RESUMO
Objective:To investigate the effect of L-carnitine on calcium oxalate-induced ferroptosis in renal tubular epithelial cells (HK-2).Methods:The effects of calcium oxalate(0, 2, 4 and 8 mmol/L) on the expression of ferroptosis-related protein long chain fatty acyl-CoA synthetase 4 (ACSL4), cystine/glutamate transporter(XCT) and glutathione peroxidase 4 (GPX4) in HK-2 cells were detected by Western blotting. The experiment was then divided into four groups: ①control group, cells were cultured in normal medium for 12 hours, then continued to use normal medium; ②L-carnitine group, cells were pretreated with medium containing 5mmol/L L-carnitine for 12 hours, then changed to medium containing 5mmol/L L-carnitine; ③calcium oxalate group, cells were cultured in normal medium for 12 hours, and then replaced with medium containing 4 mmol/L calcium oxalate; ④calcium oxalate+ L-carnitine group, the cells were pretreated with medium containing 5mmol/L L-carnitine for 12 h, and then replaced with 5mmol/L L-carnitine and 4mmol/L calcium oxalate medium. After changing the culture medium for 24 hours, the cells or supernatants were collected, and the expression levels of ferroptosis-related protein quinone oxidoreductase (NQO1), ACSL4, XCT and GPX4 were detected by Western blotting. The levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde were detected by corresponding kit, and the level of reactive oxygen species in cells was detected by reactive oxygen species kit.Results:The results of Western blotting showed that the expression of ACSL4 protein in 0, 2, 4, 8 mmol/L calcium oxalate was 0.37±0.16, 0.68±0.16, 0.73±0.09, 0.89±0.03 respectively. The expression of XCT protein was 1.11±0.10, 0.91±0.14, 0.83±0.09, 0.80±0.07, respectively. The expression of GPX4 protein was 1.23±0.13, 0.99±0.17, 0.81±0.05, 0.72±0.06, respectively. Compared with 0mmol/L group, the expression of ACSL4 protein increased and the expression of XCT and GPX4 decreased in 2, 4 and 8 mmol/L groups, and the difference was more significant between 4 mmol/L group and 0 mmol/L group. So 4 mmol/L was taken as the optimal concentration for follow-up experiment. The levels of NQO1 in control group, L-carnitine group, calcium oxalate group and calcium oxalate+ L-carnitine group were (0.36±0.06, 0.54±0.05, 0.76±0.07, 0.90±0.03) respectively. There was significant difference between L-carnitine group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of ACSL4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.66±0.10, 0.58±0.08, 0.99±0.03, 0.77±0.09) respectively. There was no significant difference between L-carnitine group and control group(P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of XCT in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.93±0.08, 0.85±0.07, 0.76±0.06, 0.99±0.05). There was no significant difference between L-carnitine group and control group (P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GPX4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (1.10±0.09, 1.09±0.09, 0.85±0.03, 0.99±0.02) respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of LDH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine were (100.00±5.37)%, (99.50±6.38)%, (153.77±6.06)% and (132.50±5.58)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The SOD levels in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±5.79)%, (105.80±3.26)%, (44.74±7.60)% and (85.01±5.15)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GSH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±4.73)%, (107.10±5.48)%, (53.49±3.98)% and (85.18±5.48)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.01). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The levels of MDA in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±2.36)%, (98.00±11.10)%, (129.11±2.59)% and (113.35±5.79)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The fluorescence intensity of ferrous ion in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (39.77±0.68) AU, (68.40±3.14) AU and (48.60±4.30) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The fluorescence intensity of reactive oxygen species in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (63.98±9.41) AU, (145.41±8.39) AU and (85.37±4.51) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). Transmission electron microscopy results showed that mitochondria were wrinkled, cristae were broken or disappeared in the calcium oxalate group compared to the control group, and a double-layer membrane structure was evident. DAPI staining showed that compared with the control group, some of the nuclei in the calcium oxalate group were significantly more damaged, while compared with the calcium oxalate group, the nuclei in the calcium oxalate + L-carnitine were significantly less damaged. The results of crystal adhesion test showed that compared with the control group, calcium oxalate crystals in the calcium oxalate group adhered to the cells in black-like particles and formed clusters. Compared with the calcium oxalate group, the calcium oxalate + L-carnitine showed less black particles adhering to the cells. Conclusions:L-carnitine may reduce the effects of oxidative stress and ferroptosis induced by calcium oxalate, thus reducing cell damage and crystal adhesion.