilitation. Indomethacin 10 mumol/L markedly attenuated the further increase in tension by anoxia but failed to inhibit the response by these vasoconstrictors.

[Histamine and early ischemic arrhythmia in anesthetized cats].

Ligation of left anterior descending coronary artery caused various arrhythmias and reduced histamine content in ischemic myocardium in anesthetized cats. Intracoronary injection of compound 48-80 100 micrograms shortened the onset of VT and VF from 13 +/- 5, 18 +/- 5 min (n = 7) in control to 7.2 +/- 1.1 (P less than 0.01), 11 +/- 5 min (P less than 0.05) (n = 6) respectively, elevated the histamine concentration of plasma after acute coronary artery occlusion (15 +/- 3, 26 +/- 10 ng/ml, P less than 0.05, before and after ligation respectively). Iv chlorpheniramine (5, 10 mg/kg) or cimetidine (20, 40 mg/kg) dose-dependently reduced arrhythmia score, incidence of VF and mortality after myocardial ischemia, but with little influence on histamine content in ischemic myocardium and plasma. These results suggest that release of histamine from the ischemic myocardium is involved in the generation of early arrhythmias through H1 and H2 receptors in anesthetized cats subjected to acute coronary artery occlusion.

Acute toxicity of dipfluzine and its effects on isolated vascular smooth muscle.

Dipfluzine (Dip) is a new derivative of cinnarizine (Cin) first developed by Department of Chemistry, Beijing University. Dip showed a dose-dependently inhibitory effect on both KCl- and NE-induced contraction in the rabbit aortic rings. It was more effective in suppressing the contractile response evoked by KCl than that by NE. Dip also inhibited the KCl-induced contraction in porcine basilar, coronary and radial arteries. Their pD2' values were 5.7 +/- 0.6, 5.4 +/- 0.4 and 4.6 +/- 0.5 respectively. The selectivity of Dip for vasodilation was proved by higher pD2' value of the basilar artery than that of the coronary and radial arteries, and this selectivity of Dip was more significant than that of Cin. The acute iv LD50 of Dip and Cin in mice were 37 and 36 mg/kg, respectively.