ABSTRACT
Objective:To investigate the protective effect of insulin on burn serum-challenged cardiomyocytes in vitro. Methods:Primary culture of cardiomyocytes from Sprague-Dawley (SD) 2-day-old neonate rats were divided into Sham group, burn group, insulin group, and insulin activation inhibitor LY294002 pretreatment group (LY group). The model of cardiomyocytes injury induced by burn serum of 3-month-old SD rats [the serum of abdominal aortic was collected at 6 hours after modelling 30% total surface area (TBSA) Ⅲ degree scald rat] was reproduced. In the insulin group, 10% burn serum and insulin (10 U/L) were added into cell culture medium, and in the LY group, LY294002 (50 μmol/L) was pretreated for 30 minutes before the addition of burn serum and insulin. Sham group was only given 10% serum of sham injured rats (sham rats were only placed in 37 ℃ warm water). After the cells were cultured for 12 hours, the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and creatine kinase (CK) were determined by enzyme-linked immunosorbent assay (ELISA). The cardiac troponin T (cTnT) protein expression was examined by Western Blot. Apoptosis of cardiomyocytes was observed after Hoechst 33258 staining.Results:Compared with the Sham group, the cardiomyocytes were damaged and released inflammatory cytokines after burn serum-challenged. The levels of TNF-α, IL-6 and CK increased [TNF-α (ng/L): 273±48 vs. 21±6, IL-6 (ng/L): 416±83 vs. 44±11, CK (U/L): 1.44±0.24 vs. 0.14±0.08, all P < 0.01], while the expression of cTnT protein decreased (cTnT/β-actin: 0.12±0.04 vs. 0.86±0.34, P < 0.01), and the cardiomyocyte apoptosis increased [(19.1±5.6)% vs. (5.2±1.3)%, P < 0.01]. Insulin could significantly reduce the damage of cardiomyocytes, decrease the release of TNF-α, IL-6 and CK induced by burn serum [TNF-α (ng/L): 105±37 vs. 273±48, IL-6 (ng/L): 176±77 vs. 416±83, CK (U/L): 0.82±0.26 vs. 1.44±0.24, all P < 0.05], the expression of cTnT protein significantly increased (cTnT/β-actin: 0.41±0.16 vs. 0.12±0.04, P < 0.05), and the cells apoptosis rate significantly decreased [(10.7±3.2)% vs. (19.1±5.6)%, P < 0.05]. Further blocking experiments showed that LY294002 could mitigate the protective effects of insulin. Conclusion:For cardiomyocytes challenged by burn serum, insulin may decrease inflammation, apoptosis and then protect the cardiomyocytes.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of activating silent information regulator 1 (SIRT1) on early myocardial damage in severely burned rats.</p><p><b>METHODS</b>Twenty-four healthy male SD rats were divided into sham injury group (SI), scald group (S), and resveratrol (RSV) treatment group (RT) according to the random number table, with 8 rats in each group. Rats in groups S and RT were inflicted with 30% TBSA full-thickness scald on the back by immersing in 95 °C water for 18 s. Immediately after injury, rats in group S were intraperitoneally injected with 10 mL normal saline (50 mL/kg) and those in group RT with 10 mL normal saline (50 mL/kg)+10 µL RSV in the concentration of 1 g/mL (50 mg/kg). Backs of rats in group SI were immersed in 20 °C room temperature water for 18 s to simulate the scald process. Heart tissues and aorta abdominalis blood samples were collected at post injury hour (PIH) 6. The histomorphology of heart tissues was observed with HE staining. The serum contents of creatine kinase (CK) and lactate dehydrogenase (LDH) were determined with ELISA. The protein expressions of SIRT1 and caspase-3 and mRNA expressions of SIRT1, caspase-3, IL-1β, and TNF-α in heart tissue specimens were determined with Western blotting and real-time fluorescent quantitative RT-PCR (with protein level denoted as gray value). Data were processed with one-way analysis of variance and LSD- t test.</p><p><b>RESULTS</b>(1) In group SI, myocardial fibers were in irregularly cylindrical shape, neatly arranged, and the transverse striation were distinct. In group S, myocardial interstitial edema, disorder of myocardial fiber arrangement, and cytoplasm destruction were observed. In group RT, the degrees of myocardial interstitial edema, disorder of myocardial fiber arrangement, and cytoplasm destruction were alleviated in comparison with those of group S. (2) The serum contents of CK and LDH of rats in group S were respectively (2 385 ± 712) and (2 551 ± 196) U/L, which were significantly higher than those in the group SI [(290 ± 59) and (759 ± 60) U/L, with t values respectively 9.466 and 25.452, P values below 0.01]. The serum contents of CK and LDH of rats in group RT were respectively (1 336 ± 149) and (2 209 ± 133) U/L, which were significantly lower than those of group S (with t values respectively -4.506 and -4.860, P values below 0.01). (3) The protein expressions of SIRT1 and caspase-3 in heart tissue of rats in group S were respectively 0.47 ± 0.11 and 0.48 ± 0.12, which were significantly higher than those in group SI [0.18 ± 0.06 and 0.09 ± 0.05, with t values respectively 4.813 and 9.014, P values below 0.01]. The protein expression of SIRT1 in heart tissue of rats in group RT was 0.74 ± 0.18, which was significantly higher than that of group S (t = 4.561, P < 0.01); the protein expression of caspase-3 in heart tissue of rats in group RT was 0.21 ± 0.08, which was significantly lower than that of group S (t = -6.239, P < 0.01). (4) The mRNA expressions of SIRT1, caspase-3, IL-1β, and TNF-α in heart tissue of rats in group S were respectively 2.33 ± 0.24, 1.96 ± 0.20, 2.46 ± 0.21, 1.89 ± 0.37, which were significantly higher than those in group SI (1.00 ± 0.07, 1.00 ± 0.06, 1.00 ± 0.08, 1.00 ± 0.09, with t values respectively 14.961, 12.823, 18.559, 6.679, P values below 0.01). The mRNA expression of SIRT1 in heart tissue of rats in group RT was 2.89 ± 0.31, which was significantly higher than that of group S (t = 3.997, P < 0.01). The mRNA expressions of caspase-3, IL-1β, and TNF-α in heart tissue of rats in group RT were respectively 1.31 ± 0.08, 1.64 ± 0.09, 1.25 ± 0.08, which were significantly lower than those of group S (with t values respectively -8.264, -10.245, -4.818, P values below 0.01).</p><p><b>CONCLUSIONS</b>The expression of SIRT1 in heart tissue is upregulated in the early stage of severely burned rats. Activation of SIRT1 by RSV can alleviate myocardial tissue injury and reduce apoptosis of cardiac myocytes and secretion of IL-1β and TNF-α.</p>