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.

Objective:To investigate the effect and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats (hereinafter referred to as burn serum).Methods:Experimental research methods were adopted. Thirty gender equally balanced Wistar rats aged 7 to 8 weeks were collected, 10 of which were used to prepare normal rat serum (hereinafter referred to as normal serum), and the other 20 were inflicted with full-thickness burn of 30% total body surface area to prepare burn serum. Primary cardiomyocytes were isolated and cultured from the apical tissue of 180 Wistar rats aged 1 to 3 days by either gender for follow-up experiments. Cells were divided into normal serum group and burn serum group treated with corresponding serum according to the random number table (the same grouping method below). Trypanosoma blue staining was performed at post treatment hour (PTH) 1, 3, 6, 9, and 12 to detect the cell survival rate. Cells were divided into burn serum alone group treated with burn serum for 6 h followed by routine culture of 30 min and 0.4 mmol/L glycine group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, 1.6 mmol/L glycine group, and 2.0 mmol/L glycine group treated with burn serum for 6 h followed by culture of 30 min with corresponding final molarity of glycine, i.e., at post intervention hour (PIH) 6.5, the cell survival rate was detected as before. Cells were divided into normal serum group, burn serum alone group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, and 1.6 mmol/L glycine group, with the same intervention of 6.5 h as before, respectively. The content of adenosine monophosphate (AMP) and adenosine triphosphate (ATP) was detected by high performance liquid chromatography, and the AMP/ATP ratio was calculated. The protein expressions of phosphorylated mammalian target of rapamycin complex 1 (p-mTORC1), phosphorylated p70 ribosomal protein S6 kinase (p-p70 S6K), phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1), and phosphorylated AMP-activated protein kinase (p-AMPK) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, 0.8 mmol/L glycine group intervened as before and 0.8 mmol/L glycine+25 ng/mL rapamycin group treated with burn serum followed by culture with two reagents. The expressions of heat shock protein 70 (HSP70), metallothionein (MT), and tubulin were detected by immunofluorescence method after 30 min of corresponding culture at PTH 1, 3, and 6, i.e., at PIH 1.5, 3.5, and 6.5, and the microtubule morphology was observed at PIH 6.5. The sample number at each time point was 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference (LSD)- t test, LSD test, and Bonferroni correction. Results:At PTH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (with t values of 4.96, 16.83, 35.51, 34.33, and 27.88, P<0.05). In burn serum group, the cell survival rate at PTH 3, 6, 9, or 12 was significantly lower than that at PTH 1 ( P<0.05), the cell survival rate at PTH 6, 9, or 12 was significantly lower than that at PTH 3 ( P<0.05), and the cell survival rate at PTH 6 was similar to that at PTH 9 ( P>0.05) but significantly higher than that at PTH 12 ( P<0.05). Treatment of 6 h was selected as the follow-up intervention time of burn serum. At PIH 6.5, compared with that in burn serum alone group, the cell survival rate in each glycine group was significantly increased ( P<0.05). The cell survival rate in 0.8 mmol/L glycine group was the highest, and 0.8, 1.2, and 1.6 mmol/L were selected as subsequent glycine intervention concentrations. At PIH 6.5, the AMP/ATP ratio of cells in burn serum alone group was significantly higher than that in normal serum group, 1.2 mmol/L glycine group, or 1.6 mmol/L glycine group ( P values all <0.05), and the AMP/ATP ratio of cells in 1.6 mmol/L glycine group was significantly lower than that in 0.8 mmol/L glycine group ( P<0.05). At PIH 6.5, the protein expressions of p-mTORC1, p-p70 S6K, and p-4E-BP1 of cells in normal serum group, burn serum alone group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, and 1.6 mmol/L glycine group were 1.001±0.037, 0.368±0.020, 1.153±0.019, 1.128±0.062, 1.028±0.037, 0.96±0.07, 0.63±0.12, 1.17±0.13, 1.13±0.16, 1.11±0.11, and 0.98±0.06, 0.45±0.08, 1.13±0.05, 0.77±0.12, 0.51±0.13. Compared with those in burn serum alone group, the protein expressions of p-mTORC1, p-p70 S6K, and p-4E-BP1 of cells in normal serum group and each glycine group were significantly increased ( P<0.05), while the protein expressions of p-AMPK were significantly decreased ( P<0.05). Compared with those in 0.8 mmol/L glycine group, the protein expression of p-4E-BP1 of cells in 1.2 mmol/L glycine group and the protein expressions of p-mTORC1 and p-4E-BP1 of cells in 1.6 mmol/L glycine group were significantly decreased ( P<0.05). Compared with those in 1.2 mmol/L glycine group, the protein expressions of p-mTORC1 and p-4E-BP1 of cells in 1.6 mmol/L glycine group were significantly decreased ( P<0.05), while the protein expression of p-AMPK was significantly increased ( P<0.05). Compared with those in normal serum group, the expression of tubulin of cells in burn serum alone group was significantly decreased at PIH 1.5, 3.5, and 6.5 ( P<0.05), while the expression of HSP70 of cells at PIH 1.5 and 3.5 and the expression of MT at PIH 3.5 and 6.5 were significantly increased ( P<0.05). The expressions of HSP70 and MT of cells at PIH 1.5, 3.5, and 6.5 and the expression of tubulin at PIH 1.5 and 3.5 in burn serum alone group and 0.8 mmol/L glycine+25 ng/mL rapamycin group were significantly lower than those in 0.8 mmol/L glycine group ( P<0.05). At PIH 6.5, compared with that in normal serum group, the cell microtubule structure in burn serum alone group was disordered; the cell boundary in 0.8 mmol/L glycine group was clearer than that in burn serum alone group, and the microtubule structure arranged neatly near the nucleus. Compared with that in 0.8 mmol/L glycine group, 0.8 mmol/L glycine+25 ng/mL rapamycin group had unclear cell boundaries and disordered microtubule structure. Conclusions:Burn serum can cause cardiomyocytes damage in rats. Glycine can significantly up-regulate mammalian target of rapamycin/p70 ribosomal protein S6 kinase/eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway through AMP-activated protein kinase, promote the synthesis of protective proteins HSP70, MT, and tubulin, stabilize the microtubule structure, and exert cardiomyocytes protection function.