CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 AFRICA 77 Statistical analysis Data are expressed as mean ± standard error of the mean (SEM). Statistical significance between multiple groups was determined by one-way analysis of variance (ANOVA) followed by the Student Newmanm–Keul post hoc test (Graph Pad lnstat). A value of p < 0.05 was considered significant. Results Cell viability was assessed by trypan blue staining as previously described.31 TNF preconditioning improved cell viability (70.6 ± 6.1 vs 43.7 ± 8.1% for ischaemic control group, p < 0.001). The administration of the NFκB inhibitor PDTC, given during the TNF preconditioning stimulus, abolished the cytoprotective effect of TNF (40.9 ± 2.8%, p < 0.001 vs TNF, Fig. 3A). However, PDTC, given at reperfusion, did not decrease the protective effect of TNF (70.7 ± 1.7%, non-significant vs TNF, p < 0.001 vs ischaemic control, Fig. 3B). PDTC alone had no effect on the cell viability. Cellular oxygen consumption (state-2 respiration) in C2C12 myotubes was measured immediately after the index simulated ischaemia (Fig. 4). Normoxic cells presented a state-2 respiration of 6.3 ± 0.4 nM of oxygen per million of viable cells/min. Following seven hours of simulated ischaemia, state-2 respiration was reduced to 2.5 ± 0.1 nM of oxygen per million of viable cells/min. TNF induced preconditioning significantly improved the state-2 respiration (5.7 ± 0.6 nM of oxygen per million of viable cells/min, p < 0.05 vs ischaemic control). Administration of PDTC during the TNF preconditioning stimulus reduced the oxygen consumption (TNF + PDTC: 3.6 ± 0.3, p < 0.05 vs TNF). Phosphorylation of IkB was measured as an indication of NFκB activation. IkB phosphorylation was measured 15 minutes into the 30-minute TNF preconditioning stimulus (Fig. 2). Preconditioning with TNF increased the phosphorylation of IkB (1.5 ± 0.2 arbitrary units for TNF vs 1.0 ± 0.0 for ischaemic control, p < 0.05) and was abolished when TNF was incubated in the presence of PDTC (0.8 ± 0.3 vs TNF, p < 0.01). Discussion The aim of our study was to explore the role of NFκB as a key component to promote cell survival in TNF-induced cytoprotection. Our findings of TNF-induced cytoprotection are in line with previous studies in isolated cells,32 perfused hearts14 and whole animals.12 Furthermore, we showed that inhibition of NFκB abrogates the protection induced by TNF. Although high doses of TNF promoted apoptosis via the activation of the pro-apoptotic factor PHLPP1 via NFκB in neonatal cardiomyocytes,10 our study demonstrated that lower cytoprotective doses of TNF promoted NFκB activation, which is required for TNF-mediated protection against simulated ischaemia–reperfusion. Our study is in agreement with other studies that found that NFκB activation is required for cytoprotection.16,18,19,33,34 While some of these studies coincide with our study in demonstrating that NFκB activation before an ischaemic insult is required CTL TNF TNF + PDTC PDTC % Cell death 80 70 60 50 40 30 20 10 0 *** ### CTL TNF TNF + PDTC PDTC % Cell death 80 70 60 50 40 30 20 10 0 *** *** Fig. 3. Effect of NFκB inhibitor (PDTC) on cell viability in TNF-induced preconditioning. A. Addition of PDTC (100 µM) during the TNF preconditioning stimulus abolished the protective effect of TNF. ***p < 0.001 vs ischaemic control group (CTL); ###p < 0.001 vs TNF; n = 9 per group. B. Addition of PDTC (100 µM) at reperfusion failed to abolish the protective effect of TNF. ***p < 0.001 vs CTL; n ≥ 10 per group. CTL TNF TNF + PDTC Oxygen consumption (mM/oxygen per million cells) 7 6 5 4 3 2 1 0 * # Fig. 4. Effect of NFκB inhibitor (PDTC) given during the TNF preconditioning stimulus on the oxygen consumption in C2C12 myotubes. PDTC (100 µM) abolished the increase of state-2 respiration induced by TNF preconditioning (TNF). *p < 0.05 vs ischaemic control group; #p < 0.05 vs TNF; n ≥ 6. A B
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