Zhonghua Shao Shang Za Zhi. 2021 Nov 25;37:1-10. doi: 10.3760/cma.j.cn501120-20210601-00208. Online ahead of print.
ABSTRACT
Objective: To investigate the effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat (hereinafter referred to as burn serum). Methods: The experimental research method was applied. Ten gender equally distributed Wistar rats aged 7-8 months were taken to prepare normal rat serum (hereinafter referred to as normal serum), another twenty gender equally distributed Wistar rats aged 7-8 months were taken to prepare burn serum after full- thickness burn injury of 30% total body surface area, and cardiomyocytes were isolated from 180 Wistar rats aged 1-3 days by either gender and used in the following experiments. The cells were divided into normal serum group and burn serum group according to the random number table (the same grouping method below), and cultured with the corresponding serum. At post culture hour (PCH) 1, 3, 6, 9, and 12, trypanosoma blue exclusion test was used to detect the cell survival rate. The cells were divided into burn serum alone group, burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group, and were treated with burn serum alone or added with the corresponding final molarity of glutamine and cultured with the time point screened in the experiment before, then the cell survival rate was dected as before. then The cells were divided into normal serum alone group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group and treated the same as before. After 30 min of culture, phosphorylation level of mammalian target of rapamycin complex 1 (mTORC1), p70 ribosomal protein S6 kinase (P70 S6k), and eIF4E-binding protein 1 (4E-BP1) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group, and treated correspondingly. At PCH 1, 3, and 6, the expressions of heat shock protein 70 (HSP70) and metallothionein (MT), and the morphology of microtubule were observed with immunofluorescence method. The number samples in each index at each time point in each group were all 10. Data were statistically analyzed with analysis of variance of factorial design, one-way analysis of variance, least significant difference t test, least significant difference test, and Bonferroni correction. Results: At PCH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (t=4.950, 16.752, 35.484, 34.428, 27.781, P<0.01). Compared within group at PCH 1, the cell survival rate was significantly decreased in burn serum group at PCH 3, 6, 9, and 12 (P<0.05). Compared within group at PCH 3, the cell survival rate was significantly decreased in burn serum group at PCH 6, 9, and 12 (P<0.05). Compared within group at PCH 6 and 9, the cell survival rate was significantly decreased in burn serum group at PCH 12 (P<0.05). There were no statistically significant differences in the cell survival rate among burn serum group at PCH 6 and 9 (P>0.05). Thus PCH 6 was selected as the subsequent intervention time of burn serum. At PCH 6, compared with burn serum alone group, the cell survival rates in burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group were significantly increased (P<0.01). There were no statistically significantl differences in cell survival rate in burn serum+12 mmol/L glutamine group and burn serum+16 mmol/L glutamine group (P>0.05). There were no statistically significantl differences in cell survival rate in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P>0.05). Thus 12, 16, and 20 mmol/L were selected as the subsequent intervention concentrations of glutamine. After 30 min of culture, the phosphorylation levels of mTORC1, P70 S6k, and 4E-BP1 of cells were respectively 1.001±0.042, 0.510±0.024, 0.876±0.022, 0.836±0.074, 0.856±0.041, 1.00±0.11, 0.38±0.09, 0.95±0.13, 0.96±0.13, 0.89±0.24, 1.00±0.07, 0.29±0.08, 0.87±0.27, 0.68±0.08, 0.60±0.21 in normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group. Compared with normal serum group, the phosphorylation levels of mTORC1, P70 S6k, and 4E-BP1 of cells were decreased in the other 4 groups (P<0.01). Compared with burn serum alone group, the phosphorylation levels of mTORC1, P70 S6k, and 4E-BP1 of cells were increased in the other 3 groups (P<0.01). The phosphorylation levels of mTORC1, P70 S6k, and 4E-BP1 of cells were similar in burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group (P>0.05). The phosphorylation level of 4E-BP1 of cells in burn serum+12 mmol/L glutamine group was significantly higher than the levels in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P<0.05). At PCH 1, 3, and 6, the expressions of HSP70 and MT of cells in burn serum alone group were significantly higher than those in normal serum group (P<0.01); the protein expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine group were significantly higher than those in burn serum alone group (P<0.05); the protein expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group were significantly lower than those in burn serum+12 mmol/L glutamine group (P<0.05). The microtubular structure was integral, network alinement, stain uniformity in normal serum group at PCH 1, 3, and 6. In burn serum alone group, some microtubules were broken and the grid arrangement was disordered at PCH 1; the microtubule structure near the nucleus was clear, while the microtubule at the distal end of the nucleus was blurred at PCH 3; the microtubule structure was blurred at PCH 6. The microtubular damage of cells was alleviated in burn serum+12 mmol/L glutamine group compared with burn serum alone group. The morphology of microtubule of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group at each time point was similar to that of burn serum alone group. Conclusions: The burn serum can lead to cardiomyocyte damage and cell survival rate decrease in mice. Glutamine can exert cell protective function through regulating the mTOR-P70 S6k-4E-BP1 signaling pathway, thus promoting the expressions of HSP70 and MT and stabilize the microtubule structure.
PMID:34839600 | DOI:10.3760/cma.j.cn501120-20210601-00208
