MicroRNA-323-3p inhibits oxidative stress and apoptosis after myocardial infarction by targeting TGF-ß2/JNK pathway.

OBJECTIVE: Myocardial infarction (MI), which causes irreversible damage and loss of cardiomyocytes, is the most important cause of death in the world. MicroRNA is an important regulator of physiological and pathological activities of cardiovascular system. The aim of this research was to study the effect of microRNA-323-3p (miR-323-3p) on MI and its underlying mechanisms of action. MATERIALS AND METHODS: A rat model of MI was established to measure the expression of miR-323-3p, Bax, Bcl-2, SOD1, and SOD2 in ischemic myocardial tissue, and the cardiac function of rats were tested at seventh day after MI. H9c2 cells were divided into control group, miRNA negative control (NC) transfection group, miR-323-3p mimic (miR-323-3p min) transfection group, and then, treated with H2O2. Oxidative stress and apoptosis of H9c2 cells were observed by Western blot, Real Time-Polymerase Chain Reaction (RT-PCR), flow cytometry, SOD activity assay, TUNEL staining, DHR dye assay, etc. RESULTS: The level of miR-323-3p was decreased in ischemic myocardium, as well as H2O2-treated H9c2 cells. MiR-323-3p overexpression greatly decreased the level of Bax and increased the levels of SOD1, SOD2, and Bcl-2. After treated with miR-323-3p mimic, TUNEL positive cells were greatly reduced, and apoptosis rate of H9c2 cells was greatly decreased. Moreover, SOD levels significantly increased, while ROS production decreased after treatment of miR-323-3p. After intravenous injection of miR-323-3p agomir in rats with MI, the cardiac function of the rats was significantly improved. Western blot and Luciferase reporter gene experiments illustrated that miR-323-3p acts by targeting TGF-ß2. CONCLUSIONS: MiR-323-3p was downregulated in ischemic myocardium and H2O2-treated H9c2 cells, and miR-323-3p overexpression reduced oxidative stress and apoptosis of cardiomyocytes. The protective function was achieved via regulation of TGF-ß2/JNK pathway.

MiR-126 regulated myocardial autophagy on myocardial infarction.

OBJECTIVE: The aim of the study is to detect the effect of miR-126 in autophagy after acute myocardial infarction (AMI). MATERIALS AND METHODS: First, the changes of miR-126 and autophagy in myocardial infarction tissue and normal heart tissue were compared by establishing the myocardial infarction rat model, so as to clarify the relationship between miR-126 and changes of myocardial infarction and autophagy. Second, the regulation of miR-126 on autophagy related protein Beclin-1 and the role in myocardial infarction were studied to clarify whether miR-126 regulates autophagy through Beclin-1 and participates in the occurrence and development of myocardial infarction. Finally, the relationship between plasma miR-126 was observed. RESULTS: MiR-126 expression can regulate Beclin-1 expression and influence the cardiac function after AMI. The results are of great significance to reveal the new mechanism of myocardial infarction. CONCLUSIONS: The down-regulation of miR-126 will lead to the over-activation of myocardial autophagy induced by Beclin-1, which is an autophagy related protein. The plasm expression of miR-126 may be a clinical marker of autophagy after AMI.

[Gas chromatography-mass spectrometry based urinary metabolomics in very low birth weight premature infants].

Objective: To investigate the urinary metabolic spectrum and pathways in very low birth weight (VLBW) premature infants. Method: A prospective case-control study was conducted to collect and compare the data of VLBW premature infants and full term infants from the Sixth Affiliated Hospital of Sun Yet-Sen University in 2014. Within 24 hours after birth, urine specimens in each group were collected. Metabolites of urine samples including amino acid, fatty acid and organic acid were detected using the urease pre-processing and gas chromatography mass spectrometry (GC-MS) technology. Using the orthogonal partial least squares discriminant analysis (OPLS-DA), the biomarkers and differences between the two groups were found. The online metabolic pathway website was explored and multivariable analysis was conducted to investigate the valuable pathways and biomarkers related to the prematurity. Result: A total of 20 VLBW premature infants were enrolled, among whom 11 were male, 9 were female; and 20 full term infants were enrolled, among whom 9 were male, 11 were female. The urinary metabolites were established and compared between the VLBW premature and term infants. The investigation showed that the following nine pathways were enriched: amino-acyl-tRNA biosynthesis(P=0.000), lysine degradation(P=0.007), fatty acid biosynthesis(P=0.008), pyrimidine metabolism(P=0.014), pantothenate and CoA biosynthesis(P=0.022), valine, leucine and isoleucine biosynthesis(P=0.022), lysine biosynthesis(P=0.031), glycerolipid metabolism(P=0.046), and valine, leucine and isoleucine degradation(P=0.031). Almost all the metabolites decreased except for the glyceric acid exhibiting a higher content in the VLBW premature infant. 12 potential biomarkers were explored with the most significant covariance and correlation, within which stearic acid, palmiticacid, myristic acid, ß-amino-isobutyric acid, and uric acid were lower, while myo-inositol, mannitol, glycine, glucose1, glucose2, glyceric acid and N-acetyl-tyrosine were higher in the VLBW premature group compared with the control group. Conclusion: There is a significant difference between the VLBW premature infants and full-term infants in the metabolic state and pathways. The urease pre-processing and GC-MS technology followed by the OPLS-DA and multivariable analysis to investigate VLBW premature infants' urinary metabolites is a valuable method to evaluate the patients' metabolism.

Hepatitis B virus suppresses the functional interaction between natural killer cells and plasmacytoid dendritic cells.

Natural killer cells (NK) are one of the key players in the eradication and control of viral infections. Infections with the hepatitis B virus (HBV) may lead to persistence in a subgroup of patients, and impaired NK cell functions have been observed in these patients. Crosstalk with other immune cells has been shown to modulate the function of NK cells. We studied the functional crosstalk between NK cells and plasmacytoid dendritic cell (pDC) and its modulation by HBV. Healthy human peripheral blood-derived NK cells and pDC were purified and cocultured in the presence or absence of HepG2.2.15-derived HBV under various in vitro conditions. The functionality of NK cells was assessed by evaluation of activation markers, cytokine production and cytotoxicity of carboxyfluorescein succinimidyl ester-labelled K562 target cells by flow cytometry or immunoassays. Additionally, the crosstalk was examined using NK and pDC from patients with chronic HBV. The activation of NK cells in cocultures with pDC, as demonstrated by CD69, CD25 and HLA-DR, was not affected by the presence of HBV. Similarly, when cocultured with pDC, the cytotoxic potential of NK cells was not influenced by HBV. However, HBV significantly inhibited pDC-induced IFN-γ production by NK cells both in the presence and in the absence of CpG. As HBV did not affect cytokine-induced IFN-γ production by NK cells cultured alone, the suppressive effect of HBV on NK cell function was mediated via interference with pDC-NK cell interaction. In contrast to other viruses, HBV does not activate pDC-NK cell interaction but inhibits pDC-induced NK cell function. In parallel with NK cells of patients with chronic HBV, which show diminished cytokine production with normal cytotoxicity, HBV specifically suppressed pDC-induced IFN-γ production by NK cells without affecting their cytolytic ability. These data demonstrate that HBV modulates pDC-NK cell crosstalk, which may contribute to HBV persistence.

Comparative study on the vasorelaxant effects of three harmala alkaloids in vitro.

Three psychological active principles from the seeds of Peganum harmala L., harmine, harmaline and harmalol, showed vasorelaxant activities in isolated rat thoracic aorta preparations precontracted by phenylephrine or KCl with rank order of relaxation potency of harmine > harmaline > harmalol. The vasorelaxant effects of harmine and harmaline (but not harmalol) were attenuated by endothelium removal or pretreatment with a nitric oxide (NO) synthase Nomega-nitro-L-arginine methyl ester. In cultured rat aortic endothelial cells, harmine and harmaline (but not harmalol) increased NO release, which was dependent on the presence of external Ca2+. In endothelium-denuded preparations, pretreatment of harmine, harmaline or harmalol (3-30 microM) inhibited phenylephrine-induced contractions in a non-competitive manner. Receptor binding assays indicated that all 3 compounds interacted with cardiac alpha1-adrenoceptors with comparable affinities (Ki value around 31 - 36 microM), but only harmine weakly interacted with the cardiac 1,4-dihydropyridine binding site of L-type Ca2+ channels (Ki value of 408 microM). Therefore, the present results suggested that the vasorelaxant effects of harmine and harmaline are attributed to their actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca2+ channels. The vasorelaxant effect of harmalol was not endothelium-dependent.

Spasmolytic effects of three harmala alkaloids on guinea-pig isolated trachea.

The present study examined and compared the spasmolytic effects of 3 harmala alkaloids, harmine, harman, and harmaline, on carbachol-, histamine-, and KCl-induced contractions of guinea-pig isolated tracheal preparations. All 3 compounds relaxed the tracheal preparations contracted by these spasmogens with similar or different EC50 values, harmine being the most potent one. The cumulative concentration-response curves of all 3 compounds for carbachol-induced contraction were shifted to the right by propranolol (1 microM) pretreatment, indicating the involvement of the activation on the beta-adrenoceptors. All 3 compounds shifted the concentration-response curves of carbachol to the right in a parallel manner with the pA2 values comparable with their relaxation EC50 values, indicating a competitive antagonism at the muscarinic receptors. Receptor binding assays indicated that all 3 compounds interacted with lung muscarinic receptors (Ki = 11-13 microM), histamine H1 receptors (Ki = 27-107 microM), and beta2-adrenoceptors (Ki = 20-51 microM). Therefore, in addition to their actions on receptor-linked and voltage-dependent Ca2+ channels as reported in other types of smooth muscle, the present study suggests that the actions on muscarinic receptors, histamine H1 receptors, and beta2-adrenoceptors are also involved in their spasmolytic effects on airway smooth muscles.

Vasorelaxant effect of harman.

The in vivo cardiovascular effect and in vitro vasorelaxant effect of harman, one of harmala alkaloids isolated from Peganum harmala, were examined in this study. Harman (1-10 mg/kg, i.v.) dose-dependently produced transient hypotension and long-lasting bradycardia in pentobarbital-anesthetized rats, which were attenuated by N(G)-nitro-L-arginine pretreatment. In isolated rat endothelium-intact thoracic aortic rings, harman concentration dependently relaxed phenylepherine-induced contractions with an IC(50) value around 9 microM. This vasorelaxant effect was attenuated by endothelium removal or N(omega)-nitro-L-arginine methyl ester pretreatment, but not by tetraethylammonium or indomethacin pretreatment. In cultured rat aortic endothelial cells, harman (1-100 microM) concentration dependently increased nitric oxide (NO) release, which was dependent on the presence of external Ca(2+). Harman pretreatment (3-30 microM) also concentration dependently inhibited the contractions induced by phenylephrine, 5-hydroxytryptamine (5-HT), and KCl in endothelium-denuded aortic rings in a non-competitive manner. In addition, harman pretreatment reduced both phasic and tonic phases of phenylephrine-induced contractions. Receptor binding assays further indicated that harman (K(i) values around 5-141 microM) interacted with the cardiac alpha(1)-adrenoceptors, brain 5-HT(2) receptors, and cardiac 1, 4-dihydropyridine binding site of L-type Ca(2+) channels. Therefore, the present results suggested that the vasorelaxant effect of harman was attributed to its actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca(2+) channels. The vasorelaxant effect may be involved in the hypotensive effect of harman.

Spasmolytic effect of water extract of Stemonae radix on the guinea-pig tracheal smooth muscle in vitro.

The present study examined the relaxing effect of a water extract of Baibu (Stemonae radix, the root tuber of Semona sessilifolia (Miq.) Franch. et Sav.) on carbachol-, histamine- and KCl-induced contractions of the guinea-pig isolated tracheal preparations. The results showed that Baibu (1-50 mg/ml) concentration-dependently relaxed the tracheal preparations contracted by these spasmogens with an IC50 value (mg/ml) of 2.0 +/- 0.1 for carbachol, 41.2 +/- 0.8 for histamine and 18.6 +/- 0.9 for KCl. The effect of Baibu was not affected by the pretreatment with a beta-adrenoceptor antagonist propranolol (10(-6) M), indicating that Baibu's effect was not due to an activation on beta-adrenoceptors. Baibu shifted the concentration-response curve of carbachol to the right in a parallel manner without changing the maximal response, having a pA2 value of 0.16 +/- 0.07 mg/ml (equivalent to a KB = 0.70 +/- 0.11 mg/ml). This indicates a competitive antagonism at the muscarinic receptors. Receptor binding assay indicated that Baibu interacted with the muscarinic receptors (Ki = 0.51 +/- 0.12 mg/ml) and the dihydropyridine (DHP) binding site of L-type Ca2+ channels (Ki = 8.0 +/- 1.9 mg/ml), but not with the histamine H1 receptors. Therefore, the present study demonstrates that Baibu contains the principle(s) acting on the muscarinic receptors and DHP binding sites, which contribute its relaxation effect on the airway smooth muscles.

Decreased tubular reabsorption of zinc in rats with chronic uremia.

Effect of chronic uremia on tubular reabsorption of zinc was studied in rats with different zinc nutritional status. Using inulin clearance to represent glomerular filtration rate and plasma ultrafiltrate to imitate glomerular filtrate in vivo, the glomerular filtered load of zinc was estimated in each rat. The amount of tubular reabsorption of zinc equals the amount of zinc filtered minus the amount of zinc excreted into the urine. Proportion of filtered zinc excreted was estimated to be 2.8-8.7% in control rats and 17.3-29.8% in uremic rats induced by five-sixths nephrectomy. Similarly, proportion of filtered zinc reabsorption decreased significantly from 91.3-97.3% in control rats to 70.1-82.7% in uremic rats. Zinc deficiency increased fractional reabsorption of zinc in uremic rats but not in control rats. Fractional excretion of zinc correlated with both urine flow rate and urinary excretion of protein. The results suggest that uremia toxins may influence tubular reabsorption of zinc even in the presence of renal adaptation to zinc deficiency.