Secretoneurin suppresses cardiac hypertrophy through suppression of oxidant stress.

The neuropeptide secretoneurin (SN) plays protective roles in myocardial ischemia. In the present study, the effect of SN in cardiac hypertrophy was investigated. We observed that, in isoproterenol (ISO) treatment induced cardiac or cardiomyocytes hypertrophy, a marked increase in the expression of endogenous SN in mouse plasma, myocardium and primary-cultured cardiomyocytes occurs. In hypertrophic mice, the heart size, heart weight/body weight (HW/BW) ratio, cardiomyocyte size, and atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) expression were significantly higher than those in controls but were effectively suppressed by SN gene therapy. Similarly, the protective effects of SN were also observed in cultured cardiomyocytes following ISO treatment. SN significantly increased the activity of catalase and superoxide dismutase (SOD) in parallel with the decrease in reactive oxygen species levels in cardiomyocytes. We observed that SN evoked the activation of all of the AMPK, P38/MAPK and ERK/MAPK pathways in cardiomyocytes, but pretreatment with only AMPK inhibitor (compound C) and ERK1/2/MAPK inhibitor (PD98059) counteracted the protective effects of SN against cardiomyocyte hypertrophy and the suppressive effects of SN on oxidant stress in cardiomyocytes. These results indicated that endogenous SN is induced in hypertrophic cardiomyocytes, and may play a protective role in the pathogenesis of cardiac hypertrophy. These results suggest that exogenous SN supplementation protects the cardiac hypertrophy induced by ISO treatment through the activation of AMPK and ERK/MAPK pathways, thus upregulating antioxidants and suppressing oxidative stress.

An enhanced targeted identification strategy for the selective identification of flavonoid O-glycosides from Carthamus tinctorius by integrating offline two-dimensional liquid chromatography/linear ion-trap-Orbitrap mass spectrometry, high-resolution diagnostic product ions/neutral loss filtering and liquid chromatography-solid phase extraction-nuclear magnetic resonance.

Targeted identification of potentially bioactive molecules from herbal medicines is often stymied by the insufficient chromatographic separation, ubiquitous matrix interference, and pervasive isomerism. An enhanced targeted identification strategy is presented and validated by the selective identification of flavonoid O-glycosides (FOGs) from Carthamus tinctorius. It consists of four steps: (i) enhanced separation and detection by offline two-dimensional liquid chromatography/LTQ-Orbitrap MS (offline 2D-LC/LTQ-Orbitrap MS) using collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD); (ii) improved identification of the major aglycones by acid hydrolysis and LC-SPE-NMR; (iii) simplified spectral elucidation by high-resolution diagnostic product ions/neutral loss filtering; and (iv) more convincing structural identification by matching an in-house library. An offline 2D-LC system configuring an Acchrom XAmide column and a BEH Shield RP-18 UPLC® column enabled much better separation of the easily co-eluting components. Combined use of CID and HCD could produce complementary fragmentation information. The intensity ratios of the aglycone ion species ([Y0-H]-/Y0- and [Y0-2H]-/Y0-) in the HCD-MS2 spectra were found diagnostic for discriminating the aglycone subtypes and characterizing the glycosylation patterns. Five aglycone structures (kaempferol, 6-hydroxykaempferol, 6-methoxykaempferol, carthamidin, and isocarthamidin) were identified based on the 1H-NMR data recorded by LC-SPE-NMR. Of the 107 characterized flavonoids, 80 FOGs were first reported from C. tinctorius. Unknown aglycones, pentose, and novel acyl substituents were discovered. A new compound thereof was isolated and fully identified, which could partially validate the MS-oriented identification. This integral strategy can improve the potency, efficiency, and accuracy in the detection of new compounds from medicinal herbs and other natural sources.

Melatonin modulates the functions of porcine granulosa cells via its membrane receptor MT2 in vitro.

Melatonin (N-acetyl-5-methoxytryptamine) is documented as a hormone involved in the circadian regulation of physiological and neuroendocrine function in mammals. Herein, the effects of melatonin on the functions of porcine granulosa cells in vitro were investigated. Porcine granulosa cells were cultivated with variable concentrations of melatonin (0, 0.001, 0.01, 0.1, 1.0, and 10ng/mL) for 48h. Melatonin receptor agonist (IIK7) and antagonist (Luzindole, 4P-PDOT) were used to further examine the action of melatonin. The results showed optimum cell viability and colony-forming efficiency of porcine granulosa cells at 0.01ng/mL melatonin for 48-h incubation period. The percentage of apoptotic granulosa cells was significantly reduced by 0.01 and 0.1ng/mL melatonin within the 48-h incubation period as compared with the rest of the treatments. Estradiol biosynthesis was significantly stimulated by melatonin supplementation and suppressed for the progesterone secretion; the minimum ratio of progesterone to estradiol was 1.82 in 0.01ng/mL melatonin treatment after 48h of cultivation. Moreover, the expression of BCL-2, CYP17A1, CYP19A1, SOD1, and GPX4 were up-regulated by 0.01ng/mL melatonin or combined with IIK7, but decreased for the mRNA levels of BAX, P53, and CASPASE-3, as compared with control or groups treated with Luzindole or 4P-PDOT in the presence of melatonin. In conclusion, the study demonstrated that melatonin mediated proliferation, apoptosis, and steroidogenesis in porcine granulosa cells predominantly through the activation of melatonin receptor MT2 in vitro, which provided evidence of the beneficial role of melatonin as well as its functional mechanism in porcine granulosa cells in vitro.

Salidroside improves doxorubicin-induced cardiac dysfunction by suppression of excessive oxidative stress and cardiomyocyte apoptosis.

Doxorubicin (DOX) is a potent available antitumor drug; however, its clinical use is limited by the cardiotoxicity. Salidroside (SLD), with strong antioxidative and cytoprotective actions, is of particular interest in the development of antioxidative therapies for oxidative injury in cardiac diseases. Now, the protection and underlying mechanisms of SLD against DOX-induced cardiotoxicity are still unknown. In the present study, we revealed both antioxidative mechanism and Bcl2-dependent survival signaling involved in SLD's protection. We observed that DOX exposure induced mortality elevation, body weight loss, and cardiac dysfunction in mice, increased lactate dehydrogenase leakage and cardiomyocyte apoptosis, but decreased cell viability and size in cardiac tissues and cultured H9c2 cells, respectively, which were effectively antagonized by SLD supplement. We further observed that SLD significantly reduced the intercellular oxidative stress level, partly by inhibiting NOX1 expression and augmenting the expression and activities of the endogenous antioxidative enzymes, catalase, and manganese superoxide dismutase. In addition, SLD treatment upregulated the antiapoptotic Bcl2 and downregulated the proapoptotic Bax and inhibited a downstream pathway of Bcl2/Bax and caspase-3 activity. Our results indicated that SLD effectively protected the cardiomyocytes against DOX-induced cardiotoxicity by suppressing the excessive oxidative stress and activating a Bcl2-mediated survival signaling pathway.