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Effect of high salt diet on vascular production of nitric oxide (NO) and reactive oxygen species (ROS) in Tff3−/−/C57BL/6N knockout mice

Introduction: Tff3 gene knockout mice (Tff3−/ −/C57BL/6N) have changes in lipid metabolism which may affect vascular function. This study aimed to asses the effect of high salt (HS) diet on vascular production of NO and ROS in carotid arteries of Tff3-/- knockout mice and their wild type controls (WT, C57BL/6N). Methods: Male, ten-weeks-old transgenic Tff3−/ −/C57BL/6N and WT/C57BL/6N (parental strain) healthy mice were divided in LS (0.4% NaCl) and HS (4% NaCl in rodent chow fed for 1 week) groups, water ad libidum. After anaesthesia (ketamine- chloride and midazolam), mice were decapitated and carotid arteries were isolated and cannulated on pressure myograph with (∆100 mmHg) or without flow (∆0 mmHg), in the absence/presence of the nitric oxide synthase (NOS) inhibitor L-NAME (10–4M) and superoxide dismutase mimetic TEMPOL (100 μmol l−1). NO production was determined by 4, 5- diaminofluorescein (DAF-2DA, 5 μM) while ROS production was determined by dihydroethidine (DHE, 20 μM) fluorescence assay. Statistical analyses were performed with One-way ANOVA test ; p<0.05 was considered significant. All experimental procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (directive 86/609) and were approved by local and national Ethical Committee (No.2158/61- 02-139/2-06 ; No.2158/61-07-14-119). Results: Basal (no-flow) production of NO was significantly decreased in WT HS mice compared to WT LS mice. Other groups exhibited similar levels of NO production under no-flow conditions. L-NAME significantly decreased NO production in all groups, except in Tff3−/− HS mice. Under flow conditions, NO production in WT HS mice was significantly lower than in WT LS mice. NO production in Tff3−/− LS knockout mice was significantly lower than in WT LS mice. NO production in Tff3−/− HS mice was similar to Tff3−/− LS mice. L-NAME significantly decreased NO production in all groups of mice, except in Tff3−/ − HS mice. Basal (no-flow) ROS production was significantly increased in WT HS compared to the WT LS group and in Tff3−/− HS compared to Tff3−/− LS group. Tempol significantly decreased ROS production in WT HS and Tff3−/− HS groups. Under flow conditions, ROS production was significantly increased in WT HS compared to WT LS group and in Tff3−/− HS compared to Tff3−/− LS group. Tempol significantly decreased ROS production in all groups. Conclusion: HS diet significantly decreased the flow-induced NO production in WT mice and increases generation of ROS in both strains. Key words: Tff3 gene, flow-induced dilation, oxidative stress, high salt diet Acknowledgements: This study was supported by the Croatian Science Foundation under the project IP- 2014-09-6380 (V-ELI Athero), VIF-2018-MEFOS-09- 1509 grant and Faculty of Medicine Osijek Institutional grant #IP-1-MEFOS2019 and #IP-2- MEFOS2020 (PI Ines Drenjančević).

Tff3 gene, flow-induced dilation, oxidative stress, high salt diet

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