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  • br Methods br Results br Discussion I R

    2020-10-28


    Methods
    Results
    Discussion I/R induces oxidative stress and increases the intracellular levels of ROS, resulting in tissue damage [31]. ROS is generated by mitochondrial electron transport chain, nicotinamide Z-YVAD-FMK dinucleotide phosphate (NADPH) oxidase complex, xanthine oxidase and NOS [32,33]. As vascular endothelium and cardiac myocytes are exposed to increased oxidant stress, NOS is converted to a ROS generator from a NO source due to “NOS uncoupling” [34]. The most important reason of NOS uncoupling is believed to be the lacking of the critical cofactor BH4 of NOS, and many reports have been published to preserve eNOS function and ameliorate heart dysfunctions by BH4 supplementation [13,35,36]. Nevertheless, BH4 is highly redox-sensitive and easy to be oxidized by one or two electron reactions to generate BH3+ radical and BH2 [37] that lack eNOS cofactor activity. It is therefore suggested that oxidative stress leads to excessive oxidation and depletion of BH4. Thus exogenous BH4 supplementation is frequently poor effective in vivo. In order to increase its activity and stability, in this study we coated BH4 with a novel encapsulation of Gum Arabic for potential application in various industries. Our results demonstrated that oral GA-coated 6R-BH4 supplementation prior to myocardial ischemia resulted in a significant enhancement in myocardial BH4 levels, compared with inefficient uncoated 6R-BH4 treatment (Fig. 4B and C). Thus, the GA-encapsulation process helps to stabilize exogenous BH4 by preventing it from being oxidized by oxidative stress during I/R. eNOS proteins are all homodimers. A functional eNOS transfers electrons from NADPH, via flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in the carboxy-terminal reductase domain, to the heme in the amino-terminal oxygenase domain, where the substrate l-arginine is oxidized to l-citrulline and NO with the essential cofactor BH4 binding [38]. As a consequence of BH4 depletion due to oxidative stress, uncoupling eNOS is converted from NO formation into a dysfunctional generating ·O2− enzyme. As shown in Fig. 5, Fig. 6, we observed ~75% loss of eNOS activity after 60 min of ischemia and even more (83%) dysfunction during early 5 min of reperfusion; subsequently, it began to recover to 50% of basal levels after 10 min of reperfusion. These results demonstrated that eNOS activity well-correlated with the myocardial BH4 levels (Fig. 4A) where a significant loss of BH4 was seen after ischemia and early reperfusion. Contributing to the increase of BH4 levels in myocardium with pre-ischemic oral GA-BH4 supplementation, eNOS activity, activated phosphorylated eNOS levels and its dimerization were well preserved during myocardial I/R. In the uncoupled state, electrons normally flowing from the reductase domain of eNOS to the oxygenase domain are diverted to molecular oxygen rather than to l-arginine; in these conditions, ·O2− rather than NO is produced [34], which indicates eNOS dysfunction. In this study, we confirmed cardiac eNOS dysfunction and ROS generation by the fluorescent indicators DCFH and DAR, respectively [28]. Intracellular NO production level can be detected by the fluorescence probe of DAR, characterized by higher photostability, longer excitation wavelength and a wide pH range. Hence, DAR compounds are useful for bioimaging NO in samples [39]. Moreover, nonfluorescent DCFH can be converted to green fluorescent DCF upon intracellular oxidation as a qualitative marker of cellular oxidative stress to estimate the formation of ROS, such as ·O2−, H2O2 and ONOO− [40]. Therefore, the use of DAR plus DCFH in our technique facilitated simultaneous detection of NO and ROS production. As shown in Fig. 7, strong DCF immunofluorescent staining for ROS was shown in hearts of I/R rats but not in NI hearts, while much weaker DAR-4M fluorescence displaying NO production was detected in I/R hearts compared with healthy ones. Oral administration of GA-BH4 ameliorated eNOS function, as determined by increased cardiac NO production and decreased cardiac ROS generation. The findings also demonstrated the existence of uncoupled eNOS in the endothelium and myocytes.