Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat
10.3760/cma.j.cn501120-20210601-00208
- VernacularTitle:谷氨酰胺对经烧伤大鼠血清干预的大鼠心肌细胞的作用及其细胞信号机制
- Author:
Shangjun LYU
1
;
Ronghui FAN
;
Dan WU
;
Xi PENG
Author Information
1. 武警陕西省总队医院烧伤整形科，西安 710086
- Keywords:
Burns;
Glutamine;
Myocytes, cardiac;
Mammalian target of rapamycin signaling pathway
- From:
Chinese Journal of Burns
2021;37(12):1149-1157
- CountryChina
- Language:Chinese
-
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 primary cardiomyocytes were isolated and cultured 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 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, which were treated with burn serum alone or burn serum added with the corresponding final molarity of glutamine and cultured for the time screened in the experiment before, and then the cell survival rate was detected as before. The cells were divided into 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 and treated the same as before. After 30 min of culture, phosphorylation levels of mammalian target of rapamycin complex 1 (mTORC1), p70 ribosomal protein S6 kinase (p70 S6K), and eukaryotic translation initiation factor 4E-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 determined with immunofluorescence method. The sample numbers in each index at each time point in each group were all 10. Data were statistically analyzed with analysis of variance for 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 the 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 the 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 the 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 rates in burn serum group between 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 significant differences in cell survival rates between burn serum+12 mmol/L glutamine group and burn serum+16 mmol/L glutamine group ( P>0.05). There were no statistically significant differences in cell survival rates 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 significantly decreased in the other 4 burn serum groups ( P<0.01). Compared with those of burn serum alone group, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were significantly increased in the other 3 burn serum groups ( P<0.01). 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). The expression of MT of cells in burn serum alone group was significantly lower than that in normal serum group at PCH 1 ( P<0.05), while the expressions of MT of cells in burn serum alone group were significantly higher than those in normal serum group at the other time points ( P<0.05). At PCH 1, 3, and 6, the expressions of HSP70 of cells in burn serum alone group were significantly higher than those in normal serum group ( P<0.05), the 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), and the 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<0.01). The microtubular structures were intact, displaying grid alinement and uniform staining in cells of normal serum group at PCH 1, 3, and 6. In burn serum alone group, some microtubules showed fracture and irregular grid arrangement at PCH 1; the microtubular structures near the nucleus were clear, while the microtubules at the distal end of the nucleus were blurry at PCH 3; the microtubular structures were blurry at PCH 6. The microtubular damage of cells was alleviated in burn serum+12 mmol/L glutamine group as compared with that in burn serum alone group at each time point of culture. The morphology of microtubules of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group at each time point of culture was similar to that of burn serum alone group. Conclusions:The burn serum can lead to damages to cardiomyocytes and significant decrease of cell survival rate in rats. Glutamine can exert cell protective function through the regulation of mTOR/p70 S6K/4E-BP1 signaling pathway, thus promoting the expressions of HSP70 and MT and stabilizing the microtubule structures.
